Infektionsimmunologie 2013 (Virologie-Teil)

Infektionsimmunologie 2013 (Virologie-Teil) Mit Blauzungenvirus (BTV) und Schmallenbergvirus (SBV) haben in den letzten Jahren die Vektor-übertragenen Viren bei uns überproportional an Bedeutung gewonnen. Als wahrscheinlich nächster Kandidat steht das West-Nil-Virus (WNV) vor den Pforten. Wir wollen uns mit diesem Thema vertieft befassen.

Aufgaben/Ziele • Jede Gruppe soll die Unterlagen konsultieren um je eine der Aufgaben (1 bis 12) kompetent lösen und darstellen zu können. • Jede Gruppe soll ein Poster erstellen, welches dazu dient, den erfragten Sachverhalt den anderen Gruppen zu erklären. Zu jedem Poster ist zudem ein Handout zu erstellen, das den Kolleg/innen eine sorgfältige Vorbereitung auf die Testatprüfung erlaubt. • Jeder Teilnehmer soll am Ende der Veranstaltung alle Aufgaben kompetent lösen können.

Aufgabenkatalog

1. West-Nil-Virus, innen und aussen Beschreiben sie das West-Nil-Virus: Morphologie, Genom, Replikation, wichtigste Proteine und ihre Funktion. Erklären sie anhand dieser Grundlagen die Diagnostik für WNV. Erläutern sie Begriffe, die im Zusammenhang wichtig sind. (Kramer et al., 2008; Pesko and Ebel, 2012) 2. Typen des West-Nil-Virus Wie unterscheiden sich die verschiedenen Typen und Linien des West-Nil-Virus und welche Bedeutung kommt den Unterschieden zu? (Bakonyi et al., 2006; Kramer et al., 2008; Pesko and Ebel, 2012) 3. Unterschiede und Gemeinsamkeiten Welche Unterschiede und Gemeinsamkeiten haben die West-Nil-Viren mit anderen Viren, z.B. FSMEV, BTV und SBV? Welche Relevanz hat dies für die Übertragung, Pathogenese, Impfung? (Colpitts et al., 2012; Kramer et al., 2008; Porträts WNV_FSME_BTV_SBV) 4. West-Nil-Virus im Vergleich betroffener Tierarten Welche Typen und Subtypen kommen vor? Welche klinischen Symptome werden beobachtet? Womit begründen sich die unterschiedlichen Symptome bei den einzelnen Tierarten? Vermehrt sich das Virus in unterschiedlichen Organen? Wie wirken sich immunologische Faktoren auf Reservoirbildung und Übertragung aus? (Hubalek and Halouzka, 1999; Jeffrey Root, 2013; Kramer et al., 2008; Kwan et al., 2012) 5. West-Nil-Virus und sein(e) Vektor(en) Welche Vektoren gibt es? Biologie und Einteilung der Vektoren? Wie vermehrt und verbreitet sich der Vektor? Wie entsteht aus dem Vektor ein Reservoir? Wie kann man den Vektor bekämpfen (theoretische und praktische Ansätze)? (Andreadis, 2012; Colpitts et al., 2012; Kramer et al., 2008) 6. Vermehrung des West-Nil-Virus im Vektor In welchen Organen vermehrt sich das Virus? Wie wird es ausgeschieden und übertragen? Welche Virusmengen sind nötig für die Vermehrung im Vektor? Wieviel Virus gibt ein Vektor bei der Übertragung ab? (Colpitts et al., 2012; Hubalek and Halouzka, 1999; Kramer et al., 2008; Kwan et al., 2012) 7. Globale Epidemiologie des West-Nil-Virus Wie hat sich die Epidemiologie von WNV in den letzten Jahrzehnten entwickelt? Fallzahlen, Prävalenz, Inzidenz, Letalität der verschiedenen Krankheitsformen? Durch welche Faktoren

wird die Epidemiologie beeinflusst? Unter welchen Voraussetzungen könnte sich das West- Nil-Virus in der Schweiz festsetzen? (Hubalek and Halouzka, 1999; Kramer et al., 2008) 8. Pathogenese des West-Nil-Virus auf Ebene Organismus Wie breitet sich das Virus im Organismus aus und wie wird es wieder ausgeschieden? Welche Verlaufsformen kommen vor? Welche Faktoren sind wichtig für die Ausprägung der einzelnen Verlaufsform? Welche Faktoren sind relevant für die Übertragung? (Colpitts et al., 2012; Kramer et al., 2008; Pesko and Ebel, 2012; Pradier et al., 2012) 9. Pathogenese des West-Nil-Virus auf zellulärer und molekularer Ebene Pathogenese: beteiligte Virusproteine, Zellen und Moleküle? Welche Rolle spielt die Immunität? (Cho and Diamond, 2012; Colpitts et al., 2012; Diamond and Gale, 2012; Kramer et al., 2008; Leis and Stokic, 2012; Pesko and Ebel, 2012; Pradier et al., 2012) 10. Impfstoffe gegen West-Nil-Virus Was für Impfstoffe stehen weltweit zur Verfügung; was für welche in der Schweiz? Was sind die Merkmale dieser Impfstoffe? Was können sie? Was können sie nicht? (De Filette et al., 2012; Kramer et al., 2008) 11. Antikörper und zelluläre Immunität gegen das West-Nil-Virus Wie entstehen sie; was bewirken sie; welche Virusproteine sind involviert? Relevanz bezüglich Schutz? Welche Antikörperklassen und –funktionen werden beobachtet; wären erwünscht? (Cho and Diamond, 2012; De Filette et al., 2012; Diamond et al., 2003; Kramer et al., 2008; Kwan et al., 2012) 12. Intrinsische und innate Abwehr gegen West-Nil-Virus Rolle und Potential im Haustier/Mensch bzw. Vektor. Positive Aspekte; negative Aspekte; Rolle in der Immunität und in der Pathogenese? (Diamond and Gale, 2012; Diamond et al., 2003; Kramer et al., 2008)

Zeitplan Wann Was Wo Mi 2. Oktober 8- 10 Uhr Einführung GHS - Gruppeneinteilung - Unterlagen - Aufgabenbesprechung - Downloaden und Sichtung der Unterlagen Fr 4. Oktober 8-10 Uhr - Studium der Unterlagen AHS - Fragen an den Tutor Mo 7. Oktober 13-15 Uhr Poster erstellen AHS Di 8. Oktober 10-12 Uhr Poster diskutieren Mikroskopiehörsaal Diagnostikzentrum

Poster • Das Poster soll die wichtigsten Sachverhalte zur Lösung der Aufgabe enthalten. • Die Darstellung soll so erfolgen, dass alle Informationen auch aus einer Entfernung von 4 bis 5 Metern aufgenommen werden können (Grösse und Dicke der Schrift bzw. der Zeichnungen). • Handschriftliche Darstellungen und eigene Zeichnungen sind Computer-Darstellungen bei Weitem vorzuziehen. Ausnahmen sind möglich, bedürfen aber einer hinreichenden Begründung. • Für die Poster-Produktion werden Packpapier und Buntstifte zur Verfügung gestellt. • Häufig ist es nützlich, das Poster mit einem Handout zu begleiten. • Jedes Gruppenmitglied muss in der Lage sein, sein Poster zu erklären.

Bibliografie Andreadis, T.G., 2012, The contribution of Culex pipiens complex mosquitoes to transmission and persistence of West Nile virus in North America. J Am Mosq Control Assoc 28, 137-151. Bakonyi, T., Ivanics, E., Erdelyi, K., Ursu, K., Ferenczi, E., Weissenbock, H., Nowotny, N., 2006, Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerg Infect Dis 12, 618-623. Cho, H., Diamond, M.S., 2012, Immune responses to West Nile virus infection in the central nervous system. Viruses 4, 3812-3830. Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 635-648. De Filette, M., Ulbert, S., Diamond, M., Sanders, N.N., 2012, Recent progress in West Nile virus diagnosis and vaccination. Vet Res 43, 16. Diamond, M.S., Gale, M., Jr., 2012, Cell-intrinsic innate immune control of West Nile virus infection. Trends Immunol 33, 522-530. Diamond, M.S., Shrestha, B., Mehlhop, E., Sitati, E., Engle, M., 2003, Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus. Viral Immunol 16, 259-278. Hubalek, Z., Halouzka, J., 1999, West Nile fever--a reemerging mosquito-borne viral disease in Europe. Emerg Infect Dis 5, 643-650. Jeffrey Root, J., 2013, West Nile virus associations in wild mammals: a synthesis. Arch Virol 158, 735-752. Kramer, L.D., Styer, L.M., Ebel, G.D., 2008, A global perspective on the epidemiology of West Nile virus. Annu Rev Entomol 53, 61-81. Kwan, J.L., Kluh, S., Reisen, W.K., 2012, Antecedent avian immunity limits tangential transmission of West Nile virus to humans. PLoS One 7, e34127. Leis, A.A., Stokic, D.S., 2012, Neuromuscular manifestations of west nile virus infection. Front Neurol 3, 37. Pesko, K.N., Ebel, G.D., 2012, West Nile virus population genetics and evolution. Infect Genet Evol 12, 181-190. Pradier, S., Lecollinet, S., Leblond, A., 2012, West Nile virus epidemiology and factors triggering change in its distribution in Europe. Rev Sci Tech 31, 829- 844.

West Nile Virus

Taxonomie Familie: Flaviviridae Genus & Bsp. für Krankheiten: Flavivirus West Nile Fieber Dengue Fieber FSME Gelbfieber Pestivirus Klassische Schweinepest Bovine Virus Diarrhoe Border Disease Hepatitis C Virus Hepatitis1

Morphologie & Genom Das West Nile Virus (WNV) ist ein behülltes Virus mit einem Durchmesser von 40-60nm und einem ikosaedrischen Nukleokapsid. Es enthält eine positiv-einzelsträngige RNA mit ca. 11‘000 Nukleotiden. Das Genom ist 9.5 bis 12kb lang. Es gibt 3 Strukturproteine und 7 Nichtstrukturproteine. Das Genom ist vom Kapsidprotein C umgeben, um das noch eine Hülle mit den eingelagerten M- und E-Proteinen ist. Das E-Protein liegt dabei als Dimer vor, das M-Protein als Monomer.2 3

Replikation Nachdem das Virus via Glykoprotein E an den zellulären Rezeptor gebunden hat, wird es mittels Endozytose in die Zelle aufgenommen. Es kommt zur Fusion der Virushülle mit der Membran des Vesikels und die genomische RNA wird ins Zytoplasma entlassen. Die (+)ssRNA wird in ein Polyprotein translatiert und in die zehn Proteine gespalten. Die Replikation findet in der Nähe der ER-Membran statt. Zuerst wird die komplementäre (-)ssRNA synthetisiert, welche als Vorlage für die Synthese neuer (+)ssRNA dient. Das Assembly findet an den ER- Membranen statt und das Virus gelangt intrazysternal via Golgi-Apparat an die Zellmembran, wo die neuen Viruspartikel durch Knospung freigesetzt werden.4 5

Wichtige Proteine Bei der Replikation des West-Nile-Virus-Genoms entsteht ein Polyprotein, welches bereits während und nach der Translation von viralen so wie von wirtseigenen Proteasen gespalten wird. Dabei entstehen: • 3 Strukturproteine, welche nötig sind für den Virus-Eintritt in die Wirtszelle (Entry & Fusion), sowie auch für die Kapsidierung des Virus-Genoms während dem Assembly - Protein E à Hüllenprotein, involviert in Rezeptorbindung, Fusion zw. Wirtszell- und Virus- membran und Virusassembly, u.A. auch für Neurovirulenz zuständig - Protein prM à Transmembranprotein, wichtig für korrekte Faltung & Funktionalität des Protein E - Protein C à Kapsidprotein; es wird eine genregulierende Funktion des Kapsides vermutet (mittels Bindung an Histone) • 7 Nichtstrukturproteine, welche unterschiedliche Funktionen ausüben - NS1 à sehr immunogen - NS3 à wirkt u.A. als virale Protease, welche andere Nichtstrukturproteine vom Polyprotein abspaltet - NS5 à fungiert als virale Polymerase - NS2A/ NS2B/ à hemmen eine oder mehrere Komponenten des nativen NS4A/ NS4B Immunsystems6 7

Diagnostik Die Inkubationsperiode bei einer Infektion mit dem WNV beträgt ungefähr 2 bis 14 Tage. Falls die klinischen Symptome eines Tieres bzw. des Menschen für WNV sprechen, sollte man das Virus durch eine Labordiagnose bestätigen.

Beispiele für Nukleinsäure-basierte Nachweisverfahren für das WNV: Probe der Wahl, um das Virus nachzuweisen, sind Blut oder Zerebrospinalflüssigkeit. Zu beachten ist, dass sich das Virus nur während den ersten Tagen isolieren lässt. Aufgrund der geringen Virusmengen im menschlichen Untersuchungsmaterial findet zunächst eine in vitro Amplifikation des genetischen Materials statt, um die Erkennungsrate einer WNV-Infektion zu erhöhen.8 Für den Nachweis des WNV auf der Basis von Nukleinsäuren gibt es verschiedene Methoden, wie beispielsweise: - Reverse Transcriptase-PCR (RT-PCR): schnelles Nachweisverfahren vom Erregergenom bei WNV- Infektionen. Es basiert auf der Vervielfachung einer DNA-Sequenz mittels DNA-Polymerase. Wobei in diesem Fall, da das Ausgangsmaterial RNA ist, diese mittels Reverser Transcriptase zuerst in DNA transkribiert werden muss.9 10 - TaqMan: Möglichkeit, während der PCR, gezielt nur das gewünschte DNA-Produkt nachzuweisen mittels TaqMan-Sonden (kurze DNA-Stücke, die mit einem mittleren Bereich der Template-DNA hybridisieren). Die TaqMan-Sonden tragen am einen Ende einen Reporterfarbstoff (R) und am anderen einen Quencher (Q), der die Fluoreszenz aus der Umgebung abfängt. Bei der Replikation, wird die TaqMan-Sonde abgebaut und somit der Reporterfarbstoff freigesetzt. Der Farbstoff gelangt so aus dem Einflussbereich des Quenchers und ist deshalb nur dann nachweisbar, wenn die Polymerase den gewünschten Strang kopiert hat.11

Beispiele für serologische AK-Nachweisverfahren von Infektionen mit WNV: Der Verdacht auf WNV erhärtet sich bei mind. 4fachem AK-Titeranstieg zwischen Akutphase und Rekonvaleszenz. Ein geeigneter Test ist z.B. ein ELISA. In der Zerebrospinalflüssigkeit des betroffenen Tieres wird nach der Anwesenheit von IgM Antikörpern gegen das WNV gesucht.12 IgM Antikörper können innerhalb von 4-7 Tagen nach Exposition nachgewiesen werden und länger als ein Jahr persistieren. Der Nutzen von IgG Antikörpern hingegen ist limitiert.13 - ELISA (indirekter ELISA): für den AK-Nachweis wird spezifisches AG an eine feste Oberfläche gekoppelt (z.B. Mikrotiterplatte). Bei der Zugabe von dem zu testenden Serum, binden die AK, falls vorhanden, an das AG auf der Platte. Anschliessend erfolgt die Zugabe von Sekundär-AK, die an die gesuchten AK binden und das nicht gebundene Material wird abgewaschen. An den Sekundär-AK ist ein Enzym gekoppelt, so dass es bei der Inkubation mit einem bestimmten Substrat zu einem Farbumschlag kommt.14 - Plaque Reduction Neutralizations Test (PRNT): zur Differenzierung zwischen dem WNV und einem anderen, eng verwandten Flavivirus (z.B. St. Louis Encephalitis Virus, Japanisches Encephalitis Virus) Mathias Ackermann 21.10.13 14:59 nach einem Nachweis im ELISA. Er wird gebraucht, wenn der Verdacht besteht, dass es zu einer Kommentar [1]: Unter den Kreuzreaktion zwischen dem WNV und einem anderen Flavivirus gekommen ist.15 beschriebenen Bedingungen ist der PRNT Bei diesem Test wird das Serum eines Patienten, in dem AK nachgewiesen werden sollen, mit Viren nicht durchführbar. Was hier beschrieben wird, entspricht dem Virus-Neutralisations- inkubiert und das Gemisch zu einer Zellkultur gegeben. Wenn nur die Viren und keine AK auf der Test (ergibt letztendlich sehr ähnliche Zellkultur sind, kommt es zu einer Zellzerstörung (cytopathischer Effekt). Sind aber neutralisierende AK Resultate). im Serum, können die Viren nicht mehr in die Zellen eindringen und es kommt zu keiner bzw. weniger Zerstörung. Beim PRNT wird nach einer bestimmten Inkubationszeit anhand der Zählung der Plaques Mathias Ackermann 21.10.13 14:59 (Regionen mit infizierten Zellen) die AK-Menge im Serum gemessen. Der AK-Titer wird bestimmt, indem Kommentar [2]: Für den Plaque- das Patientenserum mehr und mehr verdünnt wird. Als Titer wird unter der Bezeichnung PRNT50 die Reduktions-Test braucht es einen Verdünnung angegeben, die im Vergleich zum Testserum mit Viren aber ohne AK nur noch 50% der sogenannten Overlay, sodass sich die Viren nur gerade lokal von Zelle zu Zelle Plaques aufweist. Dieser Test gilt im Moment als Gold Standard bei der Messung von AK, die Viren 16 ausbreiten können, nicht aber über grössere neutralisieren. Strecken via das Medium. Wenn dieser Overlay fehlt, gibt es sogenannte sekundäre Beispiele für den AG-Nachweis von WNV: Plaques, die das Zählen der primären - VecTest: Antigen-Panel-Test, mit dem man WNV, östliche Pferdeencephalomyelitis (EEE) und St.-Louis Plaques (aufgrund des primären Encephalitis (SLE) in Mücken nachweisen kann. Dabei wird ein Nachweisteststäbchen, das mit Inokulums) verhindern. spezifischen AK überzogen ist, verwendet. Falls das AG auf die AK treffen, kommt es zu einem Ohne Overlay wird letztendlich immer der gesamte Zellrasen zerstört. Farbumschlag. Als Untersuchungsmaterial wird ein Homogenat, das aus Mücken besteht, gebraucht.17 18 - Antigen Capture ELISA (ACE)/Sandwich-ELISA: Methode für den Antigen-Nachweis mittels AK. Der Ablauf erfolgt ähnlich wie beim indirekten ELISA; mit dem Unterschied, dass auf der Oberfläche Virus-AG- spezifische AK befestigt werden. Die anschliessende Zugabe von Enzym-markierten, sekundären AK, die mit einem bestimmten Substrat zu einem Farbumschlag führen, entspricht wiederum den indirekten ELISA. Auch hier wird v.a. Serum oder Plasma als Untersuchungsmaterial genommen.19 20

1 M. Ackermann, „Beilagen zur Vorlesung Virologie 2012/2013 Teil II Taxonomie und Familienalbum der Viren“, 2012 2 M. Ackermann, „Virus-Handbuch für Veterinärmediziner“, 1. Auflage, 2013

3 M. Ackermann, H. Adler, M. Engels, C. Griot, A. Metzler, U. Müller-Doblies, D. Müller-Doblies, M. Schwyzer, N. Stäuber, M. Suter, „Beilagen zur Vorlesung Virologie Version 2007/2008 für 2012 Teil I Virus Porträts“, 2007 4 M. Ackermann, „Beilagen zur Vorlesung Virologie 2012/2013 Teil II Taxonomie und Familienalbum der Viren“, 2012 5 M. Ackermann, H. Adler, M. Engels, C. Griot, A. Metzler, U. Müller-Doblies, D. Müller-Doblies, M. Schwyzer, N. Stäuber, M. Suter, „Beilagen zur Vorlesung Virologie Version 2007/2008 für 2012 Teil I Virus Porträts“, 2007 6 T. Colpitts, M. J. Conway, R. R. Montgomery, E. Fikrig, "West Nile Virus: Biology, Transmission and Human Infection", 2012 7 M. S. Diamond, B. Shrestha, E. Mehlhop, E. Sitati, M. Engle, „Innate and Adaptive Immune Responses Determine Protection against Disseminated Infektion by West Nile Encephalitis Virus“, 2003 8 M. De Filette, Sebastian U., Mike Diamond, N. N Sanders, "Recent progress in West Nile virus diagnosis and vaccination", 2012 9 M. O. Hottiger, „Spezielle Molekularbiologie“, 2013 10M. De Filette, Sebastian U., Mike Diamond, N. N Sanders, "Recent progress in West Nile virus diagnosis and vaccination", 2012 11 http://www.roche.com/pages/facetten/pcr_d.pdf, besucht am 06.10.2013 12 T. Colpitts, M. J. Conway, R. R. Montgomery, E. Fikrig, "West Nile Virus: Biology, Transmission and Human Infection", 2012 13 M. De Filette, Sebastian U., Mike Diamond, N. N Sanders, "Recent progress in West Nile virus diagnosis and vaccination", 2012 14 H.-J. Selbitz, U. Truyen, P. Valentin-Weigand, „Tiermedizinische Mikrobiologie, Infektions- und Seuchenlehre“, 9., vollständig überarbeitete Auflage, 2011, S. 80 15 "Der Neutralisationstest (NT) ist eine Variante des Plaque-Assays, mit dem neutralisierende Antikörper gegen bestimmte Viren im Serum eines Patienten oder eines Impflings nachgewiesen werden können. Durch Bindung von Antikörpern an die Oberfläche des Virus wird seine Aufnahme in die Zelle verhindert, so dass es zu keiner Vermehrung mehr kommen kann und die Anzahl an Plaques in einer Zellkultur reduziert wird. Daher bezeichnet man den Neutralisationstest auch als Plaque-Reduktions-Assay. Antikörper, die eine Aufnahme blockieren können, nennt man neutralisierende Antikörper. Im Neutralisationstest werden nur neutralisierende Antikörper erfasst. Der NT kann auch zur Quantifizierung von Zellgiften (bakteriellen Toxinen) verwendet werden, gegen die funktionshemmende Antikörper gebildet werden.", http://de.wikipedia.org/wiki/Neutralisationstest, besucht am 06.10.2013 16 http://en.wikipedia.org/wiki/Plaque_reduction_neutralization_test, besucht am 20.10.2013 17 M. De Filette, S. Ulbert, M. Diamond, N. N Sanders, „Recent Progress in West Nile Virus diagnosis and vaccination“, 2012 18 “Intended Use: The VecTest® West Nile Virus (WNV) Antigen Assay is a rapid immunochromatographic assay intended for the qualitative determination of WNV antigen in infected mosquitoes. Results from this assay can enable public health teams to: - Continuously monitor mosquito vectors - Focus vector control and eradication efforts - Deliver cost-effective prevention of disease Principle : The VecTest® WNV Antigen Assay is based on the dual monoclonal antibody “sandwich” principle. The test is initiated by placing one VecTest® WNV dipstick into 250 ml (0.25 ml) of ground mosquito extract. Antigen present in the solution binds to the specific antibody with a gold sol particle label. As the antigen antibody-gold complexes migrate through the test zone containing immobilized WNV antibody, they bind to the immobilized antibody forming a “sandwich”. The unbound dye complexes migrate out of the test zone and can be captured later in the control zone. A reddish-purple line develops on the specific area of the test zone when antigen is present. The control line, farthest from the sample, should always develop provided the test has been carried out correctly. “http://www.afpmb.org/sites/default/files/pubs/standardlists/equipment/pdfs/6550-01-533-3943_manual.pdf, besucht am 06.10.2013 19 M. De Filette, S. Ulbert, M. Diamond, N. N Sanders, „Recent Progress in West Nile Virus diagnosis and vaccination“, 2012 20 H.-J. Selbitz, U. Truyen, P. Valentin-Weigand, „Tiermedizinische Mikrobiologie, Infektions- und Seuchenlehre“, 9., vollständig überarbeitete Auflage, 2011, S. 67 - 68

Infektionsimmunologie-Gruppenarbeit in der Virologie HS 2013

Typen des West Nile Virus

Überblick über das Paper „West Nile Virus population genetics and evolution (Pesko&Ebel 2012)“1

Es sind unterschiedliche Linien des WNV mit verschiedenen Stämmen vorhanden. Die Entstehung dieser unterschiedlichen Linien und Stämme kommt über die immense Anpassungsfähigkeit des Virus zustande: WNV hat ein sehr grosses Wirtsspektrum und kann sich über die Migration von Zugvögeln weit verbreiten. Basierend auf einer hohen Mutationsrate und unterschiedlichen Selektionskriterien (wie z.B. unterschiedliche immunologische Reaktionen in den Wirtstieren (siehe Populationsdyna- mik)) in den verschiedenen Wirtsarten etablieren sich laufend neue Mutanten, die an unterschiedliche Übertragungszyklen und Umweltbedingungen angepasst sind39.

Entsprechend sind bei den verschiedenen Stämmen eine unterschiedliche Virulenz (und Neuro- invasivität) und ein unterschiedliches Wirtsspektrum zu erwarten. Die taxonomische Einteilung der verschiedenen Isolate ist nicht einheitlich definiert. Die serologische Verwandtschaft ist bisher unge- klärt, es gibt aber Hinweise auf Kreuz-Neutralisation durch Antikörper gegen WNV-Stämme (Charrel et al 20034, Calisher et al 19895)

Folgende Grenzwerte wurden in unserem Paper angesprochen:

- >84% gemeinsame Nukleotidsequenzen (Kuno et al 19986) oder >79% (Ebel and Kramer 20097, Charrel et al., 20034 ) sind nötig für die Einteilung in einer gemeinsamen Spezies. o Laut 1. Definition müsste Linie 2 bereits eine neue Spezies darstellen, während bei der 2. erst die neu beschriebenen Linien 3-6 nicht mehr die Definition erfüllen (Bond- re et al., 20078, Vazquez et al., 201018). - Die unterschiedlichen Linien zeigen Kreuzreaktivität (Bondre et al8, Bakonyi et al3) und kön- nen sich in den gleichen Übertragungszyklen etablieren. d Linie 1: Linie 4 bestuntersuchte Linie, weltweite Verteilung - Zahlreiche Isolate aus Russland - Stamm a: beinhaltet das Isolat NY99, das - Zuerst aus Dermacentor, dann aus Mü- im Jahr 1999 in New York isoliert wurde: cken und Fröschen o NY99: zeigt verstärkte Pathoge- nität bei Vögeln o WN02 ersetzte NY99 Linie 5: - Stamm b: Kunjin Virus in Australien assoziiert mit geringerer Virulenz o attenuierte Infektionen und ver- - Isolate aus Indien, von Menschen&Culex ringerte Neuroinvasivität - Nukleotidsequenz unterscheidet sich zu - Stamm c: Indien, siehe Linie 5 20-25% von anderen Stämmen, in manchen Publikationen der Linie 1 als Stamm Linie 2: c zugeordnet. assoziiert mit weniger schweren Krankheitsver- läufen, Neuroinvasion ist seltener Linie 6 - Dennoch Verlaufsformen mit Enzephali- - In Spanien isoliert aus Culex pipiens tis in Menschen & Pferden beschrieben - Grösste Ähnlichkeit mit Linie 4 - Südliches Afrika und Madagaskar, neu auch in West- und Osteuropa, endemi- sche Zyklen in Spanien und Griechenland Linie 7 - Koutango-Virus aus Senegal wird aktuell Linie 3 als eigene Spezies gehandelt, hat aber - Rabensburg Virus, isoliert aus verschie- nur 25% Unterschiede zu anderen WNV- denen Gebieten in Tschechien Stämmen - Isoliert aus C. pipiens und Aedes rossi- - Bisher keine Infektion von Menschen mit

Gina Steiner, Jasmin Kuratli, Rahel Rigotti, Nathalie Meier, Sereina von Ah Infektionsimmunologie-Gruppenarbeit in der Virologie HS 2013

cus: konnte keine Mortalität in erwach- Koutango-Virus nachgewiesen senen Mäusen erreichen, unabhängig von der Applikationsart (Hubalek et al, 20109) Tabelle bezieht sich auf 1,2,3

Molekulare Epidemiologie

Die ursprüngliche Erkennung der Linien/Stämme beruhte auf Sequenzvergleichen und phylogeneti- sche Analysen. (Lanciotti et al., 199919)

Beispiel: - WNV-Stamm, der 1999 in NY auftauchte, zeigte grosse Ähnlichkeit mit Isolaten aus Israel und Ungarn (Zehender et al10, Lanciotti et al., 199919; Jia et al., 199920) - Vergleiche der Isolate während der ersten 2 Jahre in New England zeigten ein grosses Mass an genetischer Konservierung (àwahrscheinlich einmalige Einführung in entsprechendes Gebiet) und sehr geringe Heterogenität der WNV-Population in diesem Zeitabschnitt (Ander- son et al., 200121; Ebel et al., 200122; Lanciotti et al., 199919; reviewed in Kramer et al, 20082; Ebel and Kramer, 20097) - Später wurde WN02 in Texas isoliert, das im Vergleich zu NY99 eine AS im Hüllprotein ersetzt hatte: A159V (Beasley et al., 200323) o WN02 ersetzte NY99 sehr schnell und wurde zum dominanten Genotyp in Nordame- rika (Ebel et al, 200425; Davis et al, 200526): warum? § Es besteht eine geringere extrinsische Inkubationszeit in Mücken àerhöhte Fitness im Vergleich zu NY99 (Ebel et al, 200425; Moudy et al, 200727)

Durch die Abhängigkeit der Arboviren von verschiedenen Wirtsspezies ist die Menge an möglichen Mutationen beschränkt (sonst keine Vermehrung mehr in einer oder anderer Spezies) (Jenkins et al., 200328). Die Negativselektion in Arbovirus-Populationen ist viel wichtiger, als die Positivselektion (Bertolotti et al., 2007, 200829; McMullen et al., 201130; Armstrong et al., 201131; Amore et al., 201032;, Jerzak et al., 200533) - Positivselektion: in phylogenetischen Analysen wurden nur sehr wenige genetische Änderun- gen gefunden, die durch einen positiven Selektionsdruck gefördert werden. è viele WNV-Proteine sind Bestandteil von Positivselektionen, welche die Übertra- gungseffizienz und die Wahrscheinlichkeit für das Weiterbestehen in verschiedenen Übertragungszyklen erhöhen. è Ähnliche Veränderungen im WNV-Genom können auch die Pathogenität und die Evo- lution des Virus beeinflussen Besonders viele dieser Veränderungen sind assoziiert mit dem neuen Genotyp WN02. Womöglich besteht hier ein Zusammenhang mit anderen Mutationen, die einen Selektionsvorteil bewirken. (Pesko et al, 20121)

Populationsdynamik des Virus im Wirt11 Das WNV hat sehr unterschiedliche Wirtsspezies (Vögel und Mücken). Entsprechend daraus bestehen ganz verschiedene Selektionsdrücke, d.h. die erfolgreiche Vermehrung in Vertebraten hat andere Voraussetzungen als diejenige in Invertebraten (Jerzak et al., 200533). Die Vermehrung in Mücken führt zu einer Vergrösserung der Quasispezies, während bei der Vermehrung in Vögeln eher eine Genomrestriktion erreicht wird. Um seine Fitness zu erhalten, muss das Virus beide Vorgänge nutzen,

Gina Steiner, Jasmin Kuratli, Rahel Rigotti, Nathalie Meier, Sereina von Ah Infektionsimmunologie-Gruppenarbeit in der Virologie HS 2013 beziehungsweise überwinden können39.

Ø Vertebraten

Die 1. Reaktion auf die RNA-Viren ist Typ1 IFN (α/β). Der antivirale Status wird sofort aufgebaut, so- bald doppelsträngige RNA im Zytosol der Wirtszelle entdeckt wird. Deshalb müssen Viren den antiviralen Status umgehen – man spricht von „purifying“ Selektion -> neue Mutanten müssten ebenfalls in der Lage sein, einen antiviralen Status zu umgehen, sonst riskieren sie, im Wirt Vogel eliminiert zu werden (Ding, 201013; Jerzak et al., 200734).

Ø Invertebraten

Invertebraten, Insekten: Ihre Reaktion auf Virusinfektion verläuft v.a. über RNA-Interferenz. Dies ist getriggert durch dsRNA in Zellen: Es kommt zur sequenzspezifische Elimination von viraler RNA. Deswegen sind neue Mutanten im Vorteil, weil sie weniger effizient abgebaut werden. (Ding, 201013)

Genetische Korrelation der Pathogenese und Fitness

Viele genetische Variationen korrelieren mit erhöhter oder geminderter Pathogenität. Dies lässt sich an folgenden Beispielen verdeutlichen.

Beispiel 1: WN02 mit AS-Substitution im V159A (siehe oben).

Beispiel 2: Es bestehen unterschiedliche Glykosylierungsmotive für die Hüllproteine. Normalerweise sind diese konserviert über das ganze Flavivirus-Genus (natürliche Variation bei WNV) vorhanden. - N-bezogene Glykosylierungsstelle an der Position 154 im Hüllprotein ist assoziiert mit erhöh- ter Neuroinvasivität in Mäusen und erhöhter Virulenz und Virämie in jungen Hühnern (Shi- rato et al, 2004.12; Beasley et al., 200524; Murata et al., 201014) - Hüllprotein-Glykosylierung ist ebenso wichtig für effiziente Übertragung in manchen Mü- cken((Murata et al., 201014; Moudy et al., 200915) - Glykosylierungsmuster können auch einen Einfluss haben auf die Fähigkeit der Hüllproteine, die innate Immunantwort zu modulieren (Hanna et al., 200516; Arjona et al., 200717) o Unterschiedliche Infektionsmuster und Vermehrung in verschiedenen Zelltypen à Rolle der Glykosylierung ist Wirtsspezifisch (Hanna et al., 200516; Arjona et al., 200717) o Glykosylierte Hüllproteine können die Bildung entzündlicher Zytokine herabsetzen (Arjona et al., 200717) o Glykosylierte Hüllproteine erhöhen die Überlebensrate des Virus in saurer Umgebung (Beasley et al., 200524; Langevin et al., 201135) o Glykosylierte Hüllproteine bewirken ein erleichtertes Budding an der ER-Membran àbessere Replikationsrate als nicht-glykosyliert (Berthet et al., 199736; Shirato et al., 200412; Li et al., 200637) o Glykosylierung hat einen Einfluss auf die Bindungsfähigkeit an Rezeptoren (Davis et al., 200638)

Quellen: 1. K. N. Pesko, G. D. Ebel. 2011. West Nile virus population genetics& evolution. Infection, Genetics and Evolution 12 (2012) 181–190. 2. L. D. Kramer, L. M. Styer, and G. D. Ebel . 2007. A Global Perspective on the Epidemiology of West Nile Virus. Annu. Rev. Entomol. 2008.53:61-8. 3. T. Bakonyi , É. Ivanics, K. Erdélyi, K. Ursu, E. Ferenczi, H. Weissenböck, and N. Nowotny.2006. Lineage 1 and 2 Strains of Encepha- litic West Nile Virus, Central Europe. Emerging Infectious Diseases, Vol. 12, No. 4, April 2006. 4. Charrel, R., Brault, A., Gallian, P., Lemasson, J.J., Murgue, B., Murri, S., Pastorino, B., Zeller, H., De Chesse, R., De Micco, P., 2003. Evolu- tionary relationship between Old World West Nile virus strains: evidence for viral gene ﬂow between Africa the Middle East, and Europe. Virology 315, 381–388.

Gina Steiner, Jasmin Kuratli, Rahel Rigotti, Nathalie Meier, Sereina von Ah Infektionsimmunologie-Gruppenarbeit in der Virologie HS 2013

5. Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E., Porterfield, J.S., Westaway, E.G., Brandt, W.E., 1989. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 70, 37. 6. Kuno, G., Chang, G.J.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. Phylogeny of the genus Flavivirus. J. Virol. 72, 73–83. 7. Ebel, G.D., Kramer, L.D., 2009. West Nile virus: molecular epidemiology and diversity. In: Diamond, M.S. (Ed.), West Nile Encephalitis Virus Infection. Springer, pp. 25–43. 8. Bondre, V.P., Jadi, R., Mishra, A., Yergolkar, P., Arankalle, V., 2007. West Nile virus isolates from India: evidence for a distinct genetic lineage. J. Gen. Virol. 88,875. 9. Hubalek, Z., Rudolf, I., Bakonyi, T., Kazdova, K., Halouzka, J., Sebesta, O., Sikutova, S., Juricova, Z., Nowotny, N., 2010. Mosquito (Diptera: Culicidae) surveillance for arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Tahyna viruses in Central Europe. J. Med. Entomol. 47, 466–472. 10. Zehender, G., Ebranati, E., Bernini, F., Presti, A.L., Rezza, G., Delogu, M., Galli, M., Ciccozzi, M., 2011. Phylogeography and epidemiological history of West Nile virus genotype 1a in Europe and the Mediterranean basin. Infect. Genet. Evol. 11(5), 863–868. 11. Daffis, S., Suthar, M.S., Gale Jr., M., Diamond, M.S., 2009. Measure an countermeasure: type I IFN (IFN-a/b) antiviral response against West Nile Virus. J. Innate Immun. 1, 435. 12. Shirato, K., Miyoshi, H., Goto, A., Ako, Y., Ueki, T., Kariwa, H., Takashima, I., 2004. Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. J. Gen. Virol. 85,3637. 13. Ding, S.W., 2010. RNA-based antiviral immunity. Nat. Rev. Immunol. 10 (9), 632–644. 14. Murata, R., Eshita, Y., Maeda, A., Maeda, J., Akita, S., Tanaka, T., Yoshii, K., Kariwa, H., Umemura, T., Takashima, I., 2010. Glycosylation of the West Nile virus envelope protein increases in vivo and in vitro viral multiplication in birds. Am. J. Trop.Med. Hyg. 82, 696. 15. Moudy, R.M., Zhang, B., Shi, P.Y., Kramer, L.D., 2009. West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors. Virology 387, 222–228. 16. Hanna, S.L., Pierson, T.C., Sanchez, M.D., Ahmed, A.A., Murtadha, M.M., Doms, R.W., 2005. N-linked glycosylation of West Nile virus envelope proteins influences particle assembly and infectivity. J. Virol. 79, 13262. 17. Arjona, A., Ledizet, M., Anthony, K., Bonafé, N., Modis, Y., Town, T., Fikrig, E., 2007. West Nile virus envelope protein inhibits dsRNA- induced innate immune responses. J. Immunol. 179, 8403. 18. Vazquez, A., Sanchez-Seco, M.P.Ruiz, S., Molero, F., Hernandez, L., Moreno, J., Magallanes, A., Tejedor, C.G., Tenorio, A., 2010. Putative new lineage of west nile virus. Spain. Emerg. Infect. Dis. 16, 549-552 19. Lanciotti, R., Roehrig, J., Deubel, V., Smith, J., Parker, M., Steele, K., Crise, B., Volpe, K., Crabtree, M., Scherret, J., 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286, 2333 20. Jia, X.Y., Briese, T., Jordan, I., Rambaut, A., Chang Chi, H., Mackenzie, J.S., Hall, R.A., Scherret, J., Lipkin, W.I., 1999. Genetic analysis of West Nile New York 1999 encephalitis virus. The Lancet 354, 1971-1972. 21. Anderson, J.F., Vossbrinck, C.R., Andreadis, T.G., Iton, A., Beckwith, W.H., Mayo, D.R., 2001. A phylogenetic approach to following West Nile virus in Connecticut. Proc. Natl. Sci. 98, 12885 22. Ebel, G.D., Dupuis 2nc, A.P., Ngo, K., Nicholas, D., Kauffman, E., Jones, S.A., Young, D., Maffei, J., Shi, P.Y., Bernard, K., Kramer, L.D., 2001. Partial genetic characterization of West Nile virus strains, New York State, 2000. Emerg. Infect. Dis. 7 (4), 650-653 23. Beasley, D.W.C., Davis, C.T., Guzman, H., Valandingham, D.L., Travassos da Rosa, A., Parsons, R.E., Higgs, S., Tesh, R.B., Barrett, A.D.T., 2003. Limited evolution of West Nile virus has occurred during its southwesterly spread in the United States. Virology 309, 190-195 24. Beasley, D.W.C., Whiteman, M.C., Zhang, S., Huang, C.Y.H., Schneider, B.S., Smith, D.R., Gromowski, G.D., Higgs, S., Kinney, R.M., Barrett, A.D.T., 2005. Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. J. Virol. 79, 8339 25. Ebel, G.D., Carricaburu, J., Young, D., Bernard, K.A., Kramer, L.D., 2004. Genetic and phenotypic variation of West Nile virus in New York, 2000-2003. Am.J.Trop. Med. Hyg. 71, 493-500 26. Davis, C.T., Ebel, G.D., Lanciotti, R.S., Brault, A.C., Guzman, H., Siirin, M., Lambert, A., Parsons, R.E., Beasley, D.W., Novak, R.J., Elizondo- Quiroga, D., Green, E.N., Young, D.S., Stark, L.M., Drebot, M.A., Artsob, H., Tesh, R.B., Kramer, L.D., Barrett, A.D., 2005. Phylogentic analysis of North American West Nile virus isolates, 2001-2004: evidence for the emergence of a dominant genotype. Virology 342, 252-265 27. Moudy, R.M., Meola, M.A., Morin, L.L.L., Ebel, G.D., Kramer, L.D., 2007. A newly emergent genotype of West Nile virus is transmitted earlier and more efficiently by Culex mosquitoes. Am.J.Trop.Med.Hyg. 77, 365 28. Jenkins, G.M., Rambaut, A., Pybus, O.G., Holmes, E.C., 2002. Rates of molecular evolution in RNA viruses: a quantitative phylogenetic analysis. J.Mol.Evol. 54, 156-165 29. Bertolotti, L., Kitron, U., Goldberg, T.L., 2007. Diversity and evolution of West Nile virus in Illinois and the US, 2002-2005, Virology 360, 143-149 // Bertolotti, L., Kitron, U., Walker, E.D., Ruiz, M.O., Brawn, J.D., Loss, S.R., Hamer, G.L., Goldberg, T.L., 2008. Fine-scale genetic variation and evolution of WNV in a transmission “hot spot” in suburban Chicago, USA. Virology 374, 381-389 30. MacMullen, A.R., May, F.J., Li, L., Guzman, H., Bueno Jr., R., Dennett, J.A., Tesh, R.B., Barrett, A.D.T., 2011. Evolution of new genotype of WNV in North America, Strain, 17 31. Armstrong, P.M., Vossbrinck, C.R., Andreadis, T.G., Anderson, J.F., Pesko, K.N., Newman, R.M., Lennon, N.J., Birren, B.W., Ebel, G.D., Henn, M.R., 2011. Molecular evolution of WNV in a northern temperature region: Connecticut, USA 1999-2008, Virology 417, 203-210 32. Amore, G., Bertolotti, L., Hamer, G.I., Kitron, U.D., Walker, E.D., Ruiz, M.O., Brawn, J.D., Goldberg, T.L., 2010. Multi-year evolutionary dynamics of WNV in suburban Chicago, USA, 2005-2007, Philos.Trans.R.Soc.Lond. B. Biol. Sci. 365, 1871-1878 33. Jerzak, G., Bernard, K.A., Kramer, L.D., Ebel, G.D., 2005. Genetic variation in WNV from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection. J. gen Virol 86, 2175-2183 34. Jerzak, G.V., Bernard, K., Kramer, L.D., Shi, P.Y., Ebel, G.D., 2007. The WNV mutant spectrum in host-dependant and a determinant of mortality in mice, Virology 360, 469-476 35. Langevin, S., Bown, R., Ramey, W., Sanders, T., Maharaj, P., Fang, Y., Cornelius, J., Barker, C., Reisen, W., Beasley, D., 2011. Envelope and pre-membrane structural amino acid mutations mediate diminished avian growth and virulence of a Mexican WNV isolate, J.Gen.Virol 92 (Pt 12), 2810-1820 36. Berthet, F., Zeller, H., Drouet, M., Rauzier, J., Digouette, J., Deubel, V., 1997. Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro-African WNV. J.Gen.Virol. 78, 2293 37. Li, J., Bhuvanakantham, R., Howe, J., Ng, M.L., 2006. The glycolysation site in the envelope protein of WNV (Sarafend) plays an important role in replication and maturation processes. J.Gen.Virol. 87, 613-622 38. Davis, C.W., Ngyuyen, H.Y., Hanna, S.L., Sanchez, M.D., Doms, R.W., Pierson, T.C., 2006. WNV discriminates between DC-SIGN and DC- SIGNR for cellular attachment and infection. J.Virol. 80, 1290 39. Überarbeitet von Prof. M. Ackermann – besten Dank

Gina Steiner, Jasmin Kuratli, Rahel Rigotti, Nathalie Meier, Sereina von Ah

WNV, FSMEV, BTV, SBV: Unterschiede und Gemeinsamkeiten

A. Roentgen, K. Linder, L.Huston, L. Heimgartner, D.Brütsch

I Genom und Struktur West Nile Virus (WNV) und Frühsommermeningoenzephalitis Virus (FSEMV) besitzen ein +ssRNA Genom, Schmallenberg Virus (SBV) ein –ssRNA Genom und Bluetongue Virus (BTV) ein dsRNA Genom. Alle Viren gehören zu den Arboviren und alle ausser BTV sind behüllt. WNV und FSMEV gehören zu der Familie der Flaviviridae, Gattung Flavivirus, und weisen somit ein nicht segmentiertes Genom auf, ganz im Gegensatz zu SBV (Familie Bunyaviridae, Gattung Orthobunyavirus) und BTV (Familie Reoviridae, Gattung Orbivirus), welche beide ein segmentiertes Genom besitzen. Dies befähigt sie zur Reassortierung im Wirt und damit zur einfachen Entstehung neuer Virusstämme.

II Vektoren Obwohl alle vier Viren Arboviren sind, werden sie über verschiedene Vektoren übertragen. Während WNV von Culex übertragen wird, wird FSMEV von Zecken und BTV und SBV von Culicoides übertragen. Hinsichtlich der einzelnen Vektoren der verschiedenen Viren ergeben sich unterschiedliche Verbreitungsgebiete und unterschiedliche Verbreitungsgeschwindigkeiten. Ausserdem ist das Vektor- Habitat entscheidend hinsichtlich Infektionsrisiko: im Unterholz eines Waldes wird man eher von einer Zecke gestochen als beim Grillieren an einem Wassertümpel, wogegen ein Mückenstich am Tümpel einiges wahrscheinlicher ist. Ein Vorteil des WNV ist, dass es von sehr vielen verschiedenen Culex Arten und zusätzlich auch von einigen Aedes Arten übertragen werden kann, d.h. WNV kann sich via viele Vektoren verteilen. Culex ernähren sich von vielen verschiedenen Wirten (Säuger und Vögel) und haben die Möglichkeit des „host switching“.

III Haupt- & Endwirte WNV und FSMEV besitzen beide neben einem Hauptwirt (Vogel resp. Wildtier) einen sogenannten Endwirt. Speziell daran ist, dass die Virusmenge im Blut eines Endwirtes auch bei schwerwiegender Erkrankung so gering ist, dass Mücken nicht durch eine Blutmahlzeit mit dem Virus infiziert werden können. Somit stellen kranke Endwirte keinerlei Risiko für Artgenossen dar und müssen daher bei Verdacht auf Infektion mit WNV auch nicht von Artgenossen abgesondert werden. Ein besonderes Problem hinsichtlich der Epidemiologie von WNV (im Vergleich zu den anderen hier- Mathias Ackermann 21.10.13 09:44 Kommentar [1]: Was aber wenn sich eine genannten Viren) stellen die Zugvögel dar. Denn durch sie als Hauptwirte kann das Virus weite Distanzen Mücke im Stall aufhält? „zurücklegen“ und sich weit verbreiten. Zusätzlich sind klinisch inapparente Träger nicht selten.

IV Pathogenese, Klinik & Diagnostik WNV: Patho: 1. Replikation in Haut und regionären LK à primäre Virämie und übertritt ins Retikuloendothelial-System à sekundäre Virämie & Durchbruch durch Blut-Hirn-Schranke (BHS) Klinik (Pferd und Mensch): Häufig verläuft eine Erkrankung subklinisch. Seltener geht die Erkrankung mit hohem Fieber oder neurologischen Symptomen einher (bei Pferd und Mensch) à Diagnose (DX) anhand Symptomatik, Bestätigung durch Antikörper (AK)/Virus-Nachweis à Material: Blut, Liquor, Gehirn FSME: Patho: Siehe WNV Klinik (Hund und Mensch): Eine erste Krankheitsphase weist nur unspezifische Symptome auf. Für die darauf folgende zweite Phase sind neurologische Symptome wie hohes Fieber, Übelkeit, Tremor, Ausfall des Sensoriums, Paresen & Paralysen wegweisend. à DX: Virusisolierung (Hirn), virusspezifische RNA (PCR) & AK-Nachweis à Material: Serum, Gehirn SBV: Patho: Details unbekannt. Ausgeprägter Neurotropismus und Neuropathogenität Klinik: Beim erwachsenen Tier sind eine subklinische Erkrankung, wie auch akute Symptome (Fieber, Milchrückgang und Durchfall) möglich. Bei Infektion eines Fötus treten Abort, Missbildung oder Mumifikation auf. à DX: Verdacht bei Missbildungen/Aborten, Bestätigung durch virusspezifische RNA (PCR) & AK-Nachweis Material à Material: Abortierte Foeten, in zweiter Linie Blut BTV: Patho: Aufnahme über dendritische Zellen an der Einstichstelle und Übertritt in regionale LK à Infizierte Monozyten und T-Zellen wandern aus à Virämie à Befall von Lunge/Milz und Endothelien Klinik: Fieber, Hämorrhagien und Ödeme treten primär beim Hauptwirt „Schaf“ auf. Es gibt 3 verschiedene Verlaufsformen (akut/subakut, subklinisch und atypisch mit Fetopathien). à DX: Symptomatik & zwingend Labornachweis à Material: Virusisolation (virämisches Blut, Blutzellen), AK-Nachweis

V Impfungen Im Gegensatz zu FSMEV gibt es für WNV noch keine Schutzimpfung für den Menschen. Ein grosses Problem ist das breite Wirtsprektrum des WNV, was eine Eradikation des Virus fast verunmöglicht. Wichtig ist es bei WNV, die Vektoren zu bekämpfen. Für das Pferd gibt es derzeit jedoch mehrere Impfstoffe auf dem Markt, wovon einer in der Schweiz seit 2012 zugelassen ist. Die Impfung gegen FSMEV ist für Haustiere nicht zugelassen. Gegen BTV kann man bei Wdk impfen. Für das Schmallenbergervirus existiert noch kein Impfstoff.

Referenzen: Mathias Ackermann 22.10.13 09:11 Kommentar [2]: Bei dieser Fülle von 1. Virushandbuch für Veterinärmediziner, Mathias Ackermann, 2013 Information wäre es angezeigt, die einzelnen 2. A global perspective on the epidemiology of West Nile virus, Kramer et al., Annu. Rev. Entomol. Aussagen mit den entsprechenden 2008. 53:61–81 Referenzen zu verbinden.

WNV SBV FSME BTV Biologie (+)ssRNA, nicht-segmentiert, (-)ssRNA, segmentiert, (+)ssRNA, nicht-segmentiert, dsRNA, segmentiert, behüllt behüllt behüllt unbehüllt Familie Flaviviridae Bunyaviridae Flaviviridae Reoviridae Gattung Flavivirus Orthobunyavirus Flavivirus Orbivirus Vektoren Mücken (Culex) Gnitzen (Culicoides) Zecken (Reservoir) Gnitzen (Culicoides) • Ixodes ricinus in Europa • Ixodes persulcatus Wirte Vögel (Hauptwirte und Schaf, Rind, Ziege Wildtiere (Hauptwirte) Schaf, Rind, Ziege und Reservoir) Schafe am empfänglichsten Mensch und Hund (Endwirte) Wildwiederkäuer Mensch und Pferd (Endwirte) Reservoir: Rind, Ziegen und Schafe am empfänglichsten Sehr breites Wirtsspektrum Wildwiederkäuer Reservoir: Rind, Ziegen und Wildwiederkäuer Verbreitung Weltweit Europa Europa und Russland Gürtel bis ca 40°C nördlich und südlich des Äquators Pathogenese 1. Replikation in Haut und Details unbekannt. Siehe WNV Aufnahme über DZ an regionären Lymphknoten → Ausgeprägter Einstichstelle und primäre Virämie und übertritt Neurotropismus und Übertragung in regionäre in Retikuloendothelial-System Neuropathogenität. Lymphknotenà Infizierte → sekundäre Virämie und Monozyten und T Zellen Durchbruch der BHS wandern ausà Virämie àBefall von Lungen/Milz und Endothelien Klinik Verlauf beim Menschen: Verlauf bei Infektion von Verlauf beim Menschen: Klinische Erscheinungen • IKZ 2-6 Tage erwachsenem Tier: • IKZ: 7-14 Tage (Fieber, Hämorrhagien und • Häufig subklinisch • Akut (Fieber, • 1. Phase mit unspezifischen Ödeme) treten primär beim • Sonst Fieber oder Milchrückgang, DF) Symptomen Schaf auf neurologische Symptome • Subklinisch • 2.Phase mit Befall des ZNS, hohes Fieber, Übelkeit, 3 wichtige Verlaufsformen: Zoonose! Verlauf bei Infektion des Sensoriumsstörungen, 1. Akute und subakute Fötus: Tremor, Paresen, Paralysen klinische − Aborte Blauzungenkrankheit − Missbildungen Zoonose! 2. Atypischer Verlauf mit − Mumifikation Fetopathien 3. Subklinischer Verlauf Impfung Keine für Mensch keine Nur für den Menschen zugelassen Existiert Existiert für Pferde, einer in CH seit 2012 zugelassen

4. West-Nil-Virus im Vergleich betroffener Tierarten Welche Typen und Subtypen kommen vor? Welche klinischen Symptome werden beobachtet? Womit begründen sich die unterschiedlichen Symptome bei den einzelnen Tierarten? Vermehrt sich das Virus in unterschiedlichen Organen? Wie wirken sich immunologische Faktoren auf Reservoirbildung und Übertragung aus? (Hubalek and Halouzka, 1999; Jeffrey Root, 2013; Kramer et al., 2008; Kwan et al., 2012)

Welche Typen und Subtypen kommen vor?

Das West Nil Virus (WNV) ist ein Arbovirus aus der Familie der Flaviviren. Subtypen werden anhand von Sequenzanalysen und Serologie unterschieden und in unterschiedliche regional vorkommende Viren eingeteilt.

Typ Vorkommen Subtyp 1a kommt weltweit vor, ausser in Australien und Indien 1 Subtyp 1b (Kunjin Virus) nur in Australien Suptyp 1c nur in Indien 2 Subtyp 2 südliches Afrika und Madagaskar Auch bekannt als „Rabensburg Virus“ Czech Republic (1997, 1999, 3 2006 isoliert) 4 Russland, erstmals 1988 isoliert 5 13 Isolate aus Indien (1950 – 1980) 6 Spanien (Ähnlichkeit mit Typ 4) 7 (noch nicht definitiv ob neuer Typ) n.d. Koutango Virus, isoliert in Senegal Quellen: Kramer L..D., A global perspective on the Epidemiologie of WNV, S.64 Tibayrenc M., Infection, Genetics and Evolution, Elsevir, 3. Taxonomy & classification

Aktuell aus Zentraleuropa und Russland isolierte Typen sind noch nicht taxonomisch klar eingeteilt worden, gehören aber zu anderen Typen als die bis jetzt bekannten.

Quelle: Pesko, K., Ebel, G., 2012, West Nile virus population genetics and evolution. Infection, Genetics and Evolution 12 (2012) 181-190

Giuliana Rosato, Anita Vock, Marina Rüegg, Louise Martin, Carmen Nauer Seite 1 Vermehrt sich das Virus in unterschiedlichen Organen?

Haut: Inokulation durch Moskito, Replikation in Keratinozyten und Langerhans‘schen Zellen (1)

Regionäre Lymphknoten: - Migration der Langerhans‘schen Zellen über afferente Lymphgefässe, Replikation und Dissemina- tion über efferente Lymphgefässe und Ductus thoracicus in Blutzirkulation = Virämie - Sekundäre Infektion von v.a. Milz und Niere (1)

Im Gehirn werden folgende Wege für den Eintritt des West-Nil-Virus ins ZNS vermutet: - Retrograder Transport aus peripheren Neuronen - Durchlässigkeit der Blut-Hirn-Schranke: TNF-α (Induktion durch Aktivierung von TLR 3) mit Einfluss auf Permeabilität, Abbau extrazellulärer Matrix durch Aktivierung von Metalloproteinasen - Infektion oder passiver Transport durch Epithelzellen des Plexus choroideus - Trojanisches Pferd Mechanismus: Transport des Virus in infizierten Immunzellen (Neutrophile, CD8+ und CD4+) - Infektion der olfaktorischen Neuronen - Direkte Infektion der Endothelzellen von Blutgefässen Im Endeffekt tragen alle Faktoren zu einer fulminanten Enzephalitis bei, die sich auch selbst erhalten kann. " starke Neurotoxizität (1)

Quelle: Cho, H., Diamond, M., 2012, Immune Response to West Nile Virus Infection in the Central Nervous System. Viruses 2012, 4, 3812- 3830

Giuliana Rosato, Anita Vock, Marina Rüegg, Louise Martin, Carmen Nauer Seite 2 Welche klinischen Symptome werden beobachtet?

Mensch: Fieberhafte, grippeähnliche Erkrankung mit Kopf-, Hals-, Rücken-, Muskel- und Gelenkschmerzen, Müdigkeit, Konjunktivitis, retrobulbäre Schmerzen, Hautausschlag, Lymphadenopathie, Anorexie, Nausea, Bauchschmerzen, Durchfall und respiratorische Symptome. Gelegentlich (<15% der Fälle) kommt es zu einer akuten aseptischen Meningitis oder Enzephalitis, Myelitis, Hepatosplenomegalie, Hepatitis, Pankreatitis und Myokarditis. Die meisten Todesfälle wurden bei Patienten registriert, die älter als 50 Jahre alt waren. (2, S. 644 ff)

Pferde: Diffuse Enzephalomyelitis mit hohem Fieber, Zungen- und Lippenlähmung, Propriozeptionsdefizite, Ataxie, Hinterhandschwäche und -lähmung. Die Infektion verläuft jedoch häufig asymptomatisch. (2, S. 646)

Andere Säugetiere: Schafe: Fieber, Abort, selten Enzephalitis Schweine, Hunde: asymptomatische Infektion Kaninchen, Albino-Ratten, Meerschweinchen: resistent gegen eine WNV-Infektion Labormäuse, Hamster: deutlich anfälliger, erkranken oft an einer fatal verlaufenden Enzephalitis Affen: Fieber, Ataxie, gelegentlich Enzephalitis, Tremor, Parese oder Paralyse Die Infektion kann tödlich verlaufen oder führt in Überlebenden zu Viruspersistenz. (2, S. 646 ff)

Vögel: Infizierte Vögel zeigen meist keine Symptome. Eine natürliche Erkrankung wurde bei einer Taube in Ägypten beobachtet. Bei Inokulation bestimmter Vogelarten (Tauben, Hühner, Enten, Möwen, Gän- se, Habichte und Rabenvögel) verursacht das Virus gelegentlich Enzephalitis und Tod oder langfristige Viruspersistenz. Kükenembryonen können durch das Virus getötet werden. (2, S. 646 ff) Gewisse Vogelarten sterben jedoch an der WNV-Infektion und dienen als Warnsignal. In den USA sind es vor allem Krähen und in Ungarn und Österreich Gänse und Habichte, die als Indikatorwirte für die WNV-Überwachung genommen werden. (5)

Womit begründen sich die unterschiedlichen Symptome bei den einzelnen Tierarten? Wie wirken sich immunologische Faktoren auf Reservoirbildung und Übertragung aus?

Die Klinik hängt davon ab, ob das Virus ins ZNS eindringen kann oder nicht. Vermutlich spielen dabei die bereits erwähnten Wege (siehe S. 2) eine wichtige Rolle, jedoch auch die Effektivität der Immun- antwort. Kommt die Wirtsabwehr zu spät, d.h. erst wenn das Virus bereits ins ZNS gelangt ist, sind massive Schäden am Gehirn durch Zelluntergang die Folge, was zu neurologischen Symptomen führt. Die Gewebeschädigung kommt durch verschiedene Komponenten der Immunabwehr zustande: • Zelluntergang von infizierten Zellen durch Apoptose als Reaktion auf Typ-1-Interferon und durch die Aktivität der natürlichen Killerzellen • Zelluntergang durch zytotoxische T-Zellen • Komplementabhängige Zytolyse

Giuliana Rosato, Anita Vock, Marina Rüegg, Louise Martin, Carmen Nauer Seite 3 Um das Virus vollständig aus dem Körper zu eliminieren, ist eine adaptive zelluläre Immunantwort (Th1-Antwort) nötig. Kommt es nach Eindringen des Virus in den Wirtsorganismus zwar zu einer adä- quaten innaten Immunantwort, jedoch zu einer überwiegend humoralen Immunabwehr (Th2- Antwort), kann das Virus nicht vollständig aus dem Körper eliminiert werden. In diesem Fall zeigen die Tiere kaum Symptome, aber sie können eine persistente Virämie aufweisen, was die Vorausset- zung für eine erfolgreiche Übertragung mittels blutsaugender Vektoren darstellt. Man kann deshalb vermuten, dass die Viren ein Interesse daran haben, die Immunantwort in diese Richtung (Th2- Antwort) zu steuern, was ihnen im Hauptwirt zu gelingen scheint, jedoch nicht im Fehlwirt. (6)

Die unterschiedlichen Symptome bei den einzelnen Tierarten lassen sich damit erklären, dass einerseits die Immunantwort tierartspezifisch aber auch individuell unterschiedlich verläuft, abhängig von der Fitness der Tiere, von der Interaktion zwischen Virus und Immunsystem bzw. der ‚Manipulations- fähigkeit‘ des Virus, der Effektivität der Wirtsabwehr und der Überlebensstrategie des Virus. Ande- rerseits braucht es auch eine Empfänglichkeit des Wirtes, damit sich das Virus überhaupt im Orga- nismus etablieren kann.

Bestimmte Vogelarten dienen beim WNV als Hauptreservoir. Sie zeigen sehr hohe und lange Virä- mien, was eine Übertragung durch Mücken begünstigt. Bei Fehlwirten tritt im Gegensatz dazu eine kürzere Virämie mit geringeren Viruskonzentrationen auf. Sie bleiben damit unter dem Schwellen- wert, der nötig wäre, um Mücken zu infizieren. Jeffrey Root hat in einer neuen Studie gezeigt, dass auch verschiedene Wildsäuger genug hohe Viruskonzentrationen im Blut hätten, um das Virus auf Mücken zu übertragen. Er vermutet deshalb, dass die Wildsäugetiere eine wichtigere Rolle in der Epidemiologie von WNV spielen, als man bisher angenommen hat. Für einen Beweis braucht es jedoch weitere Untersuchungen. (3)

Jennifer L. Kwan hat sich in ihrer Studie mit der Immunitätsbildung der Vögel, der Persistenz des Vi- rus und den Krankheitsausbrüchen bei Menschen befasst. Es hat sich gezeigt, dass die Infektions- bzw. Übertragungsdynamik stark von der Herdenimmunität der Vögel abhängt. Je höher die Winter-/ Frühlingsimmunität der Vögel ist, desto geringer sind die Verbreitung des Virus und die Anzahl von Ausbrüchen bei Menschen im nächsten Sommer. Dies bedeutet, dass die Immunkompetenz des Wirts eine wichtige Rolle spielt. (4)

Quellen: (1) Cho, H., Diamond, M., 2012, Immune Response to West Nile Virus Infection in the Central Nervous System. Viruses 2012, 4, 3812- 3830 (2) Hubalek Z, Halouzka J. 1999. West Nile fever—a reemerging mosquito-borne viral disease in Europe. Emerg. Infect. Dis. 5:643–50 (3) Root, JJ., 2013, West Nile virus associations in wild mammals: a synthesis, Arch Virol (2013) 158:735–752 (4) Kwan JL, Kluh S, Reisen WK (2012) Antecedent Avian Immunity Limits Tangential Transmission of West Nile Virus to Humans. PLoS ONE 7(3): e34127. (5) Ackermann M., Virus-Handbuch für Veterinärmediziner, Haupt Verlag, 1. Auflage (2013), S. 186-191 (6) Vorträge im Rahmen der Infektionsimmunologie, HS 13, Thema ‘AK und zelluläre Immunität gegen das WNV’ und ‘Intrinsische und innate Abwehr gegen WNV’

Giuliana Rosato, Anita Vock, Marina Rüegg, Louise Martin, Carmen Nauer Seite 4

West-Nil-Virus und sein(e) Vektor(en)

1. Welche Vektoren gibt es?

Culex pipiens-Komplex: Wichtigste Vektoren

Non-Culex pipiens Komplex : Sind Opportunisten, welche Vögel bevorzugen, aber selten auch Menschen stechen (C.tarsalis, C. nigripalpus)

Non-Culex Moskitos: Dazu gehört zum Beispiel Aedes à epidemiologisch wahrscheinlich weniger von Bedeutung

Non-Moskito Vektoren: Zecken, Hippoboscidae (=Lausfliege) à spielen wahrscheinlich eine untergeordnete Rolle

2. Biologie und Einteilung der Vektoren?

è bridge vectors: Übertragen das Virus vom Vogel auf den Menschen, Pferd und andere Säuger

è maintanance vectors: Sorgen dafür, dass Virus im Reservoir (Vogel) verbleibt (z.B. C. pipiens pipiens, Culex restuans)

Culex pipiens pipiens:

Ist ein maintanance und bridge vector à Im Sommer und Frühjahr macht C. pipiens einen so genannten host-switch durch, das heisst die Mücken ernähren sich dann auch von anderen Säugern (speziell von Menschen) während sie den Rest des Jahres Vögel als Mahlzeit bevorzugen (host-switch kommt vor allem in urbanen und weniger in ruralen Regionen vor). Vermehren sich vor allem in stehendem Gewässer. (siehe Colpitts et al.)

Culex pipiens molestus:

Auch „London underground mosquito“ genannt, weil sie sich wahrscheinlich im letzten Jahrhundert an Untergrundsysteme angepasst hat. Im Gegensatz zu Culex pipiens pipiens ist sie kälteintolerant, sticht bevorzugt Menschen, Ratten und Mäuse und vermehrt sich das ganze Jahr über. (siehe http://en.wikipedia.org/wiki/London_Underground_mosquito)

Culex pipiens quniquefasciatus:

Typischer Brückenvektor à sticht Vögel und Menschen. Dringt vor allem während der Dämmerung in die Häuser ein und erreicht einen peak um Mitternacht. Sticht bevorzugt unterhalb der Knie. (siehe http://en.wikipedia.org/wiki/Culex_quinquefasciatus)

Hybridmücken:

In Nordamerika wird die Ausbreitung dadurch erleichtert, dass eine Hybridmücke aus Culex pipiens molestus und Culex pipiens als Vektor dient à diese haben keine Präferenzen bei der Blutmahlzeit und stechen sowohl Menschen wie auch Vögel. (Siehe: Emerging Vectors in the Culex pipiens Complex Dina M. Fonseca et al.)

3. Wie vermehrt und verbreitet sich der Vektor?

Vermehrung:

Die Paarung findet nach dem Schlüpfen der Imagines statt. Durch den Flügelschlag der Männchen entsteht ein Sirrton, der die Weibchen artspezifisch anlockt. Die Weibchen brauchen für die Eiproduktion Blutnahrung (die Männchen saugen kein Blut). Sie legen ihre Eier in kleinen Schiffchen auf der Wasseroberfläche von stehendem Gewässer ab. Die Larven schlüpfen und entwickeln sich über 4 Stadien zur Puppe, aus welcher schliesslich der Imago schlüpft. Dies dauert ca. 8-21 Tage. Die Weibchen leben ca. 6 Wochen.

Verbreitung:

• Mücken können mit Flugzeugen/Schiffen eingeschleppt werden • In stehenden Gewässern (z.B. auch Regentonnen, alte Autoreifen) können sich die Mücken nachdem es geregnet hat sehr schnell vermehren • Die Verbreitung ist auch abhängig von der Wirtsdichte und dem Klima

4. Wie entsteht aus dem Vektor ein Reservoir?

Begattete Weibchen können an geschützten Orten (zum Beispiel in Kellerabteilen) überwintern. Auch die Eier von Weibchen welche noch keine Blutmahlzeit aufgenommen haben, können mit dem Virus infiziert sein. Daraus kann man schliessen, dass auch eine vertikale Erregerübertragung stattfindet. Das Virus kann so persistieren und sich im Frühling wieder amplifizieren.

5. Wie kann man den Vektor bekämpfen(theoretische und praktische Ansätze)?

I. Biologisch: Entwicklung von Biopestiziden (Bacillus thuringiensis produziert Toxin welches letal ist für Mücken), Entwässerung, Vermehrung von natürlichen Fressfeinden, Schutzkleidung, Moskitonetze, Moskitoaktivtätspeaks meiden (z.B. C.pipiens 2h nach Sonnenuntergang)

II. Chemisch: Repellentien, Larvizide, Adultizide (Pyrethroide, Organophosphate) à aber hohe Resistenzen und Umweltschäden

III. Physikalisch: Ölfilme auf Gewässern, welche zum Ersticken der Larven führen, Mückenfallen mit anziehenden Lockstoffen

IV. Genetisch: Transgene Mücken, welche Virus nicht übertragen können oder selbst resistent sind gegenüber Krankheitserreger

Quellen: • Lehrbuch der Parasitologie für die Tiermedizin, Peter Deplazes et al. 2. Aufl., S 442-447 • http://de.wikipedia.org/wiki/Stechm%C3%BCckenbek%C3%A4mpfung, • Colpitts et al. 2012, Kramer et al, Andreadis, 2012 • http://en.wikipedia.org/wiki/London_Underground_mosquito • http://en.wikipedia.org/wiki/Culex_quinquefasciatus • Emerging Vectors in the Culex pipiens Complex, Dina M. Fonseca et al.

Livia Egli, Fabienne Schubnell, Lisa von Boehmer, Sarah Lais

6. Vermehrung des West-Nil-Virus im Vektor

In welchen Organen vermehrt sich das Virus? Mücken der Gattungen Culex und Aedes nehmen das Virus mit ihrer Blutmahlzeit von einem infizierten Wirtstier auf. Die Vermehrung des Virus findet anschliessend in den Zellen des Mitteldarms der Mücken statt. Über die Hämolymphe gelangen die Viren vom Mitteldarm in die Speicheldrüsen, wo die Übertagung bei der Blutmahlzeit erfolgt. Ebenso gelangen die Viren in die Geschlechtsorgane, als Voraussetzung zur vertikalen Übertragung.1 Mathias Ackermann 21.10.13 10:08 Wie wird es ausgeschieden und übertragen? Kommentar [1]: Kramer et al., 2008 beschreiben das noch viel präziser: ...vertical Beim Stich der Mücke auf einem Wirtstier werden die Viren mit dem Speichel übertragen. transmission of WNV from parent to progeny Die Mücke sticht mehrere Male in die Haut, bis sie ein geeignetes Blutgefäss gefunden hat.2 plays a significant role in the virus’s perpetuation. Flaviviruses appear to enter the Bei jedem dieser Stiche wird Speichel sezerniert. Somit gelangt nur ein kleiner Teil der Viren fully formed egg through the micropyle at the direkt in die Blutbahn, der weitaus grössere Teil wird in die Haut injiziert. time of fertilization (117). This is an Der Speichel spielt eine wichtige Rolle bei der Übertragung und Infektion, da er vasodilatativ, inefficient mechanism of vertical transmission, yet it does permit the infection of progeny immunmodulatorisch und koagulationshemmend wirkt. Dies begünstigt die Ausbreitung des following a single maternal blood meal.... Virus im Wirtstier.3 Beim Blauzungenvirus wird dieser Vorgang Eine vertikale Übertragung wurde nachgewiesen, als bei einer Untersuchung in Larven und nicht beobachtet; dort trifft das Virus auch erst nach der Verschalung der Eier im Ovar männlichen Stechmücken West Nile Virus gefunden wurden, obwohl diese kein Blut saugen. ein, wird aber dann nicht mehr ins Ei Die Übertragung erfolgt bei der Befruchtung des Eis. Dies ist ein nicht sehr effizienter aufgenommen. Mechanismus, aber er erlaubt die Infektion des Nachwuchses nach nur einer Blutmahlzeit der Adulten Mücke. Möglicherweise spielt die vertikale Übertragung eine Rolle für die Überwinterung des Virus4 Welche Virusmengen sind nötig für die Vermehrung im Vektor? Im Blut des Wirtes müssen 106 plaque forming untis/ml des Virus enthalten sein, damit eine Mücke sich bei ihrer Blutmahlzeit infiziert.5 Ausgehend von einem Mahlzeitvolumen von 0.1ml muss die Mücke also mindestens 100 pfu aufnehmen, damit sie infiziert wird. Eine andere Studie6 zeigte, dass für die Übertragung des Virus von Alligatoren auf Mücken auch Virustiter 105 pfu/ml im Alligatorenblut ausreichend sind. Wie viel Virus gibt ein Vektor bei der Übertragung ab? In einer Studie7 konnte gezeigt werden, dass bei den Stichen einer Mücke ca. 104 - 106 pfu des Virus extravaskulär (dermale Hautschichten) und nur ca. 102 pfu direkt ins Blut injiziert werden. Es konnte ebenfalls gezeigt werden, dass die injizierte Virusmenge eine wichtige Rolle für den Verlauf der Krankheit spielt, da sie die Ausprägung der Virämie und die Virenausscheidung direkt beeinflusst. Die Kenntnis der injizierten Virusmenge ist somit auch von zentraler Bedeutung in Impf- und anderen Studien.

1 Kramer et al., A Glogal Perspective on the Epidemiology Of West Nile Virus. 2008; S.69 2 Styer et al, Mosquitoes Inoculate High Doses of West Nile Virus as They Probe and Feed on Live Hosts, 2007 3 Colpitts et al., West Nile Virus: Biology, Transmission, and Human Infection, 2012; S. 638 4 Kramer et al., A Glogal Perspective on the Epidemiology Of West Nile Virus. 2008; S. 69 5 Kwan et al., Antecedent Avian Immunity Limits Tangental Transmission of West Nile Virus to Humans 2012, S.2 6 Kramer et al., A Glogal Perspective on the Epidemiology Of West Nile Virus. 2008 7 Styer et al., Mosquitoes Inoculate High Doses of West Nile Virus as They Probe and Feed on Live Hosts, 2007

Epidemiologie West Nile Virus 7 Oktober 2013

7.Globale Epidemiologie des West Nil Virus (WNV)

Entwicklung der Epidemiologie von WNV in den letzten Jahrzehnten und Faktoren, welche diese beeinflussen:

Das WNV ist das am weitesten verbreitete Arbovirus. Man findet es auf der ganzen Welt ausser in der Antarktis.

Erstmals isoliert wurde das WNV 1937 im West Nile District des nördlichen Uganda bei einer Frau mit Fieber. Durch die auftretenden Symptome wurde das WNV in Verbindung mit bereits zuvor aufgetretenen sporadischen Erkrankungen und Ausbrüchen in Eurasien, Australien und im mittleren Osten gebracht, deren Verursacher bis anhin unbekannt war.

Durch serologische Studien im Jahre 1950 konnte das Virus in Menschen, Pferden, Vögel und Moskitos in Ägypten und dem oberen Nil Delta nachgewiesen werden. In der Zeit danach wurden gelegentlich Erkrankungen im Zusammenhang mit WNV in Osteuropa detektiert. Ab dem Jahre 1990 traten zunehmend mehr Fälle von WNV auf, besonders in mediterranen Gebieten von Europa. Es wurden erstmals vermehrt schwere Enzephalitiden und neurologische Symptome beobachtet.

1996/97 trat das Virus in Bukarest/Rumänien auf, was mit mehr als 500 Krankheitsfällen verzeichnet wurde und somit einer der grössten europäischen Arbovirus Ausbrüche seit den 80er Jahren darstellte. Zwischen 1996 und 1999 traten drei grosse WNV Epidemien in Rumänien, Russland und Nordamerika auf. Die Erkrankungen äusserten sich mit starken neurologischen Symptomen und relativ hoher Sterblichkeit. Dies waren die ersten verzeichneten Epidemien in grossen urbanen Populationen. Man vermutet, dass das WNV von Tel Aviv auf dem Flugverkehrsweg in die USA kam. Eine Ärztin aus der Bronx mit Tropenkrankheitserfahrung meinte das Virus zu erkennen und meldete dies militärischen Forschungsärzten. Das Virus, welches sich als neuer Abkömmling des originären WNV darstellte, breitete sich rasant über den amerikanischen Kontinent aus. Diese Ausbreitung war jedoch nicht vergesellschaftet mit nennenswerten Mehrerkrankungen bei Mensch und Pferd oder erhöhter Vogelmortalität.

Des Weiteren erreichte das WNV auch tropische Gebiete, wie Cuba und die Cayman Islands. Die Virulenz des Virus in den Tropen scheint allerdings reduziert zu sein und Kreuzprotektion durch andere Flaviviren tritt auf. Zudem geht man davon aus, dass die Vektoren und Wirte (Vögel) weniger kompetente Überträger sind als in anderen Breitegraden. Auch kann man einen deutlichen Unterschied in der Prävalenz von WNV zwischen Nord-und Südeuropa feststellen. Dies ist vermutlich auf das Fressverhalten des Vektors (Culex Mücke) und das Klima zurückzuführen ist. Mathias Ackermann 21.10.13 10:12 Klimaveränderungen wie Erderwärmung, ökologische Nischen die Massenbrütungen der Moskitos Kommentar [1]: Gemeint ist wahrscheinlich die Wirtsspezifität der Mücken. erlauben, starke Regenfälle die zu Fluten führen und menschliche Gewohnheiten wie zum Beispiel das Bewässern von Pflanzen können zu Erhöhungen der Vektorpopulationen führen und somit zur vergrößerten Inzidenz von WNV. Die Übertragung auf den Vektor erfolgt durch langandauernde, hochgradige Virämie im Vogel, weswegen Zugvögel wesentlich zur Verbreitung des WNV beitragen. Jedoch kann sich auch ohne den Vogel ein Zyklus durch transovarielle Übertagung im Vektor aufrechterhalten. Zusätzlich können auch andere blutsaugende Arthropoden, wie zum Beispiel Zecken, zur Streuung des Erregers beitragen.

Janine Sutter, Jasmin Steiner, Mila Bucheli, Patricia Landolt, Felicia Schuler

Epidemiologie West Nile Virus 7 Oktober 2013

Diese sind befähigt dazu, in ansonsten für das Virus widriger, trockener und warmer Umgebung, einen Zyklus aufrecht zu erhalten.

Aus Säugetieren kann das Virus kaum isoliert werden. Einzig Pferde und Lemuren zeigen geringe Virämien und können dadurch zur lokalen Erhaltung des WNV beitragen. Auch Frösche können das Virus bewirten und an die Mücke weitergeben. Mathias Ackermann 21.10.13 10:15 In Europa sind zwei verschiedene Zyklen des Virus bekannt. Ein ruraler (=ländlicher) Umlauf wird Kommentar [2]: Für diesen Abschnitt mit ungewöhnliche Behauptungen wären spezifische durch wilde Wasservögel aufrechterhalten und ein urbaner Zyklus läuft über Moskitos ab, die sowohl Referenzen sehr hilfreich. am Menschen, wie auch am Vogel Blut saugen.

Fallzahlen, Prävalenz, Inzidenz, Letalität der verschiedenen Krankheitsformen?

Die Erkrankung beim Menschen äussert sich meist in milden Grippesymptomen. In 1-2% aller Fälle gelangt das Virus jedoch ins ZNS und verursacht Enzephalitiden, welche mit hoher Letalität einhergehen. Aufgrund der Ausbrüche in Bukarest, Israel und in den USA kann drauf geschlossen werden, dass sich die Todesrate bei Erkrankungen an WNV auf ca. 10% beläuft.

Die aktuelle Inzidenz von WNV in Europa ist weitgehend unbekannt. Es treten immer wieder sporadische Fälle auf. In den USA traten von 1999 bis 2006 knapp 10‘000 neurologische Fälle und ca. 14‘000 Fälle von WN Fieber auf. Davon gingen 962 Erkrankungen tödlich aus (10%ige Letalität).

Unter welchen Voraussetzungen könnte sich das Virus in der Schweiz festsetzen?

Aus oben aufgeführten Gründen, welche die Epidemiologie des WNV beeinflussen, kann das Virus jederzeit auch in die Schweiz eingetragen werden. Über Zugvögel die durch die globale Erwärmung immer weiter nach Norden gelangen, könnten virämische Vögel vor Ort von Mücken attackiert und das Virus so verbreitet werden. Ähnlich der damaligen Situation in den USA könnte so die naive Vogelpopulation der Schweiz womöglich mit WNV überrannt werden. Durch die dadurch folgende Anwesenheit des Virus im Hauptwirten und Vektor wäre eine Übertragung auf Mammalia denkbar.

Die Voraussetzung für das Festsetzen des Virus in der Schweiz ist demnach, dass das WNV durch virämische Vögel eingeschleppt wird und die anschliessende Verbreitung durch Mücken mit ausgedehnter Wirtsspezifität erfolgt (Blut saugen an Vögeln, Säugetieren und Reptilien).

Durch die Globalisierung und durch internationale (z.T. illegale) Tiertransporte gilt es auch zu beachten, dass das WNV des Weiteren auch über bereits infizierte Säugetiere oder gar durch infizierte Menschen oder Blutsauger in die Schweiz eingeschleppt werden kann.

Quellen: 1) Laura D. Kramer, Linda M. Styer & Gregory D. Ebel, 2008, A Global Perspective on the Epidemiology of West Nile Virus, Annu. Rev. Entomol. 53:61-81 2) Zdenek Hubalek & Jiri Halouzka, 1999, West Nile Fever – a Reemerging Mosquito-Borne Viral Disease in Europe, Vol. 5, No. 5 3) Mathias Ackermann, Virus-Handbuch für Veterinärmediziner, 2013, Haupt Verlag, Bern Stuttgart Wien 4) http://de.wikipedia.org/wiki/West-Nil-Virus, 07.10.2013, 23:21

Janine Sutter, Jasmin Steiner, Mila Bucheli, Patricia Landolt, Felicia Schuler

8. Pathogenese des West-Nil-Virus auf Ebene Organismus

Wie verbreitet sich das Virus im Organismus und wie wird es wieder ausgeschieden?

1. Replikation in der Haut und in regionalen Lymphknoten 2. Primäre Virämie 3. Übertritt ins retikuloendotheliale System 4. Sekundäre Virämie 5. Zum Teil Durchbruch durch die Blut-Hirn-Schranke und Replikation in den neurologischen Zellen -> dies führt zu einer zytotoxischen Immunabwehr und einer perivaskulären Entzündung

Der natürliche Übertragungszyklus von WNV mit dem Blut ist von der Mücke zum Vogel und dann wieder zur Mücke, wobei die in der Mücke teilweise auch eine transovarielle Übertragung möglich ist. Bis vor kurzem wurde die direkte Übertragung zwischen Menschen bzw. Säugetieren ausgeschlossen, d.h. Mensch und Säugetiere galten als Endwirte. Mittlerweile gilt beim Menschen aber auch eine Über- tragung mittels Blut- und Organspenden als gesichert.4 Ausserdem wird von Infektionen über die Brustmilch sowie über Aerosole (z.B. bei einer Autopsie eines Pferdes) berichtet.4

Welche Faktoren sind relevant für die Übertragung?

Mücke:

• Virusreplikation in der Mücke abhängig von Temperatur & Feuchtigkeit (vorzugsweise warm & tro- cken) • Überlebensdauer & Anzahl der Mücken • bevorzugter Wirt (z.B. Vögel) • transovarielle Übertragung in gewissen Mückenspezies

Vogel:

• Ausprägung einer genügend hohen Virämie, um Virus auf Mücken zu übertragen (wahrscheinlich auch bei gewissen Säugern möglich!) • Meist inapparente Infektion • Zugvögel verbreiten Virus weltweit 4 • Starke Hinweise auf orale Übertragung durch Aufnahme von Fäkalien oder infiziertem Aas

Welche Verlaufsformen kommen vor?

Pferd

• Asymptomatische Infektion 4 • Enzephalomyelitis mit hohem Fieber über 40°C (10% )

Mensch

4 • Asymptomatische Infektion (80% ) • Fieber über 39°C, Kopf- & Muskelschmerzen, gastrointestinale Symptome, Hautausschläge, Lym- phadenopathie • Aus den beiden oben genannten Formen kann sich eine meist gutartige Meningitis oder eine oft mit schweren Komplikationen einhergehende Encephalitis entwickeln (<15%4)

Vogel

• Normalerweise inapparent infiziert - dies ist jedoch stark abhängig von Virusstamm und Vogelspe- zies (so starben 1999 in New York tausende Tiere, vor allem Krähen und Greifvögel) Mathias Ackermann 14.10.13 08:04 Kommentar [1]: Nein, in NY waren es die Welche Faktoren sind wichtig für die Ausprägung der einzelnen Verlaufsformen? Krähen; Greifvögel bzw. Gänse spielten eine wichtige Rolle bei den Ausbrüchen und der Risikofaktoren für die Entwicklung der neuroinvasiven Form beim Mensch: Unterscheidung der Subtypen 1 und 2 in Österreich, Ungarn, Rumänien. (siehe mein 1,4 • hohes Alter Buchkapitel WNV und Referenzen darin) 1 • Kleinkind 4 • Männliches Geschlecht 4 • Bluthochdruck 4 • Diabetes mellitus 1,4 • Immunsuppression 1 • kleine Population regulatorischer T-Zellen (auch im Tiermodell)

Schutzfaktoren:

1 • effiziente Interferon-Antwort • „funktionierende“ Makrophagen: wichtige Rolle in früher Phase der Immunantwort; protektive Rolle im ZNS1 • frühe Induktion einer spezifischen, neutralisierenden IgM-Immunantwort Ø limitiert Virämie und Invasion ins ZNS4

Selbstverständlich variiert zudem die Virulenz zwischen den einzelnen Virusstämmen. Allgemein ge- sagt korrelieren die Neuroinvasivität (Virus dringt in neuronales Gewebe ein) und -pathogenität (Virus macht im neuronalen Gewebe krank) mit der Kontrolle des Interferonsystems.3

Quellen • 1 : Colpitts et al., 2012, “West Nile Virus : Biology, Transmission and Human Infection” • 2 : Kramer et al., 2008, “A Global Perspective on the Epidemiology of West Nile Virus” • 3: Pesko and Ebel, 2012, “West Nile virus population genetics and evolution” • 4: Pradier et al., 2012, “West Nile virus epidemiology and factors triggering change in its distribution in Europe”

9. Pathogenese des West-Nil-Virus auf zellulärer und molekularer Ebene

Frane Ivasovic, Sophie Peterhans, Andrea Kühler, Valentina Bottani, Bianca Berger, David Schmid.

11. Oktober 2013

1 Zyklus

• Attachment: Die Rezeptor gesteuerte Endozytose hängt ab vom Ig-like fold, die in Domäne 3 des Glykoprotein E lokalisiert ist1.

• Eintritt: Das Glykoprotein E (=Envelope) bindet an Rezeptoren der Zelle. Es gibt 3 Rezeptoren die vermutlich beteiligt sind, jedoch ist es noch nicht ganz klar. (Die vermuteten Rezeptoren sind DC-SIGN, DC-SIGNR, αvβ3-Integrin1). • Freisetzung: Die Freisetzung des Virus aus dem Vesikel beginnt mit dem Ansäuern im Innern Vesikel. Dadurch wird das Glykoprotein E, welches ursprünglich ein Dimer ist, zum Trimer. Das führt dazu, dass ein hydrophobes Peptid freigelegt wird, welches cd loop genannt wird. (Es liegt in der Domäne 2 des Glykoprotein E1.) Das Ganze führt dazu, dass die virale Membran mit der zellulären Vesikelmembran ver- schmilzt und das Genom ins Zytosol entlassen wird1.

• Translation: Die virale RNA wird sofort von der zellulären Maschine- rie zum Polypeptid translatiert1. Dieses Polypeptid wird von viralen und zellulären Proteasen in die 3 Strukturproteine und die 7 Nicht- Strukturproteine gespalten2.

• Replikation: Die positiv-Strang RNA wird repliziert zu einer negativ- Strang RNA. Diese dient als Vorlage zur Synthese von neuen positiv- Strang RNAs2.

• Austritt: Die neuen Virionen passieren den Golgi-Apparat und werden durch Exozytose ins extrazelluläre Milieu entlassen1.

1Pesko, K.N., Ebel, G.D., 2012, West Nile virus population genetics and evolution. Infect Genet Evol 12, 182. 2M. Ackermann, 2013, Das Virus-Handbuch für Veterinärmediziner, UTB GmbH, 167.

1 2 Virusproteine

WNV besteht aus 10 verschiedenen Proteinen, davon sind 3 Strukturprotei- ne und 7 Nicht-Strukturproteine3.

Strukturproteine: Kapsid, Envelope und Prämembran, diese drei sind nötig für den Virus- eintritt in die Wirtszelle, sowie für die „Enkapsidierung“ des Virusgenoms während des Assembly4.

Nicht-Strukturproteine: NSP haben verschiedene Funktion, was in Anbetracht der sehr kleinen An- zahl Proteine, die das WNV besitzt verständlich ist.

• NS1 hat sowohl eine zelluläre wie auch eine sekretierte Form und ist sehr immunogen und es wird vermutet, dass es eine Rolle bei der Vi- rusreplikation spielt

• NS3 ist die virale Protease, welche für die Abspaltung der anderen NSP vom viralen Polyprotein verantwortlich ist und andere Enzymaktivitä- ten besitzt.

• NS5 dient als virale Polymerase, sowie an Methyltransferase und ist nötig für die Virusreplikation

• NS2A, NS2B, NS4A, NS4B, für diese Proteine wurde gezeigt, dass sie eine oder mehrere Komponenten der innaten Immunabwehr gegen virale Infektionen inhibieren5.

3M. Ackermann, 2013, Das Virus-Handbuch für Veterinärmediziner, UTB GmbH, 167. 4Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 635. 5Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 635-636. 3 Pathogenese bei der Mücke

Infektion erfolgt über Blutaufnahme, die Viren gelangen zum Mitteldarm, dessen Epithel sie inﬁzieren und sich dort replizieren6. Anschliessend gelangen sie über Hämolymphe in die Speicheldrüsen7. Proteine die an diesem Vorgang beteiligt sind:

• Chitine u. andere Proteine bilden eine Barriere im Mitteldarm der Mücke die Viren an der Invasion hindern können7.

• Inﬁzierte Zellen sezernieren C-Typ Lectin, welches Virionen bindet und deren Aufnahme durch eine Phosphatase fördert8.

Immunabwehr: In der Regel sind Mücke persistent mit WMV inﬁziert, trotzdem zeigen neue Untersuchungen, dass in den Mücken eine Immunantwort induziert wird. Man nimmt an, dass die Immunantwort der Mücke aus 2 Komponenten besteht7:

• einerseits einer innaten Immunabwehr, die über drei verschieden Si- gnalwege (Toll, JAK-STAT, IMD) zu einer Expression von antimikro- biellen Peptiden (AMP’s) führen7.

• andererseits aus der RNA-Interferenz (RNAi), die durch virale doppel- strängige RNA aktiviert wird7.

6Pesko, K.N., Ebel, G.D., 2012, West Nile virus population genetics and evolution. Infect Genet Evol 12, 186. 7Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 637. 8Pesko, K.N., Ebel, G.D., 2012, West Nile virus population genetics and evolution. Infect Genet Evol 12, 182. 4 Pathogenese und Immunität beim Säugetier

• Die Mücke inokuliert beim Stich das Virus in den Wirt. Die erste Vi- rusvermehrung erfolgt lokal in den Fibroblasten und Keratinozyten. Anschliessend kommt es zu einer Virämie9.

• PAMPs (Non-self pathogen-associated molecular patterns) sind Mus- ter/Teile des Virus durch die PRR (Pathogen Recognition Receptor) der Zelle des Wirtes erkannt werden10.

• Die zwei wichtigsten PRR sind: Toll like Receptor (TLR) und cytoplas- matische RNA Helicase (RIG-1 und MDA5). Diese zwei Komponenten führen schlussendlich zur Produktion von IFN Typ I (IFN alpha und beta)11.

• Die Makrophagen-Aktivierung sowie die Produktion von IFN gilt als wichtigster Teil der Immunantwort gegen WNV10.

• Das Interferon beeinﬂusst direkt den Virustiter im Blut: hoher Titer → niedrige Viruskonzentration12.

• Unterschied zwischen kranken vs. gesunden Tieren? Gesunde Tiere können INF Typ I produzieren; kranke Tiere (Immunsupprimierte, alte Tiere) können INF Typ I nicht in genügenden Mengen ausschütten13.

• Unterschied Mücken vs. Säuger: Säugerzellen sezernieren IFN-alpha, Mückenzellen nicht (brauchen es ev. nicht?)textsuperscript11.

5 Bibliograﬁe

• M. Ackermann, 2013, Das Virus-Handbuch für Veterinärmediziner, UTB GmbH.

• Leis, A.A., Stokic, D.S., 2012, Neuromuscular manifestations of west nile virus infection. Front Neurol 3, 37.

• Cho, H., Diamond, M.S., 2012, Immune responses to West Nile virus infection in the central nervous system. Viruses 4, 3812-3830.

9Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 638. 10Diamond, M.S., Gale, M., Jr., 2012, Cell-intrinsic innate immune control of West Nile virus infection. Trends Immunol 33, 522. 11Diamond, M.S., Gale, M., Jr., 2012, Cell-intrinsic innate immune control of West Nile virus infection. Trends Immunol 33, 523. 12Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Microbiol Rev 25, 641. 13Leis, A.A., Stokic, D.S., 2012, Neuromuscular manifestations of west nile virus infection. Front Neurol 3, 37,S.6. • Colpitts, T.M., Conway, M.J., Montgomery, R.R., Fikrig, E., 2012, West Nile Virus: biology, transmission, and human infection. Clin Mi- crobiol Rev 25, 635-648.

• Diamond, M.S., Gale, M., Jr., 2012, Cell-intrinsic innate immune control of West Nile virus infection. Trends Immunol 33, 522-530.

• Kramer, L.D., Styer, L.M., Ebel, G.D., 2008, A global perspective on the epidemiology of West Nile virus. Annu Rev Entomol 53, 61-81.

• Pesko, K.N., Ebel, G.D., 2012, West Nile virus population genetics and evolution. Infect Genet Evol 12, 181-190.

• Pradier, S., Lecollinet, S., Leblond, A., 2012, West Nile virus epidemiology and factors triggering change in its distribution in Europe. Rev Sci Tech 31, 829- 844.

Impfstoffe gegen West-Nil-Virus

Das West-Nil-Virus, ein Virus aus der Familie der Flaviviridae, ist ein Vektor-übertragenes Virus, das Menschen und Pferde befällt. Vektor ist die Culex-Mücke.1

Allgemeines2 Zur Zeit sind weltweit 4 Vakzine für Pferde und 1 Vakzin für Gänse zugelassen. Impfstoffe für den Menschen existieren noch keine, sie sind aber in Entwicklung. Strategien zur Herstellung: − Inokulation von grossen Mengen an inaktiviertem Virus (Antigen) ins Tier − Expression von WNV Proteine in einem Wirtstier um eine Immunantwort hervorzurufen -> als rekombinantes subunit Vakzin -> über Inokulation von DNA-Plasmiden in eine Wirtszelle (WNV-Gene werden dann von der Zelle produziert) − Verwendung von Chimäre-Viren die PrM und E Gene enthalten, die man in ein attenuiertes Flavivirus-Gerüst eingebaut hat − Attenuierte Viren -> über induzierte Mutationen in nicht-Struktur-Genen -> über induzierte Mutationen in Kapsid-Genen

Impfstoffe gegen WNV3 Merkmale von Impfstoffen gegen WNV. Was können sie? Was können sie nicht?

Inaktivierte Vakzine: Ein formalin-inaktivierter WNV-Impfstoff funktioniert ähnlich wie formalin-inaktivierte Impfstoffe gegen andere Flaviviren (Japanisches Encephalitis Virus und FSME): 2 Wochen nach der Impfung erreichte man bei Hamstern nach zwei Applikationen einen kompletten Schutz vor einer letalen experimentellen WNV-Infektion Exposition. Nachteile: Für eine schützende Immunantwort sind mehrere Applikationsdosen nötig. Die Dauer der hervorgerufenen Immunität ist nicht bekannt. Man hat festgestellt, dass bei geimpften Tieren, die nur eine schwache oder qualitativ ungenügende humorale Immunantwort entwickelten, eine nachfolgende Infektion mit einem heterologen Flavivirus sogar verstärkt sein kann.

Subunit-Vakzine: Durch wiederholte Impfungen mit gereinigtem rekombinantem WNV E Protein erreichte man hohe Titer von neutralisierenden Antikörper gegen WNV. Nachteile: Eine genügende zelluläre Immunität kann auch bei mehrmaliger Applikation nicht erreicht werden. Man hat zwar eine gute humorale Immunität, die Impfung reicht aber nicht für einen gesamthaft guten Schutz. Experimente zeigten, dass immunisierte Mäuse nur vor niedrigdosierter WNV-Infektion vollständig geschützt sind; hohe letale Dosen überleben sie nicht.

DNA-Vakzine: Erzielt guten Schutz gegen Virämie und Mortalität (Experiment mit geimpften Pferden und Mäusen) nach einmaliger Applikation. Es handlet sich um eine Plasmid-DNA, die für das Membranprotein prM, das Envelope-Protein E oder das Kapsidprotein C des WNV kodiert.  Durch die Co-Expression von prM und E entstehen immunogene subvirale Partikel, die eine starke humorale und zelluläre Immunantwort induzieren.  Durch die Expression des Kapsidprotein C wird eine potente antigenspezifische Th1- Antwort und eine cytotoxische T-Zell-Antwort ausgelöst.

1 Vgl. Ackermann, Mathias (Hrsg.): Virus-Handbuch für Veterinärmediziner. 1. Auflage. Zürich 2013, S. 187 2 Vgl. Kramer et. al.: A Global Perspective on the Epidemiology of West Nile Virus. In: Annual Reviews, 2008, S. 71f. 3 Vgl. Diamond et alt.: Innate and Adaptive Immune Responses Determine Protection against Disseminated Infection by West Nile Encephalitis Virus. In: VIRAL IMMUNOLOGY, Volume 16, Number 3, 2003, S. 267-268

Poster 10: Impfstoffe gegen West-Nil-Virus; Anja, Chiara, Perrine, Danko, Laura, Seraina 1 Kreuzreaktive Vakzine: Die Impfung gegen Japanese Encephalitis und Dengue Virus bringt auch gegen WNV 70-100% Schutz vor Virämie und Mortalität. Beim Menschen gilt dieser Schutz nicht.

Attenuierte-Vakzine: Attenuierte WNV-Impfstoffe induzieren sowohl eine humorale wie eine zelluläre Immunantwort (ähnlich wie bei einer natürlichen Infektion). Man entwickelte zwei verschiedene attenuierte Impfstämme: 1. Attenuierter WNV-25 Stamm: Herstellung: Attenuierung der Neuroinvasivität durch serielle Passage durch eine Mosquitozellkultur Eine Impfung mit WNV-25-Impfstoff schützt Gänse gegen letale Dosen einer Infektion mit einem WNV- Isolat. 2. Chimärer WNV-YF – Impfstoff: Herstellung: Einfügen der Strukturgene für prM und prE in einen infektiösen 17D-Impfstoff-Strang gegen das Gelbfiebervirus Nach einmaliger Impfung mit dem chimären WNV-YF Virus produzierte der Impfling neutralisierende Antikörper und Komplement-bindende Antikörper. Geimpfte Hamster waren nach experimenteller Infektion mit einer letalen Dosis des virulenten WNV vollständig geschützt. Nachteile: Attenuierte Impfstoffe dürfen grundsätzlich nicht angewendet werden bei immundefizienten Individuen. Man ist aber dabei, zusätzlich attenuierte Mutationen in das chimäre WNV-YF einzubauen, so dass die Neuroinvasivität und die Neurovirulenz sicher ausgeschaltet sind.

Zugelassene Impfstoffe Weltweit zu Verfügung stehende Impfstoffe4

Vetera® WNV Vakzin − Inaktiviertes West-Nil-Virus Recombitek® − Chimäres rekombinantes Kanarienpockenvakzin − Der Impfstoff exprimiert das prM und das prE Protein (abstammend von einem 1999 New York WNV-Isolat) − Alle geimpften Pferde entwickelten neutralisierende AK gegen WNV und zeigten signifikant weniger klinische WN - Symptome nach einer nachfolgenden Infektion. West Nile-Innovator® DNA − DNA – Impfstoff − enthält Plasmid DNA, welche für prM und prE Protein kodiert

Impfstoffe in der Schweiz Wird in Endemiegebieten empfohlen. Seit neustem (ca. 2012) ein zugelassener Impfstoff für Pferde:

Duvaxyn® WNV Vakzin5 − Inaktiviertes West-Nil-Virus, Stamm VM-2 (Innovator Äquivalent) − Adjuvans: MetaStim™ (SP-Öl) − Indikation: zur aktiven Immunisierung von Pferden ab einem Mindestalter von 6 Monaten oder älter gegen die West Nil-Erkrankung, um die Anzahl virämischer Pferde zu reduzieren. Die Anwendung von Duvaxyn WNV reduziert die Zahl der virämischen Tiere nach einer natürlichen Infektion, kann sie aber nicht systematisch verhindern. − Beginn der Immunität: 3 Wochen nach der Grundimmunisierung Dauer der Immunität: 12 Monate nach der Grundimmunisierung − Besonderes: Nur gesunde Tiere impfen. Es liegen keine Informationen zur Sicherheit und Wirksamkeit der gleichzeitigen Anwendung dieses Impfstoffs mit einem anderen Impfstoff, immunologischen Produkt oder Tierarzneimittel vor.

4 Vgl. Kramer et alt.: Vaccines and Antiviral Treatments, In: Annual Reviews, 2008, S. 72f. 5 Vgl. Institut für Veterinärpharmakologie und -toxikologie: Tierarzneimittelkompendium, Duvaxyn® WNV ad us. vet. http://www.vetpharm.uzh.ch/reloader.htm?tak/00000000/00001710.V AK?inhalt_c.htm, Informationsstand: 06/2011, (Abrufdatum: 06.10.2013)

Poster 10: Impfstoffe gegen West-Nil-Virus; Anja, Chiara, Perrine, Danko, Laura, Seraina 2

11. Antikörper und zelluläre Immunität gegen das West-Nil-Virus

Antikörper

Neutralisierende Antikörper sind wichtig für die Immunantwort, da sie die Ausbreitung einer Flavivirus-Infektion verhindern können. Antikörper können freie Viren neutralisieren, indem sie das Virus spezifisch an ihr Fab-Fragment binden. Dadurch wird das Rezeptorbindungsprotein (hier E) blockiert, was eine Infektion von neuen Zellen verhindert. Das Fab-Fragment kann aber auch an nicht-neutralisierende Epitope von E und prM binden, was jedoch zu einer Konformationsänderung im Fc-Fragment des Antikörpers führt. Diese hat zur Folge, dass natürliche Killerzellen diesen Antikörper via Fc-Rezeptoren binden können, was zur Antikörper-vermittelten zellulären Zytotoxizität (engl. ADCC) führt. Die vom Antikörper markierten Zielzellen werden durch Perforine und Granzyme zerstört, welche in den Granula der natürlichen Killerzellen enthalten sind.1

Die Lyse von infizierten Zellen durch Antikörper wird ebenfalls durch Komplement unterstützt.2 Das Nicht-strukturprotein 1 (NS1) ist ein Cofaktor der Virusreplikation, von dem auch eine von der infizierten Zelle sezernierte Variante existiert, die eine immunmodulierende Wirkung hat, indem sie antagonistisch zum Toll-like-Rezeptor-Signal und zur Komplementaktivierung wirkt. Die sezernierte Variante von NS1 lagert sich wieder auf infizierten Zellen ab und bindet direkt an verschiedene Komponenten des Komplement- Systems, insbesondere an C4. Demzufolge wird C4 zu C4b abgebaut, was zum Erliegen der Komplement-Reaktion führt. Werden Antikörper gegen NS1 gebildet, so können diese natürlich nicht Virus-neutralisierend wirken, da NS1 kein Bestandteil des Virions ist. Sie können jedoch die Komplement-abbauende Wirkung von NS1 "neutralisieren", indem sie die Bindung von NS1 an C4 verhindern, was sich dann wie eine starke Komplement-Aktivierung auswirkt und einen starken, eindämmenden Effekt auf die Virusreplikation hat.3

Von dieser "Neutralisation" ausgeschlossen ist jedoch die intrazelluläre Variante von NS1, welche die TLR3-abhängige Signaltransduktion blockiert, indem es die Translokation des interferon regulatory factor 3 (IRF3) vom Zytoplasma in den Zellkern verhindert.4 Damit kann NS1 immer noch die Interferon beta Antwort der infizierten Zellen unterdrücken, eine Funktion, die von Antikörpern nicht beeinträchtigt werden kann. Allenfalls könnte dies durch Antikörper der Klasse IgA gelingen.

Die an der Immunantwort beteiligten Antikörperklassen sind IgM und IgG. Spezifische IgM erscheinen 4-7 Tage nach der Infektion und kontrollieren somit die frühe Infektion. Sie kön- nen bis zu einem Jahr persistieren. 4-5 Tage nach den ersten Krankheitssymptomen erscheinen dann IgG, welche wahrscheinlich für einen langanhaltenden Schutz gegen eine Reinfek- tion verantwortlich sind.5

1 vgl. Wikipedia, in: http://de.wikipedia.org/wiki/Antikörperabhängige_zellvermittelte_Zytotoxizität, 13.10.2013 2 vgl. Diamond et al., 2003 3 vgl. Diamond et al., 2003 4 vgl. De Filette et al., 2012 5 vgl. De Filette et al., 2012

Infektionsimmunologie Teil Virologie P. Schnetzer, S. Maier, L. Fierz, M. Rüegg, N. Studer, N. Butz Zelluläre Immunität

Die zelluläre Immunantwort ist wichtig für die Eliminierung der infizierten Zellen. 7 Zytotoxische T-Zellen (CD8+ T-Zellen) wirken über mehrere Mechanismen einer WNV- Infektion entgegen. Sie produzieren im ZNS antivrale Zytokine, insbesondere Interferon-γ, und induzieren Apoptose der Zielzellen via Perforine und Granzyme, Fas-Fas Ligand oder TRAIL (= tumor necrosis factor-related apoptosis-inducing ligand) abhängiger Weg. Die Zielzellen exprimieren MHC-I, welches mit einem Antigenfragment beladen als Komplex an die Zelloberfläche verlagert wird. Es kommt zur Antigenpräsentation, wodurch die CD8+ T- Zellen die infizierten Neuronen erkennen. Eine überschiessende CD8+ T-Zell-Antwort führt zur Zerstörung von infizierten Neuronen, wobei aber auch gesunde in Mitleidenschaft gezogen werden können. Deshalb kann dieser Schutzmechanismus auch zur Verschlimmerung des Krankheitsgeschehens führen.8

Natürlich sind neben den zytotoxischen T-Zellen auch noch Makrophagen, natürliche Killer- zellen und dendritische Zellen beteiligt, diese gehören jedoch zum innaten Immunsystem, auf welches in der Beantwortung der Frage 12 eingegangen wird. Aus diesem Grund beziehen wir uns nur auf den Teil der adaptiven Immunantwort, der durch die zytotoxischen T-Zellen sowie B-Zellen und Antikörper vermittelt wird.9 10

Fazit

Für eine effektive Immunantwort sind beide Komponenten wichtig, das heisst: Antikörper limitieren die Virämie und die Weiterverbreitung des Virus im Organismus, die vollständige Eliminierung erfolgt aber durch die zelluläre Immunität. Beteiligte Antikörper sind IgM und IgG, welche die Strukturproteine E, zum Teil M und NS1 erkennen. In den dazu durchgeführten Studien wurde gezeigt, dass vor allem spezifische IgM für die frühe effiziente Immunantwort und IgG gegen die Hüllproteine E und prM sowie gegen das nicht-Strukturprotein NS1 für den langanhaltenden Schutz erwünscht sind.

Quellenverzeichnis

• Cho, H., Diamond, M.S., 2012, Immune responses to West Nile virus infection in the central nervous system. Viruses 4, 3812-3830. • De Filette, M., Ulbert, S., Diamond, M., Sanders, N.N., 2012, Recent progress in West Nile virus diagnosis and vaccination. Vet Res 43, 16. • Diamond, M.S., Shrestha, B., Mehlhop, E., Sitati, E., Engle, M., 2003, Innate and adaptive immune responses determine protection against disseminated infection by West Nile encephalitis virus. Viral Immunol 16, 259-278. • Wikipedia (2013), in: http://de.wikipedia.org/wiki/Zelluläre_Immunantwort, 13.10.2013 • Wikipedia (2013), in: http://de.wikipedia.org/wiki/Antikörperabhängige_zellvermittelte_Zytotoxizität, 13.10.2013

7 vgl. Diamond et al., 2003 8 vgl. Cho and Diamond, 2012 9 vgl. Suter, M. (2011), Immunologie Fibel, Kap. 2.1 10 vgl. Wikipedia, in: http://de.wikipedia.org/wiki/Zelluläre_Immunantwort, 13.10.2013

Infektionsimmunologie Teil Virologie P. Schnetzer, S. Maier, L. Fierz, M. Rüegg, N. Studer, N. Butz

12. Intrinsische und innate Abwehr gegen West-Nil-Virus

Interferone Es werden drei verschiedene Interferone (IFN) unterschieden, die jeweils von verschiedenen Zelltypen gebildet werden. Zu den Typ I IFN gehören das IFN α und IFN β. Das Typ II Interferon umfasst das IFN-γ.

IFN α: gebildet unter anderem von Monozyten als Antwort auf die Erkennung viraler oder bakterieller Nukleinsäure à Aktivierung des JAK-STAT-Signalweg

Funktion der IFN α • aktiviert umliegende Virus-infizierte sowie nicht infizierte Zellen zur Proteinsynthese (antiviraler Status)à Hemmung der Proteinsynthese (also Hemmung der Translation) in infizierten Zellen sowie Abbau viraler und zellulärer RNA • Bildung MHCI & Proteasomen, welche Virus-infizierte Zellen durch T-Lymphozyten leichter angreifbar machen Mathias Ackermann 17.10.13 13:53 Kommentar [1]: Das ist mir neu. Jede Zelle • aktiviert NK-Zellen bildet und präsentiert MHC-I; jede Zelle hat Proteasomen. IFN β: Ich verstehe nicht, was sie damit aussagen wollen. Gibt es allenfalls eine Referenz dazu? gebildet von Virus-infizierten Fibroblasten (Zellen des Bgw) hat ähnliche Wirkung wie das IFN-α

ð die wichtigste Wirkung der TypI IFN ist die der Schutz vor Virusbefall ð IFN I induziert mehrere hundert Gene, deren Produkte zur wirksamen direkten und indirekten Hemmung der Virusreplikation führen. Durch die Ausschüttung von IFN werden zwar die bereits von Viren befallenen Zellen nicht gerettet, aber die Nachbarzellen werden effizient vor Virus-Befall geschützt. (Scriptum Immunologie I des Vetsuisse-Standortes Zürich für das HS 2011, Suter und Jungi)

IFN-γ : wird gebildet von aktivierten T-Zellen und NK-Zellen à aktiviert Makrophagen und stellt das wichtigste TH1-Zytokin dar Typ II IFN führt zur Synthese von proinflammatorischen und antiviralen Molekülen, unter anderem NO, was die Phagozytoseaktivität der Abwehrzellen verstärkt.

ð hauptsächliche Wirkung: Aktivierung von Makrophagen, MHCI und MHCII

Interferon I abhängige Immunreaktion ist essentiell zum Schutz vor einer Flavivirusinfektion. Die dendritischen Langerhanszellen in der Haut sind die ersten Zellen, die IFN als Reaktion auf die Infektion sezernieren.

Knock-out Mäuse welche kein Typ I IFN bilden, hatten eine 100% Mortalität nach WNV Infektion, was die Wichtigkeit von IFN beweist. (Diamond and Gale, 2012)

Komplementsystem Das Komplementsystem kann über den klassischen oder den alternativen Weg aktiviert werden. Beim klassischen Weg wird ein Antikörper-Antigenkomplex durch den Komplementfaktor C1 gebunden und startet die Kaskade. Beim alternativen Weg fungiert der Komplementfaktor C3 als Kaskade-startender Faktor. Es sind Mathias Ackermann 17.10.13 11:35 keine Antikörper nötig für die Aktivierung, sondern das C3 zerfällt spontan zu C3b welches dann Antigene Kommentar [2]: Um welches Antigen könnte es sich bei WNV handeln? binden kann. Dies führt zu:

C5-C9 = Membranangriffs-Komplex à Lysiert behüllte Viruspartikel und infizierte Zellen C3a, C5a= proInflammatorische Peptide à werden durch Komplementaktvitierung synthetisiert à Rekrutierung und Aktivierung von Monozyten und Granulozyten

C3 = proteolytisch à zerstören opsonierte Viruspartikel àerleichtert die AG-Präsentation mittels Makrophagen und Dendritischen Zellen àinduziert die Proliferation von spez. T-Zellen zur AK- Produktion

ð das Komplementsystem trägt einen grossen Beitrag zur Bekämpfung der WNV-Infektion bei Mathias Ackermann 17.10.13 12:54 (Scriptum Immunologie I des Vetsuisse-Standortes Zürich für das HS 2011, Suter und Jungi) Kommentar [3]: Sehr vage und ohne Referenz, im Immunologie-Skript wird wohl kaum auf WNV eingegangen, oder? In den verteilten Unterlagen gibt es sehr konkrete Hinweise dazu, welches WNV Protein im T-Lymphozyten Zusammenhang mit dem Komplementsystem eine zelluläre Immunität ist wichtig für die Ausmerzung von WNV-infizierten Zellen (negative) Rolle spielt. Zytotoxische T-Lymphozyten proliferieren bei Infektion und lysieren Zellen, die auf der Oberfläche AG gebunden haben à dient vor allem der Elimination von Virus-infizierten Zellen & Zellen mit intrazellulären Erregern. Mathias Ackermann 17.10.13 13:57 Kommentar [4]: Hm, bei dieser Wortwahl bin ich etwas anderer Meinung; um welche Zellen und Zudem synthetisieren sie Inflammatorische Cytokine (IL1 und IFNγ). welche Antigene würde es sich denn im Fall von (Scriptum Immunologie I des Vetsuisse-Standortes Zürich für das HS 2011, Suter und Jungi) WNV handeln? Mathias Ackermann 17.10.13 12:51 Kommentar [5]: Damit bin ich einverstanden Natürliche Killerzellen ð sind wichtig in der Bekämpfung von Virus Infektionen. Tiere mit einem NK-Defekt erkranken an schweren viralen Infektionen und sterben früh Sie haben die Fähigkeit, Virus-infizierte Zellen direkt abzutöten sowie inflammatorische Zytokine zu produzieren, damit die Infektion eingedämmt wird. Die Erkennung Virus-infizierter Zellen erfolgt mittels Fcγ-Rezeptor, der die AK-bedeckte Zielzelle erkennt (ACCD). Sie wirken nach einem missing-self Prinzip à Infizierte Zellen werden durch verändertes oder fehlendes MHC I erkannt. D.h. ist auf der Zielzelle kein MHC oder ein falsches MHC, wird der Tötungsprozess Mathias Ackermann 17.10.13 12:58 durch die NK-Zelle nicht inhibiert, was das Abtöten der Zielzelle zur Folge hat. Kommentar [6]: Meinen sie nicht ADCC? Falls ja, welches Protein von WNV wäre involviert? Falls Die NK-Zelle kann dadurch infizierte Zellen enttarnen, welche von T- Zellen nicht erkannt wurde, da sie kein nein, worum handelt es sich? MHC I aufweisen. Mathias Ackermann 17.10.13 12:56 Kommentar [7]: Das ist aber ein anderes Die NK-Zellen sind somit ein wichtiger Bestandteil der innaten Abwehr gegen WNV. Prinzip als ADCC!!! Spielt dieses Prinzip bei WNV Die Zelllyse erfolgt durch Sekretion von cytotoxischen Granula, die Perforin und Granzyme enthalten eine Rolle; falls ja, auf welchem Weg? (Scriptum Immunologie I des Vetsuisse-Standortes Zürich für das HS 2011, Suter und Jungi) Mathias Ackermann 17.10.13 08:58 Kommentar [8]: Wie beeinflusst WNV denn "das Aufweisen" von MHC-I, sodass die Zellen Makrophagen nicht von T-Zellen erkannt werden? Sie nehmen zirkulierende Viruspartikel auf, stimulieren die Zytokinproduktion und stimulieren die AG- Präsentation in B- und T-Zellen von 2° Lymph. Organen. Experimentell konnte gezeigt werden, dass das Fehlen von Makrophagen die Letalität einer WNV-Infektion erhöht. (Diamond and Gale, 2012)

Dendritische Zellen Mathias Ackermann 17.10.13 09:00 Kommentar [9]: Tststs, DAS Virus, nicht "der" Sie gehören zu den mononukleären Phagozyten und dienen der AG-Verarbeitung und -Präsentation. Virus!!!!!!!!! Durch den Stich der Mücke gelangt der Virus in die Haut, wo er von Langerhanszellen aufgenommen und in die Mathias Ackermann 17.10.13 09:01 Lymphknoten transportiert wird. Dort kann eine Vermehrung durch das innate Immunsystem schon in einer Kommentar [10]: Das würde bedeuten, dass frühen Phase verhindert werden. Der Speichel der Mücken kann dabei die wirtseigene Abwehr entscheidend die DC gar nicht Virusprotein präsentieren und beeinflussen. ( Diamond et al., 2003) damit gar keine adaptive Immunantwort einleiten können?????

ð Faz i t : Die innate Immunantwort ist nur der Anfang einer komplexen Immunantwort, die in der Elimination des WNV resultiert. Dabei spielt auch die zellintrinsische Immunantwort eine wichtige Rolle, welche durch bestimmte Gene und deren Produkte, bei der Maus durch Knock-in experimentell erforscht, den Mathias Ackermann 17.10.13 13:01 Wirt vor WNV schützen kann. (Diamond and Gale, 2012) Kommentar [11]: Haben sie diesen Begriff irgendwo gegenüber "innate" abgegrenzt?

Mathias Ackermann 17.10.13 13:00

Kommentar [12]: Sehr vage. Rolle und Potential im Haustier/Mensch bzw. Vektor

Studien (Bertolotti et al., 2007; Amore et al., 2010) haben gezeigt, dass die WNV Population in Vögeln relativ genetisch homogen ist. Daraus kann geschlossen werden, dass es innerhalb des Immunsystems der Vögel eine starke Selektion des WNV gibt. Beim Vektor, (Jerzak et al., 2005; Jerzak et al., 2007) der Culex Mücke, gibt es jedoch eine grosse genetische Variabilität. Das heisst, es muss ein Selektionsfaktor geben, der dies begünstigt. Mathias Ackermann 17.10.13 11:18 Wirbeltiere: Kommentar [13]: Nein, Mutanten entstehen bei jedem Replikationszyklus (fehlerhafte RNA Bei Wirbeltieren ist die erste Immunantwort durch IFN α /β. Im Zytoplasma wird durch die RNA-Helikasen Polymerase); beim Vogel sorgen MDA-5 und RIG-I dsRNA erkannt, was die Signalkaskade auslöst und ein antiviraler Status der Zelle induziert. Selektionsfaktoren dafür (u.a. das Immunsystem), dass nur wenige Varianten tatsächlich eines Dies fördert das Virus besonders schnell in seiner Replikation und selektioniert den fittesten Genotyp des persistierende Infektion verursachen können. Virus. Bei der Mücke FEHLEN diese Selektionsfaktoren offensichtlich, was automatisch zu einer grossen genetischen Variabilität führt. Insekten: Mathias Ackermann 17.10.13 11:22 Auch hier ist der Trigger die dsRNA im Cytosol, wobei die Antwort der Insekten durch RNA-Interference erfolgt. Kommentar [14]: Nein, der antivirale Status Dabei werden Virus stämmige small interfering RNA (viRNA) vom RNA-induced-silencing-complex (RISC) "fördert" überhaupt kein Virus in seiner gebunden und die entsprechende Gene ausgeknockt. Es ist also eine sequenzspezifische Bindung von viralen Replikation. Hingegen wären unter diesen RNAs. Gegebenheiten Viren im Vorteil, die ihre Replikation abgeschlossen haben, bevor der Mit diesem System werden die allermeisten Genotypen spezifisch ausgeschaltet. Der Nachteil an dieser antivirale Status aufgebaut ist. In der Selektion Methode ist, dass seltene Mutanten nicht im Repertoir der RNAi sind und somit einen starken Selektionsvorteil werden demzufolge "langsame" Viren eliminiert, während "schnelle" Viren sich vermehren können haben. Die zelleigenen RNAi binden die virale RNA im RISC und hindern diese somit an der Translation. (Ding, und somit die Selektion überstehen. 2010) Mathias Ackermann 17.10.13 11:24 Kommentar [15]: Ihre Kollegen vom Poster 1 erklären, WNV sei ein EINZELSTRÄNGIGES RNA Fazit: Die Mücken bilden die Grundlage für immer neue verschieden Genotypen und sorgen dafür, dass immer Virus. Wie soll denn eine doppelsträngige RNA hier eine Rolle spielen? (Fangfrage!) neue Viruslinien gebildet werden, während bei den Wirbeltieren, beziehungsweise hauptsächlich den Vögeln, Mathias Ackermann 17.10.13 13:04 die verschiedenen WNV-Genotypen nach ihrer biologischen Fitness selektioniert werden. Kommentar [16]: Diese Aussage würde aber dagegen sprechen, dass in den Mücken eine "grosse genetische Variabilität" herrscht. Rolle in der Immunität und in der Pathogenese?

Nichtstrukturproteine des WNV sind involviert in der RNA-Replikation und in der Modulation der Immunantwort der Wirtszelle. NS2a ist ein kleines, hydrophobes Transmembranprotein, das einen Teil des Replikationskomplexes darstellt und zur Inhibierung der Interferon-Induktion dient Ü IFNβ Produktion wird inhibiert (Role of Nonstructural Protein NS2A in Flavivirus Assembly†; Jason Y. Leung1) Mathias Ackermann 17.10.13 13:27 Kommentar [17]: Was heisst hier "aktivieren"? IRF3 wird konstitutiv synthetisiert, verbleibt aber Viele Viren aktivieren ein IRF3 (Interferon-Regulatorischer Faktor 3) um den antiviralen Status der Zelle und die im inaktiven Zustand im Zytoplasma. Bei der IFN α/β Produktion zu unterdrücken. Stimulierung des TLR-Signalweges wird es phosphoryliert und in den Zellkern verbracht, wo es die Expression verschiedener Interferone Beim WNV jedoch fällt diese Stimulation des IRF3 erst relativ spät aus (12-116h pi). Das erlaubt dem WNV auf erhöht. einem hohen Level zu replizieren was jedoch die Induktion von IFN α/β induziert. Dies bringt folgende Vorteile: Für mich wäre diese Phosphorylierung eine I) WNV kann sich schnell in nicht-Infizierte benachbarte Zellen ausbreiten, indem der parakrine "Aktivierung" von IRF3; wenn ich ein Virus wäre und die Interferon-Expression verhindern wollte, antivirale Effekt von IFN α/β überholt wird dann würde ich versuchen IRF3 im inaktiven II) durch Akkumulation von viralen Proteinen kann die JAK/STAT –Aktivierung gehemmt werden Zustand zu erhalten. Ich interpretiere offensichtlich Fig. 1 aus Diamond & Gale 2012 ganz anders als ihr. Ein weiterer Mechanismus des WNV, die Immunabwehr des Wirtes zu umgehen, ist die Möglichkeit, dass es erst spät vom PRR erkannt wird bzw. sich erst spät zu erkennen gibt. (How Flaviviruses Activate and Suppress the Interferon Response; Jorge L. Muñoz-Jordán)

Überwinden der Blut-Hirn-Schranke: Wie genau das WNV Ins ZNS gelangt, beziehungsweise wie es die Blut-Hirn-Schranke (BHS) überwindet, ist nicht vollständig geklärt, es existieren mehrere Theorien. Eine davon ist beispielsweise, dass TNF α (welches Mathias Ackermann 17.10.13 11:30 vor allem von Makrophagen sezerniert wird) die thight junctions der BHS so verändert, dass die Permeabilität Kommentar [18]: ... infolgedessen ist die Neutralisation der Viren im Blutstrom durch für das Virus erhöht wird. Antikörper ein wesentlicher Schutzfaktor. Wenn (Cho and Diamond, 2012) kein Virus über das Blut angeliefert wird, kann es auch nicht die BHS überwinden. Referenzen Mathias Ackermann 21.10.13 10:48 Kommentar [19]: Es fehlt die Liste der zitierten Literatur. ANRV330-EN53-04 ARI 2 November 2007 15:18

A Global Perspective on the Epidemiology of West Nile Virus

Laura D. Kramer,1,2 Linda M. Styer,1 and Gregory D. Ebel3

1The Arbovirus Laboratories, Wadsworth Center, New York State Department of Health, Slingerlands, New York 12159; email: [email protected] 2Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201 3Department of Pathology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131; email: [email protected]

Annu. Rev. Entomol. 2008. 53:61–81 Key Words Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. First published online as a Review in Advance on Culex, emerging virus, ﬂavivirus, vector-borne, zoonosis July 23, 2007

The Annual Review of Entomology is online at Abstract ento.annualreviews.org West Nile virus (WNV) (Flavivirus: Flaviviridae) is the most This article’s doi: widespread arbovirus in the world. A signiﬁcant range expansion 10.1146/annurev.ento.53.103106.093258 occurred beginning in 1999 when the virus was introduced into Copyright c 2008 by Annual Reviews. New York City. This review highlights recent research into WNV All rights reserved epizootiology and epidemiology, including recent advances in un- 0066-4170/08/0107-0061$20.00 derstanding of the host-virus interaction at the molecular, organis- mal, and ecological levels. Vector control strategies, vaccines, and antivirals, which now must be considered on a global scale, are also discussed.

61 ANRV330-EN53-04 ARI 2 November 2007 15:18

THE VIRUS a host-derived lipid bilayer bearing dimers of the viral envelope protein and the mem- Classification brane protein. Thus, the antigenic, genetic, WNV: West Nile virus West Nile virus (WNV) is a member of and three-dimensional structure of WNV and its constituent proteins are similar to several Flavivirus: genus of the Flavivirus genus of the family Flaviviri- other flaviviruses and form the basis for its predominantly dae, which contains approximately 70 mem- zoonotic bers, most of which are transmitted either by classification (91). positive-sense RNA mosquitoes or ticks (20). WNV is classified viruses Host Response to Infection approximately 11 kb within the Japanese Encephalitis serological in length complex on the basis of cross-neutralization Host cells possess a wide array of antiviral (20) and molecular genetic (71) studies. Re- mechanisms that interfere with virus replica- cent studies have extensively characterized tion (see References 16, 17, 21, and 146). Elu- the structures of intact WNV virions by cidating these mechanisms and understand- cryo-electron microscopy (Figure 1) (90) and ing how WNV subverts them are areas of found them to be similar to the structures intense research because the findings may lead of other flaviviruses (38, 151). The WNV to a better understanding of pathogenesis and virion, like that of other flaviviruses, is en- consequently the development of novel inter- veloped, spherical, approximately 40–60 nm ventions. Arboviruses, including WNV, are in diameter, with an electron-dense core (90). of particular interest because they rely on Mature virions contain a single copy of the taxonomically diverse hosts (e.g., mosquitoes viral RNA packaged within an icosahedral and birds) for perpetuation in nature. These capsid formed by the capsid protein. The hosts differ markedly in their response to genome-containing capsid is surrounded by WNV infection. Ongoing research is leading

a b E and M

5 Lipid bilayer

3 3 Core Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. c 5'UTR 3'UTR

prM EC NS12A 2BNS3 4A 4B NS5 Structural Nonstructural

Figure 1 (a, b) West Nile virion and genome. WNV structure as reconstructed by cryo-electron microscopy. (a)A surface-shaded view with one asymmetric unit of the icosahedron indicated by the triangle. (b) Central section of the reconstruction showing the concentric layers of mass density. Reproduced from Reference 90. Reprinted with permission from AAAS. (c) WNV genome, a single-stranded positive-sense RNA, approximately 11 kb in length consisting of a 5 untranslated region (UTR), a single long open-reading frame, and a 3 UTR. The open-reading frame encodes three structural and seven nonstructural proteins.

62 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

to a clearer understanding of the mechanisms Bucharest, Romania, 1996–1997, led to more for the invertebrate innate response to infec- than 500 clinical cases with a case-fatality rate tion, and it is hoped that they will someday of nearly 10% (141). This was the largest out- Neurological be manipulated to influence arbovirus trans- break of arboviral illness in Europe since Sind- disease (ND): a mission dynamics. The best-characterized in- bis virus (Alphavirus: Togaviridae) caused an clinical condition nate antiviral mechanisms in arthropods and epidemic in northern Europe in the 1980s. that may include arthropod cells are RNA interference path- Between 1996 and 1999, three major WNV meningitis and/or ways. These pathways are active in several epidemics occurred in southern Romania, the encephalitis that follows invasion of mosquito species (98, 121) and can be manip- Volga delta in southern Russia, and the north- the central nervous ulated to render mosquitoes less susceptible eastern United States, all of which involved system by a to arbovirus infection (40, 60). Although no hundreds of cases of severe ND and fatal in- microorganism published work yet addresses the WNV sys- fections. These were the first epidemics re- tem using relevant mosquitoes (i.e., Culex)or ported in large urban populations. In 2000 in their cells as hosts, much of the existing lit- Israel, a country-wide outbreak occurred with erature derived from other mosquito species a case fatality rate of 8.4% (145), and ND was should be applicable to the WNV-mosquito observed in Russia in 2001 (150) and Tunisia interaction. Sequencing of the Culex quinque- in 2003 (2). A comprehensive review of the fasciatus genome (VectorBase) will allow these history of WNV in Europe is presented by studies to proceed rapidly. Hubalek & Halouzka (49). The current epizootic/epidemic of WNV in North America appears to be the re- EPIDEMIOLOGY AND ECOLOGY sult of a single point introduction into the New York City area in 1999 (31, 73) fol- Geographic Distribution and lowed by a dramatic range expansion that Outbreaks currently encompasses the contiguous United WNV is currently the most widely distributed States, Canada, Mexico, Central America, the arbovirus in the world, occurring on all conti- Caribbean, and South America (14, 48, 67, nents except Antarctica. The virus was first 87). The southward spread of WNV in the isolated from a febrile woman in Uganda Western Hemisphere has been described well in 1937 (129) and subsequently was associ- by Komar & Clark (67) among others, and ated with sporadic cases of disease as well although extensive, the spread has not been as major outbreaks in Africa, Eurasia, Aus- accompanied by notable avian mortality or tralia, and the Middle East. Serosurveys of disease in humans or horses. This disease humans and equines and entomological stud- was previously unrecognized in the West- ies during the 1950s in Egypt and the up- ern Hemisphere and since 1999 has caused Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. per Nile delta (124, 137) led to great ad- 9843 cases of WN ND (962 deaths) and vances in understanding the ecology of the 13,489 cases of fever in the United States virus (46). Epidemics documented prior to [Centers for Disease Control and Preven- 1996 generally involved hundreds to thou- tion (CDC); Table 1]. In 1999, recognition sands of cases in mostly rural populations, of human cases was presaged by weeks by with few cases of severe neurological disease reports of dead exotic and domestic birds (ND) (47). However, beginning in the 1990s, in the New York City area (134). WN dis- outbreaks began to occur more frequently, ease also has been noted in humans in Cuba especially in the Mediterranean Basin, and (107) and the Cayman Islands (67) and in were associated with increased numbers of equines in Argentina (87), but scant evidence cases with severe disease including viral en- of human and equine morbidity and mortality cephalitis and neurological symptoms (80). has been observed in tropical America, pos- An outbreak of West Nile fever in and near sibly because of cross-protection from other

www.annualreviews.org • Epidemiology of WNV 63 ANRV330-EN53-04 ARI 2 November 2007 15:18

Table 1 Year and symptomatic classiﬁcation of WN disease casesa Neurological West Nile Unspeciﬁed Year Diseaseb Fever symptoms Total Deaths 1999 59 3 0 62 7 2000 19 2 0 21 2 2001 64 2 0 66 9 2002 2,946 1,160 50 4,156 284 2003 2,860 6,830 166 9,862 264 2004 1,142 1,269 128 2,539 100 2005 1,294 1,607 99 3,000 119 2006 1,459 2,616 194 4,269 177 Total 9,843 13,489 637 23,975 962

aData taken from CDC, as of March 6, 2007. bIncludes encephalitis and meningitis.

flaviviruses circulating in tropical regions, mission. Two major WNV lineages exist reduced virulence of WNV in the tropics (15), (Figure 2), with Lineage 1 distributed world- or less-competent arthropod and avian hosts wide and Lineage 2 occurring mainly in sub- Genotype: genetic makeup of a virus than in temperate regions in concert with the Saharan Africa (72). Recognized subclades that codes for the greater diversity of host species in the tropics. of Lineage I occur in Australia (Lineage 1b, phenotype of that Similarly, there have been no overt cases in the Kunjin virus) and in India (Lineage 1c) (72). strain United Kingdom, even with evidence of sero- Recently, strains isolated in central Europe logical conversions in sentinel chickens (19). and Russia were tentatively classified as new However, since the year 2000, after at least lineages of WNV (7, 78), but their taxonomic 35 years without disease, WNV has been de- status is currently unclear. tected regularly in neighboring France in the The introduction of WNV into North Camargue region with high levels of morbid- America in 1999 provided a unique opportu- ity in equines (8). The lack of human cases nity to prospectively observe the evolution of in northern Europe, compared to southern a novel agent in a naıve¨ environment. The Europe, may possibly be attributed to the virus introduced in 1999 was most closely re- feeding behavior of the predominant vector, lated to a strain isolated in Israel a year ear- Culex pipiens (which feeds either on humans, lier (73). Strains collected in New York in form molestus Forskal˚ 1775, or on birds, form 2000 were genetically homogeneous (31), and pipiens Linnaeus 1758, representing two dis- strains collected in Texasin 2002 were similar Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. tinct populations in northern Europe without to strains collected previously in the United hybridization, as occurs in southern Europe States (10). These studies seemed to confirm and the United States) (39; see below), as well the relative genetic stasis of arthropod-borne as other factors, especially climate. populations, but contrary evidence began to emerge when mouse-attenuated, genetically distinct WNV strains were collected in south- Molecular Epidemiology east coastal Texasin 2003 (27). Shortly there- In recent years substantial effort has been after, it was recognized that a dominant devoted to understanding the molecular genotype distinct from the introduced 1999 epidemiology of WNV in order to pro- genotype had arisen, which over the course vide a more detailed understanding of virus of three years displaced the introduced geno- spread, population dynamics, viral determi- type (28, 30, 130). This new genotype, vari- nants of pathogenesis, and mosquito trans- ously denoted the North American or WN02

64 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

WN-Romania 1996 H WN-Romania 1996 WN-South Africa WN-Israel 1952 WN-Egypt 1951 WN-France 1965 Distance WN-Senegal 1979 0.045 WN-Algeria 1968 WN-New York 1999 WN-Israel 1998 WN-C.Afr.Rep. 1989 WN-Italy 1998 91 WN-Morocco 1996 97 WN-Romania 1996 M WN-Kenya 1993 Lineage 1 WN-Senegal 1993 WN-C.Afr.Rep. 1967 WN-IvoryCoast 1981 Kunjin 1994 Kunjin 1966 Figure 2 Kunjin 1973 Phylogenetic tree Kunjin 1960 based on Kunjin 1984b E-glycoprotein Kunjin 1991 nucleic acid sequence Kunjin 1984a data (255 base pairs), WN-India 1955a as discussed in Reference 73, WN-India 1955b constructed using WN-India 1980 MEGA by WN-India 1958 neighbor-joining WN-Madagascar 1978 with Kimura two-parameter WN-Madagascar 1988 distance (scale bar). WN-Kenya Bootstrap conﬁdence WN-Madagascar 1986 level (500 replicates) WN-Uganda 1959 and a conﬁdence

Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org probability value by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. WN-WN-C.Afr.Rep. 1972a Lineage 2 based on the WN-WN-C.Afr.Rep. 1983 standard error test WN-WN-C.Afr.Rep. 1972b were calculated using WN-Nigeria MEGA. Reproduced WN-Uganda from Reference 73. Reprinted with WN-Senegal 1990 permission from JE SA 14 AAAS.

genotype, is now the only WNV genotype lope protein-coding-region sequences further recognized in the United States. Its domi- indicated the disappearance of the intro- nance appears to be related to increased trans- duced genotype and suggested that the now- mission efﬁciency in Culex spp. mosquitoes dominant genotype has reached peak preva- (30, 89). Recent analyses of 156 enve- lence in North America (130).

www.annualreviews.org • Epidemiology of WNV 65 ANRV330-EN53-04 ARI 2 November 2007 15:18

Bertolotti et al. (13) used sequence data to United States has rarely been observed ei- demonstrate that WNV is not constrained ge- ther in the Old World or in tropical America. ographically, a finding concordant with the Possible explanations include those discussed Purifying selection: selection against virus’s rapid geographic spread across the above and differences in avian populations nonsynonymous United States. Substitution rates estimated such as historical exposure of the birds to nucleotide for WNV range from 2.97 × 10−4 (130) to WNV or related flaviviruses. Whatever the substitutions 8.5 × 10−4 (13) per year, similar to estimates underlying causes of the increased avian mor- Nonsynonymous for other flaviviruses (144, 149). WNV pop- tality from WNV in the United States, it substitutions: ulations are dominated by strong purifying likely increases the intensity of transmission nucleotide selection, with nonsynonymous substitutions (i.e., R ) in the enzootic cycle partly by re- substitutions in the o genome resulting in rapidly removed from sequences relative to moving individuals who would have become amino acid synonymous substitutions (54). Finally, WNV immune. This increase in enzootic Ro might replacements populations appear to be structured as quasi- be expected to result in more human infec- Synonymous species in nature, with infection of mosquitoes tions. Third, conventional wisdom suggests substitutions: leading to greater genetic diversity than bird that only birds contribute to enzootic per- nucleotide infection and to some amount of genetic di- petuation of WNV. Evidence is accumulating substitutions in the versity shared between hosts (54, 55). that some mammals and reptiles may in fact genome that do not result in amino acid be competent amplification hosts for WNV. replacements Squirrels, for example, mount viremias suffi- Enzootic cycle: TRANSMISSION CYCLE ciently high to infect at least a low propor- continual tion of mosquitoes (116). Alligators farmed Enzootic Cycle transmission under conditions of high temperatures in a between zoonotic Throughout its worldwide distribution, crowded environment demonstrated signifi- host and arthropod WNV is maintained in nature in an enzootic cant morbidity (53) and mortality (84), and vector (enzootic vector) that leads to cycle between ornithophilic mosquitoes, pre- following experimental infection they demon- 5 −1 amplification of virus dominantly Culex (Culex) species, and birds. strated high levels of viremia, >10 pfu ml , Approximately 59 species of mosquitoes that could infect Say 1823 Ro: The basic C. quinquefasciatus reproductive rate of and 284 species of birds (48) have been (65). However, the virus may have been trans- a pathogen found infected in North America. Therefore, mitted directly between animals under these Bridge vector: an WNV is apparently an ecological generalist crowded conditions via cloacal shedding. Fi- arthropod, most compared to other arboviruses that are more nally, the importance of the experimental ob- commonly a limited in the range of vectors and vertebrates servation of cofeeding transmission, wherein a mosquito, that that they infect. This generalization has likely low proportion of uninfected mosquitoes be- carries virus from the amplification cycle to contributed to the broad geographic distri- comes infected following feeding in temporal secondary hosts bution of the virus and the human and animal and spatial proximity to infected mosquitoes, Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. disease it causes (Figure 3). is unknown in nature (82, 114). Additional Several important questions remain to studies are required to understand whether be answered regarding the dynamics of the the generalist nature of the vector and ver- WNV transmission cycle. First, the impor- tebrate host ranges that seems to be a hall- tance of corvids in the amplification cycle is mark of WNV infection is indeed critical for unresolved. Human cases have been associ- its perpetuation. ated with clusters of dead American crows in California (112) and New York(33), but blood meal analyses of engorged mosquitoes do not Bridge Vectors and Alternative indicate strong evidence of feeding on crows; Transmission Mechanisms instead other birds seem to be more impor- Bridge vectors (distinct from enzootic vec- tant hosts (5, 61). Second, it remains unclear tor species) are typically involved for signif- why the high avian mortality observed in the icant numbers of humans to become infected

66 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

Enzootic cycle Fecal-oral Epidemic

Blood transfusion Organ transplantation Vertical Breast milk transmission Intrauterine

Other vectors Epizootic

Persistence Death Immunity Cofeeding transmission? ?

Carrion Infectious viremia?

Figure 3 WNV transmission cycle. Primary enzootic amplification by birds and mosquitoes may be supplemented by bird-to-bird transmission, amplification in nonavian hosts, and transmission between cofeeding mosquitoes. Alternative vectors most likely have a less important role. Persistent infection in vertebrates may allow subsequent infection in susceptible scavengers, predators, or mosquitoes. Vertical transmission by mosquitoes provides one mechanism of virus overwintering. Equines and humans are incidental hosts; however, human-to-human transmission may occur through blood transfusion, organ transplantation, breast milk, or in utero. Solid arrows represent confirmed transmission pathways; dotted arrows represent proposed pathways that have not been confirmed in nature.

with zoonotic arboviruses such as WNV. Sev- late in the summer (i.e., in August and later eral recent observations, however, have chal- months) feed indiscriminately on either birds lenged this view. Population density and host or mammals (131, 133). C. pipiens may there- feeding studies (63, 64) and genetic anal- fore be involved in early-season amplification yses (39) have implicated C. pipiens as po- of WNV in enzootic cycles and serve as bridge Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. tentially the most important bridge vector vectors when autogenous-anautogenous hy- in the northeastern, northcentral, and mid- brids become abundant (63, 133).Recent stud- Atlantic United States, as well as in eastern ies, however, saw no difference in the pro- Europe (123) and Russia (41). C. pipiens pos- portion of mosquitoes with molestus genetic sesses atypical feeding patterns and the occur- signature during the transmission season in rence of two behaviorally and physiologically Washington D.C., but C. pipiens that fed on different forms, autogenous (C. pipiens form humans were more likely to have a moles- molestus) and anautogenous (C. pipiens form tus signature than C. pipiens that fed on birds pipiens) populations (131, 132). Pioneering (62). Population biology and feeding behav- studies by Spielman (133) hypothesized that ior of C. pipiens and WNV epidemiology are autogenous populations almost never feed on thereby linked. The importance of these fac- blood, anautogenous populations feed exclu- tors influencing WNV transmission was re- sively on birds, and hybrid offspring emerging cently demonstrated by a series of field studies

www.annualreviews.org • Epidemiology of WNV 67 ANRV330-EN53-04 ARI 2 November 2007 15:18

showing a strong temporal association be- vary in their ability to be vectored by arthro- tween shifts in C. pipiens feeding behavior pods (30) and to cause disease in animal mod- from a strong to a weak focus on American els (15, 74). Other intrinsic factors that may Vector competence: the robins and an increase in human cases (64). influence WNV epidemiology in a more pow- ability of an Two other significant enzootic vectors in the erful way include mosquito feeding behavior arthropod to become United States, C. tarsalis and C. nigripalpus, and longevity (135), which are extensively re- infected with and also may be important bridge vectors because viewed elsewhere (69). Extrinsic factors such transmit a pathogen they too switch feeding from birds to humans as temperature and rainfall patterns (35) and (32, 138), and the enzootic vectors C. quin- the density of susceptible host populations are quefasciatus and C. salinarius feed broadly on also important in determining the intensity of both avian and mammalian hosts, including WNV transmission (69). An integrated un- humans (5, 96). Bernard & Kramer (12) dis- derstanding of how extrinsic and intrinsic fac- cuss the wide array of other mosquito species tors interweave to produce WNV epidemics that may also serve as bridge vectors facilitat- requires further field- and laboratory-based ing extension of the cycle that leads to human studies. Such an understanding is a prerequi- and equine disease. Many of the secondary site for developing rational control strategies. species may be incidental vectors of little epidemiologic significance. Further research is required to determine the importance of non- LONG-TERM PERPETUATION Culex mosquitoes in the ecology and epidemi- AND SPREAD ology of WNV in North America. Re-introduction The role of nonmosquito vectors in WNV epizootiology continues to be explored. Ex- Circulation of WNV in Europe and Africa perimental transmission has been demon- occurred sporadically with limited outbreaks strated with soft ticks (1, 51), but not with until activity began to increase in 1996, partic- ixodid ticks (75). WNV has been isolated ularly in the Mediterranean basin, as has been from soft (argasid) ticks in Israel (92) and reviewed thoroughly in other papers (24, 150). hippoboscid flies in the United States (37). For example, the virus reappeared in south- Owing to the relative infrequency of these eastern France, in the Camargue district, in isolations and robust WNV-mosquito inter- 2000 after 35 years with no evidence of overt actions, these other arthropod vectors seem activity and low seroprevalence in humans and unlikely to play a critical role. There also equines (6, 24, 93). Western Mediterranean is strong evidence for nonvector routes of wetlands such as the Camargue attract birds transmission, as with bird-to-bird transmis- from central Asia, Siberia, northern and east- sion through the fecal-oral route and through ern Europe, western Africa, and the Mediter- Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. consumption of infected carrion (68). Hu- ranean basin, and numerous birds of vari- mans have become infected through blood ous species are seasonally aggregated in these transfusion, organ transplantation, and other habitats (56). Migratory birds are important novel routes (70). in spreading virus, as with the introduction of WNV into Israel (79) leading to multiple genotypes circulating concurrently, and into Other Factors Slovakia (36). Birds also have been proposed Intrinsic factors influence the epidemiology of to be the agents of long-distance movement WNV. Detailed experiments have established of WNV in the Western Hemisphere (101, that vertebrates vary in their morbidity, mor- 109), while mosquitoes and dispersing birds tality, and host competence (68, 95, 115) and may move the virus locally (108). A better that mosquito species differ in vector compe- understanding of avian migratory routes is tence (42, 122, 142) for WNV. Viral strains needed to verify models and simulations of

68 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

viral movement; however, it is probable that come infected through vertical transmission, they play a critical role in determining the dy- and they must survive the winter infected namics of the spread of this virus. An alter- with virus. However, others believe diapaus- Vertical native mechanism of virus spread is through ing Culex spp. may host-seek in nature (34) transmission: dispersal from an endemic area via migrant in- and would therefore survive the winter after transmission of virus fected mosquitoes. This mechanism of virus potentially having taken an infectious blood from a parental spread has been reviewed thoroughly (69). meal. female mosquito to Virus also might perpetuate in an enzootic its progeny without infection of the focus over time through persistent infection germline cells Maintenance within an Enzootic in birds, as has been demonstrated experi- Focus mentally in diverse tissues of various avian There are several potential mechanisms of species (68, 113). No studies to date have WNV perpetuation within an enzootic fo- demonstrated infection of mosquitoes fol- cus in habitats conducive to virus transmis- lowing feeding on vertebrates with persis- sion where mosquitoes and birds live closely tent infection; however, ingestion of persis- together. They include low-level continu- tently infected carrion by susceptible hosts ous enzootic transmission, vertical transmis- may present an alternative mode of transmission by mosquito vector(s), and chronic in- sion. The relative importance of persistence fection in birds. From 2000 to 2004 in the of WNV in vertebrates (139) needs further Camargue, most likely owing to local per- study. petuation, equine epizootics occurred accompanied by human cases and sentinel bird seroconversion. WNV has been isolated from STRATEGIES FOR CONTROL field-collected larvae and/or male C. univitta- AND TREATMENT tus mosquitoes in Kenya (83) and from larvae Vector Control (103) or diapausing adults in the United States (3, 94), among others. This discovery has Surveillance efforts have focused on mos- led to the belief that vertical transmission of quitoes, dead birds, and sentinel chickens. WNV from parent to progeny plays a sig- WNV-infected mosquito pools were the most nificant role in the virus’s perpetuation. Fla- accurate indicator of human cases when viviruses appear to enter the fully formed egg mosquito and avian surveillance approaches through the micropyle at the time of fertiliza- were compared in one study (18). Significantly tion (117). This is an inefficient mechanism higher C. quinquefasciatus infection rates and of vertical transmission, yet it does permit the incidence of human cases were found in prox- infection of progeny following a single mater- imity to clusters of dead crows in another Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. nal blood meal. Laboratory studies also have study (112). Because avian mortality is a pas- successfully demonstrated vertical transmis- sive surveillance tool that depends on pub- sion of WNV by C. tritaeniorhynchus, Aedes lic reporting, the effectiveness of this strategy albopictus, A. aegypti (9), C. pipiens (143), and may decrease over time. C. tarsalis (43); however, most of these studies Because of the large impact of WNV on used intrathoracic inoculation as the means human and animal health, it is critical to de- of infection. It remains unclear whether the velop effective methods to limit WNV trans- low vertical transmission rates observed with mission and prevent and/or treat WN disease. WNV are sufficient to allow virus to survive Currently, control measures to curtail WNV winter in temperate environments. Temper- transmission include reducing mosquito vec- ate Culex spp. enter diapause to survive winter tor populations and limiting exposure to conditions without having taken a blood meal mosquito bites with protective clothing and (85); therefore overwintering adults must be- repellents. Vector control agencies often use

www.annualreviews.org • Epidemiology of WNV 69 ANRV330-EN53-04 ARI 2 November 2007 15:18

a combination of approaches (mosquito pop- ceeded risks from exposure to mosquito insec- ulation monitoring, mosquito source reduc- ticides when compared using a risk assessment tion, larvicide and adulticide application, and (102). However, this analysis did not consider public education) to reduce mosquito popula- the effectiveness of mosquito insecticides at tions. In 2002, a program relying on surveil- reducing WN disease (i.e., a risk-benefit anal- lance and larvicide and adulticide applications ysis of insecticide use), and thus the benefits was implemented in St. Tammany Parish, of spraying may be overestimated (125). Af- Louisiana, resulting in reductions in mosquito ter widespread aerial spraying of pyrethrin populations below the five-year average and a insecticides around Sacramento, California, subsequent drop in new human WNV cases for WNV control, water and sediment sam- (99). ples were taken from nearby creeks and tested Monitoring the effectiveness of mosquito for insecticide residues and toxicity. No toxi- control programs and targeting control efforts city was detected from the active ingredient, to high-risk areas and peak mosquito activity pyrethrin; however, the synergist, piperonyl periods are critical to maximize benefits. Fol- butoxide, reacted with preexisting pyrethroids lowing the Florida hurricanes in 2004, aerial in the sediment and caused a twofold increase spraying of adulticides reduced the number of in toxicity (147). mosquitoes caught in CDC-type light traps Because of increased resistance of by 67.7% (128). On the other hand, there was mosquito populations to conventional no difference in Culex abundance at three sub- control agents, there have been renewed urban sites in Massachusetts following a sin- efforts to develop novel biopesticides. A gle application of resmethrin from vehicle- recombinant bacterial strain expressing the mounted ultra-low volume generators; this toxins of Bacillus thuringiensis israelensis and lack of control was not due to insecticide re- B. sphaericus was 20-fold-more toxic than sistance of the target population (111). Host- either of the parental strains and less likely seeking and oviposition behaviors for Culex to induce resistance in target populations mosquitoes in the northeastern United States (100). Mosquito baculoviruses, such as C. peaked approximately two hours after sunset; nigripalpus nucleopolyhedrovirus, are highly aerial application of insecticides during this virulent for certain mosquito species and time of increased flight activity is likely to im- could be developed as biopesticides (11). Ad- prove control outcomes (110). Several GIS- ditionally, recent demonstrations of Anopheles based spatial models of WNV transmission mosquito control using entomopathogenic have been developed (23, 118, 136). These fungi suggest that this technique could be a models use a variety of predictor variables, viable strategy for future control of WNV including temperature, rainfall, vegetation, vectors (57). Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. landscape, and geographic data, to predict lo- New strategies to control mosquito pop- cations of high WNV transmission risk. This ulations have been proposed. Field trials of type of information is useful for targeting a mass-trapping strategy that used mosquito mosquito control efforts, locating trapping traps with a combination of attractants (heat,

sites for surveillance, and focusing prevention CO2, octenol) demonstrated good levels of efforts. mosquito control on islands or when one Concerns have been raised regarding the species is dominant in an area (66). Mass- health and environmental effects of pesti- trapping may become a more viable strat- cides used to control WNV epidemics. After egy as knowledge of mosquito attractants im- widespread pyrethroid spraying in New York proves. Relatively simple strategies, such as City in 2000, there was no population-level using ﬂoating layers of polystyrene beads to increase in emergency department visits for obstruct the water surface, can result in suffo- asthma (59). Risks from WNV infection ex- cation of mosquito larvae because they cannot

70 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

penetrate the water surface to breathe (25). tion purposes. The first is inoculation of mul- This technique would be useful to control tiple doses of inactivated (killed) virus (119). A Culex mosquitoes breeding in enclosed spaces, second strategy involves expression of WNV such as flooded basements or cess pits. A novel viral proteins in a host to elicit an immune strategy currently pursued to control malaria response. Viral proteins can be inoculated di- and dengue involves the creation of transgenic rectly into the host, as a recombinant sub- mosquitoes that are incapable of transmitting unit vaccine (22, 76, 77), or they can be pro- pathogens (40, 52). This strategy seems more duced by host cells following inoculation of problematic for control of WNV due to the DNA plasmids (26) or virus vectors that ex- complexity of the WNV transmission cycle. press WNV genes (58). The third strategy involves the use of chimeric viruses containing the PrM and E genes of WNV within a Vaccines and Antiviral Treatments heterologous attenuated flavivirus backbone Vaccines can be used to prevent WNV in- [Yellow fever 17D (86), DEN2 PDK-53 (49) fection and antiviral treatments can be used or DEN4–3delta30 (104)]. The final vaccina- to treat severe disease (reviewed in Reference tion strategy is based on attenuated viruses. 70). There are currently four licensed WNV Several types of attenuated viruses have been vaccines for horses and one licensed vaccine created by introducing mutations into non- for domestic geese (Table 2). Although no structural genes (45, 148), resulting in reduced vaccine has been approved for use in humans, virus replication, or by introducing mutations significant progress has been made, with on- into the capsid gene (81, 126), disabling the going clinical trials of four vaccines (Table 2). release of virus from the cell. Several strategies have been employed to de- Currently there is no specific treatment for liver WNV antigens into animals for vaccina- WNV disease in humans, although several

Table 2 WNV vaccines that are licensed or in clinical trial Product name Company and/or institute Vaccine type Status Innovator® Fort Dodge Animal Health Killed virus L Recombitek® Merial Recombinant canarypox L virus PreveNileTM Intervet Chimeric virus L (WNV/YFV) NA Kimron Veterinary Killed virus L Institute/Crucell Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. NA CDC/Fort Dodge Animal Recombinant DNA L Health plasmid ChimeravaxTM-West Nile Acambis Chimeric virus CT-II (WNV/YFV) VRC-WNVDNA020–00-VP NIAID/NIH Recombinant DNA CT-I plasmid WN/DEN4–3delta30 NIAID/NIH Chimeric virus CT-I (WNV/DEN4) NA Crucell Killed virus CT-I

Abbreviations: NA, information not available; L, licensed for veterinary use; YFV, yellow fever virus; CT-II, clinical trial, phase II; CT-I, clinical trial, phase I; DEN4, dengue-4 virus. Sources of information: http://www.fortdodgelivestock.com/, http://www.merial.com/, http://www.intervetusa.com/, http://www.crucell.com/, http://www.clinicaltrials.gov/.

www.annualreviews.org • Epidemiology of WNV 71 ANRV330-EN53-04 ARI 2 November 2007 15:18

antiviral compounds and therapies are be- quence to portions of the WNV genome (an- ing tested in clinical trials. Patients with tisense therapies) have shown significant an- WNV encephalitis have been successfully tiviral activity in vitro (29, 140). The safety treated with intravenous immunoglobulin de- and efficacy of one antisense compound (AVI- rived from donor plasma with high levels of 4020) against WNV ND are being tested in WNV antibodies (127); the safety and efficacy a clinical trial. High-throughput assays devel- of this treatment are being tested in phase I/II oped by two groups (44, 105) are being used to clinical trials. Antibody therapy using human- screen compounds for antiviral activity. One ized monoclonal antibodies directed against of these assays identified triaryl pyrazoline as the WNV envelope protein is therapeuti- an inhibitor of flavivirus replication in cell cally effective in mice and hamsters. A single culture (106). dose of these antibodies given to animals at 5 days post-infection (when neurons are infected with WNV) protected animals from CONCLUSION WNV-induced mortality and resulted in de- WNV serves as a model for zoonotic diseases creased viral titers in the brain (88, 97). In- that are emerging, re-emerging, or expanding terferon therapy effectively controlled WNV their ranges globally. It is critical to conduct infection in vitro and in animal models (4, research on the underlying biological and ge- 120). A clinical trial is currently underway ographic factor(s) that allows these pathogens to test the safety and efficacy of interferon to adapt to new hosts and environments. A therapy for West Nile meningocephalitis in better understanding will allow improved pre- humans. Oligomers with complementary se- diction of risk and approaches to control.

SUMMARY POINTS 1. WNV is the most widely distributed arbovirus known; the factors explaining this are complex, including vector-virus-host interactions and climatic factors. 2. WNV is well established in the Western Hemisphere and activity will most likely continue at levels determined by virus, host, and environmental factors throughout its range. 3. The WNV cycle is complex and details vary by region, making it difﬁcult to model on a broad scale, but the predominant enzootic hosts are Culex mosquitoes and birds. Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org

by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. 4. Migratory birds are the most likely mechanism of long-distance virus spread, but mosquitoes and dispersing birds may carry the virus shorter distances. 5. Current control measures against WNV consist of mosquito population reduction and personal protective measures; however, new chemical control agents and new control strategies are being pursued. 6. Several WNV vaccines have been licensed for use in horses; signiﬁcant progress has been made in the development of WNV vaccines for humans. 7. No speciﬁc antiviral treatment exists for WNV, although several candidates are currently being tested in clinical trials.

72 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

FUTURE ISSUES 1. Sequencing of the Culex genome will allow more detailed studies of the genetic basis of vector-virus-vertebrate interactions. 2. Molecular epidemiological and fitness studies of WNV strains isolated over space and time will provide information on viral evolution and adaptation. 3. A better understanding of the role of avian migration in long-term perpetuation and spread of virus will increase the ability to predict movement of zoonotic pathogens in the future. 4. Significance of viral persistence in vertebrate hosts is important to our understanding of disease and virus perpetuation. 5. Mathematical models that integrate human infection with mosquito surveillance data should be developed to improve risk prediction. 6. An increased understanding of the impact of climate on spatial and temporal variation in virus activity, and of the drivers of spatial variation in transmission, will improve risk analyses. 7. Integration of structural biology with virology and viral genetics will allow for more detailed understanding of host-virus interactions. 8. Improved diagnostic assays are needed to distinguish among flaviviruses.

DISCLOSURE STATEMENT The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS We wish to express our gratitude to the research staff of the Arbovirus Laboratories at the Wadsworth Center, New York State Department of Health, for their contributions to many of the studies discussed in this review. We also wish to thank Elizabeth Cavosie for her assistance preparing the manuscript and Elizabeth Kauffman for her assistance with the ﬁgures. We thank A. Marm Kilpatrick for his helpful discussions on the topics reviewed. Portions of the research Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. presented were supported by the National Institute of Allergy and Infectious Disease Contract no. NO1-AI-25490.

LITERATURE CITED 1. Abbassy MM, Osman M, Marzouk AS. 1993. West Nile virus (Flaviviridae: Flavivirus)in experimentally infected Argas ticks (Acari: Argasidae). Am. J. Trop. Med. Hyg. 48:726–37 2. Abroug F, Ouanes-Besbes L, Letaief M, Ben RF, Khairallah M, et al. 2006. A cluster study of predictors of severe West Nile virus infection. Mayo Clin. Proc. 81:12–16 3. Anderson JF, Main AJ. 2006. Importance of vertical and horizontal transmission of West Nile virus by Culex pipiens in the northeastern United States. J. Infect. Dis. 194:1577–79 4. Anderson JF, Rahal JJ. 2002. Efﬁcacy of interferon alpha-2b and ribavirin against West Nile virus in vitro. Emerg. Infect. Dis. 8:107–8

www.annualreviews.org • Epidemiology of WNV 73 ANRV330-EN53-04 ARI 2 November 2007 15:18

5. Apperson CS, Hassan HK, Harrison BA, Savage HM, Aspen SE, et al. 2004. Host feeding patterns of established and potential mosquito vectors of West Nile virus in the eastern United States. Vector Borne Zoonotic Dis. 4:71–82 6. Autorino GL, Battisti A, Deubel V, Ferrari G, Forletta R, et al. 2002. West Nile virus epidemic in horses, Tuscany region, Italy. Emerg. Infect. Dis. 8:1372–78 7. Bakonyi T, Hubalek Z, Rudolf I, Nowotny N. 2005. Novel flavivirus or new lineage of West Nile virus, central Europe. Emerg. Infect. Dis. 11:225–31 8. Balenghien T, Fouque F, Sabatier P, Bicout DJ. 2006. Horse-, bird-, and human-seeking behavior and seasonal abundance of mosquitoes in a West Nile virus focus of southern France. J. Med. Entomol. 43:936–46 9. Baqar S, Hayes CG, Murphy JR, Watts DM. 1993. Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes. Am. J. Trop. Med. Hyg. 48:757–62 10. Beasley DW, Davis CT, Guzman H, Vanlandingham DL, Travassos da Rosa APA, et al. 2003. Limited evolution of West Nile virus has occurred during its southwesterly spread in the United States. Virology 309:190–95 11. Becnel JJ. 2006. Prospects for the mosquito baculovirus CuniNPV as a tool for mosquito control. J. Am. Mosq. Control Assoc. 22:523–26 12. Bernard KA, Kramer LD. 2001. West Nile virus activity in the United States, 2001. Viral Immunol. 14:319–38 13. Bertolotti L, Kitron U, Goldberg TL. 2006. Diversity and evolution of West Nile 13. Describes a lack of spatial structure virus in Illinois and The United States, 2002–2005. Virology 360:143–49 in WNV 14. Bosch I, Herrera F, Navarro JC, Lentino M, Dupuis AP, et al. 2007. Established intro- populations, adding duction of West Nile virus in Venezuela. Emerg. Infect. Dis. 13:651–53 to evidence that 15. Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff BJ, et al. 2004. Differential WNV genotypes spread rapidly virulence of West Nile strains for American crows. Emerg. Infect. Dis. 10:2161–68 throughout an 16. Brinton MA. 2001. Host factors involved in West Nile virus replication. Ann. N. Y. Acad. essentially Sci. 951:207–19 continuous 17. Brinton MA. 2002. The molecular biology of West Nile virus: a new invader of the transmission focus. Western Hemisphere. Annu. Rev. Microbiol. 56:371–402 18. Brownstein JS, Holford TR, Fish D. 2004. Enhancing West Nile virus surveillance, United States. Emerg. Infect. Dis. 10:1129–33 19. Buckley A, Dawson A, Gould EA. 2006. Detection of seroconversion to West Nile virus, Usutu virus and Sindbis virus in UK sentinel chickens. Virol. J. 3:71–76 20. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, et al. 1989. Anti- Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. genic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J. Gen. Virol. 70(Pt. 1):37–43 21. Chambers TJ, Hahn CS, Galler R, Rice CM. 1990. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44:649–88 22. Chu JH, Chiang CC, Ng ML. 2007. Immunization of flavivirus West Nile recombinant envelope domain III protein induced specific immune response and protection against West Nile Virus infection. J. Immunol. 178:2699–705 23. Cooke WH III, Grala K, Wallis RC. 2006. Avian GIS models signal human risk for West Nile virus in Mississippi. Int. J. Health Geogr. 5:36 24. Couissinier-Paris P. 2006. West Nile virus in Europe and Africa: still minor pathogen, or potential threat to public health? Bull. Soc. Pathol. Exot. 99:348–54 25. Curtis C. 2005. Insecticide-treated nets against malaria vectors and polystyrene beads against Culex larvae. Trends Parasitol. 21:504–7

74 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

26. Davis BS, Chang GJ, Cropp B, Roehrig JT, Martin DA, et al. 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays. J. Virol. 75:4040–47 27. Davis CT, Beasley DW, Guzman H, Siirin M, Parsons RE, et al. 2004. Emergence of attenuated West Nile virus variants in Texas, 2003. Virology 330:342–50 28. Davis CT, Ebel GD, Lanciotti RS, Brault AC, Guzman H, et al. 2005. Phylogenetic analysis of North American West Nile virus isolates, 2001–2004: evidence for the emergence of a dominant genotype. Virology 342:252–65 29. Deas TS, Binduga-Gajewska I, Tilgner M, Ren P, Stein DA, et al. 2005. Inhibition of flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J. Virol. 79:4599–609 30. Ebel GD, Carricaburu J, Young D, Bernard KA, Kramer LD. 2004. Genetic and 30. First study to phenotypic variation of West Nile virus in New York, 2000–2003. Am. J. Trop. Med. demonstrate Hyg. 71:493–500 transmission- 31. Ebel GD, Dupuis AP II, Ngo KA, Nicholas DC, Kauffman EB, et al. 2001. Partial genetic related phenotypic characterization of West Nile virus strains, New York State. Emerg. Infect. Dis. 7:650–53 variation in WNV and to suggest that 32. Edman JD, TaylorDJ. 1968. Culex nigripalpus: seasonal shift in the bird-mammal feeding a population ratio in a mosquito vector of human encephalitis. Science 161:67–68 displacement was 33. Eidson M. 2005. Dead crow density and West Nile virus monitoring, New York. Emerg. underway. Infect. Dis. 11:1370–75 34. Eldridge BF. 1987. Diapause and related phenomena in Culex mosquitoes: their relation to arbovirus disease ecology. Curr. Top. Vector Res. 4:1–28 35. Epstein PR. 2001. West Nile virus and the climate. J. Urban. Health 78:367–71 36. Ernek E, Kozuch O, Nosek J, Teplan J, Folk C. 1977. Arboviruses in birds captured in Slovakia. J. Hyg. Epidemiol. Microbiol. Immunol. 21:353–59 37. Farajollahi A, Crans WJ, Nickerson D, Bryant P, Wolf B, et al. 2005. Detection of West Nile virus RNA from the louse fly Icosta americana (Diptera: Hippoboscidae). J. Am. Mosq. Control Assoc. 21:474–76 38. Ferlenghi I, Clarke M, Ruttan T, Allison SL, Schalich J, et al. 2001. Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Mol. Cell 7:593–602 39. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, et al. 2004. Emerging vectors in the Culex pipiens complex. Science 303:1535–38 40. Franz AW, Sanchez-Vargas I, Adelman ZN, Blair CD, Beaty BJ, et al. 2006. Engineering Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proc. Natl. Acad. Sci. USA 103:4198–203 41. Fyodorova MV, Savage HM, Lopatina JV, Bulgakova TA, Ivanitsky AV, et al. 2006. Eval- uation of potential West Nile virus vectors in Volgograd region, Russia, 2003 (Diptera: Culicidae): species composition, bloodmeal host utilization, and virus infection rates of mosquitoes. J. Med. Entomol. 43:552–63 42. Goddard LB, Roth AE, Reisen WK, Scott TW. 2002. Vector competence of California mosquitoes for West Nile virus. Emerg. Infect. Dis. 8:1385–91 43. Goddard LB, Roth AE, Reisen WK, Scott TW. 2003. Vertical transmission of West Nile virus by three California Culex (Diptera: Culicidae) species. J. Med. Entomol. 40:743–46 44. Gu B, Ouzunov S, Wang L, Mason P, Bourne N, et al. 2006. Discovery of small molecule inhibitors of West Nile virus using a high-throughput sub-genomic replicon screen. Antiviral Res. 70:39–50

www.annualreviews.org • Epidemiology of WNV 75 ANRV330-EN53-04 ARI 2 November 2007 15:18

45. Hall RA, Nisbet DJ, Pham KB, Pyke AT, Smith GA, Khromykh AA. 2003. DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus. Proc. Natl. Acad. Sci. USA 100:10460–64 46. Hayes CG. 1989. West Nile fever. In The Arboviruses: Epidemiology and Ecology, Volume V, ed. TP Monath, pp. 59–88. Boca Raton, FL: CRC Press 47. Hayes CG. 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Annu. N. Y. Acad. Sci. 951:25–37 48. Hayes EB, Komar N, Nasci RS, Montgomery SP, O’Leary DR, Campbell GL. 2005. Epidemiology and transmission dynamics of West Nile virus disease. Emerg. Infect. Dis. 11:1167–73 49. Hubalek Z, Halouzka J. 1999. West Nile fever—a reemerging mosquito-borne 49. Comprehensive history of WNV in viral disease in Europe. Emerg. Infect. Dis. 5:643–50 Europe, where its 50. Huang CY, Silengo SJ, Whiteman MC, Kinney RM. 2005. Chimeric dengue 2 PDK- epidemiology is 53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate comparatively PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J. poorly understood. Virol. 79:7300–10 51. Hutcheson HJ, Gorham CH, Hain-Williams C, Lorono-Pino MA, James AM, et al. 2005. Experimental transmission of West Nile virus (Flaviviridae: Flavivirus)byCarios capensis ticks from North America. Vector Borne Zoonotic Dis. 5:293–95 52. Ito J, Ghosh A, Moreira LA, Wimmer EA, Jacobs-Lorena M. 2002. Transgenicanopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417:452–55 53. Jacobson ER, Johnson AJ, Hernandez JA, Tucker SJ, Dupuis AP, et al. 2005. Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American alligators (Alligator mississippiensis) in Florida. J. Wildl. Dis. 41:107–14 54. Jerzak G, Bernard KA, Kramer LD, Ebel GD. 2005. Genetic variation in West Nile virus from naturally infected mosquitoes and birds suggests quasispecies structure and strong purifying selection. J. Gen. Virol. 86:2175–83 55. Jerzak GV, Bernard K, Kramer LD, Shi PY, Ebel GD. 2007. The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice. Virology 360:469–76 56. Jourdain E, Gauthier-Clerc M, Bicout DJ, Sabatier P. 2007. Bird migration routes and risk for pathogen dispersion into western Mediterranean wetlands. Emerg. Infect. Dis. 13:365–72 57. Kanzok SM, Jacobs-Lorena M. 2006. Entomopathogenic fungi as biological insecticides Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. to control malaria. Trends Parasitol. 22:49–51 58. Karaca K, Bowen R, Austgen LE, TeeheeM, Siger L, et al. 2005. Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge. Vaccine 23:3808–13 59. Karpati AM, Perrin MC, Matte T, Leighton J, Schwartz J, Barr RG. 2004. Pesticide spraying for West Nile virus control and emergency department asthma visits in New York City, 2000. Environ. Health Perspect. 112:1183–87 60. Establishes the 60. Keene KM, Foy BD, Sanchez-Vargas I, Beaty BJ, Blair CD, Olson KE. 2004. in vivo antiviral RNA interference acts as a natural antiviral response to O’nyong-nyong virus (Al- activity of RNAi phavirus; Togaviridae) infection of Anopheles gambiae. Proc. Natl. Acad. Sci. USA pathways against 101:17240–45 arboviruses in mosquitoes. 61. Kilpatrick AM, Daszak P, Jones MJ, Marra PP, Kramer LD. 2006. Host heterogeneity dominates West Nile virus transmission. Proc. Biol. Sci. 273:2327–33

76 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

62. Kilpatrick AM, Kramer LD, Jones MA, Marra P, Daszak P, Fonseca D. 2007. Genetic influences on mosquito feeding behavior and the emergence of zoonotic pathogens. Am. J. Trop. Med. Hyg. In press 63. Kilpatrick AM, Kramer LD, Campbell SR, Alleyne EO, Dobson AP, Daszak P. 2005. West Nile virus risk assessment and the bridge vector paradigm. Emerg. Infect. Dis. 11:425– 29 64. Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Daszak P. 2006. West Nile virus epidemics in North America are driven by shifts in mosquito feeding behavior. PLoS Biol. 4:e82 65. Klenk K, Snow J, Morgan K, Bowen R, Stephens M, et al. 2004. Alligators as West Nile virus amplifiers. Emerg. Infect. Dis. 10:2150–55 66. Kline DL. 2006. Traps and trapping techniques for adult mosquito control. J. Am. Mosq. Control Assoc. 22:490–96 67. Komar N, Clark GG. 2006. West Nile virus activity in Latin America and the Caribbean. Rev. Panam. Salud Publ. 19:112–17 68. Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, et al. 2003. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg. Infect. Dis. 9:311–22 69. Kramer LD, Ebel GD. 2003. Dynamics of flavivirus infection in mosquitoes. Adv. Virus Res. 60:187–232 70. Kramer LD, Li J, Shi P-Y. 2007. West Nile virus. The Lancet Neurol. 6:171–81 71. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB. 1998. Phylogeny of the genus Flavivirus. J. Virol. 72:73–83 72. Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, et al. 2002. Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East. Virology 298:96–105 73. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, et al. 1999. Origin of the 73. Identified West Nile virus responsible for an outbreak of encephalitis in the northeastern WNV as the agent United States. Science 286:2333–37 responsible for the 74. Langevin SA, Brault AC, Panella NA, Bowen RA, Komar N. 2005. Variation in virulence outbreak of of West Nile virus strains for house sparrows (Passer domesticus). Am. J. Trop. Med. Hyg. encephalitis in New York in 1999. 72:99–102 75. Lawrie CH, Uzcategui NY, Gould EA, Nuttall PA. 2004. Ixodid and argasid tick species and West Nile virus. Emerg. Infect. Dis. 10:653–57 Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. 76. Ledizet M, Kar K, Foellmer HG, Wang T, Bushmich SL, et al. 2005. A recombinant envelope protein vaccine against West Nile virus. Vaccine 23:3915–24 77. Lieberman MM, Clements DE, Ogata S, Wang G, Corpuz G, et al. 2007. Preparation and immunogenic properties of a recombinant West Nile subunit vaccine. Vaccine 25:414–23 78. WNV is poorly 78. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov AI, Prilipov AG, et al. 2004. understood in West Nile virus and other zoonotic viruses in Russia: examples of emerging- Central Asia, despite the likely reemerging situations. Arch. Virol. Suppl. (18)85–96 importance of this 79. Malkinson M, Banet C. 2002. The role of birds in the ecology of West Nile virus in region for sampling Europe and Africa. Curr. Top. Microbiol. Immunol. 267:309–322 pathogens 80. Marfin AA, Gubler DJ. 2001. West Nile encephalitis: an emerging disease in the United trafficked by birds migrating out of States. Clin. Infect. Dis. 33:1713–19 Africa. 81. Mason PW, Shustov AV, Frolov I. 2006. Production and characterization of vaccines based on flaviviruses defective in replication. Virology 351:432–43

www.annualreviews.org • Epidemiology of WNV 77 ANRV330-EN53-04 ARI 2 November 2007 15:18

82. McGee CE, Schneider BS, Girard YA, Vanlandingham DL, Higgs S. 2007. Nonviremic transmission of West Nile virus: evaluation of the effects of space, time, and mosquito species. Am. J. Trop. Med. Hyg. 76:424–30 83. Miller BR, Nasci RS, Godsey MS, Savage HM, Lutwama JJ, et al. 2000. First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley Province, Kenya. Am. J. Trop. Med. Hyg. 62:240–46 84. Miller DL, Mauel MJ, Baldwin C, Burtle G, Ingram D, et al. 2003. West Nile virus in farmed alligators. Emerg. Infect. Dis. 9:794–99 85. Mitchell CJ. 1988. Occurrence, biology, and physiology of diapause in overwintering mosquitoes. In The Arboviruses: Epidemiology and Ecology, ed. TP Monath, pp. 191–217. Boca Raton, FL: CRC Press 86. Monath TP, Liu J, Kanesa-Thasan N, Myers GA, Nichols R, et al. 2006. A live, attenuated recombinant West Nile virus vaccine. Proc. Natl. Acad. Sci. USA 103:6694–99 87. Morales MA, Barrandeguy M, Fabbri C, Garcia JB, Vissani A, et al. 2006. West Nile virus isolation from equines in Argentina, 2006. Emerg. Infect. Dis. 12:1559–61 88. Morrey JD, Siddharthan V, Olsen AL, Roper GY, Wang H, et al. 2006. Humanized 88. Demonstrates that humanized monoclonal antibody against West Nile virus envelope protein administered after monoclonal neuronal infection protects against lethal encephalitis in hamsters. J. Infect. Dis. antibodies may be a 194:1300–8 potential treatment 89. Moudy RM, Meola MA, Morin LL, Ebel GD, Kramer LD. 2007. A newly emergent for severe WNV genotype of WestNile Virus is transmitted earlier and more efficiently by Culex mosquitoes. disease. Am. J. Trop. Med. Hyg. In press 90. Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ. 2003. Structure of West Nile virus. Science 302:248 91. Mukhopadhyay S, Kuhn RJ, Rossmann MG. 2005. A structural perspective of the flavivirus life cycle. Nat. Rev. Microbiol. 3:13–22 92. Mumcuoglu KY, Banet-Noach C, Malkinson M, Shalom U, Galun R. 2005. Argasid ticks as possible vectors of West Nile virus in Israel. Vector Borne Zoonotic Dis. 5:65–71 93. Murgue B, Murri S, Zientara S, Durand B, Durand JP, Zeller HG. 2001. West Nile outbreak in horses in southern France, 2000: the return 35 years later. Emerg. Infect. Dis. 7:692–96 94. Nasci RS, Savage HM, White D, Miller JR, Cropp BC, et al. 2001. West Nile virus in overwintering Culex mosquitoes, New York City, 2000. Emerg. Infect. Dis. 7:742–44 95. Nemeth N, Gould D, Bowen R, Komar N. 2006. Natural and experimental West Nile virus infection in five raptor species. J. Wildl. Dis. 42:1–13 Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. 96. Niebylski ML, Meek CL. 1992. Blood-feeding of Culex mosquitoes in an urban environment. J. Am. Mosq. Control Assoc. 8:173–77 97. Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, et al. 2005. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat. Med. 11:522–30 98. Olson KE, Adelman ZN, Travanty EA, Sanchez-Vargas I, Beaty BJ, Blair CD. 2002. Developing arbovirus resistance in mosquitoes. Insect Biochem. Mol. Biol. 32:1333–43 99. Palmisano CT, Taylor V, Caillouet K, Byrd B, Wesson DM. 2005. Impact of West Nile virus outbreak upon St. Tammany Parish Mosquito Abatement District. J. Am. Mosq. Control Assoc. 21:33–38 100. Park HW, Bideshi DK, Wirth MC, Johnson JJ, Walton WE, Federici BA. 2005. Recom- binant larvicidal bacteria with markedly improved efficacy against Culex vectors of West Nile virus. Am. J. Trop. Med. Hyg. 72:732–38

78 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

101. Peterson AT, Vieglais DA, Andreasen JK. 2003. Migratory birds modeled as critical transport agents for West Nile virus in North America. Vector Borne Zoonotic Dis. 3:27–37 102. Peterson RK, Macedo PA, Davis RS. 2006. A human-health risk assessment for West Nile virus and insecticides used in mosquito management. Environ. Health Perspect. 114:366–72 103. Phillips RA, Christensen K. 2006. Field-caught Culex erythrothorax larvae found naturally infected with West Nile virus in Grand County, Utah. J. Am. Mosq. Control Assoc. 22:561– 62 104. Pletnev AG, Putnak R, Speicher J, Wagar EJ, Vaughn DW. 2002. West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy. Proc. Natl. Acad. Sci. USA 99:3036–41. Erratum. 2002. Proc. Natl. Acad. Sci. USA 99:7184 105. Puig-Basagoiti F, Deas TS, Ren P, Tilgner M, Ferguson DM, Shi PY. 2005. High- throughput assays using a luciferase-expressing replicon, virus-like particles, and full- length virus for West Nile virus drug discovery. Antimicrob. Agents Chemother. 49:4980–88 106. Puig-Basagoiti F, Tilgner M, Forshey BM, Philpott SM, Espina NG, et al. 2006. Triaryl pyrazoline compound inhibits flavivirus RNA replication. Antimicrob. Agents Chemother. 50:1320–29 107. Pupo M, Guzman MG, Fernandez R, Llop A, Dickinson FO, et al. 2006. West Nile virus infection in humans and horses, Cuba. Emerg. Infect. Dis. 12:1022–24 108. Rappole JH, Compton BW, Leimgruber P, Robertson J, King DI, Renner SC. 2006. Modeling movement of West Nile virus in the Western Hemisphere. Vector Borne Zoonotic Dis. 6:128–39 109. Rappole JH, Derrickson SR, Hubalek Z. 2000. Migratory birds and spread of West Nile virus in the Western Hemisphere. Emerg. Infect. Dis. 6:319–28 110. Reddy MR, Leopore TJ, Pollack RJ, Kiszewski AE, Spielman A, Reiter P. 2007. 110. Analyzes the Early evening questing and oviposition activity by the Culex (Diptera: Culicidae) peak oviposition vectors of West Nile virus in northeastern North America. J. Med. Entomol. 44:211– and host-seeking 14 period of Culex 111. Reddy MR, Spielman A, Lepore TJ, Henley D, Kiszewski AE, Reiter P. 2006. Efficacy mosquitoes to determine the of resmethrin aerosols applied from the road for suppressing Culex vectors of West Nile optimum time for virus. Vector Borne Zoonotic Dis. 6:117–27 aerial insecticidal 112. Reisen WK, Barker CM, Carney R, Lothrop HD, Wheeler SS, et al. 2006. Role of corvids sprays to be in epidemiology of West Nile virus in southern California. J. Med. Entomol. 43:356–67 administered. 113. Reisen WK, Fang Y, Lothrop HD, Martinez VM, Wilson J, et al. 2006. Overwintering Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. of West Nile virus in Southern California. J. Med. Entomol. 43:344–55 114. Reisen WK, Fang Y, Martinez VM. 2007. Is nonviremic transmission of West Nile virus by Culex mosquitoes (Diptera: Culicadae) nonviremic? J. Med. Entomol. 44(2):299–302 115. Reisen WK, Fang Y, Martinez VM. 2005. Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission. J. Med. Entomol. 42:367–75 116. Root JJ, Oesterle PT, Nemeth NM, Klenk K, Gould DH, et al. 2006. Experimental infection of fox squirrels (Sciurus niger) with West Nile virus. Am. J. Trop. Med. Hyg. 75:697–701 117. Rosen L. 1987. Mechanism of vertical transmission of the dengue virus in mosquitoes. C. R. Acad. Sci. III 304:347–50 118. Ruiz MO, Walker ED, Foster ES, Haramis LD, Kitron UD. 2007. Association of West Nile virus illness and urban landscapes in Chicago and Detroit. Int. J. Health Geogr. 6:10

www.annualreviews.org • Epidemiology of WNV 79 ANRV330-EN53-04 ARI 2 November 2007 15:18

119. Samina I, Khinich Y, Simanov M, Malkinson M. 2005. An inactivated West Nile virus vaccine for domestic geese-efficacy study and a summary of 4 years of field application. Vaccine 23:4955–58 120. Samuel MA, Diamond MS. 2005. Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival. J. Virol. 79:13350–61 121. Sanchez-Vargas I, Travanty EA, Keene KM, Franz AW, Beaty BJ, et al. 2004. RNA interference, arthropod-borne viruses, and mosquitoes. Virus Res. 102:65–74 122. Sardelis MR, Turell MJ, Dohm DJ, O’Guinn ML. 2001. Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile virus. Emerg. Infect. Dis. 7:1018–22 123. Savage HM, Ceianu C, Nicolescu G, Karabatsos N, Lanciotti R, et al. 1999. Entomologic and avian investigations of an epidemic of West Nile fever in Romania in 1996, with serologic and molecular characterization of a virus isolate from mosquitoes. Am. J. Trop. Med. Hyg. 61:600–11. Erratum. 2000. Am. J. Trop. Med. Hyg. 62:162 124. Schmidt JR, Elmansoury HK. 1963. Natural and experimental infection of Egyptian equines with West Nile virus. Annu. Trop. Med. Parasitol. 57:415–27 125. Schofield S, Tepper M, Lalonde J. 2006. Comparing risk of West Nile virus against risk of adulticiding. Environ. Health Perspect. 114:A519 126. Seregin A, Nistler R, Borisevich V, Yamshchikov G, Chaporgina E, et al. 2006. Immuno- genicity of West Nile virus infectious DNA and its noninfectious derivatives. Virology 356:115–25 127. Shimoni Z, Niven MJ, Pitlik SD, Bulvik S. 2001. Treatmentof West Nile virus encephalitis with intravenous immunoglobulin. Emerg. Infect. Dis. 7:759 128. Simpson JE. 2006. Emergency mosquito aerial spray response to the 2004 Florida hurricanes Charley, Frances, Ivan, and Jeanne: an overview of control results. J. Am. Mosq. Control Assoc. 22:457–63 129. Smithburn KC, Hughes TP, Burke AW, Paul JH. 1940. A neurotropic virus isolated from the blood of a native of Uganda. Am. J. Trop. Med. Hyg. 20:471–73 130. Snappin KW, Holmes EC, Young DS, Bernard KA, Kramer LD, Ebel GD. 2007. 130. Analyzes the population growth Declining growth rate of West Nile virus in North America. J. Virol. 81:2531–34 rates of WNV 131. Spielman A. 1964. Studies on autogeny in Culex pipiens populations in nature. I. Re- genotypes, productive isolation between autogenous and anautogenous populations. Am. J. Hyg. demonstrating the 80:175–83 virus has reached 132. Spielman A. 1971. Studies on autogeny in natural populations of Culex pipiens. II. Seasonal peak prevalence in abundance of autogenous and anautogenous populations. 8:555–61 Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org J. Med. Entomol. by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. North America. 133. Spielman A. 2001. Structure and seasonality of nearctic Culex pipiens populations. Ann. N. Y. Acad. Sci. 951:220–34 133. Describes 134. Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, et al. 2000. Pathology of fatal Culex pipiens West Nile virus infections in native and exotic birds during the 1999 outbreak in New population York City, New York. Vet. Pathol. 37:208–24 structure and its implications for 135. Styer LM, Carey JR, Wang JL, Scott TW. 2007. Mosquitoes do senesce: departure from feeding behavior the paradigm of constant mortality. Am. J. Trop. Med. Hyg. 76:111–17 and disease 136. Tachiiri K, Klinkenberg B, Mak S, Kazmi J. 2006. Predicting outbreaks: a spatial risk epidemiology. assessment of West Nile virus in British Columbia. Int. J. Health Geogr. 5:21 137. Taylor RM, Work TH, Hurlbut HS, Rizk F. 1956. A study of the ecology of West Nile virus in Egypt. Am. J. Trop. Med. Hyg. 5:579 138. Tempelis CH. 1975. Host-feeding patterns of mosquitoes, with a review of advances in analysis of blood meals by serology. J. Med. Entomol. 11:635–53

80 Kramer · Styer · Ebel ANRV330-EN53-04 ARI 2 November 2007 15:18

139. Tesh RB, Siirin M, Guzman H, Travassos da Rosa AP, Wu X, et al. 2005. Persistent West Nile virus infection in the golden hamster: studies on its mechanism and possible implications for other ﬂavivirus infections. J. Infect. Dis. 192:287–95 140. Torrence PF, Gupta N, Whitney C, Morrey JD. 2006. Evaluation of synthetic oligonucleotides as inhibitors of West Nile virus replication. Antiviral Res. 70:60–65 141. TsaiTF, Popovici F, Cernescu C, Campbell GL, Nedelcu NI. 1998. West Nile encephalitis epidemic in southeastern Romania. Lancet 352:767–71 142. Turell MJ, Dohm DJ, Sardelis MR, Oguinn ML, Andreadis TG, Blow JA. 2005. An update on the potential of North American mosquitoes (Diptera: Culicidae) to transmit West Nile virus. J. Med. Entomol. 42:57–62 143. Turell MJ, O’Guinn ML, Dohm DJ, Jones JW. 2001. Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus. J. Med. Entomol. 38:130– 34 144. Twiddy SS, Holmes EC, Rambaut A. 2003. Inferring the rate and time-scale of dengue virus evolution. Mol. Biol. Evol. 20:122–29 145. Weinberger M, Pitlik SD, Gandacu D, Lang R, Nassar F, et al. 2001. West Nile fever outbreak, Israel, 2000: epidemiologic aspects. Emerg. Infect. Dis. 7:686–91 146. Westaway EG, Mackenzie JM, Khromykh AA. 2002. Replication and gene function in Kunjin virus. Jpn. Enceph. West Nile Viruses 267:323–51 147. Weston DP, Amweg EL, Mekebri A, Ogle RS, Lydy MJ. 2006. Aquatic effects of aerial spraying for mosquito control over an urban area. Environ. Sci. Technol. 40:5817–22 148. Yamshchikov G, Borisevich V, Seregin A, Chaporgina E, Mishina M, et al. 2004. An attenuated West Nile prototype virus is highly immunogenic and protects against the deadly NY99 strain: a candidate for live WN vaccine development. Virology 330:304–12 149. Zanotto PM, Gould EA, Gao GF, Harvey PH, Holmes EC. 1996. Population dynamics of ﬂaviviruses revealed by molecular phylogenies. Proc. Natl. Acad. Sci. USA 93:548–53 150. Zeller HG, Schuffenecker I. 2004. WestNile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the Americas. Eur. J. Clin. Microbiol. Infect. Dis. 23:147–56 151. Zhang W, Chipman PR, Corver J, Johnson PR, Zhang Y, et al. 2003. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat. Struct. Biol. 10:907–12

RELATED RESOURCES Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. Reisen W, Brault AC. 2007. West Nile virus in North America: perspectives on epidemiology and intervention. Pest. Manag. Sci. 63:641–46 Samuel MA, Diamond MS. 2006. Pathogenesis of West Nile Virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion. J. Virol. 80:9349–60

www.annualreviews.org • Epidemiology of WNV 81 AR330-FM ARI 9 November 2007 13:20

Annual Review of Entomology Contents Volume 53, 2008

Frontispiece Geoffrey G.E. Scudder ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppxiv Threads and Serendipity in the Life and Research of an Entomologist Geoffrey G.E. Scudder ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp 1 When Workers Disunite: Intraspeciﬁc Parasitism by Eusocial Bees Madeleine Beekman and Benjamin P. Oldroyd pppppppppppppppppppppppppppppppppppppppppp19 Natural History of the Scuttle Fly, Megaselia scalaris R.H.L. Disney ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp39 A Global Perspective on the Epidemiology of West Nile Virus Laura D. Kramer, Linda M. Styer, and Gregory D. Ebel pppppppppppppppppppppppppppppp61 Sexual Conﬂict over Nuptial Gifts in Insects Darryl T. Gwynne ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp83 Application of DNA-Based Methods in Forensic Entomology Jeffrey D. Wells and Jamie R. Stevens pppppppppppppppppppppppppppppppppppppppppppppppppp103 Microbial Control of Insect Pests in Temperate Orchard Systems: Potential for Incorporation into IPM Lawrence A. Lacey and David I. Shapiro-Ilan ppppppppppppppppppppppppppppppppppppppppp121 Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. Evolutionary Biology of Insect Learning Reuven Dukas pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp145 Roles and Effects of Environmental Carbon Dioxide in Insect Life Pablo G. Guerenstein and John G. Hildebrand ppppppppppppppppppppppppppppppppppppppppp161 Serotonin Modulation of Moth Central Olfactory Neurons Peter Kloppenburg and Alison R. Mercer ppppppppppppppppppppppppppppppppppppppppppppppp179 Decline and Conservation of Bumble Bees D. Goulson, G.C. Lye, and B. Darvill pppppppppppppppppppppppppppppppppppppppppppppppppp191 Sex Determination in the Hymenoptera George E. Heimpel and Jetske G. de Boer ppppppppppppppppppppppppppppppppppppppppppppppp209

vii AR330-FM ARI 9 November 2007 13:20

The Argentine Ant: Challenges in Managing an Invasive Unicolonial Pest Jules Silverman and Robert John Brightwell ppppppppppppppppppppppppppppppppppppppppppp231 Diversity and Evolution of the Insect Ventral Nerve Cord Jeremy E. Niven, Christopher M. Graham, and Malcolm Burrows pppppppppppppppppp253 Dengue Virus–Mosquito Interactions Scott B. Halstead ppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp273 Flash Signal Evolution, Mate Choice, and Predation in Fireﬂies Sara M. Lewis and Christopher K. Cratsley pppppppppppppppppppppppppppppppppppppppppppp293 Prevention of Tick-Borne Diseases Joseph Piesman and Lars Eisen pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp323 Entomological Reactions to Darwin’s Theory in the Nineteenth Century Gene Kritsky pppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppppp345 Resource Acquisition, Allocation, and Utilization in Parasitoid Reproductive Strategies Mark A. Jervis, Jacintha Ellers, and Jeffrey A. Harvey ppppppppppppppppppppppppppppppp361 Population Ecology of Insect Invasions and Their Management Andrew M. Liebhold and Patrick C. Tobin ppppppppppppppppppppppppppppppppppppppppppppp387 Medical Aspects of Spider Bites Richard S. Vetter and Geoffrey K. Isbister pppppppppppppppppppppppppppppppppppppppppppppp409 Plant-Mediated Interactions Between Whiteﬂies, Herbivores, and Natural Enemies Moshe Inbar and Dan Gerling pppppppppppppppppppppppppppppppppppppppppppppppppppppppppp431 Ancient Rapid Radiations of Insects: Challenges for Phylogenetic Analysis ppppppppppppppppppppppppppppppppppppppppppppppppppp

Annu. Rev. Entomol. 2008.53:61-81. Downloaded from www.annualreviews.org 449 by Universitat Zurich- Hauptbibliothek Irchel on 09/30/13. For personal use only. James B. Whitﬁeld and Karl M. Kjer Fruit Fly (Diptera: Tephritidae) Host Status Determination: Critical Conceptual, Methodological, and Regulatory Considerations Martín Aluja and Robert L. Mangan ppppppppppppppppppppppppppppppppppppppppppppppppppp473 Codling Moth Management and Chemical Ecology Peter Witzgall, Lukasz Stelinski, Larry Gut, and Don Thomson ppppppppppppppppppppp503 Primer Pheromones in Social Hymenoptera Yves Le Conte and Abraham Hefetz ppppppppppppppppppppppppppppppppppppppppppppppppppppp523

viii Contents Infection, Genetics and Evolution 12 (2012) 181–190

Contents lists available at SciVerse ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Review West Nile virus population genetics and evolution ⇑ Kendra N. Pesko, Gregory D. Ebel

Department of Pathology, University of New Mexico School of Medicine, 1 University of New Mexico, Albuquerque, NM 87131, USA article info abstract

Article history: West Nile virus (WNV) (Flaviviridae: Flavivirus) is transmitted from mosquitoes to birds, but can cause Received 17 October 2011 fatal encephalitis in infected humans. Since its introduction into North America in New York in 1999, Received in revised form 29 November 2011 it has spread throughout the western hemisphere. Multiple outbreaks have also occurred in Europe over Accepted 30 November 2011 the last 20 years. This review highlights recent efforts to understand how host pressures impact viral pop- Available online 27 December 2011 ulation genetics, genotypic and phenotypic changes which have occurred in the WNV genome as it adapts to this novel environment, and molecular epidemiology of WNV worldwide. Future research directions Keywords: are also discussed. West Nile virus 2011 Elsevier B.V. All rights reserved. Molecular epidemiology Ó Population genetics Pathogenesis Fitness

Contents

1. Introduction ...... 181 1.1. Molecular biology and replication ...... 181 1.2. Ecology...... 182 2. Historical perspective...... 182 3. Taxonomy and classification ...... 183 4. Molecular epidemiology ...... 183 5. Within-host population dynamics...... 185 6. Genetic correlates of pathogenesis and fitness...... 186 7. Conclusions and future research directions ...... 187 References ...... 187

1. Introduction the evolutionary implications of the host–virus interactions. In this review, we highlight recent advances in research into the popula- West Nile virus (WNV, Flaviviridae: Flavivirus) has emerged in tion and evolutionary dynamics of WNV and identify key areas recent decades as a signiﬁcant burden to public health in Europe for further research. and the Americas. This emergence, in particular the recent invasion of WNV into North America in 1999 and its subsequent spread 1.1. Molecular biology and replication throughout the new world, has stimulated intense interest in its population genetics and evolution. The dynamics of the WNV WNV is a member of the Japanese Encephalitis virus (JEV) sero- epizootic/epidemic in N. America have been of special interest logical complex of the ﬂaviviruses (Calisher et al., 1989). The virion because they provide insight into a longstanding question in is enveloped, spherical (40–60 nm in diameter) and contains a evolutionary and invasion biology: what happens when an exotic single copy of the positive-sense RNA genome (Mukhopadhyay pathogen is introduced into a naïve environment? Both observa- et al., 2003; Brinton, 2009). The WNV genome is approximately tional and laboratory studies have therefore been undertaken to 11,000 nt in length and the translated polyprotein is co- and determine the modes and direction of virus evolution and examine post-translationally cleaved by viral and host-cell proteases into three structural (capsid C, premembrane prM/M, and envelope E) ⇑ Corresponding author. Tel.: +1 505 272 3163; fax: +1 505 272 5186. and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, E-mail addresses: [email protected], [email protected] (G.D. Ebel). NS4B and NS5). C, M and E are incorporated into the mature virion,

1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.11.014 182 K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 while the nonstructural proteins assemble on host cell membranes thought to be extremely important in WNV perpetuation, but where they participate in RNA replication and suppression of the potentially significant as ‘‘bridge’’ vectors (i.e. species that feed host antivirus response (Brinton, 2009, 2001; Westaway et al., indiscriminantly) have been found infected, including Ae. albopic- 2002; Evans et al., 2011; Avirutnan et al., 2011; Ambrose and Mac- tus and Ae. vexans (Turell et al., 2002). Several laboratory studies kenzie, 2011). Overall, the genome organization of WNV, and its have established the competence of these vectors to transmit protein coding strategy are similar to other flaviviruses. WNV (Turell et al., 2005), and field studies have detected both WNV is believed to enter host cells by receptor-mediated endo- avian and mammalian blood in Ae. Vexans, although their relative cytosis that is dependent on an Ig-like fold present in domain III of importance in infecting humans and other hosts is currently un- the E glycoprotein. Virus-containing vesicles enter the endocytic clear (Kilpatrick et al., 2005; Molaei and Andreadis, 2006). WNV pathways, where acidification leads to a major reorganization of has also been detected in Culex pipiens mosquitoes that have fed E homodimers into trimers, exposing a hydrophobic peptide on human blood, indicating this mosquito may be the major bridge (termed the cd loop) contained in the distal portion of domain II vector for infecting humans (Hamer et al., 2008). Although WNV of E. Ultimately this reorganization results in fusion of the viral may infect taxonomically diverse mosquito species throughout and host cell membranes. Identifying specific host receptors for its range, certain Culex species appear to be critically important all flaviviruses has proved difficult and the literature is currently in WNV perpetuation in each geographic region where it persists. ambiguous on which host-cell molecules are so-called attachment Similarly, several bird species appear to be capable of generat- receptors and which, if any, are absolutely required for virus entry. ing sufficiently high viremias to infect mosquitoes and contribute Candidate receptors that have been proposed for WNV include DC- to virus perpetuation. American Crow (Corvus brachyrhynchos)

SIGN, DC-SIGNR and avb3 integrin (Davis et al., 2006; Chu and Ng, deaths near the Bronx Zoo in 1999 heralded the arrival of WNV, 2004a,b). In mosquito cells, a c-type lectin is secreted from infected and these birds have served as useful sentinels since then (Eidson cells and binds virions to enhance uptake involving a phosphatase et al., 2001; Kramer and Bernard, 2001). Viremia in Crows reaches homolog of human CD45, mosPTP-1 (Cheng et al., 2010). Once viral extremely high levels (>1010 PFU/mL of blood) and mortality is RNA is released into host cells, it is immediately translated by host nearly uniform (McLean et al., 2001; Komar et al., 2003). Recently machinery. The resulting viral nonstructural proteins assemble on it has become clear that other massively roosting birds, mainly host membranes and replicate the viral genome. Notably, several American Robins (Turdus migratorius) are important both in enzo- viral nonstructural proteins are multifunctional and the function otic maintenance of WNV in highly active transmission foci, and in of others are poorly defined. Excellent reviews on their roles in fla- driving a feeding shift in Culex mosquitoes that increases human vivirus replication and host cell function have been published re- risk (Kilpatrick et al., 2006). Birds also have been implicated in cently (Bollati et al., 2009). Mature virions exit cells through the spreading WNV throughout its distribution. Most importantly, trans-Golgi network and are released into the extracellular milieu migrating birds have been implicated in transportation of WNV by exocytosis and/or budding at the plasma membrane. Thus, the from Africa throughout the Middle East and into Eurasia and with- life cycle of WNV within cells is similar to other RNA viruses that in the Americas (Rappole et al., 2006; May et al., 2011; Zehender replicate cytoplasmically. However, WNV and other arboviruses et al., 2011; Dusek et al., 2009). Clearly a wide variety of birds have have evolved the ability to replicate in cells of hosts that are widely been found infected by WNV, but the species most important to taxonomically divergent (i.e. arthropods and vertebrates). This virus perpetuation may vary locally. requirement for replication in different host types exerts unique WNV is capable of being transmitted between a surprisingly evolutionary and selective pressures on the virus, which are dis- large variety of hosts. In contrast, the related Dengue virus (DENV, cussed below. Flaviviridae, Flavivirus) maintenance is mainly driven by single mosquito and host species (i.e. Aedes aegypti and human beings). 1.2. Ecology By comparison, the ability of WNV to act as an ecological generalist is quite clear, and may account, in part, for its dispersal throughout Viruses adapt to available ecological niches or they become ex- much of the tropical and temperate world. The molecular and/or tinct. A thorough understanding of what constitutes the ‘‘niche’’ for population mechanisms that form the basis for the relative lack WNV is therefore critical to formulating hypotheses regarding how of host-specificity exhibited by WNV are not fully understood, rep- the virus might evolve in order to maximize its potential to perpet- resenting a critical area for future research. uate. WNV is maintained in nature in an enzootic cycle involving birds and mosquitoes. Although the specific birds and mosquitoes most important for virus perpetuation in any given focus vary lo- 2. Historical perspective cally, they tend to include birds of the order Passeriformes and mosquitoes of the genus Culex. However, nearly 60 mosquito and 300 The evolutionary dynamics of WNV are of particular interest be- bird species have been found infected, and the species of Culex cause of the emergence of the virus as a significant health burden mosquito that is most important in a given locality is highly vari- in the last 20 years. Originally isolated in 1937 from the blood of a able. For example, in the Northeastern US, Cx. pipiens pipiens is a patient with fever in the West Nile district of Uganda (Smithburn major vector and appears to be responsible for the vast majority et al., 1940), the first outbreaks of WNV disease were associated of virus transmission (Bernard et al., 2001). In the central and wes- with relatively few cases, mild disease and rural settings (Hayes, tern US, however, Cx. tarsalis is the principal vector, while in south- 2001). Strikingly, an outbreak in Romania that occurred in 1996 ern regions of the US, Cx. p. quinquefasciatus is most important (Bell and 1997 involved over 500 reported cases, with a case-fatality et al., 2006; Molaei et al., 2010; Goldberg et al., 2010; Venkatesan rate of approximately 10% (Tsai et al., 1989, 1998). This outbreak and Rasgon, 2010). In Florida, Cx. nigripalpus is the dominant vector was also striking in that it occurred in a temperate urban region. (Vitek et al., 2008; Kramer et al., 2008). This pattern is repeated at a Shortly thereafter, epidemics were reported in the south of Roma- global scale, with the dominant Culex mosquitoes in a given local- nia and in the Volga delta region of Russia. Additional recent epi- ity driving local WNV transmission (Kramer et al., 2008). Culex spe- demics have been reported in Russia, Israel, Greece, France, cies tend to feed mainly on birds in the spring and summer, Hungary, Italy and others (Platonov, 2001; Bin et al., 2001; Papa switching focus to take more mammalian bloodmeals in the fall, et al., 2010; Balenghien et al., 2006; Depoortere et al., 2004; Kutasi when outbreaks of WNV are most likely to occur among humans et al., 2011; Bakonyi et al., 2006; Monaco et al., 2011). Generally, (Kilpatrick et al., 2006). In addition, several mosquito species not these outbreaks occurred in delta regions of major rivers including K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 183 the Volga, Rhone and Danube. Comprehensive reviews of WNV in The diversity of proposed WNV lineages worldwide reflects the Europe have been published recently (Hubalek and Halouzka, diversity of the vectors involved in virus perpetuation and suggests 1999; Zeller and Schuffenecker, 2004). that WNV or closely related agents have been introduced, and In 1999, WNV was introduced into North America in the New adapted to local transmission cycles on several occasions. York City area, resulting in an equine and avian epizootic, and asso- Taxonomic relationships are not entirely clear, and require ciated human infection, morbidity and mortality (CDC, 1999). The reevaluation, especially with the recent proposal of so many new virus rapidly spread throughout the mainland US and into Canada, WNV lineages. In terms of nucleotide identity, they may be too dis- Mexico, and as far south as Argentina. As has been amply noted, parately related to qualify as part of the same virus species, as the the introduction of WNV at a precisely defined time and place pro- cutoffs proposed by researchers are >84% pairwise sequence iden- vided a relatively unique opportunity to prospectively observe the tity (Kuno et al., 1998) or >79% for inclusion within a species (Ebel adaptation of an exotic RNA virus to an essentially naïve ecosystem. and Kramer, 2009; Charrel et al., 2003), although identity limits for Accordingly, several studies have been conducted to examine the inclusion within a lineage or species are generally arbitrary. evolution of the virus since its introduction (Anderson et al., 2001; According to the first estimate, lineage II WNV would have to be Ebel et al., 2001; Ebel et al., 2004; Beasley et al., 2003; Davis et al., separated into its own species, as it has between 17% and 20% pair- 2005; Bertolotti et al., 2007; McMullen et al., 2011; Armstrong wise distance from lineage I, while the second pairwise distance et al., 2011). Several molecular epidemiologic studies have exam- might prevent proposed lineages III–VI from inclusion in WNV, ined nucleotide sequence data from WNV strains found in birds, as most show greater than 21% pairwise distance from the first mosquitoes and human beings. The most recent of these are dis- two lineages at whole and partial genome levels (Vazquez et al., cussed in detail below and others are reviewed elsewhere (Ebel 2010; Bondre et al., 2007). These lineages appear to show some and Kramer, 2009). The ability of WNV to act as an ecological gener- cross reactivity (Bondre et al., 2007; Bakonyi et al., 2005), may per- alist, in combination with recent increases in intercontinental travel sist in similar transmission cycles, as most have been isolated from and trade, appear to have facilitated its emergence on a global scale. mosquitoes and birds, and appear to form a monophyletic clade when examined alongside Japanese encephalitis virus and Usutu virus (from the same serogroup) but a more systematic examina- 3. Taxonomy and classification tion of relationships between the different lineages is warranted, as they have not been universally accepted (Ebel and Kramer, WNV is classified as a member of the Japanese Encephalitis 2009; Vazquez et al., 2010). The relationships between these differ- complex of the Flaviviruses on the basis of serological cross-reac- ent lineages are further elucidated by Fig. 2, a phylogram based on tivity (Calisher et al., 1989). Within WNV, two major lineages the complete coding sequences for WNV lineages available in (Lineage I and II) are currently accepted, with several additional Genbank. lineages that differ from one another by 5–25% recently proposed Lineage I has been subject to the most intensive study. It is now (Vazquez et al., 2010; Bondre et al., 2007). Lineage I is distributed distributed worldwide, and includes the genotype introduced to throughout much of the world, and is further subdivided into sev- the US in 1999 (NY99). Some genotypes appear to be more patho- eral clades, one of which includes NY-99 (clade Ia), the genotype genic than others, for example NY99 shows enhanced pathogenesis introduced to the US in 1999, another includes Kunjin virus (clade in birds (see below), whereas Kunjin virus (clade Ib) is associated Ib), a variant of West Nile virus endemic to Australia (Ebel and with attenuated infection and decreased neuroinvasion (Brault Kramer, 2009; Lanciotti et al., 1999; May et al., 2011). Lineage II et al., 2007; Daffis et al., 2011). Lineage II is mainly associated with was thought to be restricted to sub-Saharan Africa until recently. less severe disease, and less frequent neuroinvasion. However, re- Since 2004, lineage II has been associated with outbreaks of West cent reports describe encephalitis produced by infection with line- Nile virus in Western and Eastern Europe, and appears to have age II strains in both humans and horses in South Africa (Venter established endemic cycles in Spain and Greece (Papa et al., and Swanepoel, 2010; Venter et al., 2009). Lineage III has only been 2010; 2011; Bakonyi et al., 2006; Vazquez et al., 2010). Lineage isolated from mosquitoes, and did not produce mortality in adult III, also known as ‘‘Rabensburg virus’’, is represented by several iso- mice infected subcutaneously, intraperitoneally, or intracranially lates made from the same region of the Czech Republic in 1997 and (Hubalek et al., 2010). Lineage V viruses from India are also associ- 1999 from Cx. pipiens mosquitoes, and 2006 from a pool of Ae. ros- ated with lower virulence (Davis et al., 2005; Bondre et al., 2007). sicus (Bakonyi et al., 2005; Hubalek et al., 2010). Lineage IV encom- WNV has thus clearly adapted to a wide array of transmission cy- passes numerous isolates made in Russia, first detected in 1988 cles and environments worldwide. This process of migration and from a Dermacentor tick, and since isolated from mosquitoes and adaptation to these environments has produced the currently ob- frogs in 2002 and 2005 in Russia (May et al., 2011). Lineage V com- served lineages. Additional studies are required in order to define prises 13 isolates from India, collected from humans and Culex differences in virulence, neuroinvasiveness, natural hosts and vec- mosquitoes from the 1950s through 1980, which differ from other tors, and basic ecology for each putative lineage. West Nile lineages by 20–25% at the nucleotide level (Bondre et al., 2007). Recent publications show these strains as basal to lineage I, comprising an independent cluster, lineage Ic (May et al., 2011). An 4. Molecular epidemiology additional, putative sixth lineage has been isolated in Spain from a pool of Cx. pipiens mosquitoes, and appears to be most closely re- Upon its introduction to the United States, WNV was initially lated to lineage IV WNV (Vazquez et al., 2010). Additionally, Kou- recognized by sequence comparisons and phylogenetic analysis tango virus (KOU), a Flavivirus isolated in Senegal, may represent (Lanciotti et al., 1999). The genotype introduced to the New World, a seventh lineage, as it is 25% divergent from other WNV isolates, dubbed NY99 for its initial isolation in New York in 1999, is most although it is currently categorized as a separate species (King closely related to isolates made in Israel in 1998 and Hungary in et al., 2011). Human infection by KOU has not been reported, and 2003 (Zehender et al., 2011; Lanciotti et al., 1999; Jia et al., its serological relationships to established WNV strains and trans- 1999). Initial sequence analysis of the WNV strains isolated during mission cycle are unclear, although partial cross neutralization the first two years in New England showed a remarkable amount of with WNV and KUN has been shown (Charrel et al., 2003; Calisher genetic conservation, indicating a single point of introduction and et al., 1989). The distribution of all described lineages of WNV is very little diversification in WNV populations during this time per- shown in Fig. 1, by country where isolations have been made. iod (Anderson et al., 2001; Ebel et al., 2001; Lanciotti et al., 1999; 184 K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190

Fig. 1. Worldwide map with countries where West Nile virus has been isolated colored as follows: Lineage Ia in light blue; lineage Ib in medium blue; lineage Ic in dark blue, lineage II in red, lineage III or ‘‘Rabensburg’’ virus in purple, lineage IV in orange, recent Spanish lineage (Vazquez et al., 2010) in green, and Koutango virus is colored yellow. Hatched coloring indicates more than one lineage has been isolated from that country. Lineage I distribution is adapted from May et al., 2011, other lineage isolates adapted from Charrel et al., 2003; Vazquez et al., 2010. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) reviewed in Kramer et al., 2008; Ebel and Kramer, 2009). Subse- 2007). Recent studies, relying on full genome sequences and quently, an additional subtype of WNV, WN02, with an amino acid encompassing samples taken over a number of years after intro- substitution in the envelope protein, A159V, was detected in sam- duction of WNV uncovered more evidence for geographical struc- ples isolated in Texas (Beasley et al., 2003). From 2001 to 2003, ture to samples (McMullen et al., 2011; Armstrong et al., 2011; WN02 rapidly displaced NY99, becoming the dominant genotype Herring et al., 2007; Grinev et al., 2008). A recent analysis indicates in North America (Ebel et al., 2004; Davis et al., 2005). WN02 sequences from the envelope coding region may not be the most strains require a shorter extrinsic incubation period in mosquitoes, phylogenetically informative, and suggests NS3 or NS5 may be bet- which appears to be the mechanism for its increased fitness rela- ter partial sequences for reconstructing the phylogenetic relation- tive to NY99 (Ebel et al., 2004; Moudy et al., 2007). Thus, shortly ships between different isolates, and can provide reconstructions after WNV was introduced into North America, the process of evo- that more closely resemble those resulting from whole genome se- lution led to increases in the basic reproductive rate of this quences (Gray et al., 2010). Several distinct genetic variants of pathogen. WNV have arisen in certain geographical areas, such as Texas As WNV became established throughout North America, the ge- (McMullen et al., 2011; Davis et al., 2004). One attenuated genetic netic diversity present in different types of data sets has led to in- lineage seems to have become extinct after being detected over the sights into its emergence and expansion. Studies have found course of two years (Davis et al., 2005, 2004; Ebel and Kramer, increased genetic diversity in mosquitoes relative to birds (Berto- 2009; Ebel, 2010). Another distinctive genotype, SW/WN03, con- lotti et al., 2007; Amore et al., 2010), perhaps due to different selec- tains several amino acid changes relative to other WN02 and is re- tive pressure from the immune pathways used by these different cently reported to be spreading through numerous states, although hosts, which will be discussed in the section on genetic diversity the phenotype associated with this new genotype has not been below (Brackney et al., 2009). Genetic diversity and therefore esti- characterized (McMullen et al., 2011). It may be that more genetic mated virus population size appeared to initially increase yearly changes accumulated in these WNV populations as they adapted to after introduction to the US, although studies suggest this may local transmission cycles. be leveling off as WNV becomes established endemically (Berto- Recent studies using full genome sequences of WNV from iso- lotti et al., 2007, 2008; Amore et al., 2010; Snapinn et al., 2007). lates made globally have uncovered phylogeographical influences Several studies (Armstrong et al., 2011)showed a lack of geo- on clade 1a distribution (May et al., 2011; Zehender et al., 2011). graphical partitioning among sequences, especially those that This clade seems to have a common ancestor that existed in sub- examined sequence data from isolates sampled immediately after Saharan Africa in the early 20th century, which had multiple the introduction of WNV to novel environments and relied primar- migrations to both Western and Eastern European countries in ily on envelope sequences (Bertolotti et al., 2007, 2008; Davis et al., the 1970s and 1980s, and single introductions to India and K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 185

flavivirus specific antiviral lycorine, and ability to overcome superinfection exclusion in replicon containing cell lines, which appears to be related to enhanced viral RNA synthesis (Mertens et al., 2010; Zou et al., 2009a,b). Adaptive evolution has also been detected at amino acid sites: E-V431I, NS2A-A224V/T, NS4A-A85T, NS5- K314R, and NS5-R422K although the functional significances of these sites are unclear (May et al., 2011; McMullen et al., 2011). Thus, several of the encoded WNV proteins are subject to positive selection that may lead to increased transmission efficiency and the likelihood for perpetuation in different transmission cycles. Synonymous changes to the WNV genome could also impact its pathogenesis and evolution. Numerous synonymous changes were associated with the new genotype, WN02, and although some of these are assumed to have become fixed by association with other mutations that might confer a selective advantage, they could also exert an effect through codon bias or changes to the RNA genomic structure. The 50 and 30 untranslated regions are well conserved and have essential roles during viral replication (Khromykh et al., 2001; Zhang et al., 2008). Additional studies have shown that other RNA genome structures present in the capsid coding region can operate to upregulate flaviviral replication (Tuplin et al., 2011; Clyde and Harris, 2006). Additionally, codon bias in flaviviruses reflect the host usage (vertebrate only and alternating seem to display vertebrate codon biases, invertebrate only have more invertebrate bias), so examination of codon bias for a given virus can provide insight into its evolutionary history (Schubert and Putonti, Fig. 2. Radial phylogram showing relationships between different lineages of WNV. 2010). Additional studies are required in order to determine the Complete coding sequences were downloaded from Genbank and aligned manually extent to which nonsynonymous variation impact RNA genomic in BioEdit. Strains used and accession numbers are as follows: JEV, NC_001437; structure in a way that influences WNV phenotype. NY99, lineage Ia, NC_009942; Kunjin, paKUN, lineage Ib, AY274505.1; Indi804994, Indian lineage Ic, DQ256376.1; 956, lineage II, NC_001563; Rabensburg, lineage III, AY765264.1; RussianLEIV, lineage IV, strain Krnd88-190, AY277251.1; Koutango virus, EU082200.1. Bayesian phylogeny is shown, generated with MrBayes 3.1.2 run 5. Within-host population dynamics with a general time reversible (GTR) model with gamma shaped rate variation and invariable sites (Ronquist and Huelsenbeck, 2003). Two Markov chain Monte Carlo Molecular epidemiologic studies such as those discussed in the (MCMC) tree searches of 5000,000 generations each were run in parallel with sampling one in every 1000 trees. Radial 50% majority-rule consensus tree is shown preceding section have provided insights into the selective forces based on the last 3750 trees. Posterior probabilities are given as numbers at each that act on WNV and shown clearly that the virus is a dynamic, node. evolving entity with the capacity to adapt to a wide range of hosts and environments. These findings have stimulated studies aimed at understanding the viral population genetic mechanisms that ac- Australia around the same time (May et al., 2011; Zehender et al., count for this, and to assess whether the two very different kinds 2011). The patterns of distribution from Africa to Europe seem to of host required for WNV perpetuation (mosquitoes and birds) follow white stork migration routes, indicating a possibly impor- influence the WNV population in different ways. Early studies sug- tant role for this bird species in the spread of WNV into that con- gested that within hosts, WNV forms a genetically complex distri- tinent (Zehender et al., 2011). Other mosquito borne Flaviviruses bution of mutants that vary in their degree of nucleotide have also apparently originated in Africa, including yellow fever divergence from the population consensus sequence. Further, and dengue virus (Bryant et al., 2007; Gaunt et al., 2001; Holmes Jerzak et al. (2005) showed that whereas WNV populations in nat- and Twiddy, 2003). urally infected birds are relatively genetically homogeneous and Arboviruses are unique in that they require replication in taxo- purifying selection is strong, in field collected WNV infected mos- nomically divergent hosts – vertebrates and invertebrates (Wea- quitoes they are very diverse, and purifying selection seems to be ver, 2006). This requirement is thought to restrict the amount of relaxed. The observations were supported by a series of laboratory mutation that can occur in arboviruses, relative to single host studies that passed WNV in colonized mosquitoes and chickens viruses (Jenkins et al., 2002). Experimental studies have shown (Jerzak et al., 2007), and cultured cells (Ciota et al., 2007). Impor- lower mutation rates in viruses serially passaged in alternating tantly, the mosquito passed virus was inoculated intrathoracically hosts, relative to those passaged in a single host type (Jerzak and whole mosquitoes were triturated to obtain passed WNV, et al., 2007; Coffey et al., 2008; Coffey and Vignuzzi, 2011). To date, bypassing putative transmission barriers in the midgut and salivary numerous studies have shown purifying or negative selection is glands (Hardy et al., 1983; Ciota et al., 2008). A highly similar study dominant in arbovirus populations, including West Nile virus (Ber- conducted using virus obtained from mosquito saliva failed to con- tolotti et al., 2007, 2008; McMullen et al., 2011; Armstrong et al., firm these results raising the possibility that infection of, or escape 2011; Amore et al., 2010; Jerzak et al., 2005). In WNV phylogenetic from salivary glands might constitute a population bottleneck in analyses, only a few genetic changes have been identified that ap- the WNV system (Ciota et al., 2008; Ciota and Kramer, 2010). None- pear to be the subject of positive selective pressure. These include theless, several studies have clearly established that mosquitoes the amino acid residue associated with increased pathogenesis and birds exert different evolutionary pressures on WNV. among North American birds, NS3 T249P and a mutation to The mechanistic basis for this difference has been addressed NS4A, or the 2 K protein (V135M or V9M) (Armstrong et al., from a variety of perspectives. First, vertebrates and invertebrates 2011; Brault et al., 2007). The valine to methionine mutation in respond to virus infections differently. In vertebrates, the earliest NS4A/2K is associated with OAS1b resistance, resistance to the responses to infection by RNA viruses are dominated by type I 186 K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 interferon (IFNa/b). This response is triggered when RIG-I senses a wave of epidemic cases among humans (Murray et al., 2010a). dsRNA in host cell cytosol, initiating signaling cascades that ulti- Studies have identified a single amino acid substitution in the mately result in an antiviral state in the cell (reviewed in (Daffis NS3 helicase coding region, T249P, that increased morbidity and et al., 2009)). Therefore, in vertebrates, WNV may be required to viral load in American crows, and appeared to be under selective essentially ‘‘outrun’’ the antiviral state in infected individuals. This pressure in areas with multiple genotypes present (Brault et al., would result in strong purifying selection that has been observed 2007, 2004). After establishment of this initial pathogenic strain after virus replication in these hosts (Ding, 2010; Jerzak et al., of WNV across the US, phylogenetic analysis of WNV sequences de- 2007), where presumably all or nearly all nonsynonymous muta- tected a new genotype, WN02, which displaced the initial strain tion results in genomes of diminished fitness. NY99 in less than 4 years (Ebel et al., 2004; Davis et al., 2005). This In contrast, insects respond to virus infection mainly through new genotype had a single amino acid substitution in the envelope RNA interference (RNAi), which is also triggered by dsRNA within coding protein, V159A, that significantly decreased the extrinsic cells (reviewed in (Ding, 2010)). Ultimately, virus-derived small- incubation time from virus infection until transmission by Cx. pipi- interfering RNAs (viRNAs) are loaded into the RNA induced ens mosquitoes important vectors in the northeastern United silencing complex (RISC) to degrade target viral RNA in a se- States (Moudy et al., 2007; Kilpatrick et al., 2008). This mutation quence-specific manner. Therefore, the antiviral state in mosquito occurs nearby the envelope glycosylation motif for WNV, which cells seems to drive WNV diversification through a mechanism is at nucleotide positions 154–156 in the envelope coding akin to negative, frequency-dependant selection, wherein rare sequence. genotypes (i.e. those that do not match common guide sequences Envelope protein glycosylation sites are conserved throughout loaded into the RISC) are favored because they are less efficiently the genus Flavivirus, although natural variation in glycosylation degraded (Brackney et al., 2009). The precise relationship between is present in populations of WNV (Adams et al., 1995; Berthet this mechanism and the observed lack of purifying selection in et al., 1997; Shirato et al., 2004; Hanna et al., 2005). An N linked mosquitoes has not been resolved or adequately addressed, and glycosylation site at position 154 in the envelope protein has been may represent two sides of the same coin. Overall, WNV popula- associated with increased neuroinvasiveness for WNV in mice, and tion biology seems to be dominated by largely opposing forces that increased virulence and viremia in young chicks (Shirato et al., exist within its natural transmission cycle. Specifically, WNV 2004; Beasley et al., 2005; Murata et al., 2010). Envelope protein undergoes alternating cycles of genetic expansion in mosquitoes glycosylation is also necessary for efficient transmission by Cx. that generates novel genotypes, and purification in birds that pipiens, Cx. tarsalis, and Cx. quinquefasciatus, but not Cx. pipiens pal- ensures that high fitness is maintained. lens, thus it influences vector competence in a species specific way Other forces that influence WNV genetic diversity also have (Murata et al., 2010; Moudy et al., 2009). Glycosylation patterns been examined recently. Population bottlenecks can stochastically from virus propagated in insect versus vertebrate cells also seem reduce population diversity and lead to fitness declines through to influence the ability of envelope protein to modulate innate im- the action of Muller’s ratchet (Duarte et al., 1992). Convention mune response, and leads to different patterns of infectivity and holds that in natural transmission cycles, arboviruses undergo pop- propagation in different cell types, thus the role of glycosylation ulation bottlenecks as they pass through mosquitoes, where they is also host specific (Hanna et al., 2005; Arjona et al., 2007). seem to sequentially infect the epithelium of the mosquito midgut, The mechanism behind envelope protein glycosylation and peripheral tissues and ultimately the salivary glands, from which modulation of WNV activity could be related to a number of differ- they are released into salivary secretions that are inoculated during ent phenomena. The ability of WNV envelope to suppress dsRNA mosquito feeding (Hardy et al., 1983). Such population bottlenecks activated innate immune response is dependent on glycosylation have been described for alphaviruses and flaviviruses. Studies status, which leads to increased inflammatory cytokine production examining early mosquito infection by Venezuelan equine enceph- for cells infected with virus lacking glycosylation (Arjona et al., alitis virus (VEEV; Togaviridae, Alphavirus) and WNV demon- 2007). Envelope glycosylation status influences ability to survive strated that only a few midgut cells are susceptible to infection, in lower pH environments (Beasley et al., 2005; Langevin et al., suggesting that anatomical bottlenecks may reduce genetic vari- 2011). Genomes lacking the envelope glycosylation site have de- ability (Smith et al., 2008). Conversely, identical non-consensus creased replication, which may be related to budding of mature WNV genomes have been detected in intrahost populations infect- virions from the lumen of the endoplasmic reticulum rather than ing birds in a single transmission focus, suggesting that population the plasma membrane (Berthet et al., 1997; Shirato et al., 2004; bottlenecks may not be as restrictive as had been assumed (Jerzak Li et al., 2006). Abolishing the N linked glycosylation site on et al., 2005), and defective DENV genomes appear to perpetuate in WNV envelope also influences receptor interactions, as it decreases transmission cycles through complementation (Aaskov et al., greatly the ability of WNV to bind DC-SIGNR (Davis et al., 2006). 2006). Supporting this, Brackney et al. recently failed to document One or a combination of these mechanisms, or perhaps a mecha- significant population bottlenecks during infection of Cx. quinque- nism yet to be uncovered may explain the increased virulence of fasciatus mosquitoes by WNV (Brackney et al., 2011). It may be that WNV with envelope N-linked glycosylation. the importance of bottlenecks during arbovirus transmission is a Strains associated with greater neuroinvasiveness and patho- function of the specific virus–host system under study, and not genesis in mice and humans tend to be better controllers of inter- consistent across systems. feron mediated responses (Daffis et al., 2011, 2009). Numerous WNV proteins may modulate the interferon signaling cascade in vertebrate hosts, including all nonstructural coding proteins 6. Genetic correlates of pathogenesis and fitness (NS1: Wilson et al., 2008, NS2A/B: Liu et al., 2006, NS3: Liu et al., 2005, NS4A/B: Muñoz-Jordán et al., 2003 NS5: Laurent-Rolle Molecular genetic and phenotypic studies of WNV mutants and et al., 2010; reviewed, Diamond et al., 2009; Samuel and Diamond, engineered clones have revealed multiple genetic variations corre- 2006). With the advent of reverse genetics, recent studies have lated with increased or decreased pathogenicity. WNV was long determined distinct amino acid changes in certain residues that thought to be a less pathogenic flavivirus, with sporadic epidemics are correlated with changed ability to control host immune producing little or no mortality in human populations up until the responses. For example, a single residue at position 653 in NS5 early 1990s (Hayes, 2001). The recent introduction of WNV to the appears to be responsible for the enhanced ability of NY99 like United States was marked by large die offs in bird populations, and viruses to suppress interferon response. In North American K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 187 genotype 1a viruses, position 653 of NS5 is a phenylalanine, emerging as a significant health issue in particular regions of the whereas in the less pathogenic Kunjin virus, this position is a ser- US (Murray et al., 2010b). The extent to which viral genetic and ine, and if these residues are switched through reverse genetics, population determinants influence this has not been adequately suppression is enhanced for Kunjin and depleted for NY99 addressed. In cell culture, establishment of persistent infection (Laurent-Rolle et al., 2010). Another example comes from studies with Flaviviruses can occur with subgenomic replicons or the of the host factor OAS1b which appears to confer natural resistance development of defective interfering particles (DIPs) (Zou et al., to WNV (Samuel and Diamond, 2006; Lucas et al., 2003). Virus cul- 2009b; Yoon et al., 2006). A second pressing matter is a reevalua- tivated in the presence of OAS1b can circumvent this factor by tion of the serological relationships within the Japanese encephali- mutating at several residues, including NS3-S365G, which seems tis serogroup. Molecular genetic studies have proposed numerous to lower the requirement of ATP for the ATPase dependent cleav- lineages of WNV beyond the traditional two lineages recognized age activity of this protease, and 2K-V9M, which generally en- previously. The basic biology, transmission cycle, host range and hances viral RNA synthesis (Mertens et al., 2010). Virus strains pathogenicity of these putative lineages should also be studied fur- with higher rates of replication may be positively selected ther. Additionally, numerous amino acid residues may be under (Armstrong et al., 2011). Studies like this that correlate genotype positive selection, but the roles of these residues are still unclear. with phenotype, and determine the underlying mechanisms, Reverse genetic studies should be undertaken to determine the should expand our understanding of virus pathogenesis and the influence of these changes on WNV biology. With the advent of forces shaping the emergence of pathogenic phenotypes. new sequencing technologies, our ability to design and conduct Attenuated genotypes of WNV emerged during the course of its experiments into immune responses of hosts to viral infection spread across the United States. In Texas, a number of small plaque and viral population biology has greatly increased. The role of RNAi variants of WNV that displayed reduced neuroinvasiveness in mice in generating viral diversity, the potential bottlenecks associated were detected in 2003 (Davis et al., 2004). Comparison to NY99 with the WNV transmission cycle, and the interaction of individu- strain followed by introduction of similar mutations into an infec- als within a viral population may all become better understood tious clone identified a combination of mutations to NS4, NS5, and with deep sequencing approaches. Finally, more collaboration be- the 30 UTR as being necessary for the attenuated phenotype found tween people studying ecology, epidemiology, molecular genetics, in one bird sample (Davis et al., 2007, 2004). Further analysis of and pathology of WNV could lead to greater insight into its overall other samples from the same region found that different amino biology. acid substitutions in these strains appear to confer attenuation, We have highlighted major advances in WNV biology over the indicating multiple pathways towards attenuated phenotypes past decade, including understanding of host specific selective (May et al., 2010). A single amino acid substitution in the central pressures on viral populations, genotypic correlates with patho- portion of NS4B, C102S, was enough to attenuate neurovirulence genic phenotypes, and phylogenetic relationships between differ- in mice (Wicker et al., 2006). Lineage II WNV has been associated ent lineages, strains, and genotypes. Molecular epidemiology with encephalitis and more severe disease only rarely, but compar- studies continue to elucidate the spread and evolutionary change ison of the strains isolated from patients with more severe disease that is ongoing in WNV populations. to less virulent strains indicates an enhanced role for NS proteins in determining virulence, relative to structural proteins, similar to findings for lineage I (Botha et al., 2008). References

Aaskov, J., Buzacott, K., Thu, H.M., Lowry, K., Holmes, E.C., 2006. Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes. Science 7. Conclusions and future research directions 311, 236. Adams, S., Broom, A., Sammels, L., Hartnett, A., Howard, M., Coelen, R., Mackenzie, J., In recent decades, WNV has emerged as one of the most inten- Hall, R., 1995. Glycosylation and antigenic variation among Kunjin virus sely studied arthropod borne viruses. The spread of WNV through- isolates. Virology 206, 49–56. Ambrose, R.L., Mackenzie, J.M., 2011. West Nile virus differentially modulates the out North America and periodic outbreaks in Eastern and Western unfolded protein response to facilitate replication and immune evasion. J. Virol. Europe have enhanced worldwide interest in understanding viral, 85, 2723. host, and ecological factors that result in outbreaks. Due to the Amore, G., Bertolotti, L., Hamer, G.L., Kitron, U.D., Walker, E.D., Ruiz, M.O., Brawn, J.D., Goldberg, T.L., 2010. Multi-year evolutionary dynamics of West Nile virus availability of an increasing number of complete genome sein suburban Chicago, USA, 2005–2007. Philos. Trans. R. Soc. Lond. B. Biol. Sci. quences (as of October 12, 2011, 479 are currently available on 365, 1871–1878. GenBank) major advances have occurred in understanding its Anderson, J.F., Vossbrinck, C.R., Andreadis, T.G., Iton, A., Beckwith, W.H., Mayo, D.R., 2001. A phylogenetic approach to following West Nile virus in Connecticut. molecular epidemiology, particularly in North America. Collec- Proc. Natl. Acad. Sci. 98, 12885. tively, these studies have clearly demonstrated that WNV is a dy- Arjona, A., Ledizet, M., Anthony, K., Bonafé, N., Modis, Y., Town, T., Fikrig, E., 2007. namic virus population that is able to perpetuate in and adapt to West Nile virus envelope protein inhibits dsRNA-induced innate immune responses. J. Immunol. 179, 8403. a wide range of ecological settings through mutation and selection. Armstrong, P.M., Vossbrinck, C.R., Andreadis, T.G., Anderson, J.F., Pesko, K.N., In addition, since the virus transmission cycle is relatively tractable Newman, R.M., Lennon, N.J., Birren, B.W., Ebel, G.D., Henn, M.R., 2011. Molecular in the laboratory using colonized mosquitoes and captive wild or evolution of West Nile virus in a northern temperate region: Connecticut, USA 1999–2008. Virology 417, 203–210. domestic birds, experimental studies have provided insights into Avirutnan, P., Hauhart, R.E., Somnuke, P., Blom, A.M., Diamond, M.S., Atkinson, J.P., how different components of the transmission cycle influence virus 2011. Binding of Flavivirus Nonstructural Protein NS1 to C4b Binding Protein population genetics and evolution. These studies have shown that Modulates Complement Activation. J. Immunol. 187, 424. mosquitoes drive WNV diversification through their innate antivi- Bakonyi, T., Hubálek, Z., Rudolf, I., Nowotny, N., 2005. Novel flavivirus or new lineage of West Nile virus, central Europe. Emerg. Infect. Dis. 11, 225–231. rus response and because purifying selection is weak in these Bakonyi, T., Ivanics, E., Erdelyi, K., Ursu, K., Ferenczi, E., Weissenbock, H., Nowotny, hosts, provide a reservoir of genetic diversity that allows the ob- N., 2006. Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. served adaptation. In contrast, infection of birds ensures that the Emerg. Infect. Dis. 12, 618–623. Balenghien, T., Fouque, F., Sabatier, P., Bicout, D., 2006. Horse-, bird-, and human- resulting variants are of high fitness through strong purifying seeking behavior and seasonal abundance of mosquitoes in a West Nile virus selection. The processes that underpin WNV evolution are thus focus of southern France. J. Med. Entomol. 43, 936–946. beginning to be understood in greater detail. Beasley, D.W.C., Davis, C.T., Guzman, H., Vanlandingham, D.L., Travassos da Rosa, A., Parsons, R.E., Higgs, S., Tesh, R.B., Barrett, A.D.T., 2003. Limited evolution of Several important areas for future research, however, remain. West Nile virus has occurred during its southwesterly spread in the United First, persistent infection of WNV in humans and vertebrates is States. Virology 309, 190–195. 188 K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190

Beasley, D.W.C., Whiteman, M.C., Zhang, S., Huang, C.Y.H., Schneider, B.S., Smith, Daffis, S., Suthar, M.S., Gale Jr., M., Diamond, M.S., 2009. Measure and D.R., Gromowski, G.D., Higgs, S., Kinney, R.M., Barrett, A.D.T., 2005. Envelope countermeasure: type I IFN (IFN-a/b) antiviral response against West Nile protein glycosylation status influences mouse neuroinvasion phenotype of Virus. J. Innate Immun. 1, 435. genetic lineage 1 West Nile virus strains. J. Virol. 79, 8339. Davis, C.T., Beasley, D.W.C., Guzman, H., Siirin, M., Parsons, R.E., Tesh, R.B., Barrett, Bell, J.A., Brewer, C.M., Mickelson, N.J., Garman, G.W., Vaughan, J.A., 2006. West Nile A.D.T., 2004. Emergence of attenuated West Nile virus variants in Texas, 2003. virus epizootiology, central Red River Valley, North Dakota and Minnesota, Virology 330, 342–350. 2002–2005. Emerg. Infect. Dis. 12, 1245–1247. Davis, C.T., Li, L., May, F.J., Bueno Jr., R., Dennett, J.A., Bala, A.A., Guzman, H., Bernard, K., Maffei, J., Jones, S., Kauffman, E., Ebel, G., Dupuis, A., 2001. West Nile Elizondo-Quiroga, D., Tesh, R.B., Barrett, A.D., 2007. Genetic stasis of dominant virus infection in birds and mosquitoes, New York State, 2000. Emerg. Infect. West Nile virus genotype, Houston, Texas. Emerg. Infect. Dis. 13, 601. Dis. 7, 679. Davis, C.W., Nguyen, H.Y., Hanna, S.L., Sánchez, M.D., Doms, R.W., Pierson, T.C., 2006. Berthet, F., Zeller, H., Drouet, M., Rauzier, J., Digoutte, J., Deubel, V., 1997. Extensive West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular nucleotide changes and deletions within the envelope glycoprotein gene of attachment and infection. J. Virol. 80, 1290. Euro-African West Nile viruses. J. Gen. Virol. 78, 2293. Davis, C.T., Ebel, G.D., Lanciotti, R.S., Brault, A.C., Guzman, H., Siirin, M., Lambert, A., Bertolotti, L., Kitron, U., Goldberg, T.L., 2007. Diversity and evolution of West Nile Parsons, R.E., Beasley, D.W., Novak, R.J., Elizondo-Quiroga, D., Green, E.N., Young, virus in Illinois and the United States, 2002–2005. Virology 360, 143–149. D.S., Stark, L.M., Drebot, M.A., Artsob, H., Tesh, R.B., Kramer, L.D., Barrett, A.D., Bertolotti, L., Kitron, U.D., Walker, E.D., Ruiz, M.O., Brawn, J.D., Loss, S.R., Hamer, G.L., 2005. Phylogenetic analysis of North American West Nile virus isolates, 2001– Goldberg, T.L., 2008. Fine-scale genetic variation and evolution of West Nile 2004: evidence for the emergence of a dominant genotype. Virology 342, 252– Virus in a transmission ‘‘hot spot’’ in suburban Chicago, USA. Virology 374, 381– 265. 389. Depoortere, E., Kavle, J., Keus, K., Zeller, H., Murri, S., Legros, D., 2004. Outbreak of Bin, H., Grossman, Z., Pokamunski, S., Malkinson, M., Weiss, L., Duvdevani, P., Banet, West Nile virus causing severe neurological involvement in children, Nuba C., Weisman, Y., Annis, E., Gandaku, D., 2001. West Nile Fever in Israel 1999– Mountains, Sudan, 2002. Tropical Med. Int. Health 9, 730–736. 2000. Ann. N. Y. Acad. Sci. 951, 127–142. Diamond, M.S., Mehlhop, E., Oliphant, T., Samuel, M.A., 2009. The host immunologic Bollati, M., Alvarez, K., Assenberg, R., Baronti, C., Canard, B., Cook, S., Coutard, B., response to West Nile encephalitis virus. Front. Biosci. 14, 3024–3034. Decroly, E., de Lamballerie, X., Gould, E.A., 2009. Structure and functionality in Ding, S.W., 2010. RNA-based antiviral immunity. Nat. Rev. Immunol. 10 (9), 632– flavivirus NS-proteins: perspectives for drug design. Antiviral Res. 644. Bondre, V.P., Jadi, R., Mishra, A., Yergolkar, P., Arankalle, V., 2007. West Nile virus Duarte, E., Clarke, D., Moya, A., Domingo, E., Holland, J., 1992. Rapid fitness losses in isolates from India: evidence for a distinct genetic lineage. J. Gen. Virol. 88, mammalian RNA virus clones due to Muller’s ratchet. Proc. Natl. Acad. Sci. 89, 875. 6015. Botha, E.M., Markotter, W., Wolfaardt, M., Paweska, J.T., Swanepoel, R., Palacios, G., Dusek, R.J., McLean, R.G., Kramer, L.D., Ubico, S.R., Dupuis, A.P., Ebel, G.D., Guptill, Nel, L.H., Venter, M., 2008. Genetic determinants of virulence in pathogenic S.C., 2009. Prevalence of West Nile virus in migratory birds during spring and lineage 2 West Nile virus strains. Emerg. Infect. Dis. 14, 222. fall migration. Am. J. Trop. Med. Hyg. 81, 1151. Brackney, D.E., Beane, J.E., Ebel, G.D., 2009. RNAi targeting of West Nile virus in Ebel, G.D., 2010. Update on Powassan virus: emergence of a North American tick- mosquito midguts promotes virus diversification. PLoS Pathog. 5, e1000502. borne flavivirus. Annu. Rev. Entomol. 55, 95–110. Brackney, D.E., Pesko, K.N., Brown, I.K., Deardorff, E.R., Kawatachi, J., Ebel, G.D., 2011. Ebel, G.D., Kramer, L.D., 2009. West Nile virus: molecular epidemiology and West Nile virus genetic diversity is maintained during transmission by Culex diversity. In: Diamond, M.S. (Ed.), West Nile Encephalitis Virus Infection. pipiens quinquefasciatus mosquitoes. PLoS One 6, e24466. Springer, pp. 25–43. Brault, A.C., Huang, C.Y.H., Langevin, S.A., Kinney, R.M., Bowen, R.A., Ramey, W.N., Ebel, G.D., Dupuis 2nd, A.P., Ngo, K., Nicholas, D., Kauffman, E., Jones, S.A., Young, D., Panella, N.A., Holmes, E.C., Powers, A.M., Miller, B.R., 2007. A single positively Maffei, J., Shi, P.Y., Bernard, K., Kramer, L.D., 2001. Partial genetic selected West Nile viral mutation confers increased virogenesis in American characterization of West Nile virus strains, New York State, 2000. Emerg. crows. Nat. Genet. 39, 1162–1166. Infect. Dis. 7 (4), 650–653. Brault, A.C., Langevin, S.A., Bowen, R.A., Panella, N.A., Biggerstaff, B.J., Miller, B.R., Ebel, G.D., Carricaburu, J., Young, D., Bernard, K.A., Kramer, L.D., 2004. Genetic and Komar, N., 2004. Differential virulence of West Nile strains for American crows. phenotypic variation of West Nile virus in New York, 2000–2003. Am. J. Trop. Emerg. Infect. Dis. 10, 2161. Med. Hyg. 71, 493–500. Brinton, M.A., 2009. Molecular Biology of West Nile Virus. In: Diamond, M. (Ed.), Eidson, M., Komar, N., Sorhage, F., Nelson, R., Talbot, T., Mostashari, F., McLean, R., West Nile Encephalitis Virus Infection: Viral Pathogenesis and the Host Immune 2001. Crow deaths as a sentinel surveillance system for West Nile virus in the Response. Springer, New York. northeastern United States, 1999. Emerg. Infect. Dis. 7, 615. Brinton, M.A., 2001. Host factors involved in West Nile virus replication. Ann. N. Y. Evans, J.D., Crown, R.A., Sohn, J.A., Seeger, C., 2011. West Nile virus infection induces Acad. Sci. 951, 207–219. depletion of IFNAR1 protein levels. Viral Immunol. 24, 253–263. Bryant, J.E., Holmes, E.C., Barrett, A.D.T., 2007. Out of Africa: a molecular perspective Gaunt, M.W., Sall, A.A., Lamballerie, X.d., Falconar, A.K.I., Dzhivanian, T.I., Gould, on the introduction of yellow fever virus into the Americas. PLoS Pathog. 3, e75. E.A., 2001. Phylogenetic relationships of flaviviruses correlate with their Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E., Porterfield, J.S., epidemiology, disease association and biogeography. J. Gen. Virol. 82, 1867– Westaway, E.G., Brandt, W.E., 1989. Antigenic relationships between 1876. flaviviruses as determined by cross-neutralization tests with polyclonal Goldberg, T.L., Anderson, T.K., Hamer, G.L., 2010. West Nile virus may have hitched a antisera. J. Gen. Virol. 70, 37. ride across the Western United States on Culex tarsalis mosquitoes. Mol. Ecol. CDC, 1999. Outbreak of West Nile-like viral encephalitis—New York, 1999. Morb. 19, 1518–1519. Mortal. Wkly. Rep. 48, 845–849. Gray, R., Veras, N., Santos, L., Salemi, M., 2010. Evolutionary characterization of the Charrel, R., Brault, A., Gallian, P., Lemasson, J.J., Murgue, B., Murri, S., Pastorino, B., West Nile Virus complete genome. Mol. Phylogenet. Evol. 56, 195–200. Zeller, H., De Chesse, R., De Micco, P., 2003. Evolutionary relationship between Grinev, A., Daniel, S., Stramer, S., Rossmann, S., Caglioti, S., Rios, M., 2008. Genetic Old World West Nile virus strains: evidence for viral gene flow between Africa, variability of West Nile virus in US blood donors, 2002–2005. Emerg Infect Dis the Middle East, and Europe. Virology 315, 381–388. 14, 436. Cheng, G., Cox, J., Wang, P., Krishnan, M.N., Dai, J., Qian, F., Anderson, J.F., Fikrig, E., Hamer, G.L., Kitron, U.D., Brawn, J.D., Loss, S.R., Ruiz, M.O., Goldberg, T.L., et al., 2008. 2010. A C-type lectin collaborates with a CD45 phosphatase homolog to Culex Pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans. facilitate West Nile virus infection of mosquitoes. Cell 142, 714–725. J. Med. Entomol. 45, 125–128. Chu, J.J., Ng, M.L., 2004a. Infectious entry of West Nile virus occurs through a Hanna, S.L., Pierson, T.C., Sanchez, M.D., Ahmed, A.A., Murtadha, M.M., Doms, R.W., clathrin-mediated endocytic pathway. J. Virol. 78, 10543–10555. 2005. N-linked glycosylation of West Nile virus envelope proteins influences Chu, J.J., Ng, M.L., 2004b. Interaction of West Nile virus with alpha v beta 3 integrin particle assembly and infectivity. J. Virol. 79, 13262. mediates virus entry into cells. J. Biol. Chem. 279, 54533–54541. Hardy, J.L., Houk, E.J., Kramer, L.D., Reeves, W.C., 1983. Intrinsic factors affecting Ciota, A.T., Kramer, L.D., 2010. Insights into arbovirus evolution and adaptation from vector competence of mosquitoes for arboviruses. Annu. Rev. Entomol. 28, 229– experimental studies. Viruses 12, 2594–2617. 262. Ciota, A.T., Lovelace, A.O., Jia, Y., Davis, L.J., Young, D.S., Kramer, L.D., 2008. Hayes, C.G., 2001. West Nile virus: Uganda, 1937, to New York City, 1999. Ann. N. Y. Characterization of mosquito-adapted West Nile virus. J. Gen. Virol. 89, 1633. Acad. Sci. 951, 25–37. Ciota, A.T., Ngo, K.A., Lovelace, A.O., Payne, A.F., Zhou, Y., Shi, P.Y., Kramer, L.D., 2007. Herring, B.L., Bernardin, F., Caglioti, S., Stramer, S., Tobler, L., Andrews, W., Cheng, L., Role of the mutant spectrum in adaptation and replication of West Nile virus. J. Rampersad, S., Cameron, C., Saldanha, J., 2007. Phylogenetic analysis of WNV in Gen. Virol. 88, 865–874. North American blood donors during the 2003–2004 epidemic seasons. Clyde, K., Harris, E., 2006. RNA secondary structure in the coding region of dengue Virology 363, 220–228. virus type 2 directs translation start codon selection and is required for viral Holmes, E.C., Twiddy, S.S., 2003. The origin, emergence and evolutionary genetics of replication. J. Virol. 80, 2170–2182. dengue virus. Infect. Genet. Evol. 3, 19–28. Coffey, L.L., Vasilakis, N., Brault, A.C., Powers, A.M., Tripet, F., Weaver, S.C., 2008. Hubalek, Z., Halouzka, J., 1999. West Nile fever – a reemerging mosquito-borne viral Arbovirus evolution in vivo is constrained by host alternation. Proc. Natl. Acad. disease in Europe. Emerg. Infect. Dis. 5, 643. Sci. 105, 6970. Hubalek, Z., Rudolf, I., Bakonyi, T., Kazdova, K., Halouzka, J., Sebesta, O., Sikutova, S., Coffey, L.L., Vignuzzi, M., 2011. Host alternation of chikungunya virus increases Juricova, Z., Nowotny, N., 2010. Mosquito (Diptera: Culicidae) surveillance for fitness while restricting population diversity and adaptability to novel selective arboviruses in an area endemic for West Nile (Lineage Rabensburg) and Tahyna pressures. J. Virol. 85, 1025. viruses in Central Europe. J. Med. Entomol. 47, 466–472. Daffis, S., Lazear, H.M., Liu, W.J., Audsley, M., Engle, M., Khromykh, A.A., Diamond, Jenkins, G.M., Rambaut, A., Pybus, O.G., Holmes, E.C., 2002. Rates of molecular M.S., 2011. The naturally attenuated Kunjin strain of West Nile virus shows evolution in RNA viruses: a quantitative phylogenetic analysis. J. Mol. Evol. 54, enhanced sensitivity to the host type I interferon response. J. Virol. 85, 5664. 156–165. K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190 189

Jerzak, G.V., Bernard, K., Kramer, L.D., Shi, P.Y., Ebel, G.D., 2007. The West Nile virus Monaco, F., Savini, G., Calistri, P., Polci, A., Pinoni, C., Bruno, R., Lelli, R., 2011. 2009 mutant spectrum is host-dependant and a determinant of mortality in mice. West Nile disease epidemic in Italy: First evidence of overwintering in Western Virology 360, 469–476. Europe? Res. Vet. Sci. 91, 321–326. Jerzak, G., Bernard, K.A., Kramer, L.D., Ebel, G.D., 2005. Genetic variation in West Nile Moudy, R.M., Meola, M.A., Morin, L.L.L., Ebel, G.D., Kramer, L.D., 2007. A newly virus from naturally infected mosquitoes and birds suggests quasispecies emergent genotype of West Nile virus is transmitted earlier and more structure and strong purifying selection. J Gen Virol 86, 2175–2183. efficiently by Culex mosquitoes. Am. J. Trop. Med. Hyg. 77, 365. Jia, X.Y., Briese, T., Jordan, I., Rambaut, A., Chang Chi, H., Mackenzie, J.S., Hall, R.A., Moudy, R.M., Zhang, B., Shi, P.Y., Kramer, L.D., 2009. West Nile virus envelope Scherret, J., Lipkin, W.I., 1999. Genetic analysis of West Nile New York 1999 protein glycosylation is required for efficient viral transmission by Culex encephalitis virus. The Lancet 354, 1971–1972. vectors. Virology 387, 222–228. Khromykh, A.A., Meka, H., Guyatt, K.J., Westaway, E.G., 2001. Essential role of Mukhopadhyay, S., Kim, B.S., Chipman, P.R., Rossmann, M.G., Kuhn, R.J., 2003. cyclization sequences in flavivirus RNA replication. J. Virol. 75, 6719–6728. Structure of West Nile virus. Science 302, 248. Kilpatrick, A.M., Meola, M.A., Moudy, R.M., Kramer, L.D., 2008. Temperature, viral Muñoz-Jordán, J.L., Sanchez-Burgos, G.G., Laurent-Rolle, M., García-Sastre, A., 2003. genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes. Inhibition of interferon signaling by dengue virus. Proc. Natl. Acad. Sci. USA 100, PLoS Pathog. 4, e1000092. 14333–14338. Kilpatrick, A.M., Kramer, L.D., Campbell, S.R., Alleyne, E.O., Dobson, A.P., Daszak, P., Murata, R., Eshita, Y., Maeda, A., Maeda, J., Akita, S., Tanaka, T., Yoshii, K., Kariwa, H., 2005. West Nile virus risk assessment and the bridge vector paradigm. Emerg. Umemura, T., Takashima, I., 2010. Glycosylation of the West Nile virus envelope Infect. Dis. 11, 425–429. protein increases in vivo and in vitro viral multiplication in birds. Am. J. Trop. Kilpatrick, A.M., Kramer, L.D., Jones, M.J., Marra, P.P., Daszak, P., 2006. West Nile Med. Hyg. 82, 696. virus epidemics in North America are driven by shifts in mosquito feeding Murray, K.O., Mertens, E., Desprès, P., 2010a. West Nile virus and its emergence in behavior. PLoS Biol. 4, e82. the United States of America. Vet. Res. 41. King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B., 2011. Virus Taxonomy: Ninth Murray, K., Walker, C., Herrington, E., Lewis, J.A., McCormick, J., Beasley, D.W., Tesh, Report of the International Committee on Taxonomy of Viruses, 1st edition. R.B., Fisher-Hoch, S., 2010b. Persistent infection with West Nile virus years after Elsevier, San Diego, CA. initial infection. J. Infect. Dis. 201, 2–4. Komar, N., Langevin, S., Hinten, S., Nemeth, N., Edwards, E., Hettler, D., Davis, B., Papa, A., Danis, K., Baka, A., Bakas, A., Dougas, G., Lytras, T., Theocharopoulos, G., Bowen, R., Bunning, M., 2003. Experimental infection of North American birds Chrysagis, D., Vassiliadou, E., Kamaria, F., 2010. Ongoing outbreak of West Nile with the New York 1999 strain of West Nile virus. Emerg. Infect. Dis. 9, 311. virus infections in humans in Greece, July–August 2010. Age 20, 20–29. Kramer, L.D., Styer, L.M., Ebel, G.D., 2008. A global perspective on the epidemiology Papa, A., Xanthopoulou, K., Gewehr, S., Mourelatos, S., 2011. Detection of West Nile of West Nile virus. Annu. Rev. Entomol. 63, 81. virus lineage 2 in mosquitoes during a human outbreak in Greece. Clin. Kramer, L.D., Bernard, K.A., 2001. West Nile virus infection in birds and mammals. Microbiol. Infect. 17 (8), 1176–1180. Ann. N. Y. Acad. Sci. 951, 84–93. Platonov, A.E., 2001. West Nile Encephalitis in Russia 1999–2001. Ann. N. Y. Acad. Kuno, G., Chang, G.J.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. Phylogeny of Sci. 951, 102–116. the genus Flavivirus. J. Virol. 72, 73–83. Rappole, J.H., Compton, B.W., Leimgruber, P., Robertson, J., King, D.I., Renner, S.C., Kutasi, O., Bakonyi, T., Lecollinet, S., Biksi, I., Ferenczi, E., Bahuon, C., Sardi, S., 2006. Modeling movement of West Nile virus in the Western hemisphere. Zientara, S., Szenci, O., 2011. Equine encephalomyelitis outbreak caused by a Vector Borne Zoonotic Dis. 6, 128–139. genetic lineage 2 West Nile virus in Hungary. J. Vet. Intern. Med. 25 (3), 586– Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference 591. under mixed models. Bioinformatics 19, 1572–1574. Lanciotti, R., Roehrig, J., Deubel, V., Smith, J., Parker, M., Steele, K., Crise, B., Volpe, K., Samuel, M.A., Diamond, M.S., 2006. Pathogenesis of West Nile virus infection: a Crabtree, M., Scherret, J., 1999. Origin of the West Nile virus responsible for an balance between virulence, innate and adaptive immunity, and viral evasion. J. outbreak of encephalitis in the northeastern United States. Science 286, 2333. Virol. 80, 9349. Langevin, S., Bowen, R., Ramey, W., Sanders, T., Maharaj, P., Fang, Y., Cornelius, J., Schubert, A.M., Putonti, C., 2010. Evolution of the sequence composition of Barker, C., Reisen, W., Beasley, D., 2011. Envelope and pre-membrane structural Flaviviruses. Infect. Genet. Evol. 10, 129–136. amino acid mutations mediate diminished avian growth and virulence of a Shirato, K., Miyoshi, H., Goto, A., Ako, Y., Ueki, T., Kariwa, H., Takashima, I., 2004. Mexican West Nile virus isolate. J. Gen. Virol. 92 (Pt 12), 2810–2820. Viral envelope protein glycosylation is a molecular determinant of the Laurent-Rolle, M., Boer, E.F., Lubick, K.J., Wolfinbarger, J.B., Carmody, A.B., Rockx, B., neuroinvasiveness of the New York strain of West Nile virus. J. Gen. Virol. 85, Liu, W., Ashour, J., Shupert, W.L., Holbrook, M.R., Barrett, A.D., Mason, P.W., 3637. Bloom, M.E., Garcia-Sastre, A., Khromykh, A.A., Best, S.M., 2010. The NS5 protein Smith, D.R., Adams, A.P., Kenney, J.L., Wang, E., Weaver, S.C., 2008. Venezuelan of the virulent West Nile virus NY99 strain is a potent antagonist of type I equine encephalitis virus in the mosquito vector Aedes taeniorhynchus: Infection interferon-mediated JAK-STAT signaling. J. Virol. 84, 3503–3515. initiated by a small number of susceptible epithelial cells and a population Li, J., Bhuvanakantham, R., Howe, J., Ng, M.L., 2006. The glycosylation site in the bottleneck. Virology 372, 176–186. envelope protein of West Nile virus (Sarafend) plays an important role in Smithburn, K., Hughes, T., Burke, A., Paul, J., 1940. A neurotropic virus isolated from replication and maturation processes. J. Gen. Virol. 87, 613–622. the blood of a native of Uganda. Am. J. Trop. Med. Hyg. 20, 471–473. Liu, W.J., Wang, X.J., Clark, D.C., Lobigs, M., Hall, R.A., Khromykh, A.A., 2006. A single Snapinn, K.W., Holmes, E.C., Young, D.S., Bernard, K.A., Kramer, L.D., Ebel, G.D., 2007. amino acid substitution in the West Nile virus nonstructural protein NS2A Declining growth rate of West Nile virus in North America. J. Virol. 81, 2531. disables its ability to inhibit alpha/beta interferon induction and attenuates Tsai, T., Popovici, F., Cernescu, C., Campbell, G., Nedelcu, N., 1998. West Nile virus virulence in mice. J. Virol. 80, 2396–2404. encephalitis epidemic in southeastern Romania. The Lancet 352, 767–771. Liu, W.J., Wang, X.J., Mokhonov, V.V., Shi, P.-Y., Randall, R., Khromykh, A.A., 2005. Tsai, T.F., Mitchell, C.J., Monath, T.P., 1989. St. Louis encephalitis. In: Anonymous Inhibition of interferon signaling by the New York 99 strain and Kunjin subtype (Ed.), The Arboviruses: Epidemiology and Ecology. CRC, Boca Raton, FL, pp. 113– of West Nile virus involves blockage of STAT1 and STAT2 activation by 143. nonstructural proteins. J. Virol. 79, 1934–1942. Tuplin, A., Evans, D., Buckley, A., Jones, I., Gould, E., Gritsun, T., 2011. Replication Lucas, M., Mashimo, T., Frenkiel, M.P., Simon-Chazottes, D., Montagutelli, X., enhancer elements within the open reading frame of tick-borne encephalitis Ceccaldi, P.E., Guénet, J.L., Desprès, P., 2003. Infection of mouse neurones by virus and their evolution within the Flavivirus genus. Nucleic Acids Res. 39 (16), West Nile virus is modulated by the interferon-inducible 20-50 oligoadenylate 7034–7048. synthetase 1b protein. Immunol. Cell Biol. 81, 230–236. Turell, M.J., Dohm, D.J., Sardelis, M.R., Oguinn, M.L., Andreadis, T.G., Blow, J.A., 2005. May, F.J., Davis, C.T., Tesh, R.B., Barrett, A.D.T., 2011. Phylogeography of West Nile An update on the potential of North American mosquitoes (Diptera: Culicidae) virus: from the cradle of evolution in Africa to Eurasia, Australia, and the to transmit West Nile virus. J. Med. Entomol. 42, 57–62. Americas. J. Virol. 85, 2964. Turell, M.J., Sardelis, M.R., O’Guinn, M.L., Dohm, D.J., 2002. Potential vectors of West May, F.J., Li, L., Davis, C.T., Galbraith, S.E., Barrett, A.D.T., 2010. Multiple pathways to Nile virus in North America. Curr. Top. Microbiol. Immunol. 267, 241–252. the attenuation of West Nile virus in south-east Texas in 2003. Virology 405, 8– Vazquez, A., Sanchez-Seco, M.P., Ruiz, S., Molero, F., Hernandez, L., Moreno, J., 14. Magallanes, A., Tejedor, C.G., Tenorio, A., 2010. Putative new lineage of west nile McLean, R., Ubico, S., Docherty, D., Hansen, W., Sileo, L., McNamara, T., 2001. West virus. Spain. Emerg. Infect. Dis. 16, 549–552. Nile virus transmission and ecology in birds. Ann. N. Y. Acad. Sci. 951, 54. Venkatesan, M., Rasgon, J.L., 2010. Population genetic data suggest a role for McMullen, A.R., May, F.J., Li, L., Guzman, H., Bueno Jr., R., Dennett, J.A., Tesh, R.B., mosquito-mediated dispersal of West Nile virus across the western United Barrett, A.D.T., 2011. Evolution of new genotype of West Nile virus in North States. Mol. Ecol. 19, 1573–1584. America. Strain, 17. Venter, M., Human, S., Zaayman, D., Gerdes, G.H., Williams, J., Steyl, J., Leman, Mertens, E., Kajaste-Rudnitski, A., Torres, S., Funk, A., Frenkiel, M.P., Iteman, I., P.A., Paweska, J.T., Setzkorn, H., Rous, G., 2009. Lineage 2 West Nile virus as Khromykh, A.A., Despres, P., 2010. Viral determinants in the NS3 helicase and cause of fatal neurologic disease in horses, South Africa. Emerg. Infect. Dis. 2K peptide that promote West Nile virus resistance to antiviral action of 20,50- 15, 877. oligoadenylate synthetase 1b. Virology 399, 176–185. Venter, M., Swanepoel, R., 2010. West Nile virus lineage 2 as a cause of zoonotic Molaei, G., Cummings, R.F., Su, T., Armstrong, P.M., Williams, G.A., Cheng, M.L., neurological disease in humans and horses in southern Africa. Vector Borne Webb, J.P., Andreadis, T.G., 2010. Vector–host interactions governing Zoonotic Dis. 10, 659–664. epidemiology of West Nile virus in Southern California. Am. J. Trop. Med. Vitek, C.J., Richards, S.L., Mores, C.N., Day, J.F., Lord, C.C., 2008. Arbovirus Hyg. 83, 1269. transmission by Culex nigripalpus in Florida, 2005. J. Med. Entomol. 45, 483. Molaei, G., Andreadis, T.G., 2006. Identification of avian-and mammalian-derived Weaver, S.C., 2006. Evolutionary influences in arboviral disease. Curr. Top. bloodmeals in Aedes vexans and Culiseta melanura (Diptera: Culicidae) and its Microbiol. Immunol. 299, 285–314. Implication for West Nile Virus transmission in Connecticut, USA. J. Med. Westaway, E.G., Mackenzie, J.M., Khromykh, A.A., 2002. Replication and gene Entomol. 43, 1088–1093. function in Kunjin virus. Curr. Top. Microbiol. Immunol. 267, 323–351. 190 K.N. Pesko, G.D. Ebel / Infection, Genetics and Evolution 12 (2012) 181–190

Wicker, J.A., Whiteman, M.C., Beasley, D.W.C., Davis, C.T., Zhang, S., Schneider, B.S., Zeller, H., Schuffenecker, I., 2004. West Nile virus: an overview of its spread in Higgs, S., Kinney, R.M., Barrett, A.D.T., 2006. A single amino acid substitution in Europe and the Mediterranean basin in contrast to its spread in the Americas. the central portion of the West Nile virus NS4B protein confers a highly Eur. J. Clin. Microbiol. Infect. Dis. 23, 147–156. attenuated phenotype in mice. Virology 349, 245–253. Zhang, B., Dong, H., Stein, D.A., Iversen, P.L., Shi, P.Y., 2008. West Nile virus genome Wilson, J.R., de Sessions, P.F., Leon, M.A., Scholle, F., 2008. West Nile virus cyclization and RNA replication require two pairs of long-distance RNA nonstructural protein 1 inhibits TLR3 signal transduction. J. Virol. 82, 8262– interactions. Virology 373, 1–13. 8271. Zou, G., Puig-Basagoiti, F., Zhang, B., Qing, M., Chen, L., Pankiewicz, K.W., Felczak, K., Yoon, S.W., Lee, S.Y., Won, S.Y., Park, S.H., Park, S.Y., Jeong, Y.S., 2006. Yuan, Z., Shi, P.Y., 2009a. A single-amino acid substitution in West Nile virus 2K Characterization of homologous defective interfering RNA during persistent peptide between NS4A and NS4B confers resistance to lycorine, a ﬂavivirus infection of Vero cells with Japanese encephalitis virus. Mol. Cells 21, 112–120. inhibitor. Virology 384, 242–252. Zehender, G., Ebranati, E., Bernini, F., Presti, A.L., Rezza, G., Delogu, M., Galli, M., Zou, G., Zhang, B., Lim, P.Y., Yuan, Z., Bernard, K.A., Shi, P.Y., 2009b. Exclusion of Ciccozzi, M., 2011. Phylogeography and epidemiological history of West Nile West Nile virus superinfection through RNA replication. J. Virol. 83, 11765– virus genotype 1a in Europe and the Mediterranean basin. Infect. Genet. Evol. 11 11776. (5), 863–868. VIRAL IMMUNOLOGY Volume 16, Number 3, 2003 © Mary Ann Liebert, Inc. Pp. 259–278

Review

Innate and Adaptive Immune Responses Determine Protection against Disseminated Infection by West Nile Encephalitis Virus

MICHAEL S. DIAMOND, BIMMI SHRESTHA, ERIN MEHLHOP, ELIZABETH SITATI, and MICHAEL ENGLE

ABSTRACT

WNV continues to spread throughout the Western Hemisphere as virus activity in insects and animals has been reported in the United States, Canada, Mexico, and the Caribbean islands. West Nile virus (WNV) infects the central nervous system and causes severe disease primarily in humans who are immunocompromised or elderly. In this review, we discuss the mechanisms by which the immune system limits dissemination of WNV infection. Recent experimental studies in animals suggest important roles for both the innate and the adaptive immune responses in controlling WNV infection. Interferons, antibody, complement components and CD81 T cells coordinate protection against severe infection and disease. These findings are analyzed in the context of recent approaches to vaccine development and immunotherapy against WNV.

INTRODUCTION

EST NILE VIRUS (WNV) is the etiologic agent of West Nile encephalitis. It is a member of the Fla- Wvivirus genus and is closely related to other arboviruses that cause human disease including dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. WNV is maintained in a natural cycle between mosquitoes and birds but also infects humans, horses, and other animals. It is endemic in parts of Africa, Europe, the Middle East, and Asia (68), and outbreaks in the North America over the past three years indicate that it has established its presence in the Western Hemisphere (103). WNV activity has now been detected in most of the continental United States and Canada (1). Hu- mans develop a febrile illness with a subset of cases progressing to meningitis, encephalitis, or a polio-like paralytic syndrome (5,68,105,106). Currently, no specific therapy or vaccine is approved for human use. WNV is an enveloped RNA virus with a single-stranded, positive-polarity 11-kilobase genome. The structural proteins include a capsid protein (C), an envelope protein (E) that functions in receptor binding, mem-

Departments of Medicine, Molecular Microbiology, Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri.

259 DIAMOND ET AL. brane fusion, and viral assembly, and a transmembrane protein (prM) that assists in proper folding and function of the E protein. The role of the nonstructural (NS) proteins is not fully delineated but these proteins form the viral protease (NS2B, NS3), NTPase (NS3), RNA helicase (NS3), and RNA-dependent RNA polymerase (NS5) (25). After binding to an uncharacterized cell surface receptor, virus uptake is believed to occur through receptor-mediated endocytosis (25). In the endosome, an acid-catalyzed conformational change in E (55,87) releases the nucleocapsid into the cytoplasm. At the endoplasmic reticulum membrane, WNV infectious RNA is translated as a polyprotein, and then cleaved into structural and non-structural proteins by virus- and host-encoded proteases (111). The structural proteins and non-structural NS1 undergo co-translational translocation, glycosylation, and membrane-associated cleavage, while the other nonstructural proteins are translated in the cytoplasm (48,85,86,121). The positive strand genomic RNA serves as a template for RNA replication to generate the negative strand RNA. This negative sense strand functions as a template for the production of additional positive strand RNA. After replication, assembly and packaging take place at the endoplasmic reticulum and viral particles are exocytosed via secretory vesicles (Fig. 1). Intense study of WNV pathogenesis and the nature of the protective immune system response have accompanied the current epidemic. Host factors clearly influence the expression of WNV disease in humans (20). Infants, the elderly, and those with impaired immune systems are at greatest risk for severe neurological disease (5,68,173). Similarly, in animals, the maturation and integrity of the immune system correlates with resistance to WNV infection (42,44,45,62,180). The purpose of this review is to outline the mechanisms by which the innate and adaptive immune systems limit dissemination of WNV infection into the central nervous system (CNS).

FIG. 1. Intracellular lifecycle of WNV. WNV attaches to susceptible cells through an as yet uncharacterized cell surface receptor and enters cells via receptor-mediated endocytosis. After acidification of the endosome, the E protein undergoes a conformational change that facilitates membrane fusion and nucleocapsid escape into the cytoplasm. Vi- ral RNA binds to ribosomes in the cytoplasm and is recruited to the rough endoplasmic reticulum where translation of the polyprotein from a single open reading frame ensues. The polyprotein is cleaved post-translationally by viral and host proteases. Translation, replication, and packaging are coupled processes and nascent viruses accumulate in membrane-derived vesicles prior to secretion by the cellular exocytosis machinery.

260 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

PATHOGENESIS OF WNV INFECTION

Infection experiments in animals have provided insight into the sequence of events that define WNV pathogenesis (Fig. 2). As with many flaviviruses, WNV infection occurs after inoculation by an infected mosquito. The initial round of replication is believed to occur in the skin in Langerhans dendritic cells (67,76,109,122,182). Based on experiments with the related dengue virus, the dendritic cell surface glycoprotein DC-SIGN (CD209) may be important for viral attachment and entry (169). Infected dendritic cells migrate to draining lymph nodes (19,75) where the risk of dissemination is countered by the development of an early immune response. Factors in the saliva of the insect vector may facilitate WNV virus transmission by altering the local and systemic host immune response. For example, sialokinin-I, a mosquito salivary protein, down-regulates IFN-g production and up-regulates the TH2 cytokines, IL-4 and IL-10 (186). After reaching secondary lymphoid tissues, a second round of WNV replication occurs, leading to its entry into the circulation via the efferent lymphatic system and thoracic duct. Viremia ensues and after spread to visceral organs (e.g., kidney, and spleen), WNV disseminates to the brain and spinal cord (42,183,184). Although several studies have defined the neuron as the principle cellular target of infection in the CNS (42,44,105,106,165,183,184), the tropism in lymphoid and visceral organs and the molecular basis of WNV attachment remain unknown. Moreover, the mechanism by which WNV disseminates into the CNS also re-

FIG. 2. WNV pathogenesis. WNV infects animals after mosquito (e.g., Culex pipens) inoculation. Infection is presumed to begin in subdermal Langerhans cells. Infected dendritic cells migrate to draining lymph nodes and produce interferons that limit spread. In the lymph node, replication occurs in a cell type that has not been definitively identified. Candidate cell types include macrophages and follicular dendritic cells. Infectious virus exits the efferent limb and enters the circulation via the thoracic duct. Natural antibodies (IgM), interferons, and complement control the initial levels of virus in the blood. Viremia allows spread to secondary visceral organs (e.g., liver, kidney, spleen) and facilitates crossing of the blood-brain barrier by an as yet uncharacterized mechanism that may involve the epithelial cells of the choroids plexus, the endothelial cells of inflamed vessels, or an infected carrier cell (e.g., monocyte or T cell).

261 DIAMOND ET AL. mains unclear. WNV may cross the blood–brain barrier via a hematogenous route (42,73,74) by passive transport across the endothelium or epithelial cells of the choroid plexus, by active replication in endothelial cells, or by a “Trojan horse” mechanism in which virus is carried into the brain by infected inflammatory cells (18,72,162). Alternatively, under certain conditions, WNV may invade the CNS directly from the olfactory mucosa (2,135) with subsequent spread to other regions by retrograde axonal transport.

INNATE IMMUNE RESPONSE TO WNV

Interferons. In vitro and in vivo studies have demonstrated that interferon-dependent innate immune responses are essential for protection against flavivirus infections. However, many of these protection experiments were performed with related flaviviruses, and not directly with WNV. Dendritic cells may be one of the first cells to produce IFN in response to infection (109). IFN binds to cell surface receptors and triggers a complex signal transduction and transcriptional response pathway that is mediated by janus kinases (JAK) and signal transducers and activators of transcription (STAT) molecules (11,164). Effector molecules are synthesized that induce an antiviral state and limit virus replication. Type I IFN (a or b) block flavivirus infection by preventing translation and replication of infectious viral RNA (39,41); this occurs at least partially through an RNAse L, Mx1, and PKR-independent mechanism (3,39,41). Type II IFN (g) inhibits flavivirus replication by signaling through the JAK-STAT signal transduction pathway, generating proinflammatory and antiviral molecules, including nitric oxide (110) and enhancing the phagocytic activity of myeloid cells. Type I (a or b) and II (g) IFN inhibit flavivirus infection in cell culture and in animals (39,41,71,107,108,110), and have been recently shown to directly inhibit WNV infection in Vero cells (3) and neurons (159). Embryonal fibroblasts that lack receptors for IFN a/b or STAT1 molecules are highly susceptible to infection with WNV (Diamond, unpublished data). The importance of IFN in preventing flavivirus infection has been confirmed in mouse models of disease. Pretreatment of mice with IFN-a prevents Saint Louis encephalitis and yellow fever virus infection (16,166) and mice that are deficient in type I and II IFN function have increased morbidity and mortality after dengue and Murray Valley encephalitis virus infections (71,116; Shresta and Harris, unpublished data). Moreover, mice that lack IFN g or IFN g receptors also have increased lethality after infection with WNV or Murray Valley encephalitis virus (116; Engle and Diamond, unpublished data). While these studies strongly suggest an inhibitory activity of IFN against WNV, definition of the precise stage in pathogenesis at which IFN exerts its inhibitory effect requires additional virologic and immunologic studies in animal models. Flavivirus resistance gene. In the mouse, an additional innate resistance to infection by WNV and other flaviviruses has been mapped to the Flv resistance gene on chromosome 5 (152,174). Resistant mice can be infected by WNV but the virus titers in tissues are 3 to 4 logs lower than in susceptible animals (35,36). The flavivirus resistant allele was recently characterized. Susceptible mouse strains have an isoform of the 2959 oligoadenylate sythetase (OAS) gene that is truncated and lacks 30% of the C-terminal sequence (125,144). Expression of the full-length OAS gene in susceptible cells conferred partial resistance to WNV infection. Although OAS activates the IFN-induced antiviral effector molecule RNAse L, this innate flavivirus resistance may be independent of IFN (15,36) since treatment with anti-IFN antibodies did not ab- rogate the resistance phenotype in animals or in cell culture. Resistance may be associated with a function of the L1 isoform of OAS that is independent of the synthesis of 2959 adenylate oligomers and activation of RNAse L (151). Indeed, other OAS isoforms have alternate functional motifs in their C-terminal regions: the 9-2 isozyme of OAS encodes a Bcl-2 homology domain that promotes apoptosis (52). Further studies are required to determine the mechanism by which the L1 isoform of OAS confers protection, and whether analogous genetic polymorphisms exist that could explain differential susceptibility to flavivirus infection in humans or other animals.

HUMORAL IMMUNE RESPONSE TO WNV

Antibody response. Humoral immunity is an essential component of the immune response to WNV and other flaviviruses as neutralizing antibodies limit dissemination of infection. The role of antibodies in the

262 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION protection against infection has been studied extensively in mouse models of flavivirus infection including WNV. Passive transfer of polyclonal or monoclonal antibodies prior to infection protects mice against lethal flavivirus challenge (42,65,66,81,88,126,150,156,158) and mice that lack B cells are vulnerable to WNV infection (29,42,62). Antibodies are speculated to protect against WNV infection by direct neutralization of receptor binding, through Fc-receptor–dependent viral clearance, by complement-mediated lysis of virus or infected cells, and by antibody-dependent cytotoxicity (ADCC). Most neutralizing antibodies recognize the structural E protein although some recognize prM (31,46,81,145,176). Several groups have generated non- neutralizing, yet protective mAbs against flavivirus NS1 (38,47,66,148,154,155,157,158), a protein that is absent from the virion. Because NS1 associates with the surface of infected cells and protective monoclonal antibodies against NS1 have strong complement fixing activity (59), complement-mediated cytolysis and ADCC have been proposed as mechanisms for protection (47,59,148,155). The importance of antibodies in the protection against WNV infection has been highlighted by recent studies in antibody-deficient mice (42). B cell–deficient mice that lack antibodies rapidly and uniformly developed encephalitis after infection with WNV; higher levels of infectious virus were detected both pe- 0 ripherally and in the CNS. Moreover, the 50% lethal dose (LD50) was markedly lower: 10 PFU for B cell- deficient mice compared to 107 PFU for wild-type mice. Antibody directly limited viremia and the dissemination of WNV early during the course of infection: B cell-deficient mice had a ,500-fold increase in serum viral load at day 4 after infection that led to a markedly increased viral burden in neurons in the CNS at day 6 and provoked a rapidly fatal encephalitis. Our most recent studies indicate that antibodies are necessary but not sufficient for eradication of infection (Engle and Diamond, unpublished data). Passive transfer of immune serum to C57BL/6 RAG1 mice that lack both B and T cells prior to infection with WNV protected mice against morbidity and mortality during the first 30 days; no viremia or viral burden was detected. However, as the titer of antibody declined, RAG1 mice developed CNS infection and succumbed to disease within 60 days of the initial virus challenge. Thus, immune antibodies control WNV dissemination, but by themselves, are unable to eliminate WNV persistence from an as yet unidentified tissue compart- ment. Specific antibodies against WNV were initially detected 4 days after infection in wild-type mice, the same time when high-grade viremia was first detected in B cell-deficient mice (42). An isotype-specific ELISA confirmed that these were exclusively IgM. Passive transfer of these low-titer IgM against WNV, derived from wild-type mice 4 days after infection, prolonged survival of B cell-deficient mice (42) and completely protected wild-type mice against WNV infection. Specific IgM may limit WNV dissemination by temporarily containing viremia and/or by triggering an adaptive IgG or T cell response that controls viral infection (138,139). Our most recent experiments with C57BL/6 mice that do not produce soluble IgM (sIgM 2/2) but have B cells that make IgG and display surface IgM (9,12,13) support this. Mice that lack soluble IgM uniformly succumb to infection (Sitati, Engle, and Diamond, unpublished data). Studies are currently underway to determine the precise mechanism by which a deficiency of soluble IgM translates into increased susceptibility—whether a lack of soluble IgM impairs the adaptive immune response against WNV or allows higher levels of WNV to enter the CNS at an earlier time after infection. Natural IgM antibodies against WNV may also have an important protective function against WNV. Nat- ural antibodies are constitutively secreted by CD51 B-1 cells without specific stimulation, have widely variable binding avidities (1023 to 10211 M, and represent an initial defense against pathogens (9,24,137). Nat- ural antibodies mediate direct neutralization of some bacteria and viruses in circulation (53,137), enhance phagocytosis of pathogens (134) and activate complement (9) to prime the immune response. Natural antibody-antigen complexes are efficiently filtered in the spleen and lymph nodes; this may diminish hematogenous spread and infection of critical end-organ targets such as the brain or spinal cord (139). Experiments, in which natural antibodies are transferred passively to mice that lack soluble IgM and/or IgG prior to WNV infection, will begin to address the significance of this group of antibodies in controlling the early phases of dissemination and triggering an early adaptive immune response. Complement and WNV. The complement cascade is an innate host defense system that participates in the control of viral infections by several mechanisms (139,177). (a) The C5-C9 membrane attack complex lyses enveloped viral particles and infected cells. (b) Pro-inflammatory peptides (C3a and C5a) are generated by complement activation; these peptides recruit and activate monocytes and granulocytes to the in-

263 DIAMOND ET AL. flammatory site. (c) The proteolytic fragments of C3 (C3b, C3bi, C3d, and C3dg) clear virus from circulation after opsonization through cells that express complement receptors. (d) By virtue of its ability to enhance viral antigen uptake (139), C3 facilitates antigen presentation by macrophages and dendritic cells and induces specific antibody production and T cell proliferation (33,92,138). Preliminary studies in our laboratory indicate that complement plays an essential role in limiting WNV infection; mice that are genetically deficient in C3 or C4 uniformly succumb to infection even at low viral doses (Mehlhop, Engle, and Dia- mond, unpublished data). Since both C3 and C4 cause a lethal phenotype, the classical and/or lectin mediated pathways of complement activation (which utilize a C4-C2 convertase to activate C3) appear important. Although the necessity of IgM for controlling infection suggests a role for the classical pathway of complement activation, studies with mice that lack C1q (32) or mannose-binding lectins (168) are planned to distinguish which pathways plays a dominant role in WNV infection. Additionally, studies with factor B 2/2 mice will directly address the function of the alternative pathway of complement activation in mediating protection against WNV infection. A deficiency of C3 or C4 could exacerbate WNV infection because of depressed C5-C9 lytic or C3 op- sonic activity that results in a failure to clear virus from circulation. Alternatively, C3 and C4 may play important roles in linking the innate and adaptive immune responses (8,23,139) against WNV (Fig. 3). C3 and

FIG. 3. Schematic model for the role of complement and IgM in priming antibody and T cell responses against WNV early in the course of infection. Virus is first found (stage A) in the regional lymph nodes after transport by migrating infected dendritic cells or after trafficking by low-titer natural IgM. In lymphoid tissue, (stage B) virus infects resident macrophages, dendritic cells or other hematopoietic cells directly or through an IgM and complement-dependent mechanism. Virus and viral antigen are shed from infected cells and bind to the B cell antibody receptor or are presented to CD41 T cells (stage C). B cell receptor cross-linking triggers specific IgM production, and signals from activated CD41 T cells induce isotype class-switching and IgG production. Activated CD41 T cells also provide stim- ulatory signals for activation of cytolytic CD81 T cells (stage D). The legend is shown in top right corner.

264 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

C4 are required for normal IgG production (33,49,138) and T cell priming (92) against influenza and herpes viruses; a deficiency in either C3 or C4 decreases opsonization and viral antigen presentation, leading to deficits in the adaptive B and T cell responses (139). Although the lytic and pro-inflammatory activity of complement may contribute to the defense against WNV, our preliminary data indicate that a deficiency in either C3 or C4 compromises the adaptive B cell immune response: mice that lack C3 or C4 have markedly depressed IgG titers against WNV (Mehlhop, Engle, and Diamond, unpublished data).

CELLULAR IMMUNE RESPONSE AGAINST WNV

T lymphocyte response. Cellular immunity is important for the eradication of flavivirus-infected cells including WNV. Antigen-restricted cytotoxic T lymphocytes (CTL) kill, proliferate, and release inflammatory cytokines after exposure to flavivirus-infected cells (43,82,96–98,100,101,112,131,167). While CTL responses are believed to be protective in vivo, their precise role in the recovery from infection by WNV and other encephalitic flaviviruses remains to be elucidated. Athymic nude mice that lack T cells have increased susceptibility to infection with Japanese encephalitis virus (102), and adoptive transfer of virus-specific CTL protected mice against lethal challenge with Japanese encephalitis virus (131). Moreover, mice that lack CD81 T cells have increased mortality after WNV infection (Shrestha and Diamond, unpublished data) and animals that are treated with drugs that impair T cell function and subsequently infected with WNV, uniformly develop encephalitis (20,30,132,133). Our most recent experiments suggest that CD81 T cells function to clear WNV from infected neurons. CD8-deficient mice that survive initial infection demonstrate WNV persistence; infectious virus was obtained from CNS tissues 1 to 2 months after initial infection of CD8-deficient but not wild-type mice (Shrestha and Diamond, unpublished data). Interestingly, increased mortality and CNS viral load was also observed in C57BL/6 mice that lack either IFN-g or perforin granules (Shrestha, Engle, and Diamond, unpublished data). Although additional studies are required, CD81 T cells may use distinct effector mechanisms to selectively target WNV-infected neurons in the CNS. Natural killer cells. Because of their capacity to directly kill virally infected cells and to produce inflammatory cytokines that limit infection, NK cells may be an important component of the initial defense against WNV (Table 1), NK cells lyse infected cells by releasing cytotoxic granules that contain perforin and granzymes, or by binding to apoptosis-inducing receptors on target cells (140). NK cell activation is finely regulated through a balance of activating receptors (Ly49D, Ly49H, and NKG2D) and inhibitory receptors (killer-cell immunoglobulin-like receptors, immunoglobulin-like inhibitory receptors, and CD94- NKG2A) (161). To control the consequences of unregulated activation of NK cells, the inhibitory receptors are expressed constitutively; these bind to host MHC class I molecules on opposing cells and transmit inhibitory signals through intracellular tyrosine-based inhibitory motifs in their cytoplasmic domains. A decrease in expression of class I MHC molecules on a cell may prompt NK cell activation by attenuating the inhibitory signals. Thus, NK cell target recognition occurs after ligation of activating receptors and repres- sion of inhibitory receptors on the cell surface. Surprisingly few experiments have been published that describe the antiviral activity of NK cells against flaviviruses. NK cells lyse dengue virus–infected target cells by both natural killing and antibody-dependent cell-mediated cytotoxicity (99). Infection of mice with Langat, West Nile and tick-borne encephalitis viruses transiently activated and then suppressed NK cell activity (175). One explanation is that, in vivo, flaviviruses, including WNV, have evolved a mechanism to evade NK cell responses. Although some DNA viruses blunt NK activity by expressing MHC class I homologues (140,172) WNV may attenuate NK cell cytotoxicity by increasing surface expression of class I MHC molecules (89,90,113,114). Expression of class I MHC molecules in WNV-infected cells is stimulated by enhancing the transport activity of TAP (115,127,130) and by NF-kB dependent transcriptional activation of MHC class I genes (83). Thus, WNV may overcome susceptibility to NK cell–mediated lysis, even at the expense of increased class I MHC expression and later recognition by an adaptive CTL response. Consistent with this, splenocytes from WNV- immunized mice had poor NK cell lytic activity (127) and mice that were genetically deficient in NK cells demonstrated no increased morbidity or mortality compared to wild type controls (Engle, Yokoyama, and Diamond, unpublished data).

265 DIAMOND ET AL.

TABLE 1. INNATE IMMUNITY TO WEST NILE VIRUS Effector molecule or cell Possible antiviral mechanism References

Interferon-a PKR/RNAse L independent (?) (39,41) Interferon-b PKR/RNAse L independent (?) (39,41) Interferon-g Nitric oxide (?) (110,116,160) L1 isoform of 2959 oAS RNAse L (?) (125,144) C3 Inflammation, opsonization Unpublished C5-C9 Inflammation, lysis ? Natural IgM Neutralization, opsonization Unpublished Induced IgM Neutralization, opsonization (42) Natural killer cells (?) Apoptosis ? Macrophages Opsonization (10)

Effector molecules and cells that likely contribute to the innate defense against WNV. IFN (a, b, and g) appear to inhibit infection through an antiviral pathway that is at least partially independent of PKR and RNAse L. C3 activation may trigger a pro-inflammatory defense via C3a or C5a or induce lysis directly by the C5-C9 membrane attack complex. Natural and induced IgM may neutralize WNV directly or facilitate opsonization and clearance through complement or FC a/m receptors on the surface of B cells, dendritic cells, and macrophages. “Unpublished” indicates our unpublished findings.

Macrophages. The role of macrophages in flavivirus infection remains controversial. Because they express high levels of Fc-g and complement receptors and facilitate antibody-dependent enhancement of infection (ADE) in cell culture, it has been speculated that they contribute to pathogenesis of secondary flavivirus infection (63). Alternatively, enhanced flavivirus uptake by macrophages in vivo could be protective as it clears virus from circulation, stimulates cytokine production, and facilitates increased antigen presentation to B and T cells in secondary lymphoid organs (120). The majority of the data support a protective role of macrophages in infection by encephalitic flavivirus, including WNV. Depletion of macrophages in vivo caused increased lethality after infection with WNV (10) and suppression of macrophage phagocytic activity in mice resulted in increased mortality after infection with tick-borne encephalitis virus (84). Some of the protection provided by macrophages may be mediated by nitric oxide although the experimental data is conflicting. Pretreatment of macrophages with agents that induce nitric oxide (NO) synthesis inhibited Japanese encephalitis virus infection (110) and treatment of mice with a NO synthetase inhibitor increased mortality after infection with Japanese encephalitis virus (110). However, only a marginal increase in mortality was observed in iNOS 2/2 mice that were infected with Murray Valley encephalitis virus (116). Oth- ers argue that that the inflammatory actions of NO may contribute to flavivirus pathogenesis; in vivo ad- minstration of a NO competitive inhibitor improved survival in mice infected with tick-borne encephalitis virus (93,94). Clearly, additional studies will be necessary to resolve the physiologic role of NO and macrophages in WNV infection. Dendritic cells. Specialized subsets of dendritic cells (DC) may control WNV infection and prime the adaptive immune response during the early phases of infection (104). In blood, at least two types of DC exist (136): CD11c1 DC’s are precursors of the Langerhans cells that reside in the skin and initially become infected by WNV after mosquito or subcutaneous inoculation. CD11c2 DC’s are precursors of the interferon-producing cells (IPC) that produce enormous quantities (up to one thousand times that typically secreted by virally infected cells [50,51]) of type I IFN in response to viral infection (6). In secondary lymphoid organs, interferon-producing DC’s may trigger both the innate and adaptive immune responses simultaneously by producing of various cytokines, activating NK cells, and by indirectly regulating T cell functions via IFN-mediated effects on other (CD11b1 or CD81) DC subsets (34,78,79,95). Studies with dengue virus (122,169,182) demonstrate that DC subsets have distinct susceptibility to infection; immature but not mature DC are permissive for viral infection. Similarly, Langerhans cells migrate to local draining lymph nodes after infection with WNV and trigger an influx of circulating leukocytes (75). Although more study is required, Langerhans DC’s may traffic WNV to interferon-producing DC’s in the lymph node, re-

266 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION sulting in a burst of interferon antiviral activity and a triggering of the adaptive immune response, mechanisms that rapidly limit the spread of viral infection.

VACCINE DEVELOPMENT AGAINST WNV

Since the WNV outbreak in North America began, significant progress has been made toward the development of an effective WNV vaccine. Attenuated and heat-killed vaccines already have been utilized to immunize exotic birds and horses with varying degrees of efficacy (37,119). Based on the mechanisms of protective immunity against WNV in animals, an effective vaccine should induce potent and durable humoral and cellular immune responses against WNV. At present, several strategies are being used to develop vaccine candidates for pre-clinical and clinical assessment. (1) Inactivated vaccines. Formalin-inactivated whole virus vaccines have been developed successfully against other flaviviruses including Japanese and tick-borne encephalitis viruses (128). Administration of one or two doses of formalin-treated WNV vaccine to geese resulted in 42% and 89% protection, respectively, at 3 weeks post-immunization (119). Similarly, at 2 weeks post-immunization with two doses of killed WNV, hamsters were completely protected from lethal WNV challenge (170). Although killed WNV vaccines could be used to vaccinate the immunocompromised, their utility may be limited. Administration of multiple vaccine doses appears to be required to elicit a protective immune response. Furthermore, it is unclear how durable the immunity will be as relatively low levels of neutralizing and complement-fixing antibodies have been detected one month after initial immunization (170). Finally, immune enhancement of heterologous flavivirus infection has been observed when animals are vaccinated with a killed or inactivated virus preparation (17,117,129). Thus, administration of a killed vaccine against WNV that generates a humoral response of low magnitude or poor quality theoretically could worsen subsequent infections with heterologous flaviviruses. (2) Subunit vaccines. Repeated immunization of C3H/HeN mice with purified, recombinant WNV E protein resulted in the development of high-titer (.1/1280) neutralizing anti-WNV antibodies. Immunized mice were completely protected against challenge with a lethal yet low (101 PFU) dose of WNV but did not survive a challenge with a high (106) inoculum of WNV (178). Although a reasonable humoral response was generated after multiple doses, the utility of these vaccines may be limited by the lack of stimulation of strong cellular immune responses against WNV. (3) Cross-reactive vaccines. The use of already existing and approved vaccines that target closely related flavivirus (e.g., Japanese encephalitis or yellow fever virus) has been proposed as a means of rapidly generating immunity against WNV. Epidemiologic and experimental evidence indicates that immunity against Japanese encephalitis and dengue virus infections provides at least partial protection against WNV (60,70,128,146,147,181). Immunization of hamsters with the live, attenuated Japanese encephalitis vaccine (SA14-2-8) or yellow fever vaccine (17D) strains reduced the severity of WNV infection (171). When hamsters were challenged with WNV 30 days after immunization, viremia and mortality rates were markedly lowered: 100% and 70% protection against mortality was generated by Japanese encephalitis and yellow fever virus vaccine strains, respectively. However, the cross-protective response in humans may be attenuated, especially when killed vaccines are used. Immunization of human subjects with the formalin-inactivated Japanese encephalitis virus vaccine or with experimental live-attenuated dengue virus vaccines failed to generate protective neutralizing antibody titers against WNV (80). Finally, the unresolved issue of immune enhancement makes the use of heterologous flavivirus vaccines to protect against WNV in humans less promising; a substantial amount of experimental evidence in animals will be required before large-scale clinical trials can be conducted. (4) DNA vaccines. Recent reports have demonstrated that immunization with a single dose of plasmid DNA that encoded the membrane (prM) and envelope (E) (37) or capsid (C) proteins (185) of WNV is protective: challenge of vaccinated horses and outbred mice with a lethal dose of WNV completely prevented viremia and mortality (37). Co-expression of prM and E in vivo, which generates immunogenic subviral particles (26–28,31,69,91), stimulates vigorous humoral and cellular immune responses against WNV, and expression of C elicits a potent antigen specific TH1 and CTL anti-WNV response.

267 DIAMOND ET AL.

(5) Live-attenuated strain vaccines. Live-attenuated viral vaccines replicate and elicit both humoral and cellular immune responses in a manner akin to infection with the natural pathogen (128). Two types of live- attenuated WNV strains have been developed as candidate vaccines, one derived by serial passage and another by chimerization. WNV-25 strain was passaged serially in cultured mosquito cells until mutations accumulated that attenuated neuroinvasiveness (10,62,118). Immunization with the WNV-25 vaccine strain protected geese against lethal challenge with a virulent WNV isolate (118). A chimeric WNV-YF viral vaccine strain has been genetically engineered by cloning: the prM and E structural genes of WNV were inserted into the infectious clone backbone of the 17D vaccine strain of yellow fever virus (4,128). Immu- nization with a single dose of chimeric WNV-YF virus resulted in production of neutralizing and complement-fixing antibodies and complete protection of hamsters from a lethal challenge of virulent WNV (170). The elderly and immunocompromised are targets for severe WNV infection and thus, may derive the greatest benefit from vaccination. However, there is a legitimate concern as to the use of live WNV strains in these at risk populations. For example, vaccination of the elderly with the “safe”17D yellow fever vaccine has caused invasive and lethal disease (123,124). To minimize this, additional attenuating mutations were engineered into the chimeric WNV-YF virus so that its neuroinvasive and neurovirulent potential were severely hampered (4). The B cell–deficient mice that were inoculated with 106 PFU of chimeric, attenuated WNV-YF virus did not become ill even though siblings inoculated with 101 PFU of wild-type virus uniformly succumbed to infection (Engle, Arroyo, Monath, and Diamond, unpublished data).

IMMUNOTHERAPY AGAINST WNV

At present, treatment for all flavivirus infections, including WNV, is supportive. Although a few agents have been proposed to have antiviral activity against WNV (3,77), none have demonstrated efficacy in vivo. Animal model studies have provided important clues as to the possible therapeutic use of antibodies against flavivirus infection: (a) protection against Saint Louis encephalitis and yellow fever virus infections with antibodies in vivo did not necessarily correlate with neutralizing activity in vitro (14,149); (b) the ability to eradicate flavivirus infection in mice depended on the dosage and time of administration of antibody (88,150); and (c) transfer of antibodies that neutralized one flavivirus did not provide durable cross-protection against other flaviviruses (17,150). Although antibody has been utilized for prophylaxis and therapy against several viral infections (153,187), there are several theoretical concerns that passive administration of anti-flavivirus antibodies could exacerbate infection in vivo. Because sub-neutralizing concentrations of antibody enhance flavivirus replication in myeloid cells in vitro (21,22,54,56,63,64,141–143), low-titer immune antibody preparations could increase viral replication and adversely affect survival. Nonetheless, despite its extensive characterization in vitro, the significance of ADE in vivo after passive administration of immune antibodies with WNV or other flaviviruses remains uncertain. Apart from or perhaps related to ADE, an “early-death” phenomenon (129) has been reported that could also limit the utility of antibody therapy against WNV. According to this model, animals that have pre-existing humoral immunity but do not respond well to viral challenge may succumb to infection more rapidly than animals without pre-existing immunity. Although this phenomenon has been described after passive acquisition of antibodies against yellow fever and Langat encephalitis viruses (7,57,58,179), it was not observed after transfer of antibodies against Japanese encephalitis virus (88). Recent animal studies suggest that passive administration of anti-WNV antibodies is both protective and therapeutic and does not cause adverse effects related to immune enhancement. Passive administration of immune serum prior to WNV infection protected wild-type, B cell–deficient (mMT), and T and B cell–deficient (RAG1) mice from infection (42), and no increased mortality was observed even when sub-neutralizing concentrations of antibodies were used. Similarly, passive administration of immune serum (170) or antiserum that recognized WNV E protein (178) protected hamsters and mice against lethal WNV infection. More recently, in therapeutic trials, we have demonstrated that immune human g-globulin partially protected mice against WNV-induced mortality (Engle and Diamond, unpublished data). Therapeutic intervention even five days after infection reduced mortality; this time point is significant because virologic data indicate that between days 4 and 5, WNV had spread to the brain and spinal cord. Thus, passive trans-

268 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION fer of immune antibody improved clinical outcome even after WNV had disseminated into the CNS. These results are consistent with earlier studies with the Sindbis alphavirus (61,163) and suggest that clinical studies with monoclonal and polyclonal antibodies may be warranted. Immunoprophylaxis and immunotherapy with neutralizing anti-WNV antibodies may be a possible intervention in the elderly and immunocompromised that respond poorly to immunization with live, attenuated WNV strains.

CONCLUSIONS

Experiments in animal models suggests that several different arms of the immune system coordinate the protective immune response against WNV; disruption of any of these can result in disseminated infection with increased mortality. It appears that IFN, complement, IgM, IgG, and CD81 T cells all have essential functions in limiting WNV infection. IFN has antiviral and immunomodulatory functions, complement may act to neutralize virus directly and to promote antigen presentation and immune system priming after opsonization, antibodies prevent virus attachment and facilitate clearance from circulation, and CD81 CTL specifically eliminate virally infected cells, especially in the CNS. Nonetheless, these inflammatory medi- ators and effector cells are just the beginning of a complicated immune response that results in control and eradication of WNV infection. Based on preliminary data from other viral systems, other aspects of the innate and adaptive immune response including toll-like receptors, CD41 T cells, DCs, and chemokines likely contribute to the prevention of disseminated infection and disease. The development of effective vaccines and therapeutics requires intensive study of both WNV pathogenesis and the mechanisms by which the immune response limits disease. As WNV likely has evolved specific evasive mechanisms to contend with the inhibitory responses of the immune responses (40), a more complete understanding of viral tropism, the pathogenesis of injury in the CNS, viral evasive strategies, and the development of short- and long-term immunity against WNV are needed. Experimentation in animal models of WNV infection should continue to provide insight into these mechanisms and explain the epidemiology of WNV-induced disease in humans. Studies that clarify the link between the innate and adaptive immune responses against WNV may suggest stages for intervention so that CNS dissemination, and the resultant pathology, can be limited.

ACKNOWLEDGMENTS

I am grateful to Sujan Shresta and Eva Harris for critical reading of the manuscript. This work was supported by grants from the Ellison Foundation for Global Infectious Diseases and the Center for Disease Control and Prevention (U50/CCU720545-03).

REFERENCES

1. Centers for Disease Control and Prevention. 2003. Provisional surveillance summary of the West Nile virus epidemic—United States, January–November 2002. JAMA 289:293–294. 2. Albrecht, P. 1968. Pathogenesis of neurotropic arbovirus infections. Curr. Top. Microbiol. Immunol. 43:44–91. 3. Anderson, J.F., and J.J. Rahal. 2002. Efficacy of interferon alpha-2b and ribavirin against West Nile virus in vitro. Emerg. Infect. Dis. 8:107–108. 4. Arroyo, J., C.A. Miller, J. Catalan, et al. 2001. Yellow fever vector live-virus vaccines: West Nile virus vaccine development. Trends Mol. Med. 7:350–354. 5. Asnis, D.S., R. Conetta, A.A. Teixeira, et al. 2000. The West Nile Virus outbreak of 1999 in New York: the Flush- ing Hospital experience. Clin. Infect. Dis. 30:413–418. 6. Barchet, W., M. Cella, B. Odermatt, et al. 2002. Virus-induced interferon alpha production by a dendritic cell subset in the absence of feedback signaling in vivo. J. Exp. Med. 195:507–516.

269 DIAMOND ET AL.

7. Barrett, A.D., and E.A. Gould. 1986. Antibody-mediated early death in vivo after infection with yellow fever virus. J. Gen. Virol. 67:2539–2542. 8. Barrington, R., M. Zhang, M. Fischer, et al. 2001. The role of complement in inflammation and adaptive immunity. Immunol. Rev. 180:5–15. 9. Baumgarth, N., O.C. Herman, G.C. Jager, et al. 2000. B-1 and B-2 cell–derived immunoglobulin M antibodies are nonredundant components of the protective response to influenza virus infection. J. Exp. Med. 192:271–280. 10. Ben-Nathan, D., I. Huitinga, S. Lustig, et al. 1996. West Nile virus neuroinvasion and encephalitis induced by macrophage depletion in mice. Arch. Virol. 141:459–469. 11. Boehm, U., T. Klamp, M. Groot, et al. 1997. Cellular responses to interferon-gamma. Annu. Rev. Immunol. 15:749–795. 12. Boes, M., C. Esau, M.B. Fischer, et al. 1998. Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM. J. Immunol. 160:4776–4787. 13. Boes, M., A.P. Prodeus, T. Schmidt, et al. 1998. A critical role of natural immunoglobulin M in immediate defense against systemic bacterial infection. J. Exp. Med. 188:2381–2386. 14. Brandriss, M.W., J.J. Schlesinger, E.E. Walsh, et al. 1986. Lethal 17D yellow fever encephalitis in mice. I. Pas- sive protection by monoclonal antibodies to the envelope proteins of 17D yellow fever and dengue 2 viruses. J. Gen. Virol. 67:229–234. 15. Brinton, M.A., H. Arnheiter, and O. Haller. 1982. Interferon independence of genetically controlled resistance to flaviviruses. Infect. Immun. 36:284–288. 16. Brooks, T.J., and R.J. Phillpotts. 1999. Interferon-alpha protects mice against lethal infection with St. Louis encephalitis virus delivered by the aerosol and subcutaneous routes. Antiviral Res. 41:57–64. 17. Broom, A.K., M.J. Wallace, J.S. Mackenzie, et al. 2000. Immunisation with gamma globulin to Murray Valley encephalitis virus and with an inactivated Japanese encephalitis virus vaccine as prophylaxis against Australian encephalitis: evaluation in a mouse model. J. Med. Virol. 61:259–265. 18. Burke, D.S., and T.P. Monath. 2001. Flaviviruses, pp. 1043–1125. In: D.M. Knipe and P.M. Howley (eds.), Fields Virology, Vol. 1, 4th ed. Lippincott, Williams & Wilkins, Philadelphia.

19. Byrne, S.N., G.M. Halliday, L.J. Johnston, et al. 2001. Interleukin-1beta but not tumor necrosis factor is involved in West Nile virus–induced Langerhans cell migration from the skin in C57BL/6 mice. J. Invest. Dermatol. 117:702–709. 20. Camenga, D. L., N. Nathanson, and G. A. Cole. 1974. Cyclophosphamide-potentiated West Nile viral encephalitis: relative influence of cellular and humoral factors. J. Infect. Dis. 130:634–641. 21. Cardosa, M.J., S. Gordon, S. Hirsch, et al. 1986. Interaction of West Nile virus with primary murine macrophages: role of cell activation and receptors for antibody and complement. J. Virol. 57:952–959. 22. Cardosa, M.J., J.S. Porterfield, and S. Gordon. 1983. Complement receptor mediates enhanced flavivirus replication in macrophages. J. Exp. Med. 158:258–263. 23. Carroll, M.C. 1998. The role of complement and complement receptors in induction and regulation of immunity. Annu Rev. Immunol. 16:545–568. 24. Casali, P., and A.L. Notkins. 1989. CD51 B lymphocytes, polyreactive antibodies and the human B-cell reper- toire. Immunol. Today 10:364–368. 25. Chambers, T.J., C.S. Hahn, R. Galler, et al. 1990. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 44:649–688. 26. Chang, G.J., B.S. Davis, A.R. Hunt, et al. Holmes, and G. Kuno. 2001. Flavivirus DNA vaccines: current status and potential. Ann. NY Acad. Sci. 951:272–285. 27. Chang, G.J., A.R. Hunt, and B. Davis. 2000. A single intramuscular injection of recombinant plasmid DNA induces protective immunity and prevents Japanese encephalitis in mice. J. Virol. 74:4244–4252. 28. Chang, G.J., A.R. Hunt, D.A. Holmes, et al. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306:170–180.

270 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

29. Charlier, N., P. Leyssen, J. Paeshuyse, et al. 2002. Infection of SCID mice with Montana myotis leukoencephalitis virus as a model for flavivirus encephalitis. J. Gen. Virol. 83:1887–1896. 30. Cole, G.A., and N. Nathanson. 1968. Potentiation of experimental arbovirus encephalitis by immunosuppressive doses of cyclophosphamide. Nature 220:399–401. 31. Colombage, G., R. Hall, M. Pavy, et al. 1998. DNA-based and alphavirus-vectored immunisation with prM and E proteins elicits long-lived and protective immunity against the flavivirus, Murray Valley encephalitis virus. Vi- rology 250:151–163. 32. Cutler, A.J., M. Botto, D. van Essen, et al. 1998. T cell–dependent immune response in C1q-deficient mice: defective interferon gamma production by antigen-specific T cells. J. Exp. Med. 187:1789–1797. 33. Da Costa, X.J., M.A. Brockman, E. Alicot, et al. 1999. Humoral response to herpes simplex virus is complement- dependent. Proc. Natl. Acad. Sci. USA 96:12708–12712. 34. Dalod, M., T. Hamilton, R. Salomon, et al. 2003. Dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon a/b. J. Exp. Med. 197:885–898. 35. Darnell, M.B., and H. Koprowski. 1974. Genetically determined resistance to infection with group B arboviruses. II. Increased production of interfering particles in cell cultures from resistant mice. J. Infect. Dis. 129:248–256. 36. Darnell, M.B., H. Koprowski, and K. Lagerspetz. 1974. Genetically determined resistance to infection with group B arboviruses. I. Distribution of the resistance gene among various mouse populations and characteristics of gene expression in vivo. J. Infect. Dis. 129:240–247. 37. Davis, B.S., G.J. Chang, B. Cropp, et al. 2001. West Nile virus recombinant DNA vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays. J. Virol. 75:4040–4047. 38. Despres, P., J. Dietrich, M. Girard, et al.1991. Recombinant baculoviruses expressing yellow fever virus E and NS1 proteins elicit protective immunity in mice. J. Gen. Virol. 72:2811–2816. 39. Diamond, M., T. Roberts, D. Edgil, et al. 2000. Modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons. J. Virol. 74:4957–4966. 40. Diamond, M.S. 2003. Evasion of innate and adaptive immunity by flaviviruses. Immunol. Cell Biol. 81:196–206. 41. Diamond, M.S., and E. Harris. 2001. Interferon inhibits dengue virus infection by preventing translation of viral RNA through a PKR-independent mechanism. Virology 289:297–311. 42. Diamond, M. S., B. Shrestha, A. Marri, et al. 2003. B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J. Virol. 77:2578–2586. 43. Douglas, M.W., A.M. Kesson, and N.J. King. 1994. CTL recognition of West Nile virus–infected fibroblasts is cell cycle dependent and is associated with virus-induced increases in class I MHC antigen expression. Immunology 82:561–570. 44. Eldadah, A.H., and N. Nathanson. 1967. Pathogenesis of West Nile Virus encepahlitis in mice and rats. II. Virus multiplication, evolution of immunofluorescence, and development of histological lesions in the brain. Am. J. Epi- demiol. 86:776–790. 45. Eldadah, A.H., N. Nathanson, and R. Sarsitis. 1967. Pathogenesis of West Nile Virus encephalitis in mice and rats. 1. Influence of age and species on mortality and infection. Am. J. Epidemiol. 86:765–775. 46. Falconar, A.K. 1999. Identification of an epitope on the dengue virus membrane (M) protein defined by cross- protective monoclonal antibodies: design of an improved epitope sequence based on common determinants present in both envelope (E and M) proteins. Arch. Virol. 144:2313–2330. 47. Falgout, B., M. Bray, J.J. Schlesinger, et al. 1990. Immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein NS1 protects against lethal dengue virus encephalitis. J. Vi- rol. 64:4356–4363. 48. Falgout, B., and L. Markoff. 1995. Evidence that flavivirus NS1–NS2A cleavage is mediated by a membrane- bound host protease in the endoplasmic reticulum. J. Virol. 69:7232–7243. 49. Fischer, M.B., M. Ma, S. Goerg, et al. 1996. Regulation of the B cell response to T-dependent antigens by classical pathway complement. J. Immunol. 157:549–556.

271 DIAMOND ET AL.

50. Fitzgerald-Bocarsly, P. 1993. Human natural interferon-alpha producing cells. Pharmacol. Ther. 60:39–62. 51. Fitzgerald-Bocarsly, P. 2002. Natural interferon-alpha producing cells: the plasmacytoid dendritic cells. Biotech- niques Suppl:16–20,22,24–29. 52. Ghosh, A., S.N. Sarkar, T.M. Rowe, et al. 2001. A specific isozyme of 29-59 oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family. J. Biol. Chem. 276:25447–25455. 53. Gobet, R., A. Cerny, E. Ruedi, et al. 1988. The role of antibodies in natural and acquired resistance of mice to vesicular stomatitis virus. Exp. Cell. Biol. 56:175–180. 54. Gollins, S., and J. Porterfield. 1984. Flavivirus infection enhancement in macrophages: radioactive and biological studies on the effect of antibody and viral fate. J. Gen. Virol. 65:1261–1272. 55. Gollins, S., and J. Porterfield. 1986. The uncoating and infectivity of the flavivirus West Nile on interaction with cells: effects of pH and ammonium chloride. J. Gen. Virol. 67:1941–1950. 56. Gollins, S.W., and J.S. Porterfield. 1985. Flavivirus infection enhancement in macrophages: an electron microscopic study of viral entry. J. Gen. Virol. 66:1969–1982. 57. Gould, E.A., P.A. Cane, and M. Doenhoff. 1987. Immune enhancement of yellow fever virus neurovirulence for mice: studies of mechanisms involved. J. Gen. Virol. 68:3105–3112. 58. Gould, E.A., and A. Buckley. 1989. Antibody-dependent enhancement of yellow fever and Japanese encephalitis virus neurovirulence. J. Gen. Virol. 70:1605–1608. 59. Gould, E.A., A. Buckley, A.D. Barrett, et al. 1986. Neutralizing (54K) and non-neutralizing (54 K and 48 K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice. J. Gen. Virol. 67:591–595. 60. Goverdhan, M.K., A.B. Kulkarni, A.K. Gupta, et al. 1992. Two-way cross-protection between West Nile and Japanese encephalitis viruses in bonnet macaques. Acta Virol. 36:277–283. 61. Griffin, D., B. Levine, W. Tyor, et al. 1997. The role of antibody in recovery from alphavirus encephalitis. Im- munol. Rev. 159:155–161. 62. Halevy, M., Y. Akov, D. Ben-Nathan, et al. 1994. Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice. Arch. Virol. 137:355–370. 63. Halstead, S.B. 1989. Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade. Rev. Infect. Dis. 11(Suppl 4):S830–S839. 64. Halstead, S.B., J.S. Porterfield, and E.J. O’Rourke. 1980. Enhancement of dengue virus infection in monocytes by flavivirus antisera. Am. J. Trop. Med. Hyg. 29:638–642. 65. Heinz, F.X., R. Berger, W. Tuma, et al. 1983. A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies. Virology 126:525–537. 66. Henchal, E.A., L.S. Henchal, and J.J. Schlesinger. 1988. Synergistic interactions of anti-NS1 monoclonal antibodies protect passively immunized mice from lethal challenge with dengue 2 virus. J. Gen. Virol. 69:2101–217. 67. Ho, L.J., J.J. Wang, M.F. Shaio, et al. 2001. Infection of human dendritic cells by dengue virus causes cell maturation and cytokine production. J. Immunol. 166:1499–1506. 68. Hubalek, Z., and J. Halouzka. 1999. West Nile fever—a reemerging mosquito-borne viral disease in Europe. Emerg. Inf. Dis. 5:643–650. 69. Hunt, A.R., C.B. Cropp, and G.J. Chang. 2001. A recombinant particulate antigen of Japanese encephalitis virus produced in stably transformed cells is an effective noninfectious antigen and subunit immunogen. J. Virol. Meth- ods 97:133–149. 70. Ilkal, M.A., Y. Prasanna, P.G. Jacob, et al. 1994. Experimental studies on the susceptibility of domestic pigs to West Nile virus followed by Japanese encephalitis virus infection and vice versa. Acta Virol. 38:157–161. 71. Johnson, A.J., and J.T. Roehrig. 1999. New mouse model for dengue virus vaccine testing. J. Virol. 73:783–786. 72. Johnson, R.T. 1982. Viral Infections of the Nervous System. Raven Press, New York.

272 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

73. Johnson, R.T., and C.A. Mims. 1968. Pathogenesis of viral infections of the nervous system. N. Engl. J. Med. 278:23–30. 74. Johnson, R.T., and G.A. Mims. 1968. Pathogenesis for viral infections of the nervous system. N. Engl. J. Med. 278:84–92. 75. Johnston, L.J., G.M. Halliday, and N.J. King. 2000. Langerhans cells migrate to local lymph nodes following cutaneous infection with an arbovirus. J. Invest. Dermatol. 114:560–568. 76. Johnston, L.J., G.M. Halliday, and N.J. King. 1996. Phenotypic changes in Langerhans’ cells after infection with arboviruses: a role in the immune response to epidermally acquired viral infection? J. Virol. 70:4761–4766. 77. Jordan, I., T. Briese, N. Fischer, et al. 2000. Ribavirin inhibits west nile virus replication and cytopathic effect in neural cells. J. Infect. Dis. 182:1214–1217. 78. Kadowaki, N., S. Antonenko, J.Y. Lau, et al. 2000. Natural interferon alpha/beta-producing cells link innate and adaptive immunity. J. Exp. Med. 192:219–226. 79. Kadowaki, N., and Y.J. Liu. 2002. Natural type I interferon-producing cells as a link between innate and adaptive immunity. Hum. Immunol. 63:1126–1132. 80. Kanesa-Thasan, N., J.R. Putnak, J.A. Mangiafico, et al. 2002. Short report: absence of protective neutralizng antibodies to West Nile virus in subjects following vaccination with Japanese encephalitis or dengue vaccines. Am. J. Trop. Med. Hyg. 66:115–116. 81. Kaufman, B.M., P.L. Summers, D.R. Dubois, et al. 1989. Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection. Am. J. Trop. Med. Hyg. 41:576–580. 82. Kesson, A.M., R.V. Blanden, and A. Mullbacher. 1987. The primary in vivo murine cytotoxic T cell response to the flavivirus, West Nile. J. Gen. Virol. 68:2001–2006. 83. Kesson, A.M., and N.J. King. 2001. Transcriptional regulation of major histocompatibility complex class I by flavivirus West Nile is dependent on NF-kappaB activation. J. Infect. Dis. 184:947–954. 84. Khozinsky, V.V., B.F. Semenov, M. Gresikova, et al. 1985. Role of macrophages in the pathogenesis of experimental tick-borne encephalitis in mice. Acta Virol. 29:194–202. 85. Khromykh, A.A., P.L. Sedlak, and E.G. Westaway. 2000. cis- and trans-acting elements in flavivirus RNA replication. J. Virol. 74:3253–3263. 86. Khromykh, A.A., A.N. Varnavski, P.L. Sedlak, et al. 2001. Coupling between replication and packaging of flavivirus RNA: evidence derived from the use of DNA-based full-length cDNA clones of Kunjin virus. J. Virol. 75:4633–4640. 87. Kimura, T., S.W. Gollins, and J.S. Porterfield. 1986. The effect of pH on the early interaction of West Nile virus with P388D1 cells. J. Gen. Virol. 67:2423–2433. 88. Kimura-Kuroda, J., and K. Yasui. 1988. Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. J. Immunol. 141:3606–3610. 89. King, N.J., and A.M. Kesson. 1988. Interferon-independent increases in class I major histocompatibility complex antigen expression follow flavivirus infection. J. Gen. Virol. 69:2535–2543. 90. King, N.J., L.E. Maxwell, and A.M. Kesson. 1989. Induction of class I major histocompatibility complex antigen expression by West Nile virus on gamma interferon–refractory early murine trophoblast cells. Proc. Natl. Acad. Sci. USA 86:911–915. 91. Konishi, E., M. Yamaoka, W. Khin Sane, et al. 1999. The anamnestic neutralizing antibody response is critical for protection of mice from challenge following vaccination with a plasmid encoding the Japanese encephalitis virus premembrane and envelope genes. J. Virol. 73:5527–5534. 92. Kopf, M., B. Abel, A. Gallimore, et al. 2002. Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection. Nat. Med. 8:373–378. 93. Kreil, T.R., and M.M. Eibl. 1996. Nitric oxide and viral infection: NO antiviral activity against a flavivirus in vitro, and evidence for contribution to pathogenesis in experimental infection in vivo. Virology 219:304–306.

273 DIAMOND ET AL.

94. Kreil, T.R., and M.M. Eibl. 1995. Viral infection of macrophages profoundly alters requirements for induction of nitric oxide synthesis. Virology 212:174–178. 95. Krug, A., R. Veeraswamy, A. Pekosz, et al. 2003. Interferon-producing cells fail to induce proliferation of naive T cells but can promote expansion and T helper 1 differentiation of antigen-experienced unpolarized T cells. J. Exp. Med. 197:899–906. 96. Kulkarni, A.B., A. Mullbacher, and R.V. Blanden. 1991. In vitro T-cell proliferative response to the flavivirus, West Nile. Viral Immunol. 4:73–82. 97. Kurane, I., M.A. Brinton, A.L. Samson, et al. 1991. Dengue virus–specific, human CD41 CD82 cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones. J. Virol. 65:1823–1828. 98. Kurane, I., and F.A. Ennis. 1994. Cytotoxic T lymphocytes in dengue virus infection. Curr. Top. Microbiol. Im- munol. 189:93–108. 99. Kurane, I., D. Hebblewaite, W.E. Brandt, et al. 1984. Lysis of dengue virus–infected cells by natural cell–mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. J. Virol. 52:223–230. 100. Kurane, I., B.L. Innis, C.H. Hoke, Jr., et al. 1995. T cell activation in vivo by dengue virus infection. J. Clin. Lab. Immunol. 46:35–40. 101. Kurane, I., B.L. Innis, A. Nisalak, et al. 1989. Human T cell responses to dengue virus antigens. Proliferative responses and interferon gamma production. J. Clin. Invest. 83:506–513. 102. Lad, V.J., A.K. Gupta, M.K. Goverdhan, et al. 1993. Susceptibility of BL6 nude (congenitally athymic) mice to Japanese encephalitis virus by the peripheral route. Acta Virol. 37:232–240. 103. Lanciotti, R.S., J.T. Roehrig, V. Deubel, et al. 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States. Science 286:2333–2337. 104. Le Bon, A., and D.F. Tough. 2002. Links between innate and adaptive immunity via type I interferon. Curr. Opin. Immunol. 14:432–436. 105. Leis, A.A., J. Fratkin, D.S. Stokic, et al. 2003. West Nile poliomyelitis. Lancet Infect. Dis. 3:9–10. 106. Leis, A.A., D.S. Stokic, J.L. Polk, et al. 2002. A poliomyelitis-like syndrome from West Nile virus infection. N. Engl. J. Med. 347:1279–1280. 107. Leyssen, P., C. Drosten, M. Paning, et al. 2003. Interferons, interferon inducers, and interferon-ribavirin in treatment of flavivirus-induced encephalitis in mice. Antimicrob. Agents Chemother. 47:777–782. 108. Leyssen, P., A. Van Lommel, C. Drosten, et al. 2001. A novel model for the study of the therapy of flavivirus infections using the modoc virus. Virology 279:27–37. 109. Libraty, D.H., S. Pichyangkul, C. Ajariyakhajorn, et al. 2001. Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis. J. Virol. 75:3501–3508. 110. Lin, Y.L., Y.L. Huang, S.H. Ma, et al. 1997. Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication. J. Virol. 71:5227–5235. 111. Lindenbach, B.D., and C.M. Rice. 2001. Flaviviridae: the viruses and their replication, pp. 991–1041. In: D.M. Knipe and P.M. Howley (eds.), Fields Virology, Vol. 1. Lippincott, Williams & Wilkins, Philadelphia. 112. Liu, Y., R.V. Blanden, and A. Mullbacher. 1989. Identification of cytolytic lymphocytes in West Nile virus–infected murine central nervous system. J. Gen. Virol. 70:565–573. 113. Liu, Y., N. King, A. Kesson, et al. 1989. Flavivirus infection up-regulates the expression of class I and class II major histocompatibility antigens on and enhances T cell recognition of astrocytes in vitro. J. Neuroimmunol. 21:157–168. 114. Liu, Y., N. King, A. Kesson, et al. 1988. West Nile virus infection modulates the expression of class I and class II MHC antigens on astrocytes in vitro. Ann. NY Acad. Sci. 540:483–485. 115. Lobigs, M., A. Mullbacher, R.V. Blanden, et al. 1999. Antigen presentation in syrian hamster cells: substrate se- lectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules. Immunogenetics 49:931–941.

274 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

116. Lobigs, M., A. Mullbacher, Y. Wang, et al. 2003. Role of type I and type II interferon responses in recovery from infection with an encephalitic flavivirus. J. Gen. Virol. 84:567–572. 117. Lobigs, M., M. Pavy, and R. Hall. 2003. Cross-protective and infection-enhancing immunity in mice vaccinated against flaviviruses belonging to the Japanese encephalitis virus serocomplex. Vaccine 21:1572–1579. 118. Lustig, S., U. Olshevsky, D. Ben-Nathan, et al. 2000. A live attenuated West Nile virus strain as a potential veterinary vaccine. Viral Immunol. 13:401–410. 119. Malkinson, M., C. Banet, Y. Khinich, et al. 2001. Use of live and inactivated vaccines in the control of West Nile fever in domestic geese. Ann. NY Acad. Sci. 951:255–261. 120. Marianneau, P., A.M. Steffan, C. Royer, et al. 1999. Infection of primary cultures of human Kupffer cells by Dengue virus: no viral progeny synthesis, but cytokine production is evident. J. Virol. 73:5201–5206. 121. Markoff, L., A. Chang, and B. Falgout. 1994. Processing of flavivirus structural glycoproteins: stable membrane insertion of premembrane requires the envelope signal peptide. Virology 204:526–540. 122. Marovich, M., G. Grouard-Vogel, M. Louder, et al. 2001. Human dendritic cells as targets of dengue virus infection. J. Invest. Dermatol. Symp. Proc. 6:219–224. 123. Martin, M., T.F. Tsai, B. Cropp, et al. 2001. Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases. Lancet 358:98–104. 124. Martin, M., L.H. Weld, T.F. Tsai, et al. 2001. Advanced age a risk factor for illness temporally associated with yellow fever vaccination. Emerg. Infect. Dis. 7:945–951. 125. Mashimo, T., M. Lucas, D. Simon-Chazottes, et al. 2002. A nonsense mutation in the gene encoding 29-59-oligoadenylate synthetase/L1 isoform is associated with West Nile virus susceptibility in laboratory mice. Proc. Natl. Acad. Sci. USA 99:11311–11316. 126. Mason, P.W., J.M. Dalrymple, M.K. Gentry, et al. 1989. Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J. Gen. Virol. 70:2037–2049. 127. Momburg, F., A. Mullbacher, and M. Lobigs. 2001. Modulation of transporter associated with antigen processing (TAP)–mediated peptide import into the endoplasmic reticulum by flavivirus infection. J. Virol. 75:5663–5671. 128. Monath, T.P. 2001. Prospects for development of a vaccine against the West Nile virus. Ann. NY Acad. Sci. 951:1–12. 129. Morens, D.M. 1994. Antibody-dependent of enhancement of infection and the pathogenesis of viral disease. Clin. Inf. Dis. 19:500–512. 130. Mullbacher, A., and M. Lobigs. 1995. Up-regulation of MHC class I by flavivirus-induced peptide translocation into the endoplasmic reticulum. Immunity 3:207–214. 131. Murali-Krishna, K., V. Ravi, and R. Manjunath. 1996. Protection of adult but not newborn mice against lethal intracerebral challenge with Japanese encephalitis virus by adoptively transferred virus–specific cytotoxic T lymphocytes: requirement for L3T41 T cells. J. Gen. Virol. 77:705–714. 132. Nathanson, N., and G.A. Cole. 1970. Immunosuppression and experimental virus infection of the nervous system. Adv. Virus Res. 16:397–448. 133. Nathanson, N., and G.A. Cole. 1971. Immunosuppression: a means to assess the role of the immune response in acute virus infections. Fed. Proc. 30:1822–1830. 134. Navin, T.R., E.C. Krug, and R.D. Pearson. 1989. Effect of immunoglobulin M from normal human serum on Leishmania donovani promastigote agglutination, complement-mediated killing, and phagocytosis by human monocytes. Infect. Immun. 57:1343–1346. 135. Nir, Y., A. Beemer, and R.A. Goldwasser. 1965. West Nile Virus infection in mice following exposure to a viral aerosol. Br. J. Exp. Pathol. 46:443–449. 136. O’Doherty, U., M. Peng, S. Gezelter, et al. 1994. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology 82:487–493. 137. Ochsenbein, A.F., T. Fehr, C. Lutz, et al. 1999. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286:2156–2159.

275 DIAMOND ET AL.

138. Ochsenbein, A.F., D.D. Pinschewer, B. Odermatt, et al. 1999. Protective T cell–independent antiviral antibody responses are dependent on complement. J. Exp. Med. 190:1165–1174. 139. Ochsenbein, A.F., and R.M. Zinkernagel. 2000. Natural antibodies and complement link innate and acquired immunity. Immunol. Today 21:624–630. 140. Orange, J.S., M.S. Fassett, L.A. Koopman, et al. 2002. Viral evasion of natural killer cells. Nat. Immunol. 3:1006–1012. 141. Peiris, J.S., and J.S. Porterfield. 1979. Antibody-mediated enhancement of Flavivirus replication in macrophage- like cell lines. Nature 282:509–511. 142. Peiris, J.S.M., S. Gordon, J.C. Unkeless, et al. 1981. Monoclonal anti-Fc receptor IgG blocks antibody-dependent enhancement of viral replication in macrophages. Nature 289:189–191. 143. Peiris, J.S.M., J.S. Porterfield, and J.T. Roehrig. 1982. Monoclonal antibodies against the flavivirus West Nile. J. Gen. Virol. 58:283–289. 144. Perelygin, A.A., S.V. Scherbik, I.B. Zhulin, et al. 2002. Positional cloning of the murine flavivirus resistance gene. Proc. Natl. Acad. Sci. USA 99:9322–9327. 145. Pincus, S., P.W. Mason, E. Konishi, et al. 1992. Recombinant vaccinia virus producing the prM and E proteins of yellow fever virus protects mice from lethal yellow fever encephalitis. Virology 187:290–297. 146. Price, W.H., and I.S. Thind. 1972. The mechanism of cross-protection afforded by dengue virus against West Nile virus in hamsters. J. Hyg. (Lond.) 70:611–617. 147. Price, W.H., and I.S. Thind. 1971. Protection against West Nile virus induced by a previous injection with dengue virus. Am. J. Epidemiol. 94:596–607. 148. Putnak, J.R., and J.J. Schlesinger. 1990. Protection of mice against yellow fever virus encephalitis by immunization with a vaccinia virus recombinant encoding the yellow fever virus non-structural proteins, NS1, NS2a and NS2b. J. Gen. Virol. 71:1697–1702. 149. Roehrig, J.T., J.H. Mathews, and D.W. Trent. 1983. Identification of epitopes on the E glycoprotein of Saint Louis encephalitis virus using monoclonal antibodies. Virology 128:118–126. 150. Roehrig, J.T., L.A. Staudinger, A.R. Hunt, et al. 2001. Antibody prophylaxis and therapy for flaviviral encephalitis infections. Ann. NY Acad. Sci. 951:286–297. 151. Samuel, C.E. 2002. Host genetic variability and West Nile virus susceptibility. Proc. Natl. Acad. Sci. USA 99:11555–11557. 152. Sangster, M.Y., N. Urosevic, J.P. Mansfield, et al. 1994. Mapping the Flv locus controlling resistance to flaviviruses on mouse chromosome 5. J. Virol. 68:448–452. 153. Sawyer, L.A. 2000. Antibodies for the prevention and treatment of viral diseases. Antiviral Res. 47:57–77. 154. Schlesinger, J.J., M.W. Brandriss, C.B. Cropp, et al. 1986. Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1. J. Virol. 60:1153–1155. 155. Schlesinger, J.J., M.W. Brandriss, J.R. Putnak, et al. 1990. Cell surface expression of yellow fever virus nonstructural glycoprotein NS1: consequences of interaction with antibody. J. Gen. Virol. 71:593–599. 156. Schlesinger, J.J., M.W. Brandriss, and E.E. Walsh. 1985. Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48. J. Immunol. 135:2805–2809. 157. Schlesinger, J.J., M.W. Brandriss, and E.E. Walsh. 1987. Protection of mice against dengue 2 virus encephalitis by immunization with the dengue 2 virus non-structural glycoprotein NS1. J. Gen. Virol. 68:853–857. 158. Schlesinger, J.J., and S. Chapman. 1995. Neutralizing F(ab9)2 fragments of protective monoclonal antibodies to yellow fever virus (YF) envelope protein fail to protect mice against lethal YF encephalitis. J. Gen. Virol. 76:217–220. 159. Shrestha, B., D.I. Gottlieb, and M.S. Diamond. 2003. Infection and injury of neurons by West Nile encephalitis virus. (submitted).

276 IMMUNE SYSTEM PROTECTION AGAINST WNV INFECTION

160. Sittisombut, N., N. Maneekarn, A. Kanjanahaluethai, et al. 1995. Lack of augmenting effect of interferon-gamma on dengue virus multiplication in human peripheral blood monocytes. J. Med. Virol. 45:43–49. 161. Smith, H.R., A.H. Idris, and W.M. Yokoyama. 2001. Murine natural killer cell activation receptors. Immunol. Rev. 181:115–125. 162. Solomon, T., and D.W. Vaughn. 2002. Pathogenesis and clinical features of Japanese encephalitis and West Nile virus infections. Curr. Top. Microbiol. Immunol. 267:171–194. 163. Stanley, J., S.J. Cooper, and D.E. Griffin. 1986. Monoclonal antibody cure and prophylaxis of lethal Sindbis virus encephalitis in mice. J. Virol. 58:107–115. 164. Stark, G.R., I.M. Kerr, B.R. Williams, et al. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67:227–264. 165. Steele, K.E., M.J. Linn, R.J. Schoepp, et al. 2000. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York. Vet. Pathol. 37:208–224. 166. Stephen, E.L., M.L. Sammons, W.L. Pannier, et al. 1977. Effect of a nuclease-resistant derivative of polyri- boinosinic–polyribocytidylic acid complex on yellow fever in rhesus monkeys (Macaca mulatta). J. Infect. Dis. 136:122–126. 167. Takada, K., H. Masaki, E. Konishi, et al. 2000. Definition of an epitope on Japanese encephalitis virus (JEV) envelope protein recognized by JEV-specific murine CD81 cytotoxic T lymphocytes. Arch. Virol. 145:523–534. 168. Takahashi, K., J. Gordon, H. Liu, et al. 2002. Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis. Microbes Infect 4:773–784. 169. Tassaneetrithep, B., T. Burgess, A. Granelli-Piperno, et al. 2003. DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells. J. Exp. Med. 197:823–829. 170. Tesh, R.B., J. Arroyo, A.P. Travassos Da Rosa, et al. 2002. Efficacy of killed virus vaccine, live attenuated chimeric virus vaccine, and passive immunization for prevention of West Nile virus encephalitis in hamster model. Emerg Infect Dis 8:1392–1397. 171. Tesh, R.B., A.P. Travassos da Rosa, H. Guzman, et al. 2002. Immunization with heterologous flaviviruses protective against fatal West Nile encephalitis. Emerg. Infect. Dis. 8:245–251. 172. Tortorella, D., B.E. Gewurz, M.H. Furman, et al. 2000. Viral subversion of the immune system. Annu. Rev. Im- munol. 18:861–926. 173. Tsai, T.F., F. Popovici, C. Cernescu, et al. 1998. West Nile encephalitis epidemic in southeastern Romania. Lancet 352:767–771. 174. Urosevic, N., J.P. Mansfield, J.S. Mackenzie, et al. 1995. Low resolution mapping around the flavivirus resistance locus (Flv) on mouse chromosome 5. Mamm Genome 6:454–458. 175. Vargin, V.V., and B.F. Semenov. 1986. Changes of natural killer cell activity in different mouse lines by acute and asymptomatic flavivirus infections. Acta Virol. 30:303–308. 176. Vazquez, S., M.G. Guzman, G. Guillen, et al. 2002. Immune response to synthetic peptides of dengue prM protein. Vaccine 20:1823–1830. 177. Volanakis, J.E. 2002. The role of complement in innate and adaptive immunity. Curr. Top. Microbiol. Immunol. 266:41–56. 178. Wang, T., J.F. Anderson, L.A. Magnarelli, et al. 2001. Immunization of mice against West Nile virus with recombinant envelope protein. J. Immunol. 167:5273–5277. 179. Webb, H.E., D.G. Wight, G.S. Platt, et al. 1968. Langat virus encephalitis in mice. I. The effect of the administration of specific antiserum. J. Hyg. 66:343–354. 180. Weiner, L.P., G.A. Cole, and N. Nathanson. 1970. Experimental encephalitis following peripheral inoculation of West Nile virus in mice of different ages. J. Hyg. (Lond.) 68:435–446. 181. Work, T.H. 1971. On the Japanese B–West Nile virus complex or an arbovirus problem of six continents. Am. J. Trop. Med. Hyg. 20:169–186.

277 DIAMOND ET AL.

182. Wu, S.J., G. Grouard-Vogel, W. Sun, et al. 2000. Human skin Langerhans cells are targets of dengue virus infection. Nat. Med. 6:816–820. 183. Xiao, S.Y., H. Guzman, H. Zhang, et al. 2001. West Nile virus infection in the golden hamster (Mesocricetus auratus): a model for West Nile encephalitis. Emerg. Infect. Dis. 7:714–721. 184. Xiao, S.Y., H. Zhang, H. Guzman, et al. 2001. Experimental yellow fever virus infection in the golden hamster (Mesocricetus auratus). II. Pathology. J. Infect. Dis. 183:1437–1444. 185. Yang, J.S., J.J. Kim, D. Hwang, et al. 2001. Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999). J. Infect. Dis. 184:809–816. 186. Zeidner, N.S., S. Higgs, C.M. Happ, et al. 1999. Mosquito feeding modulates Th1 and Th2 cytokines in flavivirus susceptible mice: an effect mimicked by injection of sialokinins, but not demonstrated in flavivirus-resistant mice. Parasite Immunol. 21:35–44. 187. Zeitlin, L., R.A. Cone, and K.J. Whaley. 1999. Using monoclonal antibodies to prevent mucosal transmission of epidemic infectious diseases. Emerg. Infect. Dis. 5:54–64.

Address reprint requests to: Michael S. Diamond, M.D., Ph.D. Departments of Medicine, Molecular Microbiology, Pathology & Immunology Washington University School of Medicine 660 South Euclid Ave., Box 8051 St. Louis, MO 63110

E-mail: [email protected]

278 This article has been cited by:

1. Prakash S. Bisen, Ruchika RaghuvanshiWest Nile Fever 316-339. [CrossRef] 2. Luisa Barzon, Monia Pacenti, Giorgio Palù. 2013. West Nile virus and kidney disease. Expert Review of Anti-infective Therapy 11:5, 479-487. [CrossRef] 3. Mirko Spiroski, Zvonko Milenkovic, Aleksandar Petlichkovski, Ljubomir Ivanovski, Irena Kondova Topuzovska, Eli Djulejic. 2013. Killer cell immunoglobulin-like receptor genes in four human West Nile virus infections reported 2011 in the Republic of Macedonia. Human Immunology 74:3, 389-394. [CrossRef] 4. Mehul S. Suthar, Michael S. Diamond, Michael Gale Jr. 2013. West Nile virus infection and immunity. Nature Reviews Microbiology 11:2, 115-128. [CrossRef] 5. Sara I. Ruiz, Elizabeth E. Zumbrun, Aysegul NalcaAnimal Models of Human Viral Diseases 927-970. [CrossRef] 6. Vandana Saxena, Thomas Welte, Xiaoyong Bao, Guorui Xie, Jia Wang, Stephen Higgs, Robert B. Tesh, Tian Wang. 2012. A hamster-derived West Nile virus strain is highly attenuated and induces a differential proinflammatory cytokine response in two murine cell lines. Virus Research 167:2, 179-187. [CrossRef] 7. Pyung Ok Lim, Tae Hee Lee, Kyung Min Chung. 2012. Potential Therapeutics Against Flaviviruses. Journal of Bacteriology and Virology 42:2, 108. [CrossRef] 8. Tian Wang. 2011. Role of γδ T cells in West Nile virus-induced encephalitis: Friend or foe?. Journal of Neuroimmunology 240-241, 22-27. [CrossRef] 9. Elena Sotelo, Jovita Fernández-Pinero, Miguel Ángel Jiménez-Clavero. 2011. La fiebre/encefalitis por virus West Nile: reemergencia en Europa y situación en España. Enfermedades Infecciosas y Microbiología Clínica . [CrossRef] 10. Melissa C. Whiteman, Jason A. Wicker, Richard M. Kinney, Claire Y.-H. Huang, Tom Solomon, Alan D.T. Barrett. 2011. Multiple amino acid changes at the first glycosylation motif in NS1 protein of West Nile virus are necessary for complete attenuation for mouse neuroinvasiveness. Vaccine . [CrossRef] 11. Thomas Welte, Judith Aronson, Bin Gong, Aparna Rachamallu, Nicole Mendell, Robert Tesh, Slobodan Paessler, Willi K. Born, Rebecca L. O'Brien, Tian Wang, Willem Eden. 2011. Vγ4+T cells regulate host immune response to West Nile virus infection. FEMS Immunology & Medical Microbiology n/a-n/a. [CrossRef] 12. Thomas Welte, Guorui Xie, Jason A. Wicker, Melissa C. Whiteman, Li Li, Aparna Rachamallu, Alan Barrett, Tian Wang. 2011. Immune responses to an attenuated West Nile virus NS4B-P38G mutant strain. Vaccine 29:29-30, 4853-4861. [CrossRef] 13. J. E. Ledgerwood, T. C. Pierson, S. A. Hubka, N. Desai, S. Rucker, I. J. Gordon, M. E. Enama, S. Nelson, M. Nason, W. Gu, N. Bundrant, R. A. Koup, R. T. Bailer, J. R. Mascola, G. J. Nabel, B. S. Graham. 2011. A West Nile Virus DNA Vaccine Utilizing a Modified Promoter Induces Neutralizing Antibody in Younger and Older Healthy Adults in a Phase I Clinical Trial. Journal of Infectious Diseases 203:10, 1396-1404. [CrossRef] 14. Julio Alonso-Padilla, Nereida Jiménez de Oya, Ana-Belén Blázquez, Estela Escribano-Romero, José M. Escribano, Juan-Carlos Saiz. 2011. Recombinant West Nile virus envelope protein E and domain III expressed in insect larvae protects mice against West Nile disease. Vaccine 29:9, 1830-1835. [CrossRef] 15. Hao Fang, Thomas Welte, Xin Zheng, Gwong-Jen J. Chang, Michael R. Holbrook, Lynn Soong, Tian Wang. 2010. γδ T cells promote the maturation of dendritic cells during West Nile virus infection. FEMS Immunology & Medical Microbiology 59:1, 71-80. [CrossRef] 16. Saguna Verma, Mukesh Kumar, Ulziijargal Gurjav, Stephanie Lum, Vivek R. Nerurkar. 2010. Reversal of West Nile virus-induced blood–brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor. Virology 397:1, 130-138. [CrossRef] 17. Kymberly A. Gyure. 2009. West Nile Virus Infections. Journal of Neuropathology and Experimental Neurology 68:10, 1053-1060. [CrossRef] 18. Lee S. Engel, Charles V. Sanders, Fred A. LopezFever and Rash in Critical Care 19-48. [CrossRef] 19. Edward B. Hayes. 2009. Zika Virus Outside Africa. Emerging Infectious Diseases 15:9, 1347-1350. [CrossRef] 20. Jian-Jong Liang, Ching-Len Liao, Jia-Teh Liao, Yi-Ling Lee, Yi-Ling Lin. 2009. A Japanese encephalitis virus vaccine candidate strain is attenuated by decreasing its interferon antagonistic ability. Vaccine 27:21, 2746-2754. [CrossRef] 21. Nicole Nemeth, Ginger Young, Christina Ndaluka, Helle Bielefeldt-Ohmann, Nicholas Komar, Richard Bowen. 2009. Persistent West Nile virus infection in the house sparrow (Passer domesticus). Archives of Virology 154:5, 783-789. [CrossRef] 22. Saguna Verma, Yeung Lo, Moti Chapagain, Stephanie Lum, Mukesh Kumar, Ulziijargal Gurjav, Haiyan Luo, Austin Nakatsuka, Vivek R. Nerurkar. 2009. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier. Virology 385:2, 425-433. [CrossRef] 23. Nicole M. Nemeth, Gail E. Kratz, Rebecca Bates, Judy A. Scherpelz, Richard A. Bowen, Nicholas Komar. 2008. Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors. EcoHealth 5:3, 298-304. [CrossRef] 24. Theodore C. Pierson, Michael S. Diamond. 2008. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection. Expert Reviews in Molecular Medicine 10. . [CrossRef] 25. Robyn S. Klein, Michael S. Diamond. 2008. Immunological headgear: antiviral immune responses protect against neuroinvasive West Nile virus. Trends in Molecular Medicine 14:7, 286-294. [CrossRef] 26. H ELGARCH, J MINKE, J REHDER, S RICHARD, C EDLUNDTOULEMONDE, S DINIC, C ANDREONI, J AUDONNET, R NORDGREN, V JUILLARD. 2008. A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse. Veterinary Immunology and Immunopathology 123:3-4, 230-239. [CrossRef] 27. Yos Priestley, Marcia Thiel, Steven B Koevary. 2008. Systemic and ophthalmic manifestations of West Nile virus infection. Expert Review of Ophthalmology 3:3, 279-292. [CrossRef] 28. Marion C. Lanteri, John W. Heitman, Rachel E. Owen, Thomas Busch, Nelly Gefter, Nancy Kiely, Hany T. Kamel, Leslie H. Tobler, Michael P. Busch, Philip J. Norris. 2008. Comprehensive Analysis of West Nile Virus–Specific T Cell Responses in Humans. The Journal of Infectious Diseases 197:9, 1296-1306. [CrossRef] 29. M FRIEMAN. 2008. SARS coronavirus and innate immunity. Virus Research 133:1, 101-112. [CrossRef] 30. Robyn S. Klein. 2008. A Moving Target: The Multiple Roles of CCR5 in Infectious Diseases. The Journal of Infectious Diseases 197:2, 183-186. [CrossRef] 31. Alexander Pereboev, Viktoriya Borisevich, George Tsuladze, Mikhail Shakhmatov, Deborah Hudman, Elena Kazachinskaia, Ivan Razumov, Viktor Svyatchenko, Valery Loktev, Vladimir Yamshchikov. 2008. Genetically delivered antibody protects against West Nile virus. Antiviral Research 77:1, 6-13. [CrossRef] 32. Julie E. Martin, Theodore C. Pierson, Sarah Hubka, Steve Rucker, Ingelise J. Gordon, Mary E. Enama, Charla A. Andrews, Qing Xu, Brent S. Davis, Martha Nason, Michael Fay, Richard A. Koup, Mario Roederer, Robert T. Bailer, Phillip L. Gomez, John R. Mascola, Gwong‐Jen J. Chang, Gary J. Nabel, Barney S. Graham. 2007. A West Nile Virus DNA Vaccine Induces Neutralizing Antibody in Healthy Adults during a Phase 1 Clinical Trial. The Journal of Infectious Diseases 196:12, 1732-1740. [CrossRef] 33. G. Dauphin, S. Zientara. 2007. West Nile virus: Recent trends in diagnosis and vaccine development. Vaccine 25:30, 5563-5576. [CrossRef] 34. James D. Brien, Jennifer L. Uhrlaub, Janko Nikolich-Žugich. 2007. Protective capacity and epitope specificity of CD8+ T cells responding to lethal West Nile virus infection. European Journal of Immunology 37:7, 1855-1863. [CrossRef] 35. Danelle M. Okeson, Shirley Yeo Llizo, Christine L. Miller, Amy L. Glaser. 2007. ANTIBODY RESPONSE OF FIVE BIRD SPECIES AFTER VACCINATION WITH A KILLED WEST NILE VIRUS VACCINE. Journal of Zoo and Wildlife Medicine 38:2, 240-244. [CrossRef] 36. A MUSHTAQ, M ELAZIZI, N KHARDORI. 2006. Category C Potential Bioterrorism Agents and Emerging Pathogens. Infectious Disease Clinics of North America 20:2, 423-441. [CrossRef] 37. Roberta L DeBiasi, Kenneth L Tyler. 2006. West Nile virus meningoencephalitis. Nature Clinical Practice Neurology 2:5, 264-275. [CrossRef] 38. David Garcia-Tapia, Christie M. Loiacono, Steven B. Kleiboeker. 2006. Replication of West Nile virus in equine peripheral blood mononuclear cells. Veterinary Immunology and Immunopathology 110:3-4, 229-244. [CrossRef] 39. Maria Rios, Ming J. Zhang, Andriyan Grinev, Kumar Srinivasan, Sylvester Daniel, Owen Wood, Indira K. Hewlett, Andrew I. Dayton. 2006. Monocytes-macrophages are a potential target in human infection with West Nile virus through blood transfusion. Transfusion 46:4, 659-667. [CrossRef] 40. Bradley S. Schneider, Lynn Soong, Yvette A. Girard, Gerald Campbell, Peter Mason, Dr. Stephen Higgs. 2006. Potentiation of West Nile Encephalitis by Mosquito Feeding. Viral Immunology 19:1, 74-82. [Abstract] [Full Text PDF] [Full Text PDF with Links] 41. Theodore C. Pierson, Melissa D. Sánchez, Bridget A. Puffer, Asim A. Ahmed, Brian J. Geiss, Laura E. Valentine, Louis A. Altamura, Michael S. Diamond, Robert W. Doms. 2006. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection. Virology 346:1, 53-65. [CrossRef] 42. Edward B. Hayes, Duane J. Gubler. 2006. West Nile Virus: Epidemiology and Clinical Features of an Emerging Epidemic in the United States*. Annual Review of Medicine 57:1, 181-194. [CrossRef] 43. Michael S Diamond. 2005. Development of effective therapies against West Nile virus infection. Expert Review of Anti-infective Therapy 3:6, 931-944. [CrossRef] 44. Ellen Averett, John S. Neuberger, Gail Hansen, Michael H. Fox. 2005. Evaluation of West Nile Virus Education Campaign. Emerging Infectious Diseases 11:11, 1751-1753. [CrossRef] 45. Patrick Luedtke, John GreenleeWest Nile Virus 20052334, 667-675. [CrossRef] 46. Edward B. Hayes, James J. Sejvar, Sherif R. Zaki, Robert S. Lanciotti, Amy V. Bode, Grant L. Campbell. 2005. Virology, Pathology, and Clinical Manifestations of West Nile Virus Disease. Emerging Infectious Diseases 11:8, 1174-1179. [CrossRef] 47. Theodore Oliphant, Michael Engle, Grant E Nybakken, Chris Doane, Syd Johnson, Ling Huang, Sergey Gorlatov, Erin Mehlhop, Anantha Marri, Kyung Min Chung, Gregory D Ebel, Laura D Kramer, Daved H Fremont, Michael S Diamond. 2005. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nature Medicine 11:5, 522-530. [CrossRef] 48. Theodore C. Pierson, Michael S. Diamond, Asim A. Ahmed, Laura E. Valentine, Carl W. Davis, Melanie A. Samuel, Sheri L. Hanna, Bridget A. Puffer, Robert W. Doms. 2005. An infectious West Nile Virus that expresses a GFP reporter gene. Virology 334:1, 28-40. [CrossRef] 49. Bradley S. Schneider, Lynn Soong, Nordin S. Zeidner, Dr. Stephen Higgs. 2004. Aedes aegypti Salivary Gland Extracts Modulate Anti-Viral and TH1/TH2 Cytokine Responses to Sindbis Virus Infection. Viral Immunology 17:4, 565-573. [Abstract] [Full Text PDF] [Full Text PDF with Links] 50. Michael S Diamond, Robyn S Klein. 2004. West Nile virus: crossing the blood-brain barrier. Nature Medicine 10:12, 1294-1295. [CrossRef] 51. Galina Yamshchikov, Victoria Borisevich, Alexey Seregin, Elena Chaporgina, Margarita Mishina, Vasiliy Mishin, Chun Wai Kwok, Vladimir Yamshchikov. 2004. An attenuated West Nile prototype virus is highly immunogenic and protects against the deadly NY99 strain: a candidate for live WN vaccine development. Virology 330:1, 304-312. [CrossRef] 52. Roy A Hall, Alexander A Khromykh. 2004. West Nile virus vaccines. Expert Opinion on Biological Therapy 4:8, 1295-1305. [CrossRef] 53. L. Hannah Gould, Erol Fikrig. 2004. West Nile virus: a growing concern?. Journal of Clinical Investigation 113:8, 1102-1107. [CrossRef] 54. Pierre-Emmanuel Ceccaldi, Marianne Lucas, Philippe Despres. 2004. New insights on the neuropathogenicity of West Nile virus. FEMS Microbiology Letters 233:1, 1-6. [CrossRef]

Review

Cell-intrinsic innate immune control of

West Nile virus infection

1,2,3 4

Michael S. Diamond and Michael Gale Jr.

Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO 63110, USA

Department of Medicine, Washington University School of Medicine, St Louis, MO 63110, USA

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA

Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195-7650, USA

West Nile virus (WNV) is an enveloped positive-stranded no approved vaccine or therapy for WNV infection in

RNA virus that has emerged over the past decade in humans. The expansion of WNV disease globally high-

North America to cause epidemics of meningitis, en- lights a need for greater understanding of mechanisms

cephalitis, and acute ﬂaccid paralysis in humans. WNV of immune control, including the cell-intrinsic processes

has broad species speciﬁcity, and replicates efﬁciently in that restrict infection.

many cell types, including those of the innate immune An effective host defense against WNV requires the

and central nervous systems. Recent studies have de- antiviral actions of type I IFN. Mice lacking the common

ﬁned the pathogen recognition receptor (PRR) and sig- type I IFN receptor (Ifnar ) exhibit 100% mortality,

naling pathways by which WNV is detected, and several accelerated spread, and expanded cellular and tissue tro-

effector mechanisms that contribute to protective cell- pism after infection with a virulent WNV isolate [1], and

intrinsic immunity. This review focuses on recent infection with attenuated WNV strains results in en-

advances in identifying the host sensors that detect hanced replication and pathogenesis [2–7]. The virulence

WNV, the adaptor molecules and signaling pathways of WNV strains has been linked directly to their ability to

that regulate the induction of interferon (IFN)-dependent antagonize IFN signaling [2,6,8,9].

defenses, and the proteins that limit WNV replication, Studies by several groups have begun to deﬁne the PRR

spread, and disease pathogenesis. and signaling pathways by which WNV and other ﬂavi-

viruses are detected, and the effector mechanisms that

Innate host defense to WNV infection contribute to protective cell-intrinsic immunity. These

To survive virus infection, the host must recognize inva- studies reveal important insights into PRR function,

sion and develop an effective antiviral immune response. PAMP discrimination, and intracellular signaling path-

This response is initiated in infected cells after detection of ways among different cell types relevant to WNV infection.

non-self pathogen-associated molecular patterns (PAMPs). This review describes the recent advances that have been

These PAMP motifs are detected by speciﬁc host PRRs, made in deﬁning the host sensors that detect WNV, the

which trigger signaling cascades that induce the activation signaling pathways and genes that regulate induction of

of latent transcription factors, including IFN regulatory IFN-dependent and -independent responses, and the pro-

factors (IRFs) and NF-kB leading to expression of virus- teins that limit WNV replication and disease pathogenesis.

responsive genes, including type I IFNs (IFN-a and b) and

hundreds of different IFN-stimulated effector genes

Innate immune cells controlling WNV are also targets of

(ISGs). ISG products include antiviral effector molecules infection

and immunomodulatory cytokines that serve to restrict Macrophages

virus replication and modulate the adaptive immune re-

Emerging data suggests that macrophages play key roles

sponse.

in orchestrating control of WNV infection. Macrophages

WNV is a single-stranded positive-polarity RNA ﬂavi-

can limit infection though direct viral clearance, enhanced

virus and is closely related to other human pathogens

antigen presentation to B and T cells, and production of

including dengue, yellow fever, Japanese encephalitis

proinﬂammatory or antiviral cytokines and chemokines.

(JEV), and tick-borne encephalitis viruses. WNV cycles

The protective role of macrophages is highlighted by stud-

in nature between mosquitoes and birds with some mam-

ies in mice, which demonstrate exacerbated WNV disease

malian species, including horses and humans, becoming

after selective macrophage depletion [10,11]. Macrophages

incidentally infected and developing disease. Although

have been reported to control infection of other ﬂaviviruses

most human infections remain asymptomatic or develop

(e.g. JEV) directly through the production of NO and other

into a mild illness, a small subset (<1%) progress to severe

reactive oxygen intermediates [12,13], although this mech-

neurological syndromes that include meningitis, acute

anism has not yet been conﬁrmed for WNV. Activation of

ﬂaccid paralysis, and encephalitis. Although veterinary

macrophages in response to WNV infection also promotes

vaccines are available commercially, there is currently

release of type I IFN, tumor necrosis factor (TNF)-a,

interleukin (IL)-1b, IL-8, and other cytokines; some of

Corresponding authors: Diamond, M.S. ([email protected]);

Gale. M., Jr ([email protected]) which have antiviral activity and reduce viral replication,

Review Trends in Immunology October 2012, Vol. 33, No. 10

at least in culture [14]. Despite their protective role in recruitment domains (CARD) at the N terminus. RIG-I

initiating innate immune defenses, macrophages also are possesses a C-terminal repressor domain, which regulates

targets of WNV [15,16]. its activity. RIG-I and MDA5 bind unique PAMPs. RIG-I

ligands include single-stranded (ss)RNA containing a 5 -

Dendritic cells ppp, short double-stranded (ds)RNA, and uridine- or aden-

Plasmacytoid (pDC) and myeloid (mDC) DC subsets can be osine-rich viral RNA motifs [26–28], whereas MDA5 binds

distinguished based on function and surface markers. short or long dsRNA structures although the particular

pDCs lack phagocytic capacity, are less efﬁcient in captur- motif is not well delineated [29].

ing and presenting antigens to T cells, but produce high Binding of viral RNA PAMP ligands by RIG-I and

levels of type I IFN in the presence of viruses or bacteria, MDA5 induces CARD–CARD interactions with the mito-

and are thus considered to have a crucial role in antiviral chondrial-associated IFN promoter stimulator (IPS)-1 (al-

immunity [17]. Low levels of WNV replication are observed so known as MAVS, VISA or CARDIF). RIG-I signaling

in pDCs, but proinﬂammatory cytokines are produced activation is dependent on RNA PAMP binding and

rapidly and can accumulate to high levels. This cytokine requires ubiquitination by Tripartite motif 25 (TRIM25)

response is not dependent on viral replication, but instead [30], oligomerization [31], and IPS-1 multimerization [32]

on endosomal Toll-like receptor (TLR)7, and can be in- on mitochondrion-associated-endoplasmic reticulum

duced by puriﬁed ﬂavivirus virion RNA [18,19]. Interest- membranes [33] and peroxisomes [34]. The proteins cas-

ingly, the host origin of WNV inﬂuences the pDC response, pase-12 and 14-3-3-e positively modulate RLR signaling of

because WNV propagated in mammalian cells is a more type I IFN production by regulating TRIM25-mediated

potent inducer of IFN-a secretion in pDCs, whereas pDCs ubiquitination of RIG-I [35,36]. Activation of IPS-1 then

fail to produce IFN-a when exposed to WNV grown in promotes activation of transcription factors through inter-

mosquito cells [20]. actions with TNF-receptor-associated factor 3 (TRAF3),

mDCs reside and circulate throughout the body, en- TANK-binding kinase 1 (TBK1), Inhibitor of kB kinase

abling them to transport antigens from peripheral sites epsilon (Ikk-e), and NF-kB essential modulator (NEMO),

of infection to lymphoid tissues. As professional antigen- driving a ‘signalosome’ that initiates the antiviral re-

presenting cells, they transmit incoming infectious signals sponse (Figure 1).

to B and T cells, to orchestrate rapid adaptive immune The precise mechanisms through which RIG-I and

responses [21]. Compared to pDCs, mDCs are more readily MDA5 recognize WNV, and the PAMPs involved in this

infected by WNV, and consequently, are thought to con- process, remain unknown. Both RIG-I and MDA5, howev-

tribute both to viral spread and early immune system er, are required for optimal antiviral responses against

activation. For example, WNV efﬁciently infects mDCs WNV [37]. Ddx58 (RIG-I) murine embryonic ﬁbroblasts

ex vivo and induces a robust type I IFN and proinﬂamma- (MEFs) support increased WNV replication and reduced

tory cytokine response, which may be compromised during IRF-3 activation, with a delayed IFN and ISG response

aging [20,22,23]. Despite an accumulating wealth of data [38,39]. In epithelial cells, a deﬁciency of both RIG-I and

on WNV infection of puriﬁed mDCs ex vivo, few studies MDA5 is required for enhanced WNV or dengue virus

have assessed their direct function in vivo. One recent infection, because loss of either gene alone had minimal

report has shown that selective genetic deletion of phenotypes [39]. The precise hierarchy of function of dif-

CD8a mDCs resulted in defective cross-presentation ferent RLRs in controlling WNV infection remains an area

and virus-speciﬁc CD8 T cell responses to WNV [24]. In of active study, although RIG-I may prime the early re-

comparison, in the skin, different inﬂammatory DC sub- sponse, with both RIG-I and MDA5 together regulating the

sets may be responsible for initiating immune responses response at later time points of infection. Of note, infection

/ /

against WNV, including myeloid cells that are rapidly of Ddx58 but not Mda5 mice with JEV results in

mobilized from the bone marrow [25]. enhanced mortality [40]. In comparison, Mavs (IPS-1)

MEFs, DCs, macrophages, and neurons sustain higher

PRR detection of WNV WNV titers and fail to induce a type I IFN response

Two major classes of PRRs have been identiﬁed in mam- [41,42]. Although Mavs mice show enhanced spread

malian cells that recognize positive-strand RNA viruses, and replication in peripheral tissues, and uniform lethality

such as WNV: (i) TLRs; and (ii) retinoic acid-inducible gene with virulent WNV strains [42], RLR alone do not exclu-

(RIG)-I-like receptors (RLRs). These PRRs sense virus sively detect WNV because Mavs mice produce higher

infections by recognizing nucleic acid PAMPs associated levels of systemic type I IFN than wild-type controls do.

with viral RNA. Members of the RLR [RIG-I and melano-

ma differentiation antigen (MDA)5] and TLR (TLR3 and Protein kinase (PK)R

TLR7) families are the dominant PRRs that detect WNV The dsRNA-dependent PKR is both a cytosolic PRR that

infection. detects dsRNA and an ISG with effector functions capable

of controlling viral replication by blocking translation of

Cytosolic RIG-I-like receptors viral and cellular mRNA. PKR can serve as a PRR by

RIG-I and MDA5 are cytoplasmic RNA helicases that binding dsRNA, which promotes its activation and signal-

recognize viral RNA products and induce transcription ing actions. PKR can regulate antiviral responses by sup-

factor activation and type I IFN gene expression. Both pressing mRNA translation through phosphorylation of

RIG-I and MDA5 are composed of helicase domains the a subunit of eukaryotic initiation factor (eIF)2 and

at the C terminus and tandem caspase-activation and by modulating IFN mRNA stability [43]. PKR has been

523

Review Trends in Immunology October 2012, Vol. 33, No. 10

IFN-α/β IFN-α/β IFN-α/β Virus IFN-α/β Type I IFN receptor

Cytoplasm (d) p p Tyk2 (a) WNV Jak1 RNA Endosome (f) p TLR7 STAT1 p MDA5 TLR3 p RIG-I STAT2 p (b) IRF-9 (e)

NS1 IFNAR IPS-1 degradation NEMO

TRAF3 IKKα IKKβ MyD88 TRIF

IKKε IRF-3 Mitochondria TBK1 IKKε p p κ P STAT1 I B IRF-7 IRF-3 p p STAT2 NF-κB P IκB IRF-7 P IRF-9 ISGF3 IRF-7 Ubq-Ubq P IRF-3 P IRF-3 P IRF-7

NS2A (c)

p p ISGs P STAT1 CBP/ IRF-3 p P NF-κB p300 IRF-3 STAT2 IRF-9 IRF-7 P ISRE IRF-7 P IFN-β Nucleus

TRENDS in Immunology

Figure 1. Detection of West Nile virus (WNV) and activation of interferon (IFN)-a/b genes and IFN-stimulated genes (ISGs). Schematic of innate immune signaling triggered

by WNV infection through cytosolic retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) or endosomal Toll-like receptors (TLRs). Solid lines indicate the primary

response. Broken lines indicate the amplification response driven by IFN regulatory factor (IRF)-7 after initial IFN production by the infected cell. P denotes phosphorylation;

Ubq denotes ubiquitin modification. Infection by WNV produces RNA intermediates in the cytosol that are detected as non-self by the host RLR. RIG-I acts as the main

sensor for WNV during the early steps of infection. RIG-I activation promotes interaction with IFN promoter stimulator (IPS)-1 that leads to the recruitment of TRAF3, TBK1

and Inhibitor of kB kinase epsilon (Ikk-e) or NF-kB essential modulator (NEMO), Ikk-a, and Ikk-b, which results in activation and nuclear translocation of IRF-3 and nuclear

factor (NF)-kB, respectively. Small amounts of constitutively produced IRF-7 also may be activated via this pathway. IRF-3, IRF-7, and NF-kB bind the IFN-b gene promoter

and induce transcription. Secretion of IFN-b by the infected cells results in paracrine type I IFN signaling through IFNAR. Activation of IFNAR induces phosphorylation of

Janus kinase (JAK)1 and Tyk2, which can promote the formation of the heterotrimer ISGF3 formed by signal transducer and activator of transcription (STAT)1, STAT2 and

IRF-9. Ultimately, translocation into the nucleus of ISGF3 induces hundreds of ISGs, including IRF-7. During late phases of infection, detection of WNV also relies on

melanoma differentiation antigen (MDA)5, whereas Ikk-e promotes specific ISG induction through phosphorylation of STAT1 on serine 708. Induction of IFN-a genes

(except IFN-a4) occurs mainly via the transcriptional activity of IRF-7. Putative mechanisms by which WNV antagonizes the type I IFN response are marked and include (a) a

delay in recognition of WNV RNA by RIG-I [38]; (b) attenuation of TLR3 signaling by the viral NS1 protein [54]; (c) reduction in IFN-b gene transcription by the viral NS2A

protein [5]; (d) blockade of phosphorylation of Tyk2 [2]; (e) downregulation of IFNAR expression through virus-induced protein degradation pathways [92]; and (f)

inactivation of the IFN-regulated JAK–STAT signaling pathway [93] through redistribution of cellular cholesterol [94].

524

Review Trends in Immunology October 2012, Vol. 33, No. 10

implicated in modulating IFN signaling during WNV in- further study but probably involves both cell-intrinsic

fection [44]. Although some studies suggest that virulent and -extrinsic effects in the CNS.

WNV infection does not directly activate PKR [45], in cells The function of TLR7 in WNV infection in vivo has been

infected with attenuated, chimeric WNV strains, viral examined in two studies using the same Tlr7 mice, and

RNA can serve as a ligand for PKR because it is not again, somewhat discordant phenotypes were described

completely protected by intracellular membranes [46]. [56,57]. One study found no difference in susceptibility

The speciﬁc role of PKR in vivo has been inferred from to WNV between wild-type and Tlr7 mice after intra-

/ / /

studies with Rnasel and Eif2ak2 (PKR) Rnasel dermal injection or feeding by infected mosquitoes. Viral

mice: more severe virological and survival phenotypes load analysis revealed similar levels of WNV RNA in the

/ /

have been observed in Eif2ak2 Rnasel mice peripheral tissues and brains of these two groups of mice

/ /

infected with WNV [47]. Eif2ak2 mice show no defect [56,57]. In the second study, Tlr7 mice were more

in type I IFN response in serum, suggesting a limited vulnerable to intraperitoneal WNV infection and showed

contribution of PKR in systemic IFN induction. Rather, increased viremia and defects in immune cell homing to

PKR appears to contribute to antiviral effector functions, WNV-infected tissues via an IL-23-dependent mechanism

because IFN-b pretreated Eif2ak2 macrophages and [56]. Both studies reported increased systemic levels of

cortical neurons are more susceptible to WNV infection proinﬂammatory cytokines (IL-6, TNF-a, and IL-12) and

relative to wild-type cells. type I IFN in Tlr7 mice when compared to wild-type

animals, which could be due to differential replication in

Endosomal TLRs key cell types. Thus, abrogation of the TLR7 signaling

Recognition of WNV by TLRs occurs in endosomes and is pathway, which eliminates type I IFN production in pDCs,

largely mediated by TLR7 and TLR3, which bind ssRNA had little systemic impact on IFN production after WNV

and dsRNA, respectively. TLR7 recruits the adaptor pro- infection. Consistent with this, recent studies with diph-

tein MyD88 and forms a complex with TRAF3, TRAF6, theria toxin receptor transgenic mice that selectively de-

interleukin-1 receptor associated kinase 1 (IRAK1) and plete pDCs have shown only small decreases in type I IFN

IRAK4. This complex recruits transforming growth fac- production after viral infection [17].

tor-b-activated kinase 1 (TAK1), a kinase that activates Two studies have reported a protective effect of MyD88

nuclear factor (NF)-kB, or TBK1 and Ikk-e kinases, which [56,58], the downstream adaptor molecule for several

in turn stimulate IRF-3 and/or IRF-7 and IFN responses. TLRs, including TLR7. Myd88 mice showed elevated

TLR3, in contrast, uses TRIF as an adaptor protein. Toll/ viral burden primarily in the brain, even though little

interleukin 1 receptor domain-containing adapter induc- effect on the systemic type I IFN response was observed.

ing IFN-b (TRIF) can stimulate an IRF-3 and IRF-7-de- In cell culture, increased WNV replication was observed in

pendent induction of type I IFN genes via its interaction Myd88 macrophages and subsets of neurons. An ab-

with TRAF3, TBK1 and Ikk-e (Figure 1). sence of MyD88 had an independent and negative effect on

Despite several studies showing that ligation of TLR3 recruitment of monocyte-derived macrophages and T cells

by dsRNA in vitro regulates IFN and other cytokine into the brain; this was associated with blunted induction

responses, its role in inducing IFN and protecting against of the chemokines that attract leukocytes, which function

viral infection in vivo remains less clear (reviewed in [48]). to clear WNV infection from the CNS. Together, these

For WNV, two studies using the same Tlr3 mice have experiments suggest that MyD88 restricts WNV by inhi-

reported distinct phenotypes. One study showed a detri- biting replication and modulating immune cell migration

mental role, because Tlr3 mice had improved survival into the CNS.

rates after WNV infection; Tlr3 mice showed a mildly

increased WNV burden in peripheral tissues with a de- Transcriptional regulation of the type I IFN response

creased systemic proinﬂammatory cytokine response that after WNV infection

was associated with reduced blood–brain barrier perme- The IRF family is composed of nine related members that

ability and entry of WNV into the brain [49]. A second function as transcription factors [59]. IRF-3 and IRF-7

study showed a protective role with decreased survival of have been identiﬁed as the key transcriptional regulators

Tlr3 mice after WNV infection, mildly elevated viral of type I IFN genes following viral infection, whereas IRF-1

titers in peripheral tissues, and early viral spread to the and IRF-5 are thought to contribute subordinately to this

central nervous system (CNS) [50]. Ex vivo studies have response. Optimal activation of the type I IFN response

shown a dispensable role of TLR3 in regulating IFN after viral infection is believed to occur following a two-step

responses and controlling WNV replication in MEFs, ampliﬁcation sequence. Viral sensing by PRR (TLR and

DCs and macrophages. TLR3 may have a more signiﬁcant RLR) triggers nuclear translocation of constitutively

role in the CNS, potentially by restricting WNV replication expressed IRF-3 with subsequent production of IFN-b

in neurons [50] or via dsRNA sensing and induction of and IFN-a4 by the infected cell. The newly synthesized

proinﬂammatory cytokines in resident cells of the CNS, IFN binds to IFNAR, activates Janus kinase–signal trans-

including microglia and astrocytes [51,52]. WNV NS1 pro- ducer and activator of transcription (JAK–STAT) signal-

tein has been reported to inhibit TLR3 signal transduction ing, and induces expression of hundreds of ISGs, including

by attenuating transcriptional activation of the IFN-b and IRF-7. Subsequent activation of IRF-7 ampliﬁes expres-

NF-kB promoters [53,54], although this ﬁnding was not sion of IFN-b and many other IFN-a subtypes through a

observed in a second study [55]. The exact contribution of positive feedback loop. Small amounts of constitutively

TLR3 in WNV protection and/or pathogenesis requires expressed IRF-7 in some cell types, however, may prime

525

Review Trends in Immunology October 2012, Vol. 33, No. 10

the type I IFN response directly [60]. Recent studies with

WNV IRF-1

WNV have helped to elucidate the role of individual IRF

IRF-3

family members in regulating the type IFN response.

Macrophage IRF-7

In vivo studies

/ /

Infection of Irf3 or Irf7 mice with WNV results in

severe pathology linked with a lack of innate antiviral

IRF-3

immunity [22,61]. Consistent with this, polymorphisms

Dendritic cell IRF-7

in the IRF-3 gene associate with symptomatic WNV infec-

/ / Other IRF?

tion in humans [62]. In both Irf3 and Irf7 mice,

enhanced viral burden is observed in peripheral tissues

(spleen, kidney, blood, and lymph nodes), which leads to

entry and sustained replication in the CNS. Analysis of IFN-

serum from Irf3 mice infected with WNV or WNV-like

IRF-3

particles has established a dispensable role of IRF-3 in Epithelial cell

IRF-7

regulating systemic production of type I IFN [22,63]. By

contrast, WNV-infected Irf7 mice show blunted type I

IFN responses [61]. The IRF-7-dependent production of

IFN in blood appears independent of TLR7, because levels

/ IRF-3

are not decreased in Tlr7 mice after WNV infection [56].

Fibroblast

Thus, it remains uncertain as to which cell type regulates IRF-7

systemic type I IFN production after WNV infection.

Recent studies have investigated the antiviral role of

IRF-1 in WNV infection, because it was originally de- TRENDS in Immunology

scribed as a regulator of type I IFN responses after virus

/ Figure 2. Induction of interferon (IFN)-b after West Nile virus (WNV) infection

infection [64]. Irf1 mice are vulnerable to lethal WNV

utilizes a different complement of IFN regulatory factor (IRF) transcription factors in

infection with enhanced viral replication in peripheral distinct cell types. In macrophages and myeloid dendritic cells (mDCs), IFN-b is

induced at least partially through IFN promoter stimulator (IPS)-1-dependent yet

tissues and rapid dissemination into the CNS [65]. IRF-

IRF-3- and IRF-7-independent pathways. In macrophages, IFN-b gene expression

1, in contrast to IRF-3 and IRF-7, also has an independent

utilizes IRF-1, whereas in mDCs, another transcription factor (possibly IRF-5) has a

effect on CD8 T cell expansion. Although markedly fewer complementing role to IRF-3 and IRF-7. The smaller text size in the Figure suggests

+ /

a subdominant role of these ancillary transcription factors compared to IRF-3 and

CD8 T cells are observed in naı¨ve animals, Irf1 mice

+ IRF-7. In comparison, in epithelial cells and fibroblasts loss of IRF-3 and IRF-7

rapidly expand their pool of WNV-speciﬁc cytolytic CD8 T

completely ablates IFN-b gene induction after WNV infection.

cells. Thus, IRF-1 restricts WNV infection by modulating

the expression of innate antiviral effector molecules while pathway [67]. Thus, at least for some macrophage subsets,

shaping the antigen-speciﬁc CD8 T cell response. the type I IFN response appears to be mediated through

the combination of IRF-1, IRF-3 and IRF-7 function.

Cell-speciﬁc innate defenses DCs have primary roles in regulating innate and adap-

Macrophages are susceptible to WNV infection [15] and are tive immune responses against viruses, and also are tar-

more permissive in the absence of IFN signaling [1]. In gets for WNV infection in vivo [57,68]. Although pDCs

macrophages, IRF-3 has been identiﬁed as an essential potently produce type I IFN after WNV infection [20],

regulator of the basal expression of host defense molecules other DC subsets are major sources of type I IFN in

including interferon induced tetratricopeptide repeat pro- response to viral infection [69]. Indeed, mDCs, when pro-

tein-1 (IFIT-1), IFIT-2, RIG-I and MDA5, potentially con- ductively infected with WNV, secrete large amounts of type

trolling the permissiveness of this cell type for WNV. The I IFN [20,70], and impaired IFN signaling in DCs is

contribution of IRF-3 and IRF-7 in macrophages has been observed in older human donors infected with WNV [23].

more fully established in subsequent studies with Irf7 The IRF-3- and IRF-7-dependent regulation of type I IFN

/ /

or Irf3 Irf7 double knockout (DKO) cells. Although gene induction in mDCs differs compared to that seen with

a deﬁciency of IRF-7 completely abrogated the IFN-a macrophages. Although the IFN-a response is analogously

response, no effect on IFN-b gene induction was observed regulated by IRF-7, the IFN-b response in mDCs is largely

/ /

in Irf7 cells. Analysis of the IFN-b response in Irf3 unaffected by the absence of IRF-1, IRF-3 or IRF-7, or a

Irf7 DKO macrophages revealed an unexpected ﬁnding: combination of IRF-3 and IRF-7 [41] (Figure 2). These

this response was completely abolished at early time points ﬁndings may reﬂect an undocumented yet important role

after WNV infection but was not substantially altered at of IRF-5 or other transcription factors in transmitting an

late time points [41]. Thus, IRF-3 and IRF-7 only partially IPS-1-dependent signal downstream of RLR recognition of

regulate the IFN-b gene and ISG expression in macro- viral RNA to induce IFN-b expression in speciﬁc cell types

phages. Consistent with this, Irf1 macrophages support such as DCs.

enhanced WNV replication and show diminished cell-in-

trinsic innate immune responses, and ectopic expression of ISGs that control WNV infection

IRF-1 inhibits WNV infection in transformed cells [65,66]. Progress has been made recently in deﬁning the speciﬁc

Moreover, IRF-1 gene expression in WNV-infected cells ISGs that limit WNV infection. Systematic investigation of

can be induced through an IRF-3 -and IRF-7-independent the antiviral functions of large groups of ISGs using ectopic

526

Review Trends in Immunology October 2012, Vol. 33, No. 10

Table 1. List of ISGs with reported antiviral effector activity against infection by WNV and other ﬂaviviruses.

ISG Activity Mechanism of action Flavivirus Refs

PKR Mice, primary cells Translation inhibition? WNV [44,47,71]

Induction of IFN

RNase L Mice, primary cells Viral RNA degradation? Generation of PAMP WNV [47,74]

0 0

Oas1b Mice, primary cells Reduction in 2 -5 oligoA production WNV, other ﬂaviviruses [75,76]

Viperin (rsad2) Mice, primary cells, ectopic Modulation of lipid biosynthesis or WNV, dengue virus [71,83]

expression droplet formation?

IFIT1 Mice, primary cells Translation inhibition? WNV [3,89]

IFIT2 Ectopic expression Translation inhibition? WNV [3]

IFITM2 Ectopic expression, primary cells Inhibition of entry? WNV, dengue virus [71,90]

IFITM3 Ectopic expression, primary cells Inhibition of entry? WNV, yellow fever [66,71,87,90]

ISG20 Ectopic expression Exonuclease? WNV, yellow fever [71,88]

MB21D1 Ectopic expression Unknown WNV [66]

HPSE Ectopic expression Unknown WNV, yellow fever [66]

NAMPT Ectopic expression Unknown WNV [66]

TRIM79a Ectopic expression, primary cells Degrades viral RNA polymerase Tick-borne encephalitis [91]

Note, ISGs that inhibit ﬂaviviruses other than WNV and their corresponding references are added for completeness but not necessarily discussed in the text.

gene or short hairpin RNA (shRNA) screens [66,71] has [82], where it is believed to inhibit infection of several

identiﬁed new genes and gene families that restrict infec- viruses by modulating cholesterol and isoprenoid biosyn-

tion of ﬂaviviruses, including WNV (Table 1). thesis, lipid raft formation, and the composition and locali-

0 0

Initial studies have shown that 2 -5 -oligoadenylate zation of lipid droplets. Viperin originally was identiﬁed as

synthase (Oas) proteins mediate intrinsic cell resistance a possible inhibitor of WNV infection in ectopic expression

0 0

to WNV. RNase L is activated by 2 -5 -linked oligoadeny- studies in cell culture [71]. Subsequent in vivo studies have

lates that are synthesized by Oas enzymes, therefore, conﬁrmed its antiviral potential, because Rsad2 mice

several groups have assessed its antiviral function in infected with WNV show increased lethality and enhanced

the context of WNV infection. RNase L inhibits viral viral replication in CNS tissues [83].

infections by cleaving viral RNA [72] and by generating Members of the IFIT family of genes (Iﬁt1, Iﬁt2, and

small self-RNA that amplify immune responses through an Iﬁt3 in mice) also probably contribute to the control of

RLR-dependent pathway [73]. Rnasel macrophages and WNV infection. Initial studies have suggested that IFIT

MEFs supported increased WNV replication [47,74] and a proteins exert their antiviral function by inhibiting protein

deﬁciency of RNase L partially rescued a WNV mutant translation through interaction with subunits of transla-

deﬁcient in the production of a noncoding subgenomic tion initiation factor eIF3 (reviewed in [84]). In cell culture

flavivirus RNA (sfRNA) [6]. Mice deficient in RNase L and mouse models of infection, WNV strongly induces Ifit1

showed moderately increased lethality following WNV gene expression in target cells via IFN-dependent and -

infection, with higher viral loads in peripheral tissues at independent signaling pathways [22,85]. Interestingly, a

early time points [47]. WNV mutant with a site-speciﬁc substitution in the NS5

Although susceptibility to ﬂaviviruses in mice has been gene (WNV-E218A) that abolishes 2 -O-methyltransferase

mapped to a mutation in Oas1b, resulting in the expression activity [86] shows wild-type levels of replication in trans-

of a truncated protein [75,76], the antiviral mechanism by formed Vero and BHK21 cells, but attenuated infection in

this gene remains unknown but appears independent of primary macrophages and wild-type mice [3]. Replication

RNase L [74,77]. Genetic variation in OAS1 also has been of WNV-E218A is rescued in the absence of IFNAR or Iﬁt1

0 0

suggested as a risk factor for initial infection with WNV in [3], therefore, 2 -O methylation of the 5 cap of WNV RNA

humans [78]. Knock-in of the wild-type Oas1b allele into a functions to subvert host antiviral responses through es-

ﬂavivirus-induced disease susceptible mouse has generat- cape of Iﬁt1-mediated suppression. Differential methyla-

ed a resistant phenotype [79], and murine cells that ectop- tion of cytoplasmic RNA probably serves as an example for

ically express wild-type Oas1b resist WNV infection by pattern recognition and restriction of propagation of some

preventing viral RNA accumulation [80]. Although bio- foreign viral RNA in host cells.

chemical studies have shown that Oas1b itself is an inac- Additional ISGs have been suggested to inhibit WNV

0 0

tive 2 -5 Oas, recent experiments have suggested that infection based on studies in cell culture. IFITM3 has

0 0

Oas1b inhibits Oas1a activity, resulting in reduced 2 -5 recently been shown to inhibit an early entry step in

0 0

oligoA production [77]. Negative regulation of 2 -5 Oas by infection of WNV in cells [87]; an observation that has

inactive Oas1b proteins may ﬁne-tune the RNase L re- been conﬁrmed by ectopic expression studies in HEK293

sponse that could cause signiﬁcant damage in cells, if it and STAT1 cells [66,71]. ISG20 also has been proposed

were not tightly controlled. to inhibit infection of ﬂaviviruses, including WNV [71,88].

The ISG viperin (rsad2) can inhibit WNV infection in Newer genetic screens have identiﬁed additional candidate

cells and in vivo. Viperin is induced in many cell types after ISGs [C6orf150 (Mab-21 domain-containing protein

exposure to type I and II IFN, TLR agonists, or virus (MB21D1)), heparanase (HPSE), and nicotinamide phos-

infection [81]. Viperin localizes to the cytosolic face of phoribosyltransferase (NAMPT)] with anti-WNV activity

the endoplasmic reticulum via an amphipathic a-helix [66,71]. Although the ﬁeld is rapidly advancing with

527

Review Trends in Immunology October 2012, Vol. 33, No. 10

beta interferon induction and attenuates virus virulence in mice. J.

respect to identifying putative antiviral ISG against WNV

Virol. 80, 2396–2404

and other ﬂaviviruses, future studies in genetically deﬁ-

6 Schuessler, A. et al. (2012) West Nile virus non-coding subgenomic

cient animals will be needed to establish the cell- and

RNA contributes to viral evasion of type I interferon-mediated antiviral

tissue-speciﬁc nonredundant effects and hierarchy in con- response. J. Virol. 86, 5708–5718

trolling infection in the context of a type I IFN response. 7 Winkelmann, E.R. et al. (2012) Intrinsic adjuvanting of a novel single-

cycle ﬂavivirus vaccine in the absence of type I interferon receptor

signaling. Vaccine 30, 1465–1475

Concluding remarks

8 Liu, W.J. et al. (2005) Inhibition of interferon signaling by the New

The innate immune response of the host is programmed to

York 99 strain and kunjin subtype of West Nile virus involves blockage

control viral replication and limit spread by recognizing of STAT1 and STAT2 activation by nonstructural proteins. J. Virol. 79,

non-self nucleic acid as PAMPs and triggering an antiviral 1934–1942

9 Perwitasari, O. et al. (2011) Inhibitor of kappaB kinase {epsilon}

response. Although type I IFN was discovered more than

(IKK{epsilon}), STAT1, and IFIT2 proteins deﬁne novel innate

50 years ago, only recently have the mechanisms by which

immune effector pathway against West Nile virus infection. J. Biol.

these molecules are induced, signal, and produce an anti-

Chem. 286, 44412–44423

viral effect been delineated. The generation and distribu- 10 Ben-Nathan, D. et al. (1996) West Nile virus neuroinvasion and

tion of mice with targeted deletions in PRRs, transcription encephalitis induced by macrophage depletion in mice. Arch. Virol.

141, 459–469

factors, IFN signaling or effector genes has allowed the

11 Purtha, W.E. et al. (2008) Early B-cell activation after West Nile virus

ﬁeld to dissect and evaluate the function of individual host

infection requires alpha/beta interferon but not antigen receptor

defense genes in the context of infection by speciﬁc viruses. signaling. J. Virol. 82, 10964–10974

WNV is a zoonotic pathogen that readily infects mice, and 12 Lin, Y.L. et al. (1997) Inhibition of Japanese encephalitis virus

infection by nitric oxide: antiviral effect of nitric oxide on RNA virus

thus has afforded a unique perspective on the interface

replication. J. Virol. 71, 5227–5235

between the mammalian cell-intrinsic innate host re-

13 Saxena, S.K. et al. (2000) Antiviral effect of nitric oxide during

sponse and viral pathogenesis. In the past few years, the

Japanese encephalitis virus infection. Int. J. Exp. Pathol. 81, 165–172

field has identified the specific PRRs and signaling path- 14 Shrestha, B. et al. (2008) Tumor necrosis factor alpha protects against

ways that detect entry and infection by WNV and initiate a lethal West Nile virus infection by promoting trafﬁcking of

mononuclear leukocytes into the central nervous system. J. Virol.

protective IFN response. Over the next ﬁve years, we

82, 8956–8964

anticipate that several important questions will be an-

15 Kong, K.F. et al. (2008) West nile virus attenuates activation of primary

swered. These include: (i) identiﬁcation of the speciﬁc

human macrophages. Viral Immunol. 21, 78–82

PAMPs on WNV that are recognized by PRRs; (ii) charac- 16 Rios, M. et al. (2006) Monocytes-macrophages are a potential target in

terization of a hierarchy of ISGs that function to control human infection with West Nile virus through blood transfusion.

Transfusion 46, 659–667

different stages of WNV infection in distinct cell types and

17 Swiecki, M. et al. (2010) Plasmacytoid dendritic cell ablation impacts

tissues; (iii) the role of viral proteins, viral RNA structural

early interferon responses and antiviral NK and CD8(+) T cell accrual.

elements, and viral noncoding RNA in modulating cell-

Immunity 33, 955–966

intrinsic innate immune responses to WNV; and (iv) the 18 Sun, P. et al. (2009) Functional characterization of ex vivo blood

basis for WNV virulence, and how it interfaces with recog- myeloid and plasmacytoid dendritic cells after infection with dengue

virus. Virology 383, 207–215

nition or evasion of cell-intrinsic immune pathways. As

19 Wang, J.P. et al. (2006) Flavivirus activation of plasmacytoid dendritic

these topics are explored, the ﬁeld undoubtedly will gain

cells delineates key elements of TLR7 signaling beyond endosomal

new insight into fundamental cellular responses as well as recognition. J. Immunol. 177, 7114–7121

mechanisms of viral pathogenesis. This work may promote 20 Silva, M.C. et al. (2007) Differential activation of human monocyte-

derived and plasmacytoid dendritic cells by West Nile virus generated

novel strategies for development of therapeutic agents that

in different host cells. J. Virol. 81, 13640–13648

activate speciﬁc innate immune processes or effector mole-

21 Steinbrink, K. et al. (2009) Myeloid dendritic cell: from sentinel of

cules to contain spread and disease of WNV, and probably

immunity to key player of peripheral tolerance? Hum. Immunol. 70,

many other viruses. 289–293

22 Dafﬁs, S. et al. (2007) Cell-speciﬁc IRF-3 responses protect against

Acknowledgments West Nile virus infection by interferon-dependent and independent

mechanisms. PLoS Pathog. 3, e106

The authors would like to acknowledge support from the National

23 Qian, F. et al. (2011) Impaired interferon signaling in dendritic cells

Institutes of Health for support of work in their laboratories (U54

from older donors infected in vitro with West Nile virus. J. Infect. Dis.

AI081680 and U54 AI057160 (Paciﬁc Northwest and Midwest Regional

203, 1415–1424

Center of Excellence for Biodefense and Emerging Infectious Diseases

24 Hildner, K. et al. (2008) Batf3 deﬁciency reveals a critical role for

Research), U19 AI083019, and R01 AI074973.

CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 322, 1097–1100

References

25 Davison, A.M. and King, N.J. (2011) Accelerated dendritic cell

1 Samuel, M.A. and Diamond, M.S. (2005) Type I IFN protects against

differentiation from migrating Ly6C(lo) bone marrow monocytes

lethal West Nile Virus infection by restricting cellular tropism and

in early dermal West Nile virus infection. J. Immunol. 186, 2382–

enhancing neuronal survival. J. Virol. 79, 13350–13361

2396

2 Keller, B.C. et al. (2006) Resistance to alpha/beta interferon is a

26 Loo, Y.M. and Gale, M., Jr (2011) Immune signaling by RIG-I-like

determinant of West Nile virus replication ﬁtness and virulence. J

receptors. Immunity 34, 680–692

Virol. 80, 9424–9434

0 27 Schlee, M. et al. (2009) Approaching the RNA ligand for RIG-I?

3 Dafﬁs, S. et al. (2010) 2 -O methylation of the viral mRNA cap evades

Immunol. Rev. 227, 66–74

host restriction by IFIT family members. Nature 468, 452–456

28 Yoneyama, M. and Fujita, T. (2008) Structural mechanism of RNA

4 Dafﬁs, S. et al. (2011) The naturally attenuated Kunjin strain of West

recognition by the RIG-I-like receptors. Immunity 29, 178–181

Nile virus shows enhanced sensitivity to the host type I interferon

29 Berke, I.C. and Modis, Y. (2012) MDA5 cooperatively forms dimers and

response. J. Virol. 85, 5664–5668

ATP-sensitive ﬁlaments upon binding double-stranded RNA. EMBO J.

5 Liu, W.J. et al. (2006) A single amino acid substitution in the West Nile

31, 1714–1726

virus nonstructural protein NS2A disables its ability to inhibit alpha/

528

Review Trends in Immunology October 2012, Vol. 33, No. 10

30 Gack, M.U. et al. (2007) TRIM25 RING-ﬁnger E3 ubiquitin ligase is 57 Welte, T. et al. (2009) Toll-like receptor 7-induced immune response to

essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 cutaneous West Nile virus infection. J. Gen. Virol. 90, 2660–2668

31 Gack, M.U. et al. (2008) Roles of RIG-I N-terminal tandem CARD and 58 Szretter, K.J. et al. (2010) The innate immune adaptor molecule

splice variant in TRIM25-mediated antiviral signal transduction. Proc. MyD88 restricts West Nile replication and spread in neurons of the

Natl. Acad. Sci. U.S.A. 105, 16743–16748 central nervous system. J. Virol. 84, 12125–12138

32 Hou, F. et al. (2011) MAVS forms functional prion-like aggregates to 59 Tamura, T. et al. (2008) The IRF family transcription factors in

activate and propagate antiviral innate immune response. Cell 146, immunity and oncogenesis. Annu. Rev. Immunol. 26, 535–584

448–461 60 Honda, K. and Taniguchi, T. (2006) IRFs: master regulators of

33 Horner, S.M. et al. (2011) Mitochondrial-associated endoplasmic signalling by Toll-like receptors and cytosolic pattern-recognition

reticulum membranes (MAM) form innate immune synapses and receptors. Nat. Rev. Immunol. 6, 644–658

are targeted by hepatitis C virus. Proc. Natl. Acad. Sci. U.S.A. 108, 61 Dafﬁs, S. et al. (2008) Interferon regulatory factor IRF-7 induces the

14590–14595 antiviral alpha interferon response and protects against lethal West

34 Dixit, E. et al. (2010) Peroxisomes are signaling platforms for antiviral Nile virus infection. J. Virol. 82, 8465–8475

innate immunity. Cell 141, 668–681 62 Bigham, A.W. et al. (2011) Host genetic risk factors for West Nile virus

35 Wang, P. et al. (2010) Caspase-12 controls West Nile virus infection via infection and disease progression. PLoS ONE 6, e24745

the viral RNA receptor RIG-I. Nat. Immunol. 11, 912–919 63 Bourne, N. et al. (2007) Early production of type I interferon during

36 Liu, H.M. et al. (2012) The mitochondrial targeting chaperone 14-3-3e West Nile virus infection: role for lymphoid tissues in IRF3-

regulates a RIG-I translocon that mediates membrane association and independent interferon production. J. Virol. 81, 9100–9108

innate antiviral immunity. Cell Host Microbe 11, 1–10 64 Fujita, T. et al. (1989) Induction of endogenous IFN-alpha and IFN-

37 Loo, Y.M. et al. (2008) Distinct RIG-I and MDA5 signaling by RNA beta genes by a regulatory transcription factor, IRF-1. Nature 337,

viruses in innate immunity. J. Virol. 82, 335–345 270–272

38 Fredericksen, B.L. and Gale, M., Jr (2006) West Nile virus evades 65 Brien, J.D. et al. (2011) Interferon regulatory factor-1 (IRF-1) shapes

activation of interferon regulatory factor 3 through RIG-I-dependent both innate and CD8 T cell immune responses against West Nile virus

and -independent pathways without antagonizing host defense infection. PLoS Pathog. 7, e1002230

signaling. J. Virol. 80, 2913–2923 66 Schoggins, J.W. et al. (2011) A diverse range of gene products are

39 Fredericksen, B.L. et al. (2008) Establishment and maintenance of the effectors of the type I interferon antiviral response. Nature 472, 481–

innate antiviral response to West Nile virus involves both RIG-I and 485

MDA5 signaling through IPS-1. J. Virol. 82, 609–616 67 Pulit-Penaloza, J.A. et al. (2012) Type 1 IFN-independent activation of

40 Kato, H. et al. (2006) Differential roles of MDA5 and RIG-I helicases in a subset of interferon stimulated genes in West Nile virus Eg101-

the recognition of RNA viruses. Nature 441, 101–105 infected mouse cells. Virology 425, 82–94

41 Dafﬁs, S. et al. (2009) Induction of IFN-beta and the innate antiviral 68 Martina, B.E. et al. (2008) DC-SIGN enhances infection of cells with

response in myeloid cells occurs through an IPS-1-dependent signal glycosylated West Nile virus in vitro and virus replication in human

that does not require IRF-3 and IRF-7. PLoS Pathog. 5, e1000607 dendritic cells induces production of IFN-alpha and TNF-alpha. Virus

42 Suthar, M.S. et al. (2010) IPS-1 is essential for the control of West Nile Res. 135, 64–71

virus infection and immunity. PLoS Pathog. 6, e1000757 69 Fitzgerald-Bocarsly, P. and Feng, D. (2007) The role of type I interferon

43 Schulz, O. et al. (2010) Protein kinase R contributes to immunity production by dendritic cells in host defense. Biochimie 89, 843–855

against speciﬁc viruses by regulating interferon mRNA integrity. 70 Dafﬁs, S. et al. (2009) Measure and countermeasure: type I IFN (IFN-

Cell Host Microbe 7, 354–361 alpha/beta) antiviral response against West Nile virus. J. Innate

44 Gilfoy, F.D. and Mason, P.W. (2007) West Nile virus-induced IFN Immun. 1, 435–445

production is mediated by the double-stranded RNA-dependent 71 Jiang, D. et al. (2010) Identiﬁcation of ﬁve interferon-induced cellular

protein kinase, PKR. J. Virol. 81, 11148–11158 proteins that inhibit west nile virus and dengue virus infections. J.

45 Elbahesh, H. et al. (2011) West Nile virus infection does not induce PKR Virol. 84, 8332–8341

activation in rodent cells. Virology 421, 51–60 72 Zhou, A. et al. (1997) Interferon action and apoptosis are defective in

0 0

46 Courtney, S.C. et al. (2012) West nile virus infections suppress early mice devoid of 2 ,5 - oligoadenylate-dependent RNase L. EMBO J. 16,

viral RNA synthesis and avoid inducing the cell stress granule 6355–6363

response. J. Virol. 86, 3647–3657 73 Malathi, K. et al. (2007) Small self-RNA generated by RNase L

47 Samuel, M.A. et al. (2006) PKR and RNAse L contribute to protection ampliﬁes antiviral innate immunity. Nature 448, 816–819

against lethal West Nile virus infection by controlling early viral 74 Scherbik, S.V. et al. (2006) RNase L plays a role in the antiviral

spread in the periphery and replication in neurons. J. Virol. 80, response to West Nile virus. J. Virol. 80, 2987–2999

7009–7019 75 Perelygin, A.A. et al. (2002) Positional cloning of the murine ﬂavivirus

48 Vercammen, E. et al. (2008) Sensing of viral infection and activation of resistance gene. Proc. Natl. Acad. Sci. U.S.A. 99, 9322–9327

innate immunity by toll-like receptor 3. Clin. Microbiol. Rev. 21, 13–25 76 Mashimo, T. et al. (2002) A nonsense mutation in the gene encoding 2 -

49 Wang, T. et al. (2004) Toll-like receptor 3 mediates West Nile virus entry 5 -oligoadenylate synthetase/L1 isoform is associated with West Nile

into the brain causing lethal encephalitis. Nat. Med. 10, 1366–1373 virus susceptibility in laboratory mice. Proc. Natl. Acad. Sci. U.S.A. 99,

50 Dafﬁs, S. et al. (2008) Toll-like receptor 3 has a protective role against 11311–11316

West Nile virus infection. J. Virol. 82, 10349–10358 77 Elbahesh, H. et al. (2011) The Flvr-encoded murine oligoadenylate

51 Town, T. et al. (2006) Microglia recognize double-stranded RNA via synthetase 1b (Oas1b) suppresses 2-5A synthesis in intact cells.

TLR3. J. Immunol. 176, 3804–3812 Virology 409, 262–270

52 Kim, H. et al. (2008) Double-stranded RNA mediates interferon 78 Lim, J.K. et al. (2009) Genetic variation in OAS1 is a risk factor for

regulatory factor 3 activation and interleukin-6 production by initial infection with West Nile virus in man. PLoS Pathog. 5, e1000321

engaging Toll-like receptor 3 in human brain astrocytes. Immunology 79 Scherbik, S.V. et al. (2007) Knock-in of the Oas1b(r) allele into a

124, 480–488 ﬂavivirus-induced disease susceptible mouse generates the resistant

53 Scholle, F. and Mason, P.W. (2005) West Nile virus replication phenotype. Virology 368, 232–237

0 0

interferes with both poly(I:C)-induced interferon gene transcription 80 Kajaste-Rudnitski, A. et al. (2006) The 2 ,5 -oligoadenylate synthetase

and response to interferon treatment. Virology 342, 77–87 1b is a potent inhibitor of West Nile virus replication inside infected

54 Wilson, J.R. et al. (2008) West Nile virus nonstructural protein 1 cells. J. Biol. Chem. 281, 4624–4637

inhibits TLR3 signal transduction. J. Virol. 82, 8262–8271 81 Fitzgerald, K.A. (2011) The interferon inducible gene: viperin. J.

55 Baronti, C. et al. (2010) Nonstructural NS1 proteins of several Interferon Cytokine Res. 31, 131–135

mosquito-borne ﬂavivirus do not inhibit TLR3 signaling. Virology 82 Hinson, E.R. and Cresswell, P. (2009) The antiviral protein, viperin,

404, 319–330 localizes to lipid droplets via its N-terminal amphipathic alpha-helix.

56 Town, T. et al. (2009) Toll-like receptor 7 mitigates lethal West Nile Proc. Natl. Acad. Sci. U.S.A. 106, 20452–20457

encephalitis via interleukin 23-dependent immune cell inﬁltration and 83 Szretter, K.J. et al. (2011) The interferon-inducible gene viperin

homing. Immunity 30, 242–253 restricts West Nile virus pathogenesis. J. Virol. 85, 11557–11566

529

Review Trends in Immunology October 2012, Vol. 33, No. 10

84 Fensterl, V. and Sen, G.C. (2011) The ISG56/IFIT1 gene family. J. 90 Chan, Y.K. et al. (2012) IFITM proteins restrict antibody-

Interferon Cytokine Res. 31, 71–78 dependent enhancement of Dengue virus Infection. PLoS ONE 7,

85 Wacher, C. et al. (2007) Coordinated regulation and widespread e34508

cellular expression of interferon-stimulated genes (ISG) ISG-49, 91 Taylor, R.T. et al. (2011) TRIM79alpha, an interferon-stimulated

ISG-54, and ISG-56 in the central nervous system after infection gene product, restricts tick-borne encephalitis virus replication

with distinct viruses. J. Virol. 81, 860–871 by degrading the viral RNA polymerase. Cell Host Microbe 10,

86 Zhou, Y. et al. (2007) Structure and function of ﬂavivirus NS5 185–196

methyltransferase. J. Virol. 81, 3891–3903 92 Evans, J.D. et al. (2011) West Nile virus infection induces depletion of

87 Brass, A.L. et al. (2009) The IFITM proteins mediate cellular resistance IFNAR1 protein levels. Viral Immunol. 24, 253–263

to inﬂuenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139, 93 Guo, J.T. et al. (2005) West Nile virus inhibits the signal transduction

1243–1254 pathway of alpha interferon. J. Virol. 79, 1343–1350

88 Zhou, Z. et al. (2011) Antiviral activities of ISG20 in positive-strand 94 Mackenzie, J.M. et al. (2007) Cholesterol manipulation by West Nile

RNA virus infections. Virology 409, 175–188 virus perturbs the cellular immune response. Cell Host Microbe 2, 229–

89 Szretter, K.J. et al. (2012) 2 -O methylation of the viral mRNA cap by 239

West Nile virus evades Iﬁt1-dependent and -independent mechanisms

of host restriction in vivo. PLoS Pathog. 8, e1002698

530 RESEARCH

Lineage 1 and 2 Strains of Encephalitic West Nile Virus, Central Europe Tamás Bakonyi,*† Éva Ivanics,‡ Károly Erdélyi,‡ Krisztina Ursu,‡ Emöke Ferenczi,§ Herbert Weissenböck,* and Norbert Nowotny*¶

Two different West Nile virus (WNV) strains caused influenzalike symptoms or no apparent disease (2); lethal encephalitis in a flock of geese and a goshawk in encephalitis and fatalities in the human population, horses, southeastern Hungary in 2003 and 2004, respectively. or poultry are spasmodic (3,8,9). In the New World, WNV During the outbreak in geese, 14 confirmed human cases exhibits increased virulence among the local wild bird of WNV encephalitis and meningitis were reported in the populations and causes more frequent severe central nerv- same area. Sequencing of complete genomes of both WNV strains and phylogenetic analyses showed that the goose- ous system symptoms and deaths in humans and horses derived strain exhibits closest genetic relationship to strains (6,10). Although exactly how WNV was introduced into isolated in 1998 in Israel and to the strain that emerged in New York is unclear, phylogenetic comparison of the viral 1999 in the United States. WNV derived from the goshawk nucleic acid sequences has shown a close relationship showed the highest identity to WNV strains of lineage 2 iso- between the American WNV isolates and strains isolated lated in central Africa. The same strain reemerged in 2005 from encephalitic geese and storks in Israel in 1998 in the same location, which suggests that the virus may (11–13). Experimental infections of rodents indicated that have overwintered in Europe. The emergence of an exotic the neurovirulence of WNV correlates with its genotype, WNV strain in Hungary emphasizes the role of migrating and the North American strains are highly neurovirulent birds in introducing new viruses to Europe. for mice (14). WNV shows relatively high levels of sequence diversi- eographically, West Nile virus (WNV) is the most ty. Comprehensive studies on the phylogenetic relatedness Gwidespread member of the Japanese encephalitis of WNV strains show that they form at least 2 main line- virus (JEV) complex within the genus Flavivirus and the ages (15–17). Lineage 1 is composed of WNV strains from family Flaviviridae. The first strain (B 956) was isolated different geographic regions, and it is subdivided into at from a human patient in the West Nile district of Uganda least 3 clades. Clade A contains strains from Europe, in 1937; later the virus was also detected in several mos- Africa, the Middle East, and America; clade B represents quito species, horses, humans, and other hosts in Africa, the Australian (Kunjin) strains; and clade C contains Europe, Asia, and Australia (where it has been named Indian WNV isolates. Lineage 2 contains the B 956 proto- Kunjin virus) (1–3). WNV was introduced into the United type strain and other strains isolated so far exclusively in States in 1999, and it spread quickly over large parts of sub-Saharan Africa and Madagascar. In addition to the 2 North America and reached Mexico (4–7). The clinical major WNV lineages, we recently proposed 2 lineages for impact of WNV varies in different regions. In the Old viruses that exhibited considerable genetic differences to World, WNV causes relatively mild infections with the known WNV lineages: lineage 3 consists of a virus strain isolated from Culex pipiens mosquitoes at the Czech Republic/Austria border (named Rabensburg virus), and *University of Veterinary Medicine, Vienna, Austria; †Szent István lineage 4 consists of a unique virus isolated in the University, Budapest, Hungary; ‡Central Veterinary Institute, Budapest, Hungary; §“Béla Johan” National Center for Caucasus. These 2 viruses, however, may also be consid- Epidemiology, Budapest, Hungary; and ¶United Arab Emirates ered independent flaviviruses within the JEV complex University, Al Ain, United Arab Emirates (18).

618 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 Encephalitic West Nile Virus, Central Europe

WNV has been known to be present in central Europe cleoside triphosphate, 10 U RNasin RNase Inhibitor for a long time. Seroprevalence in humans was reported in (Promega, Madison, WI, USA), 20 pmol of the genomic several countries, including Hungary, and WNV strains and reverse primers, 1 µL enzyme mix (containing were isolated from mosquitoes, humans, migrating birds, Omniscript and Sensiscript Reverse Transcriptases and and rodents during the last 30 years (3). Until 2003, how- HotStarTaq DNA polymerase) and 2.5 µL template RNA. ever, WNV infections in Hungary have never been associ- Reverse transcription was carried out at 50°C for 30 min, ated with clinical symptoms, although a severe outbreak of followed by a denaturation step at 95°C for 15 min. West Nile encephalitis in humans was reported in 1996 and Thereafter, the cDNA was amplified in 40 cycles of heat 1997 in neighboring Romania. denaturation at 94°C for 40 s, primer annealing at 57°C for In late summer 2003, an outbreak of encephalitis 50 s, and DNA extension at 72°C for 1 min, and the reac- emerged in a Hungarian goose flock, resulting in a 14% tion was completed by a final extension for 7 min at 72°C. death rate among 6-week-old geese (Anser anser domesti- Reactions were performed in a Perkin-Elmer GeneAmp cus). Based on histopathologic alterations, serologic inves- PCR System 2400 thermocycler (Wellesley, MA, USA) tigations, and nucleic acid detection by reverse and in a Hybaid PCR Sprint thermocycler (Thermo transcription–polymerase chain reaction (RT-PCR), WNV Electron Corporation, Waltham, MA, USA). was diagnosed as the cause of the disease (19). After RT-PCR, 10 µL of the amplicons was subjected to Chronologically and geographically related to the outbreak electrophoresis in a 1.2% Tris acetate-EDTA-agarose gel in geese, a serologically confirmed WNV outbreak was at 5 V/cm for 80 min. The gel was stained with ethidium also observed in humans, which involved 14 cases of mild bromide; bands were visualized under UV light and pho- encephalitis and meningitis (20). tographed with a Kodak DS Electrophoresis One year later, in August 2004, a goshawk (Accipiter Documentation and Analysis System using the Kodak gentilis) fledgling showed central nervous system symp- Digital Science 1D software program (Eastman Kodak toms and died in a national park in southeastern Hungary. Company, Rochester, NY, USA). Product sizes were deter- When histopathologic methods and RT-PCR were used, mined with reference to a 100-bp DNA ladder (Promega). WNV antigen and nucleic acid were detected in the organs Where clear PCR products of the previously calculated of the bird. Furthermore, the virus was isolated after injec- sizes were observed, the fragments were excised from the tion of suckling mice. Here we report the sequencing and gel, and DNA was extracted by using the QIAquick Gel phylogenetic results of these 2 encephalitic WNV strains Extraction Kit (Qiagen). Fluorescence-based direct that emerged recently in central Europe. sequencing was performed in both directions on PCR products. Sequencing of PCR products was carried out Materials and Methods with the ABI Prism Big Dye Terminator cycle sequencing Brain specimens from one 6-week-old goose, which ready reaction kit (Perkin-Elmer), according to the manu- died during the encephalitis outbreak in a Hungarian goose facturer’s instructions, and an ABI Prism 310 genetic ana- flock, and brain samples from a goshawk, which also died lyzer (Perkin-Elmer) automated sequencing system. from encephalitis, were used for WNV nucleic acid deter- Nucleotide sequences were identified by Basic Local mination. The brain samples were homogenized in ceram- Alignment Search Tool (BLAST, www.ncbi.nlm.nih. ic mortars by using sterile quartz sand, and the gov/blast) search against gene bank databases. Based on homogenates were suspended in RNase-free distilled the sequence information obtained from the amplification water. Samples were stored at –80°C until nucleic acid products, complete WNV sequences that exhibited the extraction was performed. highest nucleotide identities with the Hungarian genotypes Viral RNA was extracted from 140 µL of brain were selected from the GenBank database to design homogenates by using the QIAamp viral RNA Mini Kit primers that amplify overlapping RT-PCR products cover- (Qiagen, Hilden, Germany) according to the manufactur- ing the entire genome of the strains. Oligonucleotide er’s instructions. First, a universal JEV-group specific primers were designed with the help of the Primer oligonucleotide primer pair designed on the nonstructural Designer 4 for Windows 95 (Scientific and Educational protein 5 (NS5) and 3′-untranslated regions (UTR) of Software, Version 4.10; Microsoft, Redmond, WA, USA) WNV (forward primer: 5′-GARTGGATGACVACRGAA- and were synthesized by GibcoBRL Life Technologies, GACATGCT-3′ and reverse primer: 5′-GGGGTCTCCTC- Ltd. (Paisley, Scotland, UK). Detailed information on all TAACCTCTAGTCCTT-3′; [21]) was applied on the RNA primers is available as an online appendix (http://www. extracts in a continuous RT-PCR system employing the cdc.gov/ncidod/EID/vol12no04/05-1379_app.htm). PCR QIAGEN OneStep RT-PCR Kit (Qiagen). Each 25-µL amplification products were directly sequenced in both µ × reaction mixture contained 5 L of 5 buffer (final MgCl2 directions; the sequences were compiled and aligned to concentration 2.5 mmol/L), 0.4 mmol/L of each deoxynu- complete genome sequences of selected representatives of

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 619 RESEARCH

WNV lineages 1a, 1b, 2, and putative lineages 3 and 4 addition, phylogenetic analysis was performed to indicate (listed in Table). Phylogenetic analysis was performed by the relationships between the Hungarian goose–derived using the modified neighbor-joining method (ClustalX; WNV strain and selected representatives of WNV clades [22]), and trees were constructed to demonstrate the rela- and clusters. The resulting phylogenetic tree (Figure) con- tionship between the Hungarian WNVs and other WNV firmed the results of the BLAST search, i.e., the Hungarian strains (Figure). goose–derived WNV strain is clustering close to the previ- The nucleotide sequences of the Hungarian WNV ously mentioned WNV strains isolated in the United States strains goose-Hungary/03 (Hu03) and goshawk-Hungary/ and Israel, which belong to lineage 1a of WNV. Other 04 (Hu04) were submitted to the GenBank database. They European WNV strains (isolated in Italy, France, and are available under accession numbers DQ118127 and Romania) are more distant to the Hungarian strain; they DQ116961, respectively. form a separate cluster consisting of a Romanian/Russian and a French/Italian subcluster. Results The complete nucleotide sequence of the goshawk- In this study, the complete genome sequences of WNV Hungary/04 WNV strain is composed of 11,028 nt and strains derived from a 6-week-old goose, which died in contains 1 open reading frame between nucleotide posi- 2003 during an outbreak of encephalitis in a Hungarian tions 97 and 10,401, coding for a 3,434-aa putative goose flock (strain goose-Hungary/03), and from a polyprotein precursor. In BLAST search, the strain showed goshawk, which also died from encephalitis in the same the highest (96% nt and 99% aa) identity to the WNV pro- region 1 year later (strain goshawk-Hungary/04), were totype strain B 956. Consequently, as the phylogram also determined, aligned, and phylogenetically analyzed. The indicates (Figure), this virus belongs to lineage 2 of WNV. genome of the goose-Hungary/03 strain is composed of Alignments of the available partial sequences from the E 10,969 nucleotides (nt) and contains 1 open reading frame protein coding regions of other representatives of this clus- between nucleotide positions 97 and 10,398, coding for a ter showed even higher identities (97%–98% nt and 100% 3,433 amino acid (aa)–long putative polyprotein precursor. aa) with WNV strains isolated in central Africa in 1972 The complete genomic sequence of the virus was subject- (AnB3507, AF001563) and in 1983 (HB83P55, ed to a BLAST search against gene bank databases. The AF001557), respectively (15). highest identity rates (98% at the nucleotide and 99% at More recently (in early August 2005), additional lethal the amino acid level) were found with WNV strains isolat- cases of encephalitis occurred in birds of prey in the same ed in 1998 in Israel and in 1999 in the United States. In place in which the goshawk died of West Nile encephalitis

620 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 Encephalitic West Nile Virus, Central Europe in 2004, involving up to a total of 3 goshawks and 2 sparrow hawks (A. nisus); 2 of the goshawks and 1 sparrow hawk died. Preliminary investigations detected WNV-specific nucleic acid in the brains of the birds. The partial nucleotide sequence of the 2005 virus (1,000 bp at the NS5′–3′-UTR regions) showed 99.9% identity with the goshawk-Hungary/04 strain (only 1 substitution at nucleotide position 9,376 [g→a] has been observed, which did not influence the putative amino acid sequence). Additional observation of the outbreak and investigations of the cases are in progress.

Discussion The primary aim of our investigations was to show the genetic relatedness of the WNV strains detected in Hungary in the last 2 years and to estimate their clinical and epidemiologic impact. The phylogenetic analysis emphasizes the close genetic relationship of the goose- Hungary/03 strain with a WNV strain isolated in Israel in 1998 and the WNV strain introduced in New York in 1999, since the 3 WNVs form 1 single cluster within clade 1a of lineage 1. These strains caused outbreaks in birds, humans, and horses. Previous European WNV isolates exhibited lower identity values, e.g., the strain that was responsible Figure. Phylogenetic tree based on the complete nucleotide for the Romanian outbreak(s) in 1996 and 1997 showed sequences of selected West Nile virus strains demonstrating the only 96% nt identity with the Hungarian goose-2003 genetic relatedness of these strains (abbreviations are listed in strain, and in the phylogenetic tree the other European iso- Table.) Boxes indicate different lineages and clades. The lates form a separate cluster consisting of 2 subclusters Hungarian strains reported in this article are highlighted with gray (Figure). The earliest representatives of the Israel/USA/ background). RabV, Rabensburg virus; JEV, Japanese encephalitis virus. Scale bar depicts degree of relatedness. goose-Hungary/03 cluster were reported by Malkinson et al. (23) from ill and dead white storks (Ciconia ciconia) in Israel in 1998. These storks, however, had hatched in cen- When a WNV infection was detected in 2004 in a tral Europe, and during their autumn migration south- goshawk fledgling, which died from encephalitis in the wards, strong winds had blown them off course, from their same region of Hungary in which the outbreak in geese usual route to Africa, to southern Israel. Malkinson et al. and humans occurred during the previous year, we antici- suspected that these birds introduced the neurovirulent pated a WNV strain more or less identical to the genotype genotype of WNV to Israel from their hatching place. The detected there in 2003. The genomic sequence of this strain wetlands of southeastern Hungary are foraging and nesting was not closely related to the sequence of the WNV strain habitats for storks and many other wild bird species, and detected in geese in the year before, however, but belonged the goose farm, where the WNV outbreak occurred in to the group of central African lineage 2 WNV strains. A 2003, is located in this region. These facts, together with closely related strain from this cluster (ArB3573, the close phylogenetic relatedness of the Israeli/US/ AF001565, and AF458349) was identified as a neuroinva- Hungarian WNV strains, strongly support the theory that sive strain of WNV in a mouse model (14). To our knowl- storks carried the neurovirulent WNV strain from central edge, this report is the first on the emergence of a lineage Europe (that is, from Hungary) to Israel, which sheds new 2 WNV strain outside Africa. Migratory birds that had light on the introduction of WNV to New York. This virus overwintered in central Africa probably introduced this could have originated in Israel (which is the generally exotic strain to the wetlands of Hungary. On the other accepted although not proven theory) or central Europe. In hand, as the goshawk is not a migratory species, and infec- both cases, however, the virus seems to have its true origin tion occurred in August, the African WNV strain must in Europe. In a recent publication, Lvov et al. suggested have already successfully adapted to local mosquito vec- that WNV could have been introduced into New York by tors. Consequently, this neurotropic, exotic WNV strain ships traveling from Black Sea ports (24). may become a resident pathogen in Europe with all the possible public health consequences.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 621 RESEARCH

Our results indicate that the WNV strains that emerged global warming, that may have enhanced the recent emer- in 2 consecutive years and caused avian deaths in Hungary gence of viruses, which had previously been restricted to are epidemiologically unrelated. Genetically distinct WNV Africa, in new habitats and continents. Improved observa- strains are circulating simultaneously yet independently in tion, reporting, and detection methods have also con- local birds and thus most likely also in local mosquito pop- tributed to the apparent increasing emergence of these ulations within the same region. They cause sporadic cases viruses. of encephalitis and also raise the possibility of spreading to other European countries or even to other continents, as Acknowledgments happened in 1999 with another WNV strain, which result- We thank Róbert Glávits, Csaba Drén, and Vilmos Palya for ed in a public health catastrophe in America. their help in detecting and identifying the goose WNV strain. In addition to the above 2 novel WNVs, we recently This study was partially supported by grant OTKA D characterized another novel flavivirus of so far unknown 048647. human pathogenicity named Rabensburg virus, which has been isolated from Culex pipiens mosquitoes in 1997 and Dr Bakonyi is lecturer of virology at the Faculty of 1999 at the Czech Republic–Austria border, only a few Veterinary Science, Budapest, and also works as a guest hundred kilometers from the region where the Hungarian researcher at the University of Veterinary Medicine, Vienna. He WNVs emerged. After the entire genome was sequenced, is interested in the molecular diagnosis and epidemiology of ani- Rabensburg virus turned out to represent either a new mal and human viruses. (third) lineage of WNV or a novel flavivirus of the JEV group (18). Thus, several distinct WNV strains seem to cir- References culate in central Europe. In 2001 another flavivirus of the JEV group, Usutu virus, which has never previously been 1. Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus observed outside Africa, emerged in Austria and resulted isolated from the blood of a native of Uganda. Am J Trop Med Hyg. 1940;20:471. in deaths in several species of birds, especially Eurasian 2. Peiris JSM, Amerasinghe FP. West Nile fever. In: Beran GW, Steele blackbirds (Turdus merula) (21). This virus became a resi- JH, editors. Handbook of zoonoses. Section B: Viral. 2nd ed. Boca dent pathogen in Austria and continues to disperse and Raton (FL): CRC Press; 1994. p. 139–48. cause deaths in blackbirds and other species of birds 3. Hubálek Z, Halouzka J. West Nile fever—a reemerging mosquito- borne viral disease in Europe. Emerg Infect Dis. 1999;5:643–50. (25,26). 4. Anderson JF, Andreadis TG, Vossbrink CR, Tirrell S, Wakem EM, The snowy winter and rainy spring of 2005 resulted in French RA, et al. Isolation of West Nile virus from mosquitoes, serious floods in the area in which the Hungarian WNV crows, and a Cooper’s hawk in Connecticut. Science. strains were identified. Since the floodplains and polders 1999;286:2331–3. 5. Blitvich BJ, Fernandez-Salas I, Contreras-Cordero JF, Marlenee NL, were under water, the conditions for mosquito develop- Gonzalez-Rojas JI, Komar N, et al. Serologic evidence of West Nile ment were ideal. The summer was also very rainy, which virus infection in horses, Coahuila State, Mexico. Emerg Infect Dis. resulted in more floods in the region and continuous mos- 2003;9:853–6. quito gradation. The most recent data imply that the line- 6. Glaser A. West Nile virus and North America: an unfolding story. Rev Sci Tech. 2004;23:557–68. age 2 WNV strain may have overwintered in Hungary, 7. Spielman A, Andreadis TG, Apperson CS, Cornel AJ, Day JF, Edman causing several clinical cases of encephalitis in Accipiter JD, et al. Outbreak of West Nile virus in North America. Science. species in 2005 as well. 2004;306:1473–5. The routine diagnostic techniques in most of the 8. Zeller HG, Schuffenecker I. West Nile virus: an overview of its spread in Europe and the Mediterranean basin in contrast to its spread in the European public health and veterinary laboratories are Americas. Eur J Clin Microbiol Infect Dis. 2004;23:147–56. designed to detect lineage 1 WNV strains. In a recent PCR 9. Van der Meulen KM, Pensaert MB, Nauwynck HJ. West Nile virus in external quality assurance multicenter test, <40% of the the vertebrate world. Arch Virol. 2005;150:637–57. involved laboratories could detect lineage 2 WNV strains 10. Campbell GL, Marfin AA, Lanciotti RS, Gubler DJ. West Nile virus. Lancet Infect Dis. 2002;2:519–29. (Matthias Niedrig, pers. comm.). Therefore, a major goal 11. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et of this article is to increase the scientific and public aware- al. Origin of the West Nile virus responsible for an outbreak of ness of this potential public health threat for Europe and, encephalitis in the northeastern United States. Science. perhaps, America. Furthermore, comprehensive investiga- 1999;286:2333–7. 12. Giladi M, Metzkor-Cotter E, Martin DA, Siegman-Igra Y, Korczyn tions on the occurrence, ecology, and epidemiology of the AD, Rosso R, et al. West Nile encephalitis in Israel, 1999: the New different WNV strains circulating in central Europe, as York connection. Emerg Infect Dis. 2001;7:654–8. well as the development of monitoring and surveillance 13. Banet-Noach C, Malkinson M, Brill A, Samina I, Yadin H, Weisman programs, must be of highest priority. One may also spec- Y, et al. Phylogenetic relationships of West Nile viruses isolated from birds and horses in Israel from 1997 to 2001. Virus Genes. ulate on environmental factors, such as climate change or 2003;26:135–41.

622 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 Encephalitic West Nile Virus, Central Europe

14. Beasley DW, Li L, Suderman MT, Barrett AD. Mouse neuroinvasive 22. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. phenotype of West Nile virus strains varies depending upon virus The CLUSTAL_X windows interface: flexible strategies for multiple genotype. Virology. 2002;296:17–23. sequence alignment aided by quality analysis tools. Nucleic Acids 15. Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte JP, Deubel V. Res. 1997;25:4876–82. Extensive nucleotide changes and deletions within the envelope gly- 23. Malkinson M, Banet C, Weisman Y, Pokamunski S, King R, Drouet coprotein gene of Euro-African West Nile viruses. J Gen Virol. MT, et al. Introduction of West Nile virus in the Middle East by 1997;78:2293–7. migrating white storks. Emerg Infect Dis. 2002;8:392–7. 16. Lanciotti RS, Ebel GD, Deubel V, Kerst AJ, Murri S, Meyer R, et al. 24. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov AI, Prilipov Complete genome sequences and phylogenetic analysis of West Nile AG, Kinney R, et al. West Nile virus and other zoonotic viruses in virus strains isolated from the United States, Europe, and the Middle Russia: examples of emerging-reemerging situations. Arch Virol East. Virology. 2002;298:96–105. Suppl. 2004;18:85–96. 17. Charrel RN, Brault AC, Gallian P, Lemasson JJ, Murgue B, Murri S, 25. Bakonyi T, Gould EA, Kolodziejek J, Weissenböck H, Nowotny N. et al. Evolutionary relationship between Old World West Nile virus Complete genome analysis and molecular characterization of Usutu strains. Evidence for viral gene flow between Africa, the Middle East, virus that emerged in Austria in 2001: comparison with the South and Europe. Virology. 2003;315:381–8. African strain SAAR-1776 and other flaviviruses. Virology. 18. Bakonyi T, Hubalek Z, Rudolf I, Nowotny N. Novel flavivirus or new 2004;328:301–10. lineage of West Nile virus, central Europe. Emerg Infect Dis. 26. Weissenböck H, Kolodziejek J, Fragner K, Kuhn R, Pfeffer M, 2005;11:225–31. Nowotny N. Usutu virus activity in Austria, 2001-2002. Microbes 19. Glávits R, Ferenczi E, Ivanics É, Bakonyi T, Mató T, Zarka P, et al. Infect. 2003;5:1132–6. Occurrence of West Nile Fever in a circovirus infected goose flock in Hungary. Avian Pathol. 2005;34:408–14. Address for correspondence: Norbert Nowotny, Zoonoses and Emerging 20. Ferenczi E, Rácz G, Faludi G, Czeglédi A, Mezey I, Berencsi G. Infections Group, Clinical Virology, Clinical Department of Diagnostic Natural foci of classical and emerging viral zoonoses in Hungary. In: Berencsi G, Khan AS, Halouzka J, editors. Emerging biological Imaging, Infectious Diseases and Clinical Pathology, University of threat. Amsterdam: IOS Press; 2005. p. 43–9. Veterinary Medicine, Vienna, Veterinärplatz 1, A-1210 Vienna, Austria; 21. Weissenböck H, Kolodziejek J, Url A, Lussy H, Rebel-Bauder B, fax: 43-1-25077-2790; email: [email protected] Nowotny N. Emergence of Usutu virus, an African mosquito-borne flavivirus of the Japanese encephalitis virus group, central Europe. Emerg Infect Dis. 2002;8:652–6.

ANOTHER DIMENSION

Bedside Manners Christopher Wiseman

How little the dying seem to need– We panic to do more for them, A drink perhaps, a little food, And especially when it's your father, A smile, a hand to hold, medication, And his eyes are far away, and your tears A change of clothes, an unspoken Are all down your face and clothes, Understanding about what's happening. And he doesn't see them now, but smiles You think it would be more, much more, Perhaps, just perhaps because you're there. Something more difficult for us How little he needs. Just love. More Love. To help with in this great disruption, But perhaps it's because as the huge shape Rears up higher and darker each hour They are anxious that we should see it too And try to show us with a hand-squeeze.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 4, April 2006 623

RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

RNA / Bunyaviridae

"Schmallenberg" Virus (SBV)

1. Einleitung

Das "Schmallenberg" Virus (SBV), auch europäisches Orthobunyavirus genannt, ist ein 2011 neu aufgetretener Erreger einer Grippe-ähnlichen Erkrankung von Wiederkäuern (Hoffmann et al., 2012). Werden Tiere während der Trächtigkeit inﬁziert, kann dies mit nachfolgenden Aborten, Totgeburten und Missbildungen neu geborener Tiere einher gehen. Schafe gelten als am Meisten betroffen, gefolgt von Ziegen und Rindern. Die Bedeutung des Virus für Wildwiederkäuer ist noch nicht geklärt, obwohl die Infektion serologisch bei verschiedenen Wildtierarten nachgewiesen werden konnte (Conraths et al., 2013).

2. Besonderheiten

Viren der Familie Bunyaviridae werden typischerweise von Arthropoden übertragen und sind deshalb unter den Arboviren eingereiht. SBV zeichnet sich durch einen ausgeprägten Neurotropismus aus (Varela et al., 2013). Ausserdem wird SBV auch vertikal auf ungeborene Foeten übertragen. Innerhalb der Familie gibt es Hunderte verschiedener Viren mit einem relativ breiten Wirtsspektrum. Sowohl Tiere als auch Pﬂanzen sind von Infektionen mit Bunyaviren betroffen. Glücklicherweise gibt es bislang keine Hinweise darauf, dass das Schmallenberg Virus beim Menschen eine Krankheit verursachen könnte.

3. Geschichte

Ab Sommer 2011 häuften sich die Berichte deutscher Bauern (Nordrhein-Westfalen; nahe der Grenze zu den Niederlanden) bezüglich einer ungewöhnlich hohen Frequenz von ﬁeberhaften Erkrankungen bei Kühen, die mit Fressunlust und Milchrückgang gepaart waren (Hoffmann et al., 2012). Im Spätherbst begannen sich dann auch Berichte über Aborte und Totgeburten bei denselben Tieren sowie kongenitale Missbildungen der Neugeborenen zu häufen. Mitarbeiter des Friedrich-Löfﬂer Instituts (FLI) konnten im Blut eines erkrankten Tieres dann ein Bunyavirus nachweisen, dem sie, nach dem Ort des ersten bekannten Falles, den Namen "Schmallenberg" Virus gaben. In der Folge wurde das Virus sowie dessen Krankheitsproblematik rasch auch im restlichen Europa festgestellt, inklusive in der Schweiz (Juli 2012) und in Österreich (September 2012). Während zunächst die meisten Fälle von SBV bei Schafen festgestellt wurden, nahm die Inzidenz bei dieser Tierart ab Mitte Februar 2012 ab, während sie gleichzeitig bei Rindern anstieg.

4. Verbreitung

Die Verbreitung von SBF umfasst ganz Europa, inklusive Grossbritannien, Skandinavien und Südeuropa. Der Ursprungsort der SBV Epidemie wird in der gleichen Gegend (Grenzregion zwischen Belgien, Deutschland und den Niederlanden) vermutet, wo 2006 bereits der Seuchenzug des Blauzungenvirus (Serotyp 8) seinen Anfang genommen hatte (Hoffmann et al., 2012). Retrospektive serologische Untersuchungen ergaben, dass SBV vor 2011 in jener Gegend nicht vorgekommen war (Conraths et al., 2013). Die Minimalliste mit den Ländern, aus denen SBV-Ausbrüche gemeldet wurden umfasst: Deutschland, Niederlande, Belgien, Luxemburg, England, Schottland, Irland,

Autoren: Ackermann M. 1 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

Frankreich, Spanien, Italien, Schweiz, Österreich, Dänemark, Finnland, Schweden, Polen sowie die Türkei.

Abbildung 1: Schematische Darstellung eines Bunyavirus. Die wichtigsten Strukturkomponenten sind bezeichnet. Im Schema erscheinen die einzelnen Segmente als zirkuläre Moleküle, was in Wirklichkeit aber nicht vollständig zutrifft: Die Enden der einzelnen Segmente sind zwar über Wasserstoffbrücken, nicht aber durch kovalente Bindungen, miteinander verbunden. Abdruck mit freundlicher Genehmigung von ViralZone: www.expasy.org/viralzone; Swiss Institute of Bioinformatics.

5. Erreger

SBV ist ein Mitglied der sogenannten Simbu Serogruppe der Orthobunyaviren. Formell ist es in die Familie Bunyaviridae und das Genus Orthobunyavirus eingeteilt. Das Virion ist kugelig geformt, behüllt und weist einen Durchmesser von 80-120 nm auf. Das Genom besteht aus einer einzelsträngigen, negativ-polaren RNA (-ssRNA), die in drei Segmente unterteilt ist. Das kleinste Segment S (ca. 1'000 Basen) kodiert für das Nukleoprotein (N) sowie ein Nicht-

Strukturprotein (NSS), welches durch alternative Translations-Initiation von derselben Vorlage translatiert wird. Das Mittel-grosse Segment M (ca. 4'500 Basen) kodiert für das Glykoprotein G, aus welchem durch proteolytische Spaltung die beiden Hüllproteine Gn und Gc sowie ein weiteres Nicht-

Strukturprotein (NSM) entstehen. Das grösste Segment L (ca. 6'900 Basen) kodiert für die RNA- abhängige RNA-Polymerase L.

Autoren: Ackermann M. 2 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

Abbildung 2: Genom eines Bunyavirus mit Angabe der Segmentbezeichnung und -grösse sowie der darin kodierten Genprodukte. Weitere Details im Text. Abdruck mit freundlicher Genehmigung von ViralZone: www.expasy.org/viralzone; Swiss Institute of Bioinformatics.

6. Virusvermehrung und Genexpression

Bunyaviren binden via Dimere des Gn-Gc Glykoproteins an zelluläre Rezeptoren und werden durch endozytotische Vorgänge aufgenommen. Die Virusmembran fusioniert dann mit der Membran des Transportvesikels, sodass die genomischen RNA Segmente ins Zytoplasma entlassen werden. Dort beginnt die Polymerase L mit der Transkription, indem sie zunächst an eine Promoter-Region der genomischen RNA bindet und die einzelnen Segmente zu nicht-polyadenylierter mRNA abschreibt. Diese mRNAs werden durch L während des Abschreibevorgangs mit einer cap-Struktur versehen, sodass sie von den zellulären Ribosomen als translatierbare Moleküle wahrgenommen werden. Die Genom-Replikation und Enkapsidierung der neu gebildeten Segmente ﬁndet im Zytoplasma in sogenannten "viral factories" statt. Die so gebildeten Nukleokapside migrieren anschliessend zu den Golgi-Membranen, wo mittlerweile ebenfalls die Glykoproteine hin gekommen sind. Die Virushülle bildet sich durch Budding ins Lumen des Golgi Apparates. Fertige Viruspartikel werden in Vesikeln an die Zelloberﬂäche transportiert und frei gelassen.

7. Epidemiologie

Gemäss bisheriger Analysen geschah die grösste Anzahl der Neuinfektionen genau in jenem Zeitraum, der vorher typisch für die Vektor-basierte Übertragung des Blauzungenvirus bekannt gewesen war. Aufgrund dieser Beobachtung sowie der Zugehörigkeit des SBV zur Simbu Serogruppe der Bunyaviren geht man davon aus, dass SBV in erster Linie durch Stechmücken übertragen wird. Tatsächlich konnte SBV in Stechmücken (Culicoides spp.) dokumentiert werden, während bislang eine direkte Übertragung des SBV mittels Tierkontakt nicht belegt werden konnte. Selbst die experimentelle oro-nasale Inokulation von Versuchskälbern mit SBV war nicht erfolgreich (Conraths et al., 2013). Hingegen gilt die vertikale Übertragung von inﬁzierten Muttertieren auf ihre ungeborenen Foeten als gesichert. Im Gefolge der Neuinfektion trächtiger Tiere wird eine transplazentare Übertragung zwischen der vierten und der sechsten Trächtigkeitswoche der Schafe bzw. zwischen 70. und 150. Trächtigkeitstag der Rinder verantwortlich gemacht für das Auftreten der beobachteten Missbildungen bei Neugeborenen.

8. Desinfektion

Genaue Angaben liegen noch nicht vor, aber aufgrund von Analogieschlüssen zur Blauzungen- krankheit kann man davon ausgehen, dass die Desinfektion eine untergeordnete Rolle spielt. Allenfalls sollten Detergens-haltige Mittel genügen.

Autoren: Ackermann M. 3 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

9. Pathogenese

Die Inkubationszeit sowie Details der Pathogenese sind noch nicht bekannt. Allerdings steht fest, dass SBV über eine ausgeprägte Neurotropie verfügt und sich zudem neuropathogen äussert, indem es die inﬁzierten Neuronen des Gehirns und des Rückenmarks via eine vakuolisierende Nekrose mit entzündlichen Begleitsymptomen dem Untergang zuführt (Varela et al., 2013). Die bei den Foeten beobachteten Missbildungen sind im Zusammenhang mit diesen ZNS-Läsionen zu erklären. Bei der Infektion überwindet SBV die Abwehrreaktionen des nativen Immunsystems, indem es die Synthese von Typ I Interferon hemmt (Funktion des NSs Proteins). Es ist noch ungeklärt auf welchen Wegen das Virus ins ZNS gelangt und welche Faktoren zur anhaltenden Virämie beitragen. Gemäss bisheriger Erfahrung ist eine nachweisbare Virämie nur von relativ kurzer Dauer (maximal 5 Tage), was sich negativ auf die Diagnostizierbarkeit der Infektion am lebenden Tier auswirkt. Diese Beobachtung widerspricht eigentlich auch ein Bisschen der Theorie, dass SBV besonders efﬁzient durch Vektoren übertragen wird.

10. Klinik

Die Infektion kann klinisch oder subklinisch verlaufen. In der Anfangsphase der Epidemie beobachtete man in erster Linie einen Rückgang der Milchproduktion, allenfalls verbunden mit Fieber, Appetitlosigkeit, einer generellen Malaise sowie Diarrhöe. Meist erholen sich die betroffenen Tiere innerhalb von einer bis zwei Wochen wieder von der Krankheit. Unterdessen werden die subklinischen Fälle immer häuﬁger. Transplazentare Infektion. Wird das Virus vom Muttertier auf den Foeten übertragen, so hängt dessen Schicksal stark vom Zeitpunkt der Übertragung ab. So kann es zum unbemerkten Fruchtabgang kommen oder zum Abort oder zur Geburt missgebildeter, lebensunfähiger Neugeborener. Im ersten Fall wäre klinisch eine Unfruchtbarkeit zu bemerken durch Erhöhung der Return-rate nach erfolgter Begattung oder künstlicher Bsamung. Zu den typischen SBV-assoziierten Missbildungen zählen: Arthrogryposis, Torticollis, Brachygnatia inferior, Hydranenzephalopathie und cerebelläre Hypoplasie (Conraths et al., 2013). Versteifte Verkrümmungen von Wirbelsäule und Gliedmassen bei den Foeten können zu erheblichen Störungen des Geburtsvorgangs führen. Video der Tierärztlichen Hochschule Hannover zur Klinik von SBV: http://www.animal-health- online.de/gross/2012/02/01/tiho-hannover-videopodcast-zum-schmallenberg-virus/20036/

11. Immunreaktion

Inﬁzierte Tiere entwickeln innerhalb von 12 bis 14 Tagen nachweisbare Antikörper gegen SBV. Dies geht mit grosser Wahrscheinlichkeit einher mit einer belastbaren Immunität, da Muttertiere von missgebildeten Foeten nach einer erneuten Trächtigkeit erfahrungsgemäss dann normale Jungtiere zur Welt bringen. Man weiss jedoch noch nicht, wie lange diese Immunität anhält. Je nach Zeitpunkt der transplazentaren Infektion können betroffene Foeten auch schon Antikörper gegen SBV bilden (Präkolostrale Ak: wichtig für die Diagnose).

12. Prophylaxe

Impfstoffe sind in Entwicklung, stehen aber noch nicht zur Verfügung.

Autoren: Ackermann M. 4 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

13. Diagnose

Beim Auftreten von Missbildungen bei neugeborenen Wiederkäuern sowie bei unspeziﬁschen Fruchtbarkeitsstörungen an SBV denken.

14. Labordiagnose

Das Virus kann auf vielen Zellkulturen für die weitere Analyse angezüchtet werden. Schneller und direkter Virusnachweis mittels Reverse-Transkription-PCR (RT-PCR). Für den Antikörpernachweis sind verschiedene Tests beschrieben, inklusive Serumneutralisationstest (SNT), indirekte Immunﬂuoreszenz, ELISA (Breard et al., 2013). Sowohl indirekte wie kompetitive ELISA-Formate sind kommerziell erhältlich.

15. Differentialdiagnose

Andere Viren: Bovine Virus-Diarrhoe (BVDV), Border Disease (BDV), Blauzungenkrankheit (BTV), Maul- und Klauenseuche (MKSV), Rift Valley Fieber Virus (Genus Phlebovirus der Bunyaviridae). Infektionen durch bakterielle Aborterreger sowie Ernährungs- und Stoffwechsel-bedingte Ursachen müssen ebenfalls in Betracht gezogen werden.

16. Bei Verdacht

Die Einsendung von Untersuchungsmaterial grundsätzlich mit dem Labor absprechen. Deutschland: Friedrich-Loefﬂer-Institut, Bundesforschungsinstitut für Tiergesundheit, Südufer 10, 17493 Greifswald-Insel Riems. Telefon: +49 38351 7–-10. Österreich: Österreichische Agentur für Gesundheit und Lebensmittelsicherheit (AGES), Institut für veterinärmedizinische Untersuchungen, Mödling, Telefon: +43 (0)50555-38100. Schweiz: Institut für Viruskrankheiten und Immunprophylaxe (IVI), Telefon: +41 31 848 9211.

17. Untersuchungsmaterial

Abortierte Foeten oder deren Organe, insbesondere Grosshirn und Kleinhirn. In zweiter Priorität können auch Milz und Blut sowie Amnionsﬂüssigkeit für die Untersuchung eingeschickt werden. Blutproben der abortierten Foeten sind sehr wichtig, erstens für die Untersuchung auf präkolostrale Antikörper, zweitens auch für den Virusnachweis mittels RT-PCR. Blutproben von postnatal angesteckten Tieren sind hingegen infolge der relativ kurzen Virämie eher ungeeignet.

18. Therapie

Nicht verfügbar.

19. Staatliche Massnahmen

Da die Langzeitbedeutung von SBV bisher noch relativ unklar ist, wurden in Österreich und der Schweiz bislang noch keine verbindlichen staatlichen Massnahmen angeordnet. Deutschland hingegen führte im März 2012 eine amtliche Meldepﬂicht ein.

Autoren: Ackermann M. 5 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages RNA / Bunyaviridae! Virus Porträts / Virologisches Institut UZH "Schmallenberg" Virus (SBV)

20. Literatur

Breard, E., Lara, E., Comtet, L., Viarouge, C., Doceul, V., Desprat, A., Vitour, D., Pozzi, N., Cay, A.B., De Regge, N., Pourquier, P., Schirrmeier, H., Hoffmann, B., Beer, M., Sailleau, C., Zientara, S., 2013, Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies. PLoS One 8, e53446. Conraths, F.J., Peters, M., Beer, M., 2013, Schmallenberg virus, a novel orthobunyavirus infection in ruminants in Europe: potential global impact and preventive measures. N Z Vet J 61, 63-67. Hoffmann, B., Scheuch, M., Hoper, D., Jungblut, R., Holsteg, M., Schirrmeier, H., Eschbaumer, M., Goller, K.V., Wernike, K., Fischer, M., Breithaupt, A., Mettenleiter, T.C., Beer, M., 2012, Novel orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis 18, 469-472. Varela, M., Schnettler, E., Caporale, M., Murgia, C., Barry, G., McFarlane, M., McGregor, E., Piras, I.M., Shaw, A., Lamm, C., Janowicz, A., Beer, M., Glass, M., Herder, V., Hahn, K., Baumgartner, W., Kohl, A., Palmarini, M., 2013, Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host. PLoS Pathog 9, e1003133.

Autoren: Ackermann M. 6 / 6 Version 1 / Mai 2013 RNA_Bunyaviridae_Schmallenbergvirus.pages Antecedent Avian Immunity Limits Tangential Transmission of West Nile Virus to Humans

Jennifer L. Kwan1, Susanne Kluh2, William K. Reisen1* 1 Center for Vectorborne Diseases, Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America, 2 Greater Los Angeles County Vector Control District, Santa Fe Springs, California, United States of America

Abstract

Background: West Nile virus (WNV) is a mosquito-borne flavivirus maintained and amplified among birds and tangentially transmitted to humans and horses which may develop terminal neuroinvasive disease. Outbreaks typically have a three-year pattern of silent introduction, rapid amplification and subsidence, followed by intermittent recrudescence. Our hypothesis that amplification to outbreak levels is contingent upon antecedent seroprevalence within maintenance host populations was tested by tracking WNV transmission in Los Angeles, California from 2003 through 2011.

Methods: Prevalence of antibodies against WNV was monitored weekly in House Finches and House Sparrows. Tangential or spillover transmission was measured by seroconversions in sentinel chickens and by the number of West Nile neuroinvasive disease (WNND) cases reported to the Los Angeles County Department of Public Health.

Results: Elevated seroprevalence in these avian populations was associated with the subsidence of outbreaks and in the antecedent dampening of amplification during succeeding years. Dilution of seroprevalence by recruitment resulted in the progressive loss of herd immunity following the 2004 outbreak, leading to recrudescence during 2008 and 2011. WNV appeared to be a significant cause of death in these avian species, because the survivorship of antibody positive birds significantly exceeded that of antibody negative birds. Cross-correlation analysis showed that seroprevalence was negatively correlated prior to the onset of human cases and then positively correlated, peaking at 4–6 weeks after the onset of tangential transmission. Antecedent seroprevalence during winter (Jan – Mar) was negatively correlated with the number of WNND cases during the succeeding summer (Jul–Sep).

Conclusions: Herd immunity levels within after hatching year avian maintenance host populations ,10% during the antecedent late winter and spring period were followed on three occasions by outbreaks of WNND cases during the succeeding summer. Because mosquitoes feed almost exclusively on these avian species, amplification was directly related to the availability of receptive non-immune hosts.

Citation: Kwan JL, Kluh S, Reisen WK (2012) Antecedent Avian Immunity Limits Tangential Transmission of West Nile Virus to Humans. PLoS ONE 7(3): e34127. doi:10.1371/journal.pone.0034127 Editor: Clive Shiff, Johns Hopkins University, United States of America Received December 22, 2011; Accepted February 22, 2012; Published March 23, 2012 Copyright: ß 2012 Kwan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grant R01-AI055607 from the National Institutes of Allergy and Infectious Diseases, National Institutes of Health; funds and resources were provided by the Greater Los Angeles County Vector Control District, and supplemental funds for Extended Laboratory Capacity from the Center for Disease Control and the California Department of Public Health. WKR acknowledges support from the RAPIDD program of the Science and Technology Directorate, Department of Homeland Security, and the Fogarty International Center, National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction transmission cycles in urban landscapes to a few key species [3]. The population dynamics of these host species, in turn, may The epidemiology of mosquitoborne arboviral zoonoses is dictate the frequency of recurrent outbreaks due to the acquisition complex. Frequently extensive maintenance and amplification and persistence of population or ‘herd’ immunity. Zoonotic transmission is required prior to spillover or tangential transmis- mosquito-borne arboviruses seem to rely on two divergent, but sion to humans or domestic animals. The efficiency of amplifica- often concurrent, strategies for persistence: high virulence/high tion depends upon the frequency of blood feeding by competent mortality in amplifying host species that may become regionally mosquito vectors upon immunologically naı¨ve and competent depopulated, or moderate virulence/low mortality in host species hosts during favorable climatic conditions [1] that decrease the that acquire herd immunity. Therefore, the timing and intensity of duration of the gonotrophic cycle increasing the frequency of amplification transmission and the occurrence of human outbreaks transmission and that decrease the extrinsic incubation period seems contingent upon host population recruitment to either reducing the chronological age of the vector when transmission repopulate or dilute immunity in affected host populations. can occur [2]. Despite this potential complexity, landscape The invasion of North America by West Nile virus (family homogeneity, reduced host and vector diversity, and focused Flaviviridae, genus Flavivirus, WNV) has provided a unique natural host-selection by the primary vectors frequently simplifies experiment to investigate these processes, because transmission

PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus intensity seems greatest in urban/periurban environments where Herein, we have extended these data into the 2011 outbreak cycles are simplified and frequently involve only a few key vector season, and test the hypothesis that seroprevalence levels in and avian species [3]. During the invasion of North America, maintenance hosts during late winter determines the efficiency of WNV repeatedly has exhibited a three year pattern of silent enzootic amplification of WNV during the subsequent summer introduction, explosive amplification to epidemic levels, and then season and therefore whether or not an outbreak of human disease rapid subsidence [4]. Although subsidence may be attributed to will occur. Specifically our study investigated: 1) differences in multiple factors, immunity within key avian host species seemed species specific seroconversion patterns between hatching and critical in slowing or delaying vernal amplification during the year after hatching year birds, 2) the impact of WNV infection on the following outbreaks and thereby reducing or preventing spill over survivorship of banded birds, 3) antibody persistence in naturally or tangential transmission to humans; however, data to substan- infected birds, and 4) the level of herd immunity or seroprevalence tiate this paradigm has been difficult to obtain. In addition, the necessary to inhibit WNV amplification and human cases. levels of herd immunity required for subsidence and recrudescence Understanding herd immunity in maintenance host populations have yet to be determined. In Los Angeles, California, elevated is important not only for a better understanding of WNV seroprevalence in key peridomestic maintenance hosts, the House epidemiology, but also for predicting outbreak risk and organizing Finch (Carpodacus mexicanus) and the House Sparrow (Passer preventive intervention in a timely manner. domesticus) [5], and concurrent depopulation of the highly susceptible amplifying host, the American Crow [6,7], were Methods associated with outbreak subsidence during 2005 and low level transmission during subsequent years. Waning seroprevalence in The ecology of the invasion and persistence of WNV in Los these peridomestic passerines was followed by WNV resurgence to Angeles, descriptions of our principal study areas, sampling outbreak levels during 2008 and 2011, indicating that there may methods, and temporal and spatial trends in surveillance data be thresholds of winter/spring immunity that suppress mainte- from 2003–2008 were summarized previously [5]. The current nance transmission, following outbreak years. In agreement, Culex paper extended our data from 2009 into 2011 and focused on the bloodmeal identification studies in California repeatedly have how the dynamics of WNV infection in House Finch and House documented that during late winter and spring almost all blood Sparrow populations affected tangential transmission to humans. meals are taken from House finches and House sparrows [8–11]. Before nesting, these populations are composed entirely of after Avian Serology hatching year birds, many of which may have acquired protective Birds were collected by grain-baited drop-down or Australian immunity during previous seasons. crow traps [22], with inlet apertures reduced to limit ingress to Late summer communal American Crow roosts may be critical small birds. Traps were placed at each of eight sites and were for rapid WNV amplification to outbreak levels, spatially closed for 24 hours biweekly. Birds were aged as juvenile, delimiting the distribution of Culex infection and human incidence hatching-year and after hatching-year categories by plumage, [12], and for seeding virus into residential areas [13,14], whereas and sexed based on plumage [23]. Birds then were banded with abundant and widely distributed peridomestic passerines may be USGS bands, and 0.1 ml of blood was collected by 28 g needle important as maintenance hosts initiating vernal amplification and syringe from each bird by jugular venipuncture and expelled into continuing epidemic transmission in and around residential 0.9 ml of sterile saline. Samples were clarified by centrifugation habitats. Both House Finches and House Sparrows are competent and the diluted sera tested by enzyme immunoassay (EIA) for hosts. Experimentally infected House Finches exhibited viremias western equine encephalomyelitis virus (WEEV) or flavivirus .6 log10 plaque forming units (PFU)/mL for 4–5 days [15], a titer antibody [24,25]. Because antibodies against WNV cross-react sufficient to infect Culex quinquefasciatus, the main vector present in with closely related St. Louis encephalitis virus (SLEV) [15], EIA the Los Angeles area [16]. Mortality in these experimentally results with positive over negative antigen well optical density infected birds was 65% [15] and field population abundance has ratios $2 were confirmed and the infecting virus identified by end been shown to have declined after the arrival of WNV in point plaque reduction neutralization tests (PRNT), using the California [6]. In agreement 26% of dead House Finches NY99 strain of WNV and the KERN217 strain of SLEV. Positive submitted for testing to the California Department of Public PRNTs neutralized .80% of .75 plaque forming units (PFU) of Health’s Dead Bird testing program [17] from Los Angeles were WNV or SLEV grown on Vero cells in 6 well plates at a dilution of positive for WNV RNA [5]. House Sparrow viremias following $1:20. For specific virus identification, titers exceeded 46 the experimental infection ranged from 8–10 log10 PFU/mL for 4 competing virus. days in Colorado [18] to 4–6 log10 PFU/mL for 2–6 days in Serological test results were used to calculate seroprevalence California [19], with 38 and 16% mortality, respectively. In proportions, as the total number of EIA positive birds/total agreement, the California Dead Bird program reported that 14% number of birds bled on each bleed date. To estimate of carcasses from Los Angeles were positive for WNV RNA [5]. seroconversions, new infections were identified as antibody- Humoral immunity following WNV infection in House Sparrows positive birds known from recapture data to have been previously from Colorado has been demonstrated to last 36 months, with negative at the most recent previous bleeding. No time period was limited decrease in neutralizing antibody titers [20], and similar specified between blood sampling for conversion to an antibody- results were reported for House Finches and House Sparrows from positive state. California for up to 8 months [21]. These data indicated that WNV infection should decrease population size and that birds Sentinel chickens surviving infection should be protected for life from conspecific As described previously [26], flocks of 10 white leghorn hens viral infection thereby dampening subsequent transmission. that were 16–18 weeks of age were deployed annually at each of Our detailed investigation of WNV epidemiology and ecology six sites that were near 6 of the 8 bird sampling sites. Blood in Los Angeles included the systematic monitoring of antibody samples (0.1 mL) were collected every 2 weeks by brachial seroprevalence within House Finch and House Sparrow popula- venipuncture and placed on filter paper strips [27]. The strips tions at multiple locations during the 2003–2009 period [5]. were sent to the California Department of Public Health in

PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

Table 1. Number of sera tested (proportion positive for West Nile virus antibodies) in Los Angeles summarized by species and year.

Species 2003 2004 2005 2006 2007 2008 2009 2010 2011 Total

Brown-headed 5 70 (0.01) 32 (0.13) 59 (0.05) 77 (0.01) 42 71 (0.03) 64 47 (0.02) 467 (0.03) Cowbird California Towhee 6 37 (0.05) 31 (0.03) 49 (0.10) 25 (0.04) 35 (0.06) 23 (0.17) 16 17 239 (0.06) House Finch 639 1,285 (0.14) 869 (0.26) 1,045 (0.14) 1,943 (0.09) 1,399 (0.14) 2,515 (0.20) 1,766 (0.07) 1,213 (0.03) 12,674 (0.12) House Sparrow 800 1,416 (0.19) 790 (0.09) 827 (0.04) 670 (0.03) 692 (0.08) 766 (0.08) 615 (0.03) 469 (0.02) 7,045 (0.08) Mourning Dove 35 86 (0.33) 32 (0.34) 1 (1.00) 154 (0.26) Nutmeg Manakin 1 6 (0.17) 39 (0.03) 24 90 (0.01) 46 (0.02) 337 (0.01) 322 60 925 (0.01) Red-winged Blackbird 13 (0.08) 5 (0.20) 2 2 22 (0.09) Song Sparrow 7 18 (0.11) 32212 35(0.06) White-crowned 33 56 58 (0.02) 62 223 (0.01) 131 (0.03) 228 (0.02) 121 (0.02) 89 1001 (0.01) Sparrow Totals* 1,524 2,979 (0.16) 1,907 (0.17) 2,100 (0.09) 3,067 (0.07) 2,347 (0.11) 3,945 (0.14) 2,908 (0.05) 1,895 (0.02) 22,672 (0.10)

Only frequently sampled birds included. A more complete listing is presented in Kwan et al. (2010b). *Included within yearly totals were 8 positives from 110 sera collected from 29 species of birds. doi:10.1371/journal.pone.0034127.t001

Richmond, California, for testing by EIA and immunofluores- determine the impact of herd immunity. The herd immunity cence assay (IFA) for presence of antibody to WNV, WEEV, and threshold was defined as the value of seroprevalence that best SLEV [28]. Chickens within flocks were replaced after five or correlated with the cessation of WNV activity as measured by new more chickens seroconverted to WNV. Chicken seroconversions WNND cases and sentinel chicken seroconversions. Correlation previously were found to provide a concordant measure of analyses were performed using SAS version 9.2 Software (SAS tangential transmission based on the onset of human cases [26]. Institute Inc., Cary, NC).

Human case reports Ethics Human cases of West Nile neuroinvasive disease (WNND) were The collection, banding, and bleeding of wild birds was done monitored by the Los Angeles County Department of Health and under protocols 11184, 12889 and 15893 approved by the Human Services, Acute Communicable Disease Control, through Institutional Animal Care and Use Committee of the University of passive case detection and reporting. WNND cases were limited to California, Davis; Master Station Federal Bird Banding permit those that matched the Center for Disease Control and Prevention 22763 issued by the U.S. Geological Survey, California and (CDC) definition for WNV-associated neuroinvasive illness and Resident Scientific Collection permits by the State of California had been laboratory-confirmed, typically by demonstration of Department of Fish and Game. The husbandry and bleeding of immunoglobulin M (IgM) antibody in sera or spinal fluid by EIA sentinel chickens was done under protocols 11186, 12878 and (http://www.cdc.gov/ncidod/dvbid/westnile/clinicians/clindesc. 15892 approved by the Institutional Animal Care and Use htm). Febrile cases were not included in our study, because of the progressive decline in testing and reporting after 2004 as indicated by decrease in the ratio of febrile to neuroinvasive Table 2. Total numbers (proportion positive for West Nile cases (data not shown). Additional human infections were virus antibodies) of House Finches and House Sparrows discovered through blood donor programs and were included if collected at eight study areas in Los Angeles. they developed acute symptoms.

Analysis Site Name House Finches House Sparrows Time series graphs were constructed at monthly intervals for Machado Lake 1,274 (0.03) 4 catch per trap-day and seroprevalence. Chi square tests of Rowland Heights 2,110 (0.13)A 5 (0.60) homogeneity were performed for the birds sampled by infection Whittier Narrows 1,628 (0.12) 2,195 (0.02) status and species as well as by site using SAS version 9.2 software B (SAS Institute Inc., Cary, NC). To assess the impact of infection Santa Fe Springs 2,372 (0.25) 2,608 (0.15) history, banded birds were grouped by species and serological Griffith Park 1,838 (0.12) 170 (0.1) status indicating if they were ever infected, and time retained Sylmar 1,119 (0.01) 29 within our study from banding to last recapture. A linear Santa Clarita 1,494 (0.03) 227 regression was fitted to the numbers collected per 10 week time Encino 547 (0.37)A,B 1,677 (0.05) step transformed by ln (y+1) as a function of time in weeks, presuming constant population loss due to emigration and death. Proportions followed by a letter were significantly different by Chi square test Survivorship was estimated as the backtransformed slope of the for homogeneity. LS Means for significant difference. fitted regression function. Ap value = 0.04. Time series and correlation analyses of seroprevalence vs. Bp value = 0.02. human cases and sentinel chicken seroconversions were used to doi:10.1371/journal.pone.0034127.t002

PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

Figure 1. Total numbers of House Finches and House Sparrows collected per month and the proportion banded or recaptured (recap). doi:10.1371/journal.pone.0034127.g001

Committee of the University of California, Davis. Use of Of the total EIA positives, 1,946 (87%) were confirmed by PRNT, arboviruses was approved under Biological Use Authorizations 112 were PRNT negative, and 209 were not retested. WNV was #0554 and #0873 issued by the Environmental Health and identified as the infecting virus for all EIA positive birds with Safety Committee of the University of California, Davis, and PRNT titers $1:40; none had been infected previously with USDA permit #47901. Human data used in this project were SLEV. The displacement of SLEV by WNV throughout granted an exemption from informed consent protocols by the California since 2003 was supported by human case, sentinel Institutional Review Board at the University of California, Davis chicken serology and mosquito pool diagnostics [5,29]. Because (Approval # 201018171-1). few other bird species were collected or frequently tested positive, further analyses focused on House Finches and House Sparrows. Results Seroprevalence Sera collected Temporal changes in seroprevalence for young of the year birds A total of 22,672 sera were collected from 38 species of birds, of classified as juvenile or hatching year and for after hatching year which 87% were House Finches and House Sparrows (Table 1). birds are shown in Figure 3 for House Finches and House Other frequently bled birds included small-sized species trapped Sparrows. During the outbreak years of 2004 and 2008 young concurrently, such as Nutmeg Manakins and White-crowned birds exhibited increased seroprevalence, whereas during inter- Sparrows, Rock Doves collected as part of bird removal programs, vening years mostly after hatching year birds were seropositive, and species such as Mourning Doves sampled at bird rehabilita- and the overall seroprevalence levels subsequently declined as tion centers. House Finches were abundant at all of our sampling these birds were replaced by immunologically naı¨ve hatching year locations, whereas most House Sparrows (93%) were collected birds. Data shown were seroprevalence by month for different from 3 of 8 trap locations (Table 2). However, when overall species and age categories, and included birds captured on seroprevalence was compared spatially, there were minimal multiple occasions. We attempted to also show changes in virus statistical differences. The number collected varied markedly over activity among years as seroconversions in Table 3. Here, the time (Figure 1), ranging from 13 to 352 House Finches and from 1 numbers of banded birds recaptured that previously tested to 242 House Sparrows per month, but the catch of these species negative were reported by the year that they first tested positive. per month was significantly correlated over time (r = 0.39, df = 95, However, these data were confounded, because the year of first P,0.01). The number of House sparrows caught per month remained relatively similar among years, whereas there was a progressive increase in the catch of House Finches (Figure 2), leading to a significant species by year interaction term (F = 6.53, df = 8, 184; P,0.001) in a two-way ANOVA comparing species and years. There were no significant temporal relationships among catch per month and the proportions of these birds that were recaptured (Figure 1). Of the 22,672 sera tested by EIA, 2,267 were positive against flavivirus antigen when tested by EIA, including 1,521 House Finches and 563 House Sparrows (92% of total EIA positives). The proportion of House Finch sera positive for WNV (0.12) was slightly, but significantly (X2 = 76.4, P,0.0001), greater than the proportion of House Sparrow sera positive (0.08). Mourning doves and other birds from rehabilitation centers frequently were positive during 2004 and 2005, but were sampled inconsistently at low numbers and were not tested after 2005. Other species such Figure 2. Mean number of House Finches and House Sparrows as feral Nutmeg Manakins and winter resident White-crowned collected per month during each year. Sparrows were collected frequently, but rarely were positive (0.01). doi:10.1371/journal.pone.0034127.g002

PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

Figure 3. Proportion of after hatching year (AHY) and juvenile/hatching year (JUV/HY) House Finches and House Sparrows positive for antibodies against WNV based on EIA results. Seroprevalence was cumulative and based on all birds regardless of recapture status. doi:10.1371/journal.pone.0034127.g003 positive recapture was not always well aligned with the year of House Finch abundance (Figure 2) may have been due to actual infection, and many AHY House Finches were most likely enhanced recruitment or the progressive acceptance of our traps recorded as seroconversions after their year of infection. as routine feeding stations. Some long-lived birds were recaptured on multiple occasions Survivorship over several years (Figure 5). For example, House Finch 2 was Seroprevalence between outbreak years declined (Figure 3) as a captured on 50 occasions and House Sparrow 5 on 57 occasions. function of recruitment and survivorship. The number of birds These long term recaptures allowed us to examine antibody recaptured was plotted as a function of weeks between the first and persistence under field conditions. All 6 of the House Finches that last date of capture grouped by species and infection status and seroconverted remained positive throughout the study, although transformed to natural logarithms (Figure 4). Numbers of birds House Finch 6 lost neutralizing antibody and several birds recaptured or surviving per 10 week time interval for each group exhibited unexplained intermittent negative test results. House decreased as a significant linear function (P,0.001) of weeks. Sparrows 2, 5 and 6 that were initially positive by both EIA and Interestingly, the slope values for the fitted regression equations for PRNT reverted to seronegative over time, and all birds exhibited infected birds of both species were significantly less (P,0.05) than intermittent negative test results. Serum samples were assigned the slope values for non-infected birds, indicating they survived sequential numbers in the field, and laboratory staff did not know significantly longer due to acquired immune status (Table 4). In a the band numbers, so these samples were tested ‘blind’. We 2-way ANOVA of weeks in study grouped by species and infection initially suspected that these test discrepancies were due to status, House finches lived significantly longer (F = 16.65, df = 1, laboratory assay inconsistency; however, when multiple specimens 2383, P,0.001) than House Sparrows, and birds ever positive for were retested the discrepancies shown in Figure 5 remained. In WNV infection lived significantly longer (F = 158.5, df = 1, 2383, addition, paired tests from 44 experimentally infected birds that P,0.001) than never infected birds. In agreement with the were known to be negative, infected once, or challenged with the similarity in regression slopes, the interaction term in this ANOVA same virus provided satisfactory EIA and PRNT results (Figure 6), was not significant (P.0.05). Population losses for both infected although none of these birds had infections for longer than 6 and non-infected birds included death and emigration; however, weeks. the uninfected birds also suffered mortality from their initial WNV infection and may have had a greater emigration rate as HY birds Seroprevalence departed the study area after fledging. There were no significant Results from House Finches and House Sparrows were differences in regression slopes between species, so the increase in combined to examine the effects of cumulative seroprevalence or

PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus 2011 2011 0/185 8/771 1/160 0/99 2010 2010 1/168 8/1028 1/246 1/202

Figure 4. Number of House Finches (HOFI) and House Sparrows (HOSP) ever testing positive (POS) or negative (NEG) for West Nile virus antibodies transformed to ln(y+1) and plotted as a function time retained within our study area grouped into 10 week intervals. 2009 2009 8/237 70/1385 15/446 5/260 doi:10.1371/journal.pone.0034127.g004

herd immunity on tangential transmission to sentinel chickens and humans (Figure 7). Seroprevalence here was antibody positive birds over total birds bled per month, combined over species and age, and therefore was comparable to the cumulative seroconver- 2008 2008 2/134 32/654 10/110 18/295 sions in sentinel chickens within flocks. The increase in seroprevalence commenced concurrent with seroconversions of sentinel chickens and the onset of human cases, but typically peaked 4–6 weeks later, as shown by cross-correlation analyses (Figure 8). It appeared, however, that once seroprevalence or ‘herd immunity’ exceeded ca. 0.25, the numbers of new human cases 2007 2007 3/244 26/972 3/256 0/230 subsided and remained low during subsequent years until seroprevalence declined to #0.10 during late winter/early spring (Figure 9). Overall, the number of WNND cases during the summer transmission season (Jul–Sep) was inversely correlated (r = 20.709, df = 6, P,0.05) with combined seroprevalence during 2006 2006 5/229 19/420 3/134 2/349 Table 4. The numbers of House Finches or House Sparrows that ever tested positive (pos) or negative (neg) for WNV antibody transformed by ln(y+1) and regressed as a function of time retained within study areas grouped in 10 week intervals. 7/336 37/415 2005 2005 7/181 6/56 House Finch House Sparrow

Statistic pos neg pos neg

Intercept 3.480 5.410 3.032 4.709 Slope 20.011 20.024 20.014 20.023 LL 20.014 20.029 20.013 20.028 37/744 48/697 2004 2004 22/324 13/83 UL 20.009 20.020 20.009 20.017 R2 0.816 0.892 0.805 0.822 Survival 0.989 0.976 0.986 0.978 Mean age 52.6 26.0 43.8 18.3 SE 3.33 0.95 3.68 0.90 n 294 1146 118 825

All slopes were significant (P,0.001) when tested by ANOVA. LL and UL are the

Total seroconversions/recaptures detected per year for after hatching year (AHY) and juvenile and hatching year (JUV/HY) House Finches and House Sparrows. lower and upper 95% confidence limits about the slope; slopes with non- overlapping limits were significantly different (P,0.05). R2 is the coefficient of determination. Survivorship was estimated as was the backtransformed slope and measured retention within the study, with losses due to mortality and emigration. Mean age was expressed as weeks remaining within the study area. AHY AHY JUV/HY JUV/HY House Sparrows Table 3. House Finches Seroconversions were listed withindoi:10.1371/journal.pone.0034127.t003 the first year they were detected for recaptured birds. doi:10.1371/journal.pone.0034127.t004

PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

Figure 5. Number of times recaptured birds tested negative, EIA positive, and EIA and PRNT positive. Data are shown for 6 House Finches and 6 House Sparrows collected on multiple occasions. doi:10.1371/journal.pone.0034127.g005 the previous winter (Jan–Mar). Winter seroprevalence #0.10 Discussion during 2004, 2008 and 2011 was followed by outbreaks of human WNND reported to the Los Angeles Department of Public Health. Elevated herd immunity in peridomestic House Finch and House Sparrow populations impacted WNV transmission dynamics in Los Angeles in several ways. First, the accumulation of seropositve birds to .25% of the total during outbreak years seemed to dampen or even arrest tangential transmission during late summer (Figure 7), as measured by new WNND cases and seroconversions in sentinel chickens as well as the infection rate in Cx. p. quinquefasciatus mosquitoes and in dead American Crows reported by the public [5]. Temperatures in Los Angeles during September and October usually remained warm and conducive to transmission [5,30], and American Crows at communal roosts remained reasonably abundant, despite mortality due to WNV infection. These data implied that even though viremic corvids may have been critical in driving infection into the Culex vector population [12], transmission at large communal roosts may not have been sufficient to continue tangential transmission without a receptive passerine population to support peridomestic transmission [13,14]. Interestingly, the level of protective herd immunity in these maintenance hosts seen here for a complex zoonotic arbovirus was far less than the estimated 75–85% required for vaccination to protect humans from directly transmitted pathogens Figure 6. Inverse of plaque reduction neutralization test [31]. However, further field studies are needed to establish the (PRNT) titers per mL plotted as a function of enzyme levels of corvid abundance and infection at late summer immunoassay positive over negative well optical density ratios (EIA P/N) for WNV experimentally infected and uninfected communal roosts that are needed to support outbreaks of WNV. House Finches and House Sparrows (n = 44). Secondly, although the mechanisms of WNV overwintering in doi:10.1371/journal.pone.0034127.g006 California have not been fully resolved, several paradigms have

PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

Figure 7. West Nile neuroinvasive disease (WNND) human cases, proportion seroprevalence of House Finches and House Sparrows combined, and cumulative sentinel chicken seroconversions plotted by monthly intervals, Los Angeles, California. doi:10.1371/journal.pone.0034127.g007 been supported by field data, including persistent infection in and House Sparrows [10,33], and therefore elevated herd quiescent female and vertically infected Cx. p. quinquefasciatus and in immunity in these species would suppress transmission and delay chronically infected birds, and continued low level transmission amplification until after the recruitment of naı¨ve hatching year during periods of warm weather [32]. Regardless of the (HY) birds. As indicated by the reduced number of seroconver- overwintering mechanism, transmission most probably commenc- sions (Table 3) as well as the low seroprevalence in HY birds es in late winter when the weather warms, Cx. p. quinquefasciatus (Figure 3), years with decreased transmission produced few new resume gonotrophic activity, and resident passerines begin infections, and during these subsidence years seroprevalence was reproductive behavior. At this time most Culex in maritime associated with surviving AHY birds infected during previous California blood feed on after hatching year (AHY) House Finches years.

Figure 8. Cross correlations for House Finch (HOFI), House Sparrow (HOSP) and combined seroprevalence against A) Human cases of West Nile neuroinvasive disease and B) sentinel chicken seroconversions. doi:10.1371/journal.pone.0034127.g008

PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

birds (0.35) were much less than post-WNV estimates of 0.47 per year in Sacramento, perhaps reflecting the impact of greater infection rates in Los Angeles. Antibody persistence waned over time in naturally infected birds, contrasting laboratory studies [20,37] and outdoor flight cage studies [21] that showed long term retention of PRNT titers in House Sparrows and House Finches. Field data for 12 especially long-lived birds showed that some individuals intermittently reverted to antibody-negative over time, agreeing with previous results for SLEV in naturally-infected field birds [38]. Our short term field data for House Finches and House Sparrows agreed well with several laboratory host competence experiments [15,19] that showed good agreement between EIA and PRNT results for up to 6 weeks. Although data coding errors by mis-reading band numbers in the field cannot be discounted or double checked, it Figure 9. West Nile neuroinvasive disease (WNND) cases appears that some birds may undergo changes in immunity with during July–September plotted as a function of combined age leading to changes in test results. Future studies will address antecedent House Finch and House Sparrow seroprevalence the impact of these immune changes on virus recrudescence in during January–March for each year from 2003–2011. Values chronically infected birds. were inversely correlated (r, P,0.05). In addition to ambient temperature [2], the level of herd doi:10.1371/journal.pone.0034127.g009 immunity within peridomestic passerine populations during late Acquired immunity significantly increased avian survivorship winter and spring seemed critical in delineating the timing and and the mean duration of life within our study areas (Table 4), and slope of the WNV amplification curve, in establishing the may have slowed the decline of seroprevalence following outbreak amplitude of the curve during summer, and ultimately in years, requiring more than one season to dilute seroprevalence to determining if sufficient tangential transmission occurred to low enough levels to allow early season amplification. In precipitate an outbreak of human disease. Although these agreement, WNV recrudescence occurred in 2008, 3 years after conclusions were well-supported by data for Los Angeles, the 2004 outbreak, and in 2011, 2 years after the 2008 outbreak. additional studies are needed in other habitats such Bakersfield The shorter period of subsidence after 2008 may have related to in Kern County where outbreaks have recurred during successive the lower peak seroprevalence during the outbreak (,30%) and years despite moderate herd immunity in House Finches and the more rapid return to ,10% than after 2004, when Western Scrub-jays [39] or in habitats with high avian diversity seroprevalence peaked at 51% during December. Although and low corvid abundance such as Coachella Valley [29] where difficult to measure, both species populations also were probably continued low herd immunity has failed to result in outbreaks of impacted heavily by mortality associated with WNV infection, human disease. because experimentally infected House Finches and House Sparrows showed 65 and 38% mortality, respectively. This Acknowledgments mortality may have contributed to the survivorship differences seen between seropositive and negative birds. Interestingly, We thank the Scientific-Technical Department of the Greater Los Angeles County Vector Control District for assistance with the collection and although calculated differently, our survivorship estimates were sampling of the birds and chickens. Wild bird sera were tested by S Garcia greater than those for a smaller cohort of House Finches and and Y Fang at the UC Davis Center for Vectorborne Diseases. Sentinel House Sparrows banded and recaptured in Kern County [34] chicken sera were tested by the Viral and Rickettsial Disease Laboratory when they were infected at a low level with WEEV and SLEV and the Vector-borne Disease Section of the California Department of [35]. Similar to our data, they found that House Finches lived Public Health. Human case reports were compiled by the Los Angeles longer than House Sparrows, and that some especially long-lived County Department of Public Health. birds were recaptured 55 and 66 months after banding, respectively. In addition, House Finches in Sacramento County Author Contributions were found to have an annual survival rate of 0.59 before and 0.47 Conceived and designed the experiments: WKR JLK. Performed the after the arrival of WNV [36]. Annual survivorship estimates for experiments: SK JLK. Analyzed the data: JLK WKR. Contributed seropositive birds in LA were similar to pre-WNV estimates in reagents/materials/analysis tools: SK. Wrote the paper: WKR JLK. Sacramento of 0.59, but estimates for Los Angeles seronegative

References 1. Reisen WK (2010) Landscape epidemiology of vector-borne diseases. Annu Rev 6. Wheeler SS, Barker CM, Armijos MV, Carroll BD, Husted SR, et al. (2009) Entomol 55: 461–483. Differential impact of West Nile virus on California birds. Condor 111: 1–20. 2. Hartley DM, Barker CM, Le Menac’h A, Niu T, Gaff HD, et al. (2011) The 7. Koenig WD, Marcus L, Scott TW, Dickinson JL (2007) West Nile virus and effects of temperature on the emergence and seasonality of West Nile virus in California breeding bird declines. Ecohealth 4: 18–24. California. Am J Trop Med Hyg [in press]. 8. Tempelis CH, Reeves WC, Bellamy RE, Lofy MF (1965) A three-year study of 3. Kilpatrick AM (2011) Globalization, land use, and the invasion of West Nile the feeding habits of Culex tarsalis in Kern County, California. Am J Trop Med virus. Science 334: 323–327. Hyg 14: 170–177. 4. Hayes EB, Komar N, Nasci RS, Montgomery SP, O’Leary DR, et al. (2005) 9. Thiemann TC, Lemenager D, Kluh S, Carroll B, Lothrop HD, et al. (2011) Epidemiology and transmission dynamics of West Nile virus disease. Emerg Host selection patterns of Culex tarsalis and the Culex pipiens in California. Proc Infect Dis 11: 1167–1173. Mosq Vector Control Assoc Calif 79. 5. Kwan JL, Kluh S, Madon MB, Reisen WK (2010) West Nile virus emergence 10. Molaei G, Cummings RF, Su T, Armstrong PM, Williams GA, et al. (2010) and persistence in Los Angeles, California, 2003–2008. Am J Trop Med Hyg 83: Vector-host interactions governing epidemiology of West Nile virus in southern 400–412. california. Am J Trop Med Hyg 83: 1269–1282.

PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e34127 Avian Immunity to West Nile Virus

11. Tempelis CH, Reeves WC, Nelson RL (1976) Species identification of blood 26. Kwan JL, Kluh S, Madon MB, Nguyen DV, Barker CM, et al. (2010) Sentinel meals from Culex tarsalis that had fed on passeriform birds. Am J Trop Med Hyg chicken seroconversions track tangential transmission of West Nile virus to 25: 744–746. humans in the greater Los Angeles area of California. Am J Trop Med Hyg 83: 12. Reisen WK, Barker CM, Carney R, Lothrop HD, Wheeler SS, et al. (2006) Role 1137–1145. of corvids in epidemiology of West Nile virus in southern California. J Med 27. Reisen WK, Lin J, Presser SB, Enge B, Hardy JL (1993) Evaluation of new Entomol 43: 356–367. methods for sampling sentinel chickens for antibodies to WEE and SLE viruses. 13. Nielsen CF, Reisen WK (2007) Dead birds increase the risk of West Nile Virus Proc Calif Mosq Vector Control Assoc 61: 33–36. infection in Culex mosquitoes (Diptera: Culicidae) in Domestic Landscapes. J Med 28. Patiris PJ, Oceguera LF, III, Peck GW, Chiles RE, Reisen WK, et al. (2008) Entomol 44: 1007–1013. Serologic diagnosis of West Nile and St. Louis encephalitis virus infections in 14. Nasci RS, Komar N, Marfin AA, Ludwig GV, Kramer LD, et al. (2002) domestic chickens. Am J Trop Med Hyg 78: 434–441. Detection of West Nile Virus-infected mosquitoes and seropositive juvenile birds 29. Reisen WK, Lothrop HD, Wheeler SS, Kennsington M, Gutierrez A, et al. in the vicinity of virus-positive dead birds. Am J Trop Med Hyg 67: 492–496. (2008) Persistent West Nile virus transmission and the apparent displacement St. 15. Fang Y, Reisen WK (2006) Previous infection with West Nile or St. Louis Louis encephalitis virus in southeastern California, 2003–2006. J Med Entomol encephalitis viruses provides cross protection during reinfection in house finches. 45: 494–508. Am J Trop Med Hyg 75: 480–485. 30. Reisen WK, Fang Y, Martinez VM (2006) Effects of temperature on the 16. Reisen WK, Barker CM, Fang Y, Martinez VM (2008) Does variation in Culex transmission of West Nile virus by Culex tarsalis (Diptera: Culicidae). J Med (Diptera: Culicidae) vector competence enable outbreaks of West Nile virus in Entomol 43: 309–317. California? J Med Entomol 45: 1126–1138. 31. Fine PEM (1993) Herd immunity: history, theory and practice. Epidemiol Rev 15: 265–302. 17. McCaughey K, Miles SQ, Woods L, Chiles RE, Hom A, et al. (2003) The 32. Reisen WK, Fang Y, Lothrop HD, Martinez VM, Wilson J, et al. (2006) California West Nile virus dead bird surveillance program. Proc Mosq Vector Overwintering of West Nile virus in Southern California. J Med Entomol 43: Control Assoc Calif 71: 38–43. 344–355. 18. Langevin SA, Brault AC, Panella NA, Bowen RA, Komar N (2005) Variation in 33. Thiemann T (2011) Bloodfeeding patterns of Culex tarsalis and the Culex pipiens virulence of West Nile virus strains for house sparrows (Passer domesticus). complex in California [dissertation]. University of California, Davis. Am J Trop Med Hyg 72: 99–102. 34. Milby MM, Wright ME (1976) Survival of house sparrows and house finches in 19. Reisen WK, Fang Y, Martinez VM (2005) Avian host and mosquito (Diptera: Kern County, California., 1963–1973. Bird Banding 47: 119–122. Culicidae) vector competence determine the efficiency of West Nile and St. 35. Milby MM, Reeves WC (1990) Natural infection in vertebrate hosts other than Louis encephalitis virus transmission. J Med Entomol 42: 367–375. man. In: Reeves WC, ed. Epidemiology and control of mosquito-borne 20. Nemeth NM, Oesterle PT, Bowen RA (2009) Humoral immunity to West Nile arboviruses in California, 1943–1987. Sacramento, Calif: Calif. Mosq. Vector virus is long-lasting and protective in the house sparrow (Passer domesticus). Control Assoc. pp 26–65. Am J Trop Med Hyg 80: 864–869. 36. Pellegrini AR, Wright S, Reisen WK, Treiterer B, Ernest HB (2011) Annual 21. Wheeler SS, Reisen WK (2009) Persistent West Nile Virus infections in avian survival of house finches in relation to West Nile virus. The Condor 113: hosts: a possible overwintering mechanism for WNV? Proc Mosq Vector 233–238. Control Assoc Calif 77: 42–43. 37. Nemeth N, Young G, Ndaluka C, Bielefeldt-Ohmann H, Komar N, et al. (2009) 22. McClure HE (1984) Bird Banding. Pacific Grove, Calif: Boxwood Press. Persistent West Nile virus infection in the house sparrow (Passer domesticus). Arch 23. Pyle P, Howell SNG, Yunick RP, DeSante DF (1987) Identification guide to Virol 154: 783–789. North American Passerines. Bolinas, Calif: Slate Creek Press. 38. Gruwell JA, Fogarty CL, Bennett SG, Challet GL, Vanderpool KS, et al. (2000) 24. Chiles RE, Reisen WK (1998) A new enzyme immunoassay to detect antibodies Role of peridomestic birds in the transmission of St. Louis encephalitis virus in to arboviruses in the blood of wild birds. J Vector Ecol 23: 123–135. southern California. J Wildl Dis 36: 13–34. 25. Ebel GD, DuPuis AP, Nicholas D, Young D, Maffei J, et al. (2002) Detection by 39. Reisen WK, Carroll BD, Takahashi R, Fang Y, Garcia S, et al. (2009) Repeated Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds. West Nile virus epidemic transmission in Kern County, California, 2004–2007. Emerg Infect Dis 8: 979–982. J Med Entomol 46: 139–157.

PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e34127 Synopses

West Nile Fever—a Reemerging Mosquito-Borne Viral Disease in Europe

Zdenek Hubálek and Jirí Halouzka Academy of Sciences, Brno, Czech Republic

West Nile virus causes sporadic cases and outbreaks of human and equine disease in Europe (western Mediterranean and southern Russia in 1962-64, Belarus and Ukraine in the 1970s and 1980s, Romania in 1996-97, Czechland in 1997, and Italy in 1998). Environmental factors, including human activities, that enhance population densities of vector mosquitoes (heavy rains followed by floods, irrigation, higher than usual temperature, or formation of ecologic niches that enable mass breeding of mosquitoes) could increase the incidence of West Nile fever.

The 1996-97 outbreak of West Nile fever in and near Bucharest, Romania, with more than 500 clinical cases and a case-fatality rate approaching 10% (1-3), was the largest outbreak of arboviral illness in Europe since the Ockelbo- Pogosta-Karelian fever epidemic caused by Sindbis virus in northern Europe in the 1980s. This latest outbreak reaffirmed that mosquito- borne viral diseases may occur on a mass scale, even in temperate climates. West Nile virus is a member of the Japanese encephalitis antigenic complex of the genus Flavivirus, family Flaviviridae (4). All known members of this complex (Alfuy, Japanese encephalitis, Kokobera, Koutango, Kunjin, Mur- ray Valley encephalitis, St. Louis encephalitis, Stratford, Usutu, and West Nile viruses) are transmissible by mosquitoes and many of them can Figure. European distribution of West Nile virus, based on the virus isolation from mosquitoes or cause febrile, sometimes fatal, illnesses in humans. vertebrates, including humans (black dots), labora- West Nile virus was first isolated from the tory-confirmed human or equine cases of West Nile blood of a febrile woman in the West Nile district fever (black squares), and presence of antibodies in of Uganda in 1937 (5) and was subsequently vertebrates (circles and hatched areas). isolated from patients, birds, and mosquitoes in Egypt in the early 1950s (6-7). The virus was Egypt, Ethiopia, India, Israel, Kazakhstan, soon recognized as the most widespread of the Madagascar, Morocco, Mozambique, Nigeria, flaviviruses, with geographic distribution in- Pakistan, Senegal, South Africa, Tajikistan, cluding Africa and Eurasia. Outside Europe Turkmenia, Uganda, and Uzbekistan. Further- (Figure), the virus has been reported from more, West Nile virus antibodies have been Algeria, Asian Russia, Azerbaijan, Botswana, detected in human sera from Armenia, Borneo, Central African Republic, Côte dIvoire, Cyprus, China, Georgia, Iraq, Kenya, Lebanon, Malay- Democratic Republic of Congo (former Zaire), sia, the Philippines, Sri Lanka, Sudan, Syria, Thailand, Tunisia, and Turkey (8-10). Kunjin Address for correspondence: Z. Hubálek, Institute of virus is closely related to West Nile virus (11,12), Vertebrate Biology, Academy of Sciences, Kláterní 2, CZ- 69142 Valtice, Czech Republic; fax: 420-627-352-387; e-mail: representing a counterpart or subtype for [email protected]. Australia and Southeast Asia; some West Nile

Vol. 5, No. 5, SeptemberOctober 1999 643 Emerging Infectious Diseases Synopses

virus seroreactions in Southeast Asia may, in maintaining transmission cycles of the virus in fact, represent antibodies to Kunjin virus. ecosystems. Only horses and lemurs (20) have moderate viremia and seem to support West Nile West Nile Virus Ecology virus circulation locally. Frogs (Rana ridibunda) also can harbor the virus, and their donor ability Arthropod Vectors for Cx. pipiens has been confirmed (21). Mosquitoes, largely bird-feeding species, are the principal vectors of West Nile virus. The Transmission Cycles virus has been isolated from 43 mosquito species, Although Palearctic natural foci of West Nile predominantly of the genus Culex (Table 1). In virus infections are mainly situated in wetland Africa and the Middle East, the main vector is ecosystems (river deltas or flood plains) and are Cx. univittatus (although Cx. poicilipes, Cx. neavei, characterized by the bird-mosquito cycle, argasid Cx. decens, Aedes albocephalus, or Mimomyia spp. and amblyommine ticks may serve as substitute play an important role in certain areas). In Europe, vectors and form a bird-tick cycle in certain dry the principal vectors are Cx. pipiens, Cx. modestus, and warm habitats lacking mosquitoes. Even a and Coquillettidia richiardii, and in Asia, frog-mosquito cycle (21) may function under Cx. quinquefasciatus, Cx. tritaeniorhynchus, and certain circumstances. Cx. vishnui predominate. Successful experimen- In Europe, West Nile virus circulation is tal transmission of the virus has been described confined to two basic types of cycles and in Culiseta longiareolata, Cx. bitaeniorhynchus, ecosystems: rural (sylvatic) cycle (wild, usually and Ae. albopictus (8,13). Transovarial transmis- wetland birds and ornithophilic mosquitoes) and sion of the virus has been demonstrated in Cx. urban cycle (synanthropic or domestic birds and tritaeniorhynchus, Ae. aegypti, and Ae. albopictus, mosquitoes feeding on both birds and humans, though at low rates. mainly Cx. pipiens/molestus). The principal Virus isolations have occasionally been cycle is rural, but the urban cycle predominated reported from other hematophagous arthropods in Bucharest during the 1996-97 outbreak (2,3). (e.g., bird-feeding argasid [soft] or amblyommine Circulation of West Nile fever in Europe is [hard] ticks) (Table 1), and experimental similar to that of St. Louis encephalitis in North transmission has been observed in Ornithodoros America, where the rural cycle of exoanthropic savignyi, O. moubata, O.maritimus, O. erraticus, birdsCx. tarsalis alternates with the urban Rhipicephalus sanguineus, R. rossicus, Derma- cycle of synanthropic birdsCx. pipiens/ centor reticulatus, and Haemaphysalis leachii quinquefasciatus. (8,13). West Nile Fever in Humans and Other Vertebrate Hosts Vertebrates Wild birds are the principal hosts of West Nile virus. The virus has been isolated from a Humans number of wetland and terrestrial avian species West Nile fever in humans usually is a in diverse areas (7-10,14-16). High, long-term febrile, influenzalike illness, characterized by an viremia, sufficient to infect vector mosquitoes, abrupt onset (incubation period is 3 to 6 days) of has been observed in infected birds (7,17,18). The moderate to high fever (3 to 5 days, infrequently virus persists in the organs of inoculated ducks biphasic, sometimes with chills), headache (often and pigeons for 20 to 100 days (18). Migratory frontal), sore throat, backache, myalgia, arthral- birds are therefore instrumental in the introduc- gia, fatigue, conjunctivitis, retrobulbar pain, tion of the virus to temperate areas of Eurasia maculopapular or roseolar rash (in approxi- during spring migrations (12,14-16,19). mately half the cases, spreading from the trunk Rarely, West Nile virus has been isolated to the extremities and head), lymphadenopathy, from mammals (Arvicanthis niloticus, Apodemus anorexia, nausea, abdominal pain, diarrhea, and flavicollis, Clethrionomys glareolus, sentinel respiratory symptoms (9). Occasionally (<15% of mice and hamsters, Lepus europaeus, Rousettus cases), acute aseptic meningitis or encephalitis leschenaulti, camels, cattle, horses, dogs, Galago (associated with neck stiffness, vomiting, senegalensis, humans) in enzootic foci (8-10). confusion, disturbed consciousness, somnolence, Mammals are less important than birds in tremor of extremities, abnormal reflexes,

Emerging Infectious Diseases 644 Vol. 5, No. 5, SeptemberOctober 1999 Synopses

Table 1. Isolations of West Nile virus from hematophagous arthropods (7-10) Species No. Countries Mosquitoes Culex antennatusa 6 Egypt, Madagascar decens group 8 Madagascar ethiopicus 1 Ethiopia guiarti 1 Côte dIvoire modestus 3 France, Russia neavei 4 Senegal, South Africa nigripes 1 Central African Republic perexiguus 1 Israel perfuscus group 3 Central African Republic, Senegal pipiensa 7 South Africa, Egypt, Israel, Romania, Czechland, Bulgariab poicilipes 29 Senegal pruina 1 Central African Republic quinquefasciatusa 7 India, Pakistan, Madagascar scottii 1 Madagascar theileria 4 South Africa tritaeniorhynchusa 3 Pakistan, India, Madagascar univittatusa 51 Egypt, Israel, South Africa, Madagascar vishnuia group 6 India, Pakistan weschei 1 Central African Republic sp. 3 Egypt, Algeria, Central African Republic Coquillettidia metallica 1 Uganda microannulata 1 South Africa richiardii 5 South Russia, Bulgariab Mansonia uniformis 1 Ethiopia Aedes aegyptia 1 Madagascar africanus 1 Central African Republic albocephalus 35 Madagascar albothorax 1 Kenya cantans 7 Slovakia, Ukraine, Bulgariab caspiusa 1 Ukraine circumluteolus 2 South Africa, Madagascar excrucians 1 Ukraine juppi+caballus 1 South Africa madagascarensis 1 Madagascar vexans 3 Senegal, Russia Anopheles brunnipes 1 Madagascar coustani 1 Israel maculipalpis 1 Madagascar maculipennis 3 Portugal, Ukraine subpictus 1 India sp. 1 Madagascar Mimomyia hispida 8 Senegal lacustris 4 Senegal splendens 6 Senegal sp. 2 Senegal Aedeomyia africana 1 Senegal Soft ticks Argas hermannia 3 Egypt Ornithodoros capensisa 5 Azerbaijan Hard ticks Hyalomma marginatum 5 Astrakhan, Azerbaijan detritum 1 Turkmenistan Rhipicephalus turanicus 1 Azerbaijan muhsamae 1 Central African Republic Amblyomma variegatum 1 Central African Republic Dermacentor marginatusa 1 Moldavia aExperimental transmission of the virus also demonstrated. bDetected in mosquitoes by immunofluorescence assay.

Vol. 5, No. 5, SeptemberOctober 1999 645 Emerging Infectious Diseases Synopses

convulsions, pareses, and coma), anterior myelitis, infection seen in pigs and dogs (9,28). Rabbits, hepatosplenomegaly, hepatitis, pancreatitis, adult albino rats, and guinea pigs are resistant to and myocarditis occur. Laboratory findings West Nile virus infection, but laboratory mice involve a slightly increased sedimentation rate and Syrian hamsters are markedly susceptible; and a mild leukocytosis; cerebrospinal fluid in they often become ill with fatal encephalitis, patients with central nervous system involve- even when inoculated peripherally (8). Adult ment is clear, with moderate pleocytosis and rodents stressed or immunosuppressed by cold, elevated protein. The virus can be recovered from isolation, cyclophosphamide, corticosterone, or the blood for up to 10 days in immunocompetent bacterial endotoxin contract fatal encephalitis, febrile patients, as late as 22 to 28 days after even when an attenuated viral strain is given infection in immunocompromised patients; peak (29). Inoculation of rhesus and bonnet monkeys viremia occurs 4 to 8 days postinfection. (but not cynomolgus monkeys or chimpanzees) Recovery is complete (less rapid in adults than in causes fever, ataxia, and prostration with children, often accompanied by long-term myalgias occasional encephalitis, tremor of extremities, and weakness), and permanent sequelae have pareses, or paralysis. Infection may be fatal or not been reported. Most fatal cases have been cause long-term virus persistence in survivors recorded in patients older than 50 years. Many of (5,6,30). the West Nile fever symptoms have been reproduced in volunteers with underlying Birds neoplastic disease who had been inoculated with Birds usually do not show any symptoms virus to achieve pyrexia and oncolysis (22). when infected with West Nile virus. However, Hundreds of West Nile fever cases have been natural disease due to the virus has been described in Israel and South Africa. The largest observed in a pigeon in Egypt (7), and inoculation African epidemic, with approximately 3,000 of certain avian species (e.g., pigeons, chickens, clinical cases, occurred in an arid region of the ducks, gulls, and corvids) causes occasional Cape Province after heavy rains in 1974 (23). An encephalitis and death or long-term virus outbreak with approximately 50 patients, eight persistence (7,10,17,18). Chick embryos may be of whom died, was described in Algeria in 1994 killed by the virus (8). (1). Other cases or outbreaks have been observed in Azerbaijan, Central African Republic, Demo- West Nile Virus and Fever in Europe cratic Republic of Congo (former Zaire), Egypt, In Europe, the presence of West Nile virus Ethiopia, India, Madagascar, Nigeria, Pakistan, was indicated in 1958, when two Albanians had Senegal, Sudan, and in a few European countries. specific West Nile virus antibodies (31). The first European isolations of the virus were recorded in Horses 1963 from patients and mosquitoes in the Rhône Equine disease, called Near Eastern equine Delta (32) and from patients and Hyalomma encephalitis in Egypt and lourdige in France, marginatum ticks in the Volga Delta (33,34). was observed and experimentally reproduced as West Nile virus was subsequently isolated in fever and diffuse encephalomyelitis with a Portugal (35), Slovakia (36), Moldavia (37), moderate to high fatality rate in Egypt (24), Ukraine (38), Hungary (39), Romania (2), France (c. 50 cases in 1962-65) (25), Italy (14 Czechland (40), and Italy (V. Deubel, G. Ferrari, cases in 1998, six died or were euthanised) (R. pers. comm.). Lelli, G. Ferrari, pers. comm.), Portugal (26) and The incidence of West Nile fever in Europe is Morocco (42 of 94 affected horses died) (27). In largely unknown. In the 1960s, cases were the 1960s, the biphasic, encephalomyelitic form, observed in southern France (25), southern which causes staggering gait and weakness to Russia (41), Spain (26), southwestern Romania paralysis of the hind legs, was apparent among (42), in the 1970s, 1980s, and 1990s in Belarus infected semiferal horses in Camargue (25). (43), western Ukraine (44), southeastern Romania (1,2), and Czechland (45). West Nile Other Mammals fever in Europe occurs during the period of Inoculation of sheep with West Nile virus maximum annual activity of mosquito vectors results in fever, abortion in pregnant ewes, and (July to September) (Table 2). rare encephalitis, in contrast to the asymptomatic

Emerging Infectious Diseases 646 Vol. 5, No. 5, SeptemberOctober 1999 Synopses

Table 2. West Nile Virus in Europe, 1960-1998 Country Year Species infected HIa (%) Neutralization (%) Ref. Portugal, southern 1967-1970 Cattle, sheep 15 26, 35 Horses 29 Humans 3 Mosquitoes (1 isolate) Wild birds 5 Spain, northern 1979 Rodents 3 Northwestern 1960s Humans 17 17 26 Doñana National Park Humans + Ebro Delta 1979 Epidemic of influenza-like illness 8-30 France, southern 1962 Humans, 10 severe cases (2 isolates) 19 25,32,46 Horses, 50 encephalomyelitis cases (1 isolate) 9 30 Camargue 1962-1965 Mosquitoes (2 isolates) Wild birds 6 1975-1980 Humans 5 Horses 2 Corsica 1965 Humans 30 55 Italy, Tuscany 1998 Horses, 14 cases, 2 fatal (1 isolate) 40 47 Northeastern 1967-1969 Migrating birds 10 Humans 5 b Northwestern Humans 23 Central Humans 2-8 Domestic mammals 8 Rodents 8 Chickens 20 Southern Humans 2-5 Goats 2-13 1981 Rodents 1 Former Yugoslavia Serbia Humans 1-8 48 Croatia Humans 1-3 Montenegro Humans 1 Bosnia, Kosovo Humans 1 Albania 1958 Humans 2 31,49 Domestic animals Greece 1970-1978 Humans 1-27 1 50,51 Domestic animals 7 Rabbits 4 Birds 22 Bulgaria 1960-1970 Humans 3 52,53 Eastern Wetland birds 2 10 Domestic animals 1 1978 Mosquitoes (virus detected) Romania 1-3,42,54,55 Bucharest, SE lowlands 1996 Humans, 453 clin. cases, 9% fatality rate (1 isolate) 17 1997 Human, 14 cases, 2 fatal Domestic & wild mammals 2-23 c Dogs 19-45 Wild birds 22 1966-70 Mosquitoes (1 isolate) Banat (SW) Humans (cases) 17 Southern 1980-1995 Humans 2-12 Hungary 1970s Rodents (2 isolates) 39,56 Cattle 4-9 Humans 4-6 Slovakia 1972 Mosquitoes (1 isolate) 16, 36, 57-60 1970-1973 Migrating birds (4 isolates) 1-13 Game animals 1-8 Cattle, dogs 8 Sheep 1 Pigeons 5 Humans 1-4 Austria 1964-1977 Wetland passerines 1-3 61,62 Reptiles Wild mammals Domestic animals 7-33 Humans 1-6 Czechland 1978 Domestic animals 2 40,45,63-67 Southern Bohemia 1978 Hares 5 Southern Moravia 1980s Game animals 8 1985 Wetland birds 4-10 1990 Cormorants 10 1997 Mosquitoes (1 isolate) Humans (5 cases) 2 Poland, near Warsaw 1996 Sparrows 3-12 68 Belarus 1977 Humans (cases in Brest area) 1 43 1972-1973 Wild birds 3 Ukraine 38,44,69 Southern, western Birds (7 isolates), mosquitoes (3 isolates) Southern 1970s Human cases (4 isolates) Western 1985 Humans, 38 cases, encephalitis in 16 Moldavia 1970s Ticks, mosquitoes (several isolates) 37, 70 Humans 3 Russia, 33,34,41,55 Volga Delta 1963-1968 Humans (>10 cases, 3 isolates) 7-31 Ticks (4 isolates) Water birds (2 isolates) 4-59 2-11 Mosquitoes (2 isolates) aHemagglutination inhibition. bQ. Ferrari, R. Lelli, pers. comm. c C. Ceianu, pers. comm.

Vol. 5, No. 5, SeptemberOctober 1999 647 Emerging Infectious Diseases Synopses

The Future 2. Tsai TF, Popovici F, Cernescu C, Campbell GL, Nedelcu West Nile virus can cause sporadic human NI. West Nile encephalitis epidemic in southeastern cases, clusters, or outbreaks of West Nile fever, Romania. Lancet 1998;352:767-71. 3. Savage HM, Ceianu C, Nicolescu G, Karabatsos N, even in temperate Europe. Environmental Lanciotti R, Vladimirescu A, et al. Entomologic and factors, including human activities that enhance avian investigations of an epidemic of West Nile fever in vector population densities (irrigation, heavy Romania, 1996, with serologic and molecular rains followed by floods, higher than usual characterization of a virus isolate from mosquitoes. Am temperatures, and formation of ecologic niches J Trop Med Hyg. In press 1999. 4. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, enabling the mass breeding of mosquitoes) allow Porterfield JS, Westaway EG, et al. Antigenic the reemergence of this mosquito-borne disease. relationships between flaviviruses as determined by For instance, global warming scenarios hypoth- cross-neutralization tests with polyclonal antisera. J esize warmer, more humid weather that may Gen Virol 1989;70:37-43. produce an increase in the distribution and 5. Smithburn KC, Hughes TP, Burke AW, Paul JH. A neurotropic virus isolated from the blood of a native of abundance of mosquito vectors (71). Surveillance Uganda. Am J Trop Med Hyg 1940;20:471-92. for West Nile fever (monitoring population 6. Melnick JL, Paul JR, Riordan JT, Barnett VHH, densities and infection rates of principal vectors, Goldblum N, Zabin E. Isolation from human sera in serosurveys on vertebrates and exposed human Egypt of a virus apparently identical to West Nile virus. groups, and routine diagnosis of human Proc Soc Exp Biol Med 1951;77:661-5. 7. Taylor RM, Work TH, Hurlbut HS, Rizk F. A study of infections) should therefore be carried out in the ecology of West Nile virus in Egypt. Am J Trop Med affected areas. Hyg 1956;5:579-620. The mechanism of West Nile virus persis- 8. Karabatsos N, editor. International catalogue of tence in disease-endemic foci of temperate arboviruses, including certain other viruses of vertebrates. Europe presents a challenge for further 3rd ed., and Supplements 1986-98. San Antonio: American Society of Tropical Medicine and Hygiene; 1985. research. General hypotheses of how an 9. Peiris JSM, Amerasinghe FP. West Nile fever. In: arbovirus could overwinter under adverse Beran GW, Steele JH, editors. Handbook of zoonoses. climatic conditions have already been postulated Section B: Viral. 2nd ed. Boca Raton (FL): CRC Press; (72). The virus could persist in hibernating 1994. p. 139-48. female Culex spp.; transovarially infected Culex 10. Hubálek Z, Halouzka J. Arthropod-borne viruses of vertebrates in Europe. Acta Scientiarum Naturalium spp. progeny; or chronically infected vertebrate Brno 1996;30(no.4-5):1-95. hosts, perhaps birds or frogs. Alternatively, the 11. Zanotto PM de A, Gould EA, Gao GF, Harvey PH, virus may be reintroduced by chronically Holmes EC. Population dynamics of flaviviruses infected migratory birds from tropical or revealed by molecular phylogenies. Proc Natl Acad Sci subtropical foci at irregular intervals. These U S A 1996;93:548-53. 12. Berthet FX, Zeller HG, Drouet MT, Rauzier J, Digoutte issues have to be addressed, because present JP, Deubel V. Extensive nucleotide changes and data substantiate all particular mechanisms and deletions within the envelope glycoprotein gene of their combinations. For instance, the hibernat- Euro-African West Nile viruses. J Gen Virol ing vector idea has been supported by a few field 1997;78:2293-7. and experimental data on female Cx. univittatus 13. Hurlbut HS. West Nile virus infection in arthropods. Am J Trop Med Hyg 1956;5:76-85. (7,73). On the other hand, if the reintroduction 14. Nir Y, Goldwasser R, Lasowski Y, Avivi A. Isolation of scheme is correct, a greatly increased activity of arboviruses from wild birds in Israel. Am J Epidemiol West Nile virus in Africa should be followed by 1967;86:372-8. an epidemic occurrence of West Nile fever in 15. Watson GE, Shope RE, Kaiser MN. An ectoparasite and Europe in the next few years. virus survey of migratory birds in the eastern Mediterranean. In: Cherepanov IA, editor. Zdenek Hubálek and Jirí Halouzka are scientists Transcontinental connections of migratory birds and at the Academy of Sciences of the Czech Republic. They their role in the distribution of arboviruses. Novosibirsk: Nauka; 1972 p.176-80. are interested in the ecology of arthropodborne human 16. Ernek E, Kozuch O, Nosek J, Teplan J, Folk C. pathogenic viruses and bacteria. Arboviruses in birds captured in Slovakia. Journal of Hygiene, Epidemioloigy, Microbiology and Immunology References (Prague) 1977;21:353-9. 17. Work TH, Hurlbut HS, Taylor RM. Indigenous wild birds of 1. Le Guenno B, Bougermouh A, Azzam T, Bouakaz R. the Nile Delta as potential West Nile virus circulating West Nile: A deadly virus? Lancet 1996;348:1315. reservoirs. Am J Trop Med Hyg 1955;4:872-8.

Emerging Infectious Diseases 648 Vol. 5, No. 5, SeptemberOctober 1999 Synopses

18. Semenov BF, Chunikhin SP, Karmysheva VY, 34. Butenko AM, Chumakov MP, Stolbov DN. Serological and Yakovleva NI. Studies of chronic arbovirus infections in virological examinations in a natural focus of West Nile birds. 1. Experiments with West Nile, Sindbis, Bhanja fever in the Astrakhan region (in Russian). Voprosy and SFS viruses (in Russian). Vestnik Akademii Medicinskoi Virusologii 1967;1:208-11. Medicinskikh Nauk SSSR (Moskva) 1973;2:79-83. 35. Filipe AR. Isolation in Portugal of West Nile virus from 19. Hannoun C, Corniou B, Mouchet J. Role of migrating Anopheles maculipennis mosquitoes. Acta Virol birds in arbovirus transfer between Africa and Europe. 1972;16:361. In: Cherepanov AI, editor. Transcontinental connections 36. Labuda M, Kozuch O, Greíková M. Isolation of West of migratory birds and their role in the distribution of Nile virus from Aedes cantans mosquitoes in west arboviruses. Novosibirsk: Nauka; 1972; p. 167-72. Slovakia. Acta Virol 1974;18:429-33. 20. Rodhain F, Petter JJ, Albignac R, Coulanges P, Hannoun 37. Chumakov MP, Bashkirtsev VN, Golger EI, Dzagurova C. Arboviruses and lemurs in Madagascar: experimental TK, Zavodova TI, Konovalov YN, et al. Isolation and infection of Lemur fulvus with yellow fever and West Nile identification of Crimean hemorrhagic fever and West Nile viruses. Am J Trop Med Hyg 1985;34:816. fever viruses from ticks collected in Moldavia (in Russian). 21. Kostyukov MA, Alekseev AN, Bulychev VP, Gordeeva Medicinskaya Virusologia 1974;22:45-9. ZE. Experimental infection of Culex pipiens mosquitoes 38. Vinograd IA, Obukhova VR. Isolation of arboviruses from with West Nile virus by feeding on infected Rana birds in western Ukraine (in Russian). Sbornik Trudov ridibunda frogs and its subsequent transmission (in Instituta Virusologii (Moskva) 1975;3:84-7. Russian). Med Parazitol (Mosk) 1986;6:76-8. 39. Molnár E, Gulyas MS, Kubinyi L, Nosek J, Kozuch O, 22. Southam CM, Moore AE. Induced virus infections in Ernek E, et al. Studies on the occurrence of tick-borne man by the Egypt isolates of West Nile virus. Am J Trop encephalitis in Hungary. Acta Veterinaria Academiae Med Hyg 1954;3:19-50. Scientiarum Hungaricae 1976;26:419-38. 23. McIntosh BM, Jupp PG, Dos Santos I, Meenehan GM. 40. Hubálek Z, Halouzka J, Juricová Z, ebesta O. First Epidemics of West Nile and Sindbis viruses in South Africa isolation of mosquito-borne West Nile virus in the with Culex (Culex) univittatus Theobald as vector. South Czech Republic. Acta Virol 1998;42:119-20. African Journal of Science 1976;72:295-300. 41. Butenko AM, Chumakov MP, Bashkirtsev VN, 24. Schmidt JR, El Mansoury HK. Natural and Tkachenko EA, Rubin SG, Stolbov DN. New experimental infection of Egyptian equines with West investigations of West Nile virus infections in the Nile virus. Am J Trop Med Hyg 1963;57:415-27. USSRAstrakhan region (in Russian). Materialy XV 25. Hannoun C, Panthier R, Corniou B. Epidemiology of West Nauchnoi Sessii Instituta Poliomielita i Virusnykh Nile infections in the South of France. In: Bárdo V, editor. Encefalitov (Moskva) 1968;3:175-6. Arboviruses of the California complex and the Bunya- 42. Topciu V, Rosiu N, Arcan P. Contribution to the study mwera group. Bratislava: Publ House SAS; 1969. p. 379-87. of arboviruses in Banat. Revue Roumaine 26. Filipe AR, De Andrade HR. Arboviruses in the Iberian dInframicrobiologie 1971;8:101-6. Peninsula. Acta Virol 1990;34:582-91. 43. Voinov IN, Rytik PG, Grigoriev AI. Arbovirus infections 27. Abdelhaq AT. West Nile fever in horses in Morocco. in Belarus (in Russian). In: Drozdov SG, editor. Virusy Bulletin de lOffice International des Épizooties i virusnyje infektsii. Moskva: Inst Poliomiel Virus Enc; 1996;108:867-9. 1981. p. 86-7. 28. Barnard BJH, Voges SF. Flaviviruses in South Africa: 44. Buletsa BA, Turak JA, Korol MJ, Ignatovich II, Vitvitskyi pathogenicity for sheep. Onderstepoort J Vet Res AA. Neurological manifestations of West Nile fever in the 1986;53:235-8. Transcarpathian region, Ukrainian SSR (in Russian). 29. Ben-Nathan D, Lustig S, Feuerstein G. The influence of Zhurnal Nevrologii Psikhiatrii 1989;89:29-30. cold or isolation stress on neuroinvasiveness and 45. Hubálek Z, Halouzka J, Juricová Z. West Nile fever in virulence of an attenuated variant of West Nile virus. Czechland. Emerg Infect Dis 1999;5:594-5. Arch Virol 1989;109:1-10. 46. Rollin PE, Rollin D, Martin P, Baylet R, Rodhain F, 30. Pogodina VV, Frolova MP, Malenko GV, Fokina GI, Hannoun C. Résultats denquetes séroépidemiologiques Koreshkova GV, Kiseleva LL, et al. Study on West Nile récentes sur les arboviroses en Camargue: populations virus persistence in monkeys. Arch Virol 1983;75:71-86. humaines, équines, bovines et aviaires. Médecine et 31. Bárdo V, Adamcová J, Dedei S, Gjini N, Rosický B, Maladies Infectieuses 1982;12:77-80. imková A. Neutralizing antibodies against some 47. Verani P, Balducci M, Lopes MC. Arboviruses in Italy. neurotropic viruses determined in human sera in In: Kurstak E, editor. Arctic and tropical arboviruses. Albania. Journal of Hygiene, Epidemiology, Microbiology New York: Academic Press; 1979. p. 101-21. and Immunology (Prague) 1959;3:277-82. 48. Vesenjak-Hirjan J, Punda-Poli V, Dobec M. Geographical 32. Hannoun C, Panthier R, Mouchet J, Eouzan JP. distribution of arboviruses in Yugoslavia. Journal of Isolement en France du virus West Nile à partir de Hygiene, Epidemiology, Microbiology and Immunology malades et du vecteur Culex molestus Ficalbi. Compte (Prague) 1991;35:129-40. Rendu de lAcadémie des Sciences 1964;D259:4170-2. 49. Eltari E, Gina A, Bitri T, Sharofi F. Some data on 33. Chumakov MP, Belyaeva AP, Butenko AM. Isolation arboviruses, especially tick-borne encephalitis, in and study of an original virus from Hyalomma Albania. Giornale de Malattie Infettive e Parassitaire plumbeum plumbeum ticks and from the blood of a 1993;45:404-11. febrile patient in the Astrakhan region (in Russian). 50. Koptopoulos G, Papadopoulos O. A serological survey for Materialy XI Nauchnoi Sessii Instituta Poliomielita i tick-borne encephalitis and West Nile viruses in Greece. Virusnykh Encefalitov (Moskva) 1964:5-7. Zentralblatt für Bakteriologie 1980;Suppl 9:185-8.

Vol. 5, No. 5, SeptemberOctober 1999 649 Emerging Infectious Diseases Synopses

51. Antoniadis A, Alexiou-Daniel S, Malissiovas N, Doutsos 62. Greíková M, Thiel W, Batiková M, Stünzner D, J, Polyzoni T, LeDuc JW, et al. Seroepidemiological Sekeyová M, Sixl W. Haemagglutination-inhibiting survey for antibodies to arboviruses in Greece. Arch antibodies against arboviruses in human sera from Virol 1990;Suppl 1:277-85. different regions in Steiermark (Austria). Zentralblatt 52. Rusakiev M. Studies on the distribution of für Bakteriologie 1973;224:298-302. arboviruses transmitted by mosquitoes in Bulgaria. 63. Greíková M, Sekeyová M, Vota J, Hanák P. In: Bárdo V, editor. Arboviruses of the California Haemagglutination-inhibiting antibodies to some complex and the Bunyamwera group. Bratislava: arboviruses in human and animal sera from Ceské Publ House SAS; 1969. p. 389-92. Budejovice. In: Sixl W, editor. Naturherde von 53. Katsarov G, Vasilenko S, Vargin V, Butenko S, Infektionskrankheiten in Zentraleuropa. Graz-Seggau: Tkachenko E. Serological studies on the distribution of Hyg Inst Univ; 1979. p. 25-9. some arboviruses in Bulgaria. Problems of Infectious 64. Juricová Z. Antibodies to arboviruses in game animals and Parasitic Diseases 1980;8:32-5. in Moravia, Czechland (in Czech). Veterinární 54. Draganescu N, Gheorghiu V. On the presence of group Medicina (Praha) 1992;37:633-6. B arbovirus infections in Romania. Investigations on 65. Juricová Z, Hubálek Z, Halouzka J, Machácek P. the incidence of West Nile antibodies in humans and Virological examination of cormorants for arboviruses (in certain domestic animals. Revue Roumaine Czech). Veterinární Medicina (Praha) 1993;38:375-9. dInframicrobiologie 1968;5:255-8. 66. Hubálek Z, Jur icová Z, Halouzka J, Pellantová J, Hudec 55. Antipa C, Girjabu E, Iftimovici R, Draganescu N. K. Arboviruses associated with birds in southern Serological investigations concerning the presence of Moravia, Czechoslovakia. Acta Scientiarum Naturalium antibodies to arboviruses in wild birds. Revue Brno 1989;23(7):1-50. Roumaine de Médicine - Virologie 1984;35:5-9. 67. Juricová Z, Halouzka J. Serological examination of 56. Molnár E, Greíková M, Kubászová T, Kubínyi L, Szabó domestic ducks in southern Moravia for antibodies against JB. Arboviruses in Hungary. Journal of Hygiene, arboviruses of the groups A, B, California and Epidemiology, Microbiology and Immunology (Prague) Bunyamwera (in Czech). Biológia (Bratisl) 1993;48:481-4. 1973;17:1-10. 68. Juricová Z, Pinowski J, Literák I, Hahm KH, 57. Kozuch O, Nosek J, Labuda M. Arboviruses occurring in Romanowski J. Antibodies to Alphavirus, Flavivirus, western Slovakia and their hosts. In: Labuda M, and Bunyavirus arboviruses in house sparrows (Passer Calisher CH, editors. New aspects in ecology of domesticus) and tree sparrows (P. montanus) in Poland. arboviruses. Bratislava: Inst Virol SAS; 1980. p. 323-33. Avian Dis 1998;42:182-5. 58. Kozuch O, Nosek J, Greíková M, Ernek E. 69. Vinograd IA, Beletskaya GV, Chumachenko SS, Surveillance on mosquito-borne natural focus in Omelchenko GA, Lozinski IN, Yartys OS, et al. Záhorská lowland. In: Sixl W, Troger H, editors. Ecological aspects of arbovirus studies in the Ukrainian Naturherde von Infektionskrankheiten in SSR (in Russian). In: Lvov DK, Gaidamovich SY, Zentraleuropa. Graz: Hyg Inst Univ; 1976. p. 115-8. editors. Ecology of viruses and diagnostics of arbovirus 59. Sekeyová M, Greíková M, Kozuch O. Haemagglutination- infections. Moscow: Acad Med Sci USSR; 1989. p. 21-7. inhibition antibodies to some arboviruses in sera of pigeons 70. Chumakov MP, Spasskiy AA, Uspenskaya IG, Tikhon trapped in Bratislava. In: Sixl W, Troger H, editors. EI, Zaitsev NA, Konovalov YN, et al. Virological, Naturherde von Infektionskrankheiten in Zentraleuropa. serological, zoological, and ecological investigations of Graz: Hyg Inst Univ; 1976. p. 187-9. natural foci of arbovirus infections in Moldavian SSR 60. Greíková M, Sekeyová M. Haemagglutination-inhibiting (in Russian). Abstr Conf Viruses and virus infections of antibodies against arboviruses in the population of humans. Moskva 1981, p. 101. Slovakia. Journal of Hygiene, Epidemiology, Microbiology 71. Reeves WC, Hardy JL, Reisen WK, Milby MM. and Immunology (Prague) 1967;11:278-85. Potential effect of global warming on mosquito-borne 61. Aspöck H, Kunz C, Picher O, Böck F. Virologische und arboviruses. J Med Entomol 1994;310:323-32. serologische Untersuchungen über die Rolle von Vögeln 72. Reeves WC. Overwintering of arboviruses. Prog Med als Wirte von Arboviren in Österreich. Zentralblatt für Virol 1974;17:193-220. Bakteriologie 1973;A224:156-67. 73. Cornel AJ, Jupp PG, Blackburn NK. Effect of environmental temperature on the vector competence of Culex univittatus (Diptera, Culicidae) for West Nile virus. J Med Entomol 1993;30:449-56.

Emerging Infectious Diseases 650 Vol. 5, No. 5, SeptemberOctober 1999 West Nile Virus: Biology, Transmission, and Human Infection

Tonya M. Colpitts, Michael J. Conway, Ruth R. Montgomery and Erol Fikrig Clin. Microbiol. Rev. 2012, 25(4):635. DOI: 10.1128/CMR.00045-12.

Downloaded from

Updated information and services can be found at: http://cmr.asm.org/content/25/4/635

These include: http://cmr.asm.org/ REFERENCES This article cites 237 articles, 106 of which can be accessed free at: http://cmr.asm.org/content/25/4/635#ref-list-1

CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» on September 5, 2013 by UNIVERSITAT ZURICH

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ West Nile Virus: Biology, Transmission, and Human Infection

Tonya M. Colpitts,a Michael J. Conway,a Ruth R. Montgomery,b and Erol Fikriga,c Department of Internal Medicine, Section of Infectious Diseasesa and Section of Rheumatology,b Yale University School of Medicine, New Haven, Connecticut, USA, and Howard Hughes Medical Institute, Chevy Chase, Maryland, USAc

INTRODUCTION ...... 635 Downloaded from BIOLOGY ...... 635 Flaviviridae...... 635 Structure and Proteins...... 635 Life Cycle ...... 636 VECTOR-VIRUS RELATIONSHIP ...... 636 Vector Preference ...... 636 Host Reservoirs ...... 636 Vector Acquisition ...... 636 Vector Response to Infection...... 637

Transmission to Vertebrate Host ...... 638 http://cmr.asm.org/ Mosquito Saliva Factors ...... 638 MAMMALIAN INFECTION ...... 639 Epidemiology and Clinical Features ...... 639 Diagnostics ...... 639 Immune Response ...... 640 Genetic Determinants of Disease...... 641 Therapeutics...... 642 CONCLUSIONS AND FUTURE DIRECTIONS ...... 642 ACKNOWLEDGMENTS...... 642

REFERENCES ...... 642 on September 5, 2013 by UNIVERSITAT ZURICH

INTRODUCTION and there are 10 serologic/genetic complexes (30, 101, 118). The geographic distribution of the mosquito-borne flaviviruses largely est Nile virus (WNV) is a neurotropic human pathogen that depends on the habitat of the preferred mosquito vector, with is the causative agent of West Nile fever and encephalitis. W Culex mosquitoes transmitting encephalitic flaviviruses mainly in WNV was introduced into the Western Hemisphere during the the Northern Hemisphere. late summer of 1999, when infected individuals were diagnosed in New York State (104, 125). In 2000, the epizootic expanded to 12 states and the District of Columbia (125), and WNV can now be Structure and Proteins found in many avian and mosquito species throughout North WNV is an enveloped virion containing a single-stranded, posi- America (72, 73). From 1999 to 2010, more than 2.5 million peo- tive-sense RNA genome. The genome consists of a single open ple were infected, with over 12,000 reported cases of encephalitis reading frame of approximately 11 kb with no polyadenylation tail or meningitis and over 1,300 deaths (93). at the 3= end. Both the 5= and 3= noncoding regions of the genome The purpose of this review is to present and summarize recent form stem-loop structures that aid in replication, transcription, discoveries about the acquisition and transmission of WNV by translation, and packaging (63, 92, 196). The viral RNA is trans- mosquitoes as well as insights into human infection. We discuss lated as a single polyprotein that is post- and cotranslationally and review data collected and presented over the last decade, and cleaved by both host and viral proteases, resulting in three struc- we present future directions of research. tural (capsid, envelope, and premembrane) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins BIOLOGY (174). The 5= end of the genome encodes the structural proteins, which are necessary for virus entry and fusion as well as encapsi- Flaviviridae dation of the viral genome during assembly (118). The nonstruc- The family Flaviviridae contain 3 genera: the flaviviruses, which tural proteins have many diverse functions, which is understand- include WNV, dengue virus (DENV), and yellow fever virus able as the virus has a very limited number of proteins and they (YFV); the hepaciviruses, which include hepatitis B and C viruses; must each serve multiple purposes during infection. NS1 has both and the pestiviruses, which affect hoofed mammals. Within the a “cellular” form and a secreted form and is highly immunogenic Flavivirus genus, which contains more than 70 viruses, viruses can be further classified into tick-borne and mosquito-borne virus groups. The mosquito-borne viruses may be roughly sorted into Address correspondence to Erol Fikrig, erol.fi[email protected]. the encephalitic clade, or the JE serocomplex, which includes Copyright © 2012, American Society for Microbiology. All Rights Reserved. WNV and Japanese encephalitis virus (JEV), and the nonencepha- doi:10.1128/CMR.00045-12 litic or hemorrhagic fever clade, which includes DENV and YFV,

October 2012 Volume 25 Number 4 Clinical Microbiology Reviews p. 635–648 cmr.asm.org 635 Colpitts et al. but has no described role in virion assembly, though it has been several recent studies have shown that immature WNV particles suggested to play a role in replication (234). NS3 is the viral pro- can be highly immunogenic and infectious in vitro and in vivo tease responsible for cleaving other nonstructural proteins from when bound by antibodies against the E or prM protein (43, 51, the viral polyprotein and encodes enzyme activities, and these 179). These antibody-bound immature virus particles enter im- functions have been widely characterized (118). The NS5 protein mune cells via the Fc receptor, resulting in productive infection. serves as the viral polymerase and encodes a methyltransferase, Further work remains to be done to determine the role that im- and it is necessary for viral replication (117, 174). Several of the mature particles play in viral pathogenesis and disease in both nonstructural proteins, including NS2A, NS2B, NS4A, and NS4B, vector and mammalian hosts. have been shown to inhibit one or more components of the innate immune response against viral infection (116, 121, 122, 139). VECTOR-VIRUS RELATIONSHIP The West Nile virus virion is an icosahedral particle with the Vector Preference Downloaded from capsid protein associating with the RNA genome to form the nu- The ability of different mosquito species to acquire and transmit cleocapsid, which is surrounded by a lipid bilayer. A high propor- WNV is highly variable. Culex mosquitoes are accepted as the tion of capsid protein localizes to the nucleus, while viral assembly primary global transmission vector; C. tarsalis is a main mosquito takes place in the cytoplasm, with budding in the endoplasmic vector of WNV in the western United States and can feed on a reticulum (ER) (17, 41, 183). Although the nuclear functions of variety of avian and mammalian species (95, 163). Other vectors capsid are not fully understood, recent evidence suggests a role in shown to have competence for both infection and transmission of gene regulation through binding with histone proteins (41). Dur- West Nile virus are C. quinquefasciatus, C. stigmatosoma, C. thri- ing virus assembly, the envelope protein embeds in the lipid bi- ambus, C. pipiens, and C. nigripalpus; to date, over 65 mosquito http://cmr.asm.org/ layer of the virus and is exposed to the virion surface. The envelope species have been shown to be infected by WNV (79, 164, 222). protein is responsible for binding the receptor on the cell surface There is evidence that C. pipiens in the eastern United States may for viral entry (134). The prM protein is also known to embed in feed on mammals and humans instead of birds during the late the lipid bilayer and is thought to protect E from undergoing summer and early fall, and this “host switching” has also been premature fusion upon virus exocytosis to the cell surface. During reported with C. tarsalis in the western United States (96, 212). infection, the virus population contains both mature and imma- There are several reports of WNV in Aedes mosquitoes, though ture virus particles containing a varying number of immature prM they are not considered a primary vector in nature (46, 58, 83, 184, protein molecules on the surface (57, 239). 216, 221). WNV has also been detected in field-collected male A. on September 5, 2013 by UNIVERSITAT ZURICH triseriatus and C. salinarius (219), which not only points to vertical Life Cycle transmission of virus, as only females feed on animal blood, but Entry of WNV is through receptor-mediated endocytosis after also further supports that WNV has the ability to infect Aedes virus attachment to the cell surface. Several molecules have been mosquitoes in nature. The ability to infect various mosquito spe- implicated as receptors for West Nile virus, including DC-SIGN, cies, the geographic range of mosquito species, and the ability of mannose receptor, and several glycosaminoglycans (52, 110, 211). mosquitoes to feed on and transmit virus to particular hosts all The virus-containing endosome matures during internalization play a role in WNV vector preference. from the cell surface, with the pH dropping from neutral to slightly acidic in the early endosome and becoming more acidic Host Reservoirs during maturation to the late endosome. Within the late endo- WNV is maintained in nature in a cycle between mosquitoes and some, the envelope protein will undergo a conformational change animal hosts (Fig. 1 shows a schematic of mosquito-mammal resulting in fusion of the viral lipid membrane with the endocytic transmission), with the predominant and preferred reservoir be- membrane and the release of the viral RNA genome into the cell ing birds (3, 75, 136, 162). Birds of some species become ill, show cytoplasm (134). Following capsid disassociation, the RNA ge- symptoms of disease, and may die, while others become infected nome is replicated and virus assembly is initiated following a well- and serve as carriers without showing signs of disease. Although documented program (118). The viral polyprotein is translated house sparrows and crows are highly susceptible to WNV, they and processed on intracellular membranes, resulting in the ex- make up a small fraction of analyzed mosquito blood meals and pression of the 10 viral proteins. The original viral RNA is repli- may be of minor importance in transmission. The American robin cated by viral and cellular proteins into multiple copies to be used is instead thought to be the main host species responsible for the in the production of new virions. The structural proteins assemble maintenance and transmission of WNV in the United States due onto membranes in the endoplasmic reticulum, associate with the to the feeding preference for robins by the dominant viral vectors nucleocapsid, and bud into the cytoplasm via the Golgi network. (80, 91, 94). Bird-bird transmission has been demonstrated in the The virus travels to the cell surface in an exocytic vesicle and ma- laboratory, with several species proving to be capable of contact tures as cellular enzymes cleave the prM, resulting in the release of transmission (99). Humans are considered “dead-end” hosts for mature virus from the cell surface (174). WNV, as the low level of viremia in mammals is usually not suf- There has been a recent spike in interest in the role of partially or ficient to be transmitted to mosquitoes, thereby ending the trans- fully immature flavivirus particles during infection. These imma- mission cycle (20). The ability of mammals to act as hosts could ture flavivirus particles form when there is inefficient cleavage of change, though, should Aedes mosquitoes, which feed primarily the prM protein from the virion surface during maturation and on humans, become primary transmission vectors for WNV. budding (237). Immature or partially mature flavivirus particles of both DENV and WNV have been shown to account for up to as Vector Acquisition much as 40% of the total virus population in a given infection Mosquitoes acquire WNV after taking a blood meal from a vire- (135). While they were traditionally thought to be noninfectious, mic animal. The stages of infection and replication in the mos-

636 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

posed to limit the dissemination of WNV throughout the mosquito body (220). There is also evidence of a midgut barrier to secondary ﬂavivirus infection, where mosquitoes which acquired more than one virus showed no evidence of dissemination of the second virus, which would prevent transmission (151). Research supports the existence of both physical and immune midgut barriers to WNV infection, and the list of genes both required for and inhibitory to acquisition is sure to increase with further experimentation.

Vector Response to Infection Downloaded from There have been many recent studies aimed at elucidating the transcriptomic and proteomic response to ﬂavivirus infection in the mosquito vector. Although WNV establishes a persistent infection in mosquito cells in vitro and in live mosquitoes, there is growing evidence that the mosquito does mount some immune response to virus infection. Most of what is known about the insect immune system comes from experiments with Drosophila melanogaster, though current examination of the mosquito im- http://cmr.asm.org/ mune response is starting to reveal corresponding proteins and pathways. The mosquito antiviral response is thought to consist of two pathways: the innate immune pathway and the RNA interference (RNAi) pathway (7). The innate immune response is com- prised of three signaling pathways: Toll, JAK-STAT, and IMD. The Toll and IMD pathways both culminate in NF-␬B-mediated expression of antimicrobial peptides (AMPs), and IMD signaling

FIG 1 Schematic of West Nile virus transmission from mosquito to mammal has been shown to control RNA virus infection in Drosophila (44). on September 5, 2013 by UNIVERSITAT ZURICH and host factors known to be involved in infection. Not much is known regarding the role of mosquito AMPs in antiviral immunity, though their expression is often induced by viral infection. Both Toll signaling and the JAK-STAT pathways have quito have been well described (68, 126, 175). The virus must then been shown to play a role in the control of DENV infection in infect and replicate in cells of the mosquito midgut as the blood Aedes aegypti (161, 202) and may also be significant during infec- meal is being processed. After replication in the midgut epithelia, tion of Culex with WNV. The RNAi pathway in mosquitoes is the virus travels through the mosquito hemolymph to the salivary activated by viral double-stranded RNA and has been shown to be glands. Accumulation of virus in the salivary glands will eventually crucial for controlling alphavirus infection in both Aedes and result in high viremia in the saliva, from where it can then be Anopheles (32, 90). The RNAi pathway is known to be induced transmitted to mammalian hosts during feeding. The mosquito during WNV infection of Culex pipiens (21), and another RNAi midgut can serve as a barrier to infection due to the presence of pathway, PIWI, may participate in the mosquito response to virus, certain chitins and other proteins as well as a strong immune as it was shown to be involved in limiting WNV infection in Dro- response to the virus (194). The peritrophic matrix, which consists sophila (39). Infection with dengue virus was also found to actively of chitin microfibrils embedded in a proteoglycan matrix, has suppress mosquito immune responses in vitro (200). been shown to play a role in reducing pathogen invasion of the Evidence for a transcriptomic signature of flavivirus infection midgut epithelium, though its role in flavivirus infection is not was found during a comprehensive study of Aedes aegypti infected entirely understood (89). A recent study looking at alteration in with WNV, DENV, and YFV (42). Genes involved in transcription midgut gene expression in C. pipiens quinquefasciatus during and ion binding were found to be downregulated, and genes cod- WNV infection found 21 genes to be upregulated and 5 genes ing for proteases and cuticle proteins were found to be upregu- downregulated after mosquitoes fed on infected blood. Most of lated, during infection with all three viruses (42). Serine proteases the genes were not canonical immune genes, though a putative had previously been shown to be important for viral propagation Toll-like receptor (TLR) with increased expression during infec- and blood digestion, though there have been varying reports re- tion was identified (201). Proteins that have significantly in- garding their impact on flaviviral infection in the mosquito (22, creased or reduced levels in the mosquito midgut during WNV 138). Another global study of flaviviral infection in Drosophila infection may play a role in disease acquisition or viral spread identified many insect host factors relevant during dengue virus throughout the mosquito, and many are under active investiga- infection of the mosquito, including a putative NADH dehydro- tion as virulence factors. For example, a recent study found that a genase and proteins involved in vesicular transport and endocy- C-type lectin from mosquitoes facilitated WNV entry into mos- tosis (193). Adding to our knowledge of the mosquito response to quito cells by directly binding the virion and aiding interaction WNV infection, a recent transcriptomic analysis of Culex quin- with a mosquito CD45 receptor homolog on the cell surface (36). quefasciatus revealed that many genes involved in metabolism and These molecules may prove to be important for virus acquisition transport are upregulated during infection (14). Given that the in the mosquito midgut. In mosquitoes that are refractory to in- virus must infect a variety of cell types and organs in the mosquito fection, apoptosis in infected midgut epithelial cells has been pro- vector, as well as optimize the cellular environment to benefit its

October 2012 Volume 25 Number 4 cmr.asm.org 637 Colpitts et al. life cycle, there are likely a large number of differentially regulated ivary gland extract (SGE). Importantly, enhanced viremia was not genes, proteins, and other host factors important to WNV infec- observed when SGE was inoculated in a distal site, supporting that tion of the mosquito that have yet to be discovered. mosquito saliva exerts its effect locally (206). Due to the complex nature of mosquito saliva, multiple activi- Transmission to Vertebrate Host ties may lead to the enhancement of early virus infection. Further, WNV is transmitted to its vertebrate hosts by an infected mos- due to the intense selective pressures exerted on mosquito saliva quito vector during the probing process of blood feeding. Mos- proteins by the host immune systems, successful viruses likely quitoes probe host skin using their proboscis in order to inject coevolve with their mosquito vectors in order to coopt unique pharmacologically active saliva proteins and to locate a blood saliva protein activities. For example, A. aegypti SGE reduced mu- source (84, 171, 172). Although many hematophagous insects can rine splenocyte proliferation and production of both Th1 and Th2 obtain a blood meal without functional salivary glands, the effi- cytokines while C. quinquefasciatus SGE did not have this activity Downloaded from ciency of blood feeding is severely limited (84, 171, 172). In order (224). These data suggest that the reduction of splenocyte prolif- to combat the host’s hemostatic system, all hematophagous in- eration and Th1/Th2 cytokine production may be critical for virus sects inject at least one vasodilator, one coagulation inhibitor, and transmission and predict that C. quinquefasciatus would be less one platelet inhibitor, and often the saliva includes immuno- efficient at transmitting virus. The adaptation that has taken place modulatory, digestive, and antimicrobial proteins as well (167, between a virus and its vector’s saliva proteins may contribute to 169, 170, 186). While numerous proteins in the saliva of hema- vector competence, although these mechanisms remain poorly tophagous insects have been described, many remain that have defined. not been characterized, especially with respect to viral infection. Multiple reports have suggested that immunomodulatory ac- http://cmr.asm.org/ During probing, mosquito saliva is injected mostly extravascu- tivities in mosquito saliva could result in enhanced early infection larly in the skin’s dermal layer (205). Dermal blood vessels are the (45, 188, 190, 224, 231). These reports suggest that saliva modu- targets for hematophagous insects. In order to locate these struc- lates skin-resident immune cells. In one report, A. aegypti saliva tures, the proboscis must navigate through a very elastic environ- was able to decrease beta interferon (IFN-␤) and inducible nitric ment that has a high tensile strength. To efficiently move through oxide synthase in macrophages ex vivo (188). Recruitment of T this environment, mosquito saliva may contain components that cells was also reduced when WNV was inoculated during mos- liquefy the bite site. A salivary endonuclease with a proposed func- quito feeding, rather than by syringe, suggesting that saliva hin- tion to facilitate probing in host skin has been identified in C. ders infiltration of these cells into the inoculation site (188). These on September 5, 2013 by UNIVERSITAT ZURICH quinquefasciatus (31). effects correlated with enhanced expression of interleukin-10 (IL- Host skin acts as an important barrier to many infections, 10), which has anti-inflammatory activities, including the down- though WNV antigen has been detected in skin at multiple phases regulation of Th1 cytokines, major histocompatibility complex of infection. WNV replication was observed in skin tissue at the (MHC) class II molecules, and costimulatory molecules on mac- inoculation site at 1 and 3 days postinfection (189), and WNV has rophages (188). While this study is limited by the use of A. aegypti also been shown to spread to areas of skin contralateral to the site SGE, which is not the primary vector for WNV, it is likely that of inoculation (27). Infectious WNV has been shown to persist in some Culex salivary proteins act to enhance WNV infection. skin at the inoculation site for at least 14 days postinfection (5). It is unknown whether Culex sp. SGEs have similar immuno- Many reports document that both keratinocytes and fibroblasts modulatory activities; however, C. pipiens SGE was able to en- are permissive to WNV infection in vitro and in vivo (8, 37, 38, 55, hance Cache Valley fever virus infection, and C. tarsalis saliva was 60, 62, 86, 87, 102, 109, 115, 165, 185, 195, 233). By immunohis- able to enhance WNV infection in a mouse model (56, 206). Ad- tochemistry and fluorescence-activated cell sorter (FACS) analy- ditionally, saliva from C. tarsalis and C. pipiens was able to en- sis, WNV antigen was detected in keratinocytes at 4 and 5 days hance WNV infection in chickens (204). The fact that saliva from postinfection, and virus presence in a small subset of skin cells that multiple species in both the Aedes and Culex genera was able to lacked the keratin marker K10 suggests that skin cells other than enhance virus infectivity would suggest either that the relevant keratinocytes may also be important early reservoirs (115). saliva proteins are highly conserved or that a similar activity has convergently evolved in multiple mosquito vectors. If all Culex Mosquito Saliva Factors spp. modulate a specific component of the host immune system to Saliva from hematophagous insects has been shown to alter the facilitate blood feeding, WNV may have evolved to benefit from transmissibility of many pathogens (1, 50, 160, 178, 187, 206, 223, this universal mosquito saliva activity. In addition, differences in 231). Saliva from both A. aegypti and C. tarsalis has been shown to salivary gland protein activities could alter the ability of a mos- alter transmissibility in a WNV mouse model (189, 206). Specifi- quito species to enhance pathogen transmission. Multiple activi- cally, when mice were fed on by uninfected A. aegypti prior to ties that differ between Aedes and Culex mosquitoes have been intradermal inoculation with WNV, more progressive infection, noted (166, 169, 170, 173, 223). Since such dramatically different higher viremia, and accelerated neuroinvasion occurred. Even at a saliva activities exist between Aedes and Culex spp., direct compar- low dose of infection, mice that were previously fed on by mos- isons of mosquito saliva activities that are responsible for the en- quitoes had a lower survival rate after WNV infection (189). Sim- hancement of WNV transmission need to be performed for each ilar experiments with C. tarsalis showed that mice infected with Culex sp. that is able to vector WNV. WNV through the bite of a single mosquito had viremia and tissue Though mosquito saliva has been shown to enhance WNV titers that were 5- to 10-fold higher postinoculation and showed infection, the precise mechanisms as well as the specific saliva faster neuroinvasion than those in animals infected by syringe proteins involved remain to be investigated. In one example, inoculation (206). Enhanced early infection was also observed hyaluronidase from sand fly saliva was found to be important when mice were inoculated with WNV mixed with mosquito sal- for the enhancement of Leishmania infectivity in mice (223).

638 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

Saliva hyaluronidase may enlarge the feeding lesion and serve MAMMALIAN INFECTION as a spreading factor for other pharmacologically active factors Epidemiology and Clinical Features present in saliva (223). This activity was also found in C. quinquefasciatus saliva and may also affect the spread of WNV and The emergence of WNV in North America was first documented in other saliva components as well as influence the local host im- the fall of 1999 in New York City following an outbreak of mosquito- mune response (168, 223). In another example, Salp15 from borne encephalitis responsible for the death of humans, birds, and horses (3, 26, 104, 145, 232). Over the next decade, WNV spread tick saliva was able to directly interact with the surface of Bor- throughout the United States and into Canada, Mexico, and the Ca- relia burgdorferi and facilitated evasion from host B cell-medi- ribbean (75). From 2005 to 2009, 12,975 cases were reported to the ated immunity (160), and immunization against Salp15 pro- CDC, including 496 fatalities, and 35% of reported cases were the tected mice from Lyme disease (50). Another study identified more severe forms of neuroinvasive disease, including encephalitis Downloaded from two tick saliva proteins that functioned to inhibit polymorpho- (119). As detailed above, in most cases the virus is transmitted by the nuclear leukocyte recruitment during infection of mice with Culex mosquito vector (4), but transmission may occur through Borrelia burgdorferi, likely increasing the spirochete burden blood transfusion, organ transplantation, breast-feeding, or intra- and enhancing infection (77). Identification of proteins in uterine exposure, and laboratory-acquired infection has also been mosquito saliva that are responsible for the enhancement of reported (35a, 81, 85, 103, 177). WNV transmission is under way, and these investigations may Infections in humans are predominantly subclinical, but re- provide novel nonvirus targets for vaccine design. ported infection manifestations may range from fever and myal-

Multiple negative salivary gland factors that limit flavivirus gias to meningoencephalitis and death (152). Encephalitis occurs http://cmr.asm.org/ transmission have been identified (42, 124). In one example, mi- in only a small subset of patients; progression to severe neurolog- croarray analysis of DENV-infected and uninfected salivary gland ical illness may induce acute flaccid paralysis after meningitis or mRNAs showed an upregulation of a putative antibacterial, ce- encephalitis, with rapidly progressing symptoms that may involve cropin-like peptide (i.e., AAEL000598), which showed antiviral all four limbs (111). Severe poliomyelitis-like syndrome can occur activity against both DENV and Chikungunya virus (124). A re- and has a poor long-term outcome (191). Elderly individuals are cent comparative microarray analysis of mRNAs from DENV-, more susceptible to neurological involvement that may result in YFV-, and WNV-infected and uninfected whole A. aegypti iden- death, and among those older adults who survive, as many as 50%

tified multiple genes that were downregulated by all three viruses may have significant postillness morbidity for at least a year fol- on September 5, 2013 by UNIVERSITAT ZURICH (42). Genes downregulated by day 14 postinfection likely play a lowing infection (33) and may have an increased risk of death for role in salivary gland invasion or virus transmission. Among up to 3 years after acute illness (120). Among individuals over 70 those, a recombinant pupal cuticle protein was able to directly years of age, the case-fatality rate ranges from 15% to 29% (152). interact with WNV envelope protein and inhibit infection in vitro Higher fatality is also seen in infected infants and immunocom- and prevent lethal WNV encephalitis in mice (42). Although these promised patients (73). Risk factors for encephalitis and death proteins were expressed in salivary glands, they have yet to be include being homeless, a history of cardiovascular disease or formally identified in saliva. chronic renal disease, hepatitis C virus infection, and immuno- Transgenic traits and introduced factors can also alter the trans- suppression (140, 192). In addition, in some cases convalescent patients may have persistent or chronic infection detected mission of vector-borne pathogens and may play a role in the through PCR of the urine, which suggested ongoing viral replica- future control of virus-infected mosquito populations. Trans- tion in renal tissue (141, 143). Although persistence of WNV has genic mosquito populations that can be selected to either block also been noted in several animal models (156, 199, 213), it has not transmission, block acquisition, decrease host seeking, decrease been uniformly evident in assays of urine (66). probing and biting, increase background mortality, or increase mosquito infection-induced mortality are in development (1, 59, Diagnostics 98, 128, 147, 148). To date, most studies have focused on produc- The diagnosis of WNV infection is based largely on clinical criteria ing transgenic mosquitoes that block transmission. For example, and testing for antibody responses (28). The incubation period for experimental strains of A. aegypti that inhibit flavivirus replication WNV infection is thought to range from about 2 to 14 days (143). in the midgut and consequent migration to the salivary gland have The presence of anti-WNV IgM, particularly from cerebrospinal been engineered (59, 98, 147, 148). Another gene that is responsi- fluid (CSF), is used for diagnosis. Cross-reactivity with related ble for host seeking behavior has been identified (203). Many flaviviruses (Japanese encephalitis virus, St. Louis encephalitis vi- strategies that lead to increased background mortality have been rus, YFV, and DENV), if suspected, can be assessed through implemented, and field trials have already begun to test the effec- plaque neutralization assays (143). Replication of WNV has been tiveness of these transgenic mosquitoes in reducing wild mosquito documented in human monocytes in vitro and with even higher populations (9, 64, 65, 214). Laboratory infection with Wolbachia efficiency in polymorphonuclear leukocytes; this could lead to bacteria also reduces the life span of mosquitoes (127). This strat- transmission via transfusion of blood (10, 177). Thus, several egy has also been tested in field trials to reduce wild mosquito rapid tests have been developed for blood donor screening using populations (82). The release of insect-specific densoviruses also nucleic acid testing (NAT), an amplification-based transcription shows high mortality in mosquito populations and may be used as technique, which identifies WNV-infected individuals before they a control strategy (34). The advantage of using Wolbachia or become symptomatic and may be used to safeguard the blood Densovirus infection as opposed to insecticide treatment is that supply (238). Of note, 45% of NAT-positive subjects were subse- these pathogens are expected to replicate and spread through the quently not confirmed, and in one study, only 4 to 5% of the wild mosquito populations (128). patients received a diagnosis of WNV infection (238).

October 2012 Volume 25 Number 4 cmr.asm.org 639 Colpitts et al.

TABLE 1 In vivo function of murine genes in WNV infection Gene Survivala Viremia Brain viral load Remarks Reference(s) Myd88Ϫ/Ϫ S Up Up Reduced leukocytes in CNS 209, 218 Tlr3Ϫ/Ϫ R Up Up Reduced viral entry into CNS 230 Tlr7Ϫ/Ϫ S Up Up Defective leukocyte homing 218 Il10Ϫ/Ϫ R Down Down Enhanced antiviral response 9a Irf7Ϫ/Ϫ S Up Up Defective type I IFN production 48 Casp12Ϫ/Ϫ S Up Up Defective type I IFN production 226 Ifn␣␤RϪ/Ϫ SUpUp 181 Ifn␤Ϫ/Ϫ SUpUp 109 Ϫ Ϫ Ϫ Ϫ Ifn␥R / or Ifn␥ / SUpUp 53a Downloaded from Ips1Ϫ/Ϫ S Up Up Lack of regulatory T cells 208 PkrϪ/Ϫ or RnaseLϪ/Ϫ SUpUp 182, 185 Mmp9Ϫ/Ϫ R Equivalent Down Reduced viral entry into CNS 227 Irf3Ϫ/Ϫ S Up Up Impaired IFN-stimulated gene expression in macrophages 47 C3Ϫ/Ϫ S Up Up Impaired CD8ϩ T/antibodies 131 Compl R1/2Ϫ/Ϫ S Up Up Impaired protective antibodies 129 Ccr5Ϫ/Ϫ S Equivalent Up Reduced T cells, NK cells, macrophages in CNS 69 Cxcr2Ϫ/Ϫ R Down Down 10 Cxcr3Ϫ/Ϫ S Equivalent Up Impaired CD8ϩ T cell recruitment to brain 97a http://cmr.asm.org/ Cxcl10Ϫ/Ϫ S Equivalent Up Impaired CD8ϩ T cell recruitment to brain 114a Ccr2Ϫ/Ϫ S Equivalent Up Fewer inflammatory monocytes in CNS 197a sIgMϪ/Ϫ S Up Up Reduced WNV-specific IgG, no IgM 197b Casp3Ϫ/Ϫ R Equivalent Equivalent Reduced neuron apoptosis 182 Icam1Ϫ/Ϫ R Equivalent Down Reduced viral entry into CNS 50 Cd8aϪ/Ϫ MHCclass1aϪ/Ϫ S Equivalent Up Increased viral loads (spleen), persistent infection in 197 surviving mice Cd4Ϫ/Ϫ MHCclass2Ϫ/Ϫ S Equivalent Up Impaired WNV-specific IgM and IgG production, 200a

persistent infection on September 5, 2013 by UNIVERSITAT ZURICH Cd40Ϫ/Ϫ S Equivalent Up Impaired WNV-speciﬁc IgM/IgG production, reduced 200b CD8ϩ cells in CNS Il22Ϫ/Ϫ R Equivalent Down Reduced viral entry into the CNS 225 Dhx58Ϫ/Ϫ S Equivalent Up Reduced CD8ϩ T cell expansion 208a TRAILϪ/Ϫ S Equivalent Equivalent CD8ϩ T cells use TRAIL to limit infection 236a a R and S, mice are more resistant or susceptible, respectively, to lethal WNV infection than their wild-type controls.

Antibody testing in patients follows an expected timetable of vivo, and thus NS5 antibody cannot be used to distinguish re- median times of 3.9 days from RNA detection to IgM serocon- cent from past WNV infection (157). version and 7.7 days from RNA detection to IgG seroconversion (28). RNA generally became undetectable after 13.2 days, Immune Response although it rarely was found to persist for Ͼ40 days. IgM and Control of WNV infection by the human and murine hosts has been IgA antibodies fell signiﬁcantly, although not universally, while investigated for both innate and adaptive immune responses. the IgG level remained elevated for Ͼ1 year after detection of Through integrating these results, a picture of critical elements in viremia (28, 141, 149, 180). Antibody to WNV NS5 persists in immune responses to WNV is emerging (Tables 1 and 2). Sensing

TABLE 2 Genes and corresponding SNPs important in human WNV infection Gene(s) SNP(s) Comparison groups (n) Study results Reference OASL rs3213545 WNVϩ cases (33) vs healthy controls (16) Associated with increased susceptibility to 236 WNV infection CCR5 ⌬32 deletion WNVϩ cases (395) vs WNVϪ (1,463) Increased risk of symptomatic WNV infection 69 WNVϩ cases (224) vs healthy controls (1,318) Increased risk of symptomatic WNV infection 113 WNVϩ cases (634) vs WNVϪ (422) Not a risk factor for WNV initial infection; 114 associated with symptomatic WNV infection OAS1 rs10774671 WNVϩ cases (501) vs healthy controls (552) A risk factor for initial infection with WNV 112 IRF3, MX1, OAS1 rs2304207, rs7280422, Symptomatic cases (422) vs asymptomatic Associated with symptomatic WNV infection 19 rs34137742 cases (331) RFC1, SCN1A, ANPEP rs2066786, rs2298771, Severe WNV cases (560) vs mild WNV cases Associated with neuroinvasive disease in 123 rs25651 (950) patients infected with WNV

640 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

WNV pathogen-associated molecular patterns through pathogen (150). Examination of memory T cells from 40 patients months after recognition receptors such as Toll-like receptors (TLRs) and cyto- infection showed persistence of the memory phenotype and WNV- plasmic RNA helicases is critical for early detection and activation of specific polyfunctional CD8ϩ T cell responses. More cytolytic mem- innate immune pathways that facilitate early control of viral replica- ory T cells were found in patients with neurological disease (154). tion (48, 61, 208–210, 218, 226, 230). This early response is mediated Indeed, CD8ϩ T cells have been shown to be important for control of largely by macrophages; WNV infection of macrophage-depleted viral load in mouse models of WNV infection, at least in part due to a mice results in increased mortality, higher and extended viremia, and role for perforin (97a, 197, 197a, 200a). WNV-specific murine CD4 T substantially shortened survival. Moreover, in mice, even a nonneu- cells produced IFN-␥ and IL-2 and also showed direct antiviral activ- rotrophic WNV strain may cross the blood-brain barrier (BBB) in the ity (25, 197b, 200b). Tregs play an important role in protecting absence of macrophage clearance of virus (16). Macrophages express against severe disease, and it has been shown in both human patients TLRs, mediate clearance of opsonized viral particles, produce proin- and animal models that symptomatic patients show a lower fre- Downloaded from flammatory cytokines, and upregulate costimulatory proteins that quency of Tregs despite having similar systemic T cell responses link innate to adaptive immune responses (114a, 215). Macrophages (108). are also a major component in inflamed central nervous system Complement has also been indicated as an important compo- (CNS) tissues and are considered protective against WNV infection. nent of the host innate immune response to flavivirus infection. The control of WNV by macrophages has been linked both to con- However, while complement traditionally limits the spread of stitutive expression of innate immune genes, such as those for RIG-I, many pathogens, it appears to have both protective and patho- MDA5, PKR, and RNase L, and to direct effector mechanisms such as genic roles during flavivirus infection. Whether or not comple- the production of radical oxygen species and type I IFN (47,49,61,67, ment is protective or pathogenic depends on a variety of factors, http://cmr.asm.org/ 181, 182, 208a, 226, 229). including the specific virus, the phase of infection, and the under- Although cellular immune mechanisms remain incompletely lying immune status of the host (40, 130, 131). explained, innate immunity and in particular interferon responses A paradoxical role for polymorphonuclear cells (PMNs) in have been shown to be critical in resistance to WNV (7, 9a, 181, WNV infection has been described, where PMNs are recruited to 197b). Patients who mount a robust IFN-␣ response show lower the site inoculated with WNV (10). It was determined by depleting viral loads, even before IgM seroconversion, concomitant with PMNs prior to WNV infection that recruitment of PMNs to the significant upregulation of IFN-␥ during the viremic phase (217). inoculation site was associated with enhanced WNV replication.

Permeability of the blood-brain barrier (BBB), which is enhanced However, if PMNs were depleted after WNV infection, mice de- on September 5, 2013 by UNIVERSITAT ZURICH by cytokine responses, has been shown in murine models to be veloped higher viremia and mortality. Thus, infiltrating PMNs critical to resistance to WNV infection (230), and elements which may serve as an early reservoir of WNV replication (10). Dendritic decrease the integrity of the BBB contribute to susceptibility to cells express DC-SIGN, suggesting that they may also be early infection with WNV (7, 226, 227). Entry to the CNS may be af- cellular targets in host skin (211). WNV infection of dendritic cells forded by trafficking of infected CD45ϩ leukocytes and CD11b leads to production of IFN. Interestingly, studies with dendritic macrophages (218), T cells (228), or neutrophils (225). Mice lack- cells from human donors showed that type I IFN expression in ing TLR3 show improved survival over wild-type animals due to a response to WNV in vivo is lower in cells from older donors than lower cytokine response and protection from BBB permeability in those from younger donors, which may contribute to older (100, 230). Human studies show a role for CXCL10 and CCL2 in individuals being more susceptible to WNV disease (159). control of early infection and an important role for IFN-mediated These innate pathways are critical not only for immediate anti- innate immunity in resolving acute WNV infection (217). RNAi viral defense pathways such as the upregulation of type I interfer- studies in human cell lines have indicated that interferon-induc- ons but also for the generation of an effective adaptive T and B ible transmembrane protein (IFITM) inhibits the early replication cell-mediated sustained immune response (24, 53a, 129, 131, 155, of WNV (23). 181, 198). The ␥␦ T cell population rapidly expands after WNV Infection with flaviviruses leads to upregulation of MHC class I, infection. Mice that lack ␥␦ T cells have higher viremia and in- MHC class II, and adhesion molecules, which may enhance infec- creased mortality (229). Soon after infection, ␥␦ T cells produce tion through reducing NK cell activity, or enhance a transient IFN-␥, which correlates with an increase in perforin expression in autoimmunity in early infection (97). It is clear that CD8ϩ T cells splenic T cells. Bone marrow chimera reconstitution experiments are critical in the response to flavivirus infections. Overall T cell in mice support that IFN-␥ production by ␥␦ T cells is critical for responses in humans revealed that multiple peptide regions of the early control of WNV infection (229). ␥␦ T cells also promote WNV proteins are recognized by T cells, with a subset of 8 pep- a protective adaptive immune response by facilitating dendritic tides predominating, and the highest magnitude of specific T cell cell maturation, providing an important link between the innate responses was from CD8ϩ cells (105). The immunodominant T and adaptive immune responses against WNV infection (229). cell epitopes which elicited both highest-frequency and highest- magnitude responses included sequences from WNV M, E, NS3, Genetic Determinants of Disease and NS4 proteins and, furthermore, were equivalent between Specific human genetic factors that influence the severity of infec- symptomatic and asymptomatic subjects in this cohort (105). tion with WNV and the antiviral innate immune response have During infection with WNV, CD8ϩ T cells expand dramatically been identified (Table 2). Certain HLA types appear to be associ- and migrate to the site of CNS infection (97, 236a). Examination ated with risk of a more severe outcome (HLA-A*68 and C*08) or of immune responses from WNV patients shows that memory T better resistance to infection (B*40 and C*03) (107). Single nucle- cell responses to WNV are mainly due to CD8ϩ T cells with a otide polymorphism (SNP) studies have detected SNPs in key defined set of epitopes; these were quite constant over 12 months regulators of immune function, including interferon pathway el- of observation and were not apparently related to disease severity ements. In particular, polymorphisms in IRF3 and MX-1 were

October 2012 Volume 25 Number 4 cmr.asm.org 641 Colpitts et al. associated with symptomatic infection, and an SNP in the oligo- CONCLUSIONS AND FUTURE DIRECTIONS adenylate synthetase 1b (OAS-1) gene, an interferon-regulated WNV has now persisted and become established in North Amer- gene involved in RNA degradation, was associated with an in- ica. Of particular significance is the expansion of the mosquito creased risk for initial infection with WNV and severe neurologi- vectors harboring WNV to include Aedes albopictus, a common cal disease (Ͼ750 subjects) (19, 112). Notably, the 2=,5=-oligoad- mammal-biting mosquito (2, 73, 136). It is hoped that the increase enylate synthase (2=-5=-OAS) gene has also been identified as a in our knowledge of the interactions of WNV with the mosquito susceptibility factor in WNV in horses and as a contributing factor vector will lead to new avenues for therapeutics and preventative for severity of neurological disease in tick-borne encephalitis virus measures. Mosquito responses at the levels of protein and gene (13, 176, 236). A dominant negative splice variant of RNase L, expression as well as a more complete understanding of viral pathogenesis in the vector, especially with regard to the immune which functions in the antiproliferative roles of interferon, was Downloaded from detected more often in WNV patients than in control patients response, may point to novel targets to focus our efforts to inhibit (236). Another genomic study investigated Ͼ1,500 symptomatic or block WNV infection in both mosquitoes and mammals. subjects (with severe versus mild disease) and showed more severe For example, a single-chain human monoclonal antibody de- neurological disease to be associated with SNPs in the genes for veloped through phage display directed against the fusion loop of RFC1 (a replication factor), SCN1a (a sodium channel), and the envelope protein showed both pan-flaviviral protection and ANPEP (an aminopeptidase), although even more differences therapeutic efficacy when tested in the murine model (71, 207). Recent advances in nanoparticle technology have also been em- might have been revealed when comparing asymptomatic and ployed in vaccination studies of murine WNV infection and show symptomatic cases (123). In addition, a deletion in CCR5, which is http://cmr.asm.org/ promising efficacy of TLR9-targeted biodegradable nanoparticles, known to be protective in infection with HIV, while not associated which produce a high number of circulating effector T cells and with susceptibility to WNV, did correspond to severity of infec- antigen-specific lymphocytes (53). Potential relevant viral suscep- tion, presumably due to reduced function of CCR5 pathways in tibility mechanisms, including host antagonism of chemokine re- infected hosts (69, 113, 114). As more host factors are identified, sponses as has been noted in infection with the related flavivirus there are sure to be a number of new determinants of WNV infec- hepatitis C virus (35), may reveal infectious mechanisms used by tion. WNV and other mosquito-borne flaviviruses. The pace of discovery of vector, virus, and host components of pathogenesis contin- Therapeutics ues to provide critical insights for the successful development of on September 5, 2013 by UNIVERSITAT ZURICH controls and treatments for WNV. Current therapeutic options against WNV are mainly supportive; there are no FDA-approved vaccines or treatments available (54). ACKNOWLEDGMENTS Investigations to identify individual susceptibility markers, re- We are grateful to our long-standing colleague John F. Anderson of the combinant antibodies, peptides, RNA interference, and small Connecticut Agricultural Experiment Station and to members of the Yale molecules with the ability to directly or indirectly neutralize WNV IMAGIN and HIPC teams for valuable discussions. have been reported; however, an effective drug is still lacking (6, This work was supported in part by the NIH (grants HHS 12, 70, 71, 74, 146, 158). There are currently four USDA-licensed N272201100019C, U19AI089992, and U01AI 070343). E.F. is an In- vaccines available for equines (two are inactivated whole WNV, vestigator of the Howard Hughes Medical Institute. T.M.C. is supported by grant 2T32HL007974-11, and M.J.C is supported by grant one is a nonreplicating live canary pox recombinant vector vac- 5T32AI07019-35. cine, and one is an inactivated flavivirus chimeric vaccine). Though passive immunization has been used in a few cases, it has REFERENCES serious limitations, such as inadvertent transfer of blood-borne 1. Ader DB, et al. 2004. Modulation of dengue virus infection of dendritic pathogens, inconsistent quality of the donor antisera, cost, and cells by Aedes aegypti saliva. Viral Immunol. 17:252–265. allergic reactions (78). A case study of two WNV encephalitis pa- 2. Anderson JF, Andreadis TG, Main AJ, Ferrandino FJ, Vossbrinck CR. 2006. West Nile virus from female and male mosquitoes (Diptera: Cu- tients treated with alpha interferon, the standard of care for infec- licidae) in subterranean, ground, and canopy habitats in Connecticut. J. tion with the related flavivirus hepatitis C virus, showed substan- Med. Entomol. 43:1010–1019. tial improvement and an improved convalescence course (88). 3. Anderson JF, et al. 1999. Isolation of West Nile virus from mosquitoes, Several approaches are being pursued for the development of a crows, and a Cooper’s hawk in Connecticut. Science 286:2331–2333. 4. Andreadis TG, Anderson JF, Vossbrinck CR, Main AJ. 2004. Epide- vaccine in humans that may prove valuable for use by targeted miology of West Nile virus in Connecticut: a five-year analysis of mos- populations. Investigations include live attenuated vaccines, re- quito data 1999–2003. Vector Borne Zoonotic Dis. 4:360–378. combinant subunit vaccines, vectorized vaccines, DNA vaccines 5. Appler KK, et al. 2010. Persistence of West Nile virus in the central nervous system and periphery of mice. PLoS One 5:e10649. doi:10.1371/ with constructs that express the WNV E protein, live recombinant journal.pone.0010649. vaccines, and an attenuated strain based on nonglycosylated E and 6. Arjona A, et al. 2007. Abrogation of macrophage migration inhibitory mutant NS1 proteins (15, 235). A neutralizing, WNV-specific factor decreases West Nile virus lethality by limiting viral neuroinvasion. monoclonal antibody, E16 (MGAWN1), which penetrates the J. Clin. Invest. 117:3059–3066. 7. Arjona A, Wang P, Montgomery RR, Fikrig E. 2011. Innate immune CNS in animal models, produced neutralizing antibodies in phase control of West Nile virus infection. Cell. Microbiol. 13:1648–1658. I trials (15). Very promising results were seen with a chimeric 8. Arnold SJ, Osvath SR, Hall RA, King NJ, Sedger LM. 2004. Regulation vaccine based on the WNV prM and E proteins inserted into the of antigen processing and presentation molecules in West Nile virus- yellow fever 17D vaccine moiety (ChimeriVax-WN02). It was infected human skin fibroblasts. Virology 324:286–296. 9. Atkinson MP, et al. 2007. Analyzing the control of mosquito-borne shown to be safe and immunogenic in phase II clinical trials, with diseases by a dominant lethal genetic system. Proc. Natl. Acad. Sci. high seroconversion rates, but it is no longer available (18). U. S. A. 104:9540–9545.

642 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

9a.Bai F, et al. 2009. IL-10 signaling blockade controls murine West Nile 36. Cheng G, et al. 2010. A C-type lectin collaborates with a CD45 phos- virus infection. PLoS Pathog. 5:e1000610. phatase homolog to facilitate West Nile virus infection of mosquitoes. 10. Bai F, et al. 2010. A paradoxical role for neutrophils in the pathogenesis Cell 142:714–725. of West Nile virus J. Infect. Dis. 202:1804–1812. 37. Cheng Y, King NJ, Kesson AM. 2004. Major histocompatibility com- 11. Reference deleted. plex class I (MHC-I) induction by West Nile virus: involvement of 2 12. Bai F, et al. 2007. Antiviral peptides targeting the West Nile virus enve- signaling pathways in MHC-I up-regulation. J. Infect. Dis. 189:658–668. lope protein. J. Virol. 81:2047–2055. 38. Cheng Y, King NJ, Kesson AM. 2004. The role of tumor necrosis factor 13. Barkhash AV, et al. 2010. Variability in the 2=-5=-oligoadenylate synthe- in modulating responses of murine embryo fibroblasts by flavivirus, tase gene cluster is associated with human predisposition to tick-borne West Nile. Virology 329:361–370. encephalitis virus-induced disease. J. Infect. Dis. 202:1813–1818. 39. Chotkowski HL, et al. 2008. West Nile virus infection of Drosophila 14. Bartholomay LC, et al. 2010. Pathogenomics of Culex quinquefasciatus melanogaster induces a protective RNAi response. Virology 377:197– and meta-analysis of infection responses to diverse pathogens. Science 206. 330:88–90. 40. Chung KM, et al. 2006. West Nile virus nonstructural protein NS1 Downloaded from 15. Beasley DW. 2011. Vaccines and immunotherapeutics for the preven- inhibits complement activation by binding the regulatory protein factor tion and treatment of infections with West Nile virus. Immunotherapy H. Proc. Natl. Acad. Sci. U. S. A. 103:19111–19116. 3:269–285. 41. Colpitts TM, Barthel S, Wang P, Fikrig E. 2011. Dengue virus capsid 16. Ben-Nathan D, Huitinga I, Lustig S, van Rooijen N, Kobiler D. 1996. protein binds core histones and inhibits nucleosome formation in hu- West Nile virus neuroinvasion and encephalitis induced by macrophage man liver cells. PLoS One 6:e24365. doi:10.1371/journal.pone.0024365. depletion in mice. Arch. Virol. 141:459–469. 42. Colpitts TM, et al. 2011. Alterations in the Aedes aegypti transcriptome 17. Bhuvanakantham R, Chong MK, Ng ML. 2009. Specific interaction of during infection with West Nile, dengue and yellow fever viruses. PLoS capsid protein and importin-alpha/beta influences West Nile virus pro- Pathog. 7:e1002189. doi:10.1371/journal.ppat.1002189. duction. Biochem. Biophys. Res. Commun. 389:63–69. 43. Colpitts TM, et al. 2011. prM-antibody renders immature West Nile 18. Biedenbender R, Bevilacqua J, Gregg AM, Watson M, Dayan G. 2011. virus infectious in vivo. J. Gen. Virol. 92:2281–2285. http://cmr.asm.org/ Phase II, randomized, double-blind, placebo-controlled, multicenter 44. Costa A, Jan E, Sarnow P, Schneider D. 2009. The Imd pathway is study to investigate the immunogenicity and safety of a West Nile virus involved in antiviral immune responses in Drosophila. PLoS One vaccine in healthy adults. J. Infect. Dis. 203:75–84. 4:e7436. doi:10.1371/journal.pone.0007436. 19. Bigham AW, et al. 2011. Host genetic risk factors for West Nile virus 45. Cross ML, Cupp EW, Enriquez FJ. 1994. Differential modulation of infection and disease progression. PLoS One 6:e24745. doi:10.1371/ murine cellular immune responses by salivary gland extract of Aedes journal.pone.0024745. aegypti. Am. J. Trop. Med. Hyg. 51:690–696. 20. Bowen RA, Nemeth NM. 2007. Experimental infections with West Nile 46. Cupp EW, et al. 2007. West Nile virus infection in mosquitoes in the virus. Curr. Opin. Infect. Dis. 20:293–297. mid-south USA, 2002-2005. J. Med. Entomol. 44:117–125. 21. Brackney DE, Beane JE, Ebel GD. 2009. RNAi targeting of West Nile 47. Daffis S, Samuel MA, Keller BC, Gale M, Jr, Diamond MS. 2007. virus in mosquito midguts promotes virus diversification. PLoS Pathog. Cell-specific IRF-3 responses protect against West Nile virus infection by on September 5, 2013 by UNIVERSITAT ZURICH 5:e1000502. doi:10.1371/journal.ppat.1000502. interferon-dependent and -independent mechanisms. PLoS Pathog. 22. Brackney DE, Foy BD, Olson KE. 2008. The effects of midgut serine 3:e106. doi:10.1371/journal.ppat.0030106. proteases on dengue virus type 2 infectivity of Aedes aegypti. Am. J. Trop. 48. Daffis S, Samuel MA, Suthar MS, Gale M, Jr, Diamond MS. 2008. Med. Hyg. 79:267–274. Toll-like receptor 3 has a protective role against West Nile virus infection. 23. Brass AL, et al. 2009. The IFITM proteins mediate cellular resistance to J. Virol. 82:10349–10358. influenza A H1N1 virus, West Nile virus, and Dengue virus. Cell 139: 49. Daffis S, et al. 2008. Interferon regulatory factor IRF-7 induces the 1243–1254. antiviral alpha interferon response and protects against lethal West Nile 24. Brien JD, et al. 2011. Interferon regulatory factor-1 (IRF-1) shapes both virus infection. J. Virol. 82:8465–8475. innate and CD8(ϩ) T cell immune responses against West Nile virus 50. Dai J, et al. 2009. Antibodies against a tick protein, Salp15, protect mice infection. PLoS Pathog. 7:e1002230. doi:10.1371/journal.ppat.1002230. from the Lyme disease agent. Cell Host Microbe 6:482–492. 25. Brien JD, Uhrlaub JL, Nikolich-Zugich J. 2008. West Nile virus-specific 51. da Silva Voorham JM, et al. 2012. Antibodies against the envelope CD4 T cells exhibit direct antiviral cytokine secretion and cytotoxicity glycoprotein promote infectivity of immature dengue virus serotype 2. and are sufficient for antiviral protection. J. Immunol. 181:8568–8575. PLoS One 7:e29957. doi:10.1371/journal.pone.0029957. 26. Briese T, Jia XY, Huang C, Grady LJ, Lipkin WI. 1999. Identification 52. Davis CW, et al. 2006. West Nile virus discriminates between DC-SIGN of a Kunjin/West Nile-like flavivirus in brains of patients with New York and DC-SIGNR for cellular attachment and infection. J. Virol. 80:1290– encephalitis. Lancet 354:1261–1262. 1301. 27. Brown AN, Kent KA, Bennett CJ, Bernard KA. 2007. Tissue tropism 53. Demento SL, et al. 2010. TLR9-targeted biodegradable nanoparticles as and neuroinvasion of West Nile virus do not differ for two mouse strains immunization vectors protect against West Nile encephalitis. J. Immu- with different survival rates. Virology 368:422–430. nol. 185:2989–2997. 28. Busch MP, et al. 2008. Virus and antibody dynamics in acute West Nile 53a.Diamond MS, Shrestha B, Marri A, Mahan D, Engle M. 2003. B cells virus infection. J. Infect. Dis. 198:984–993. and antibody play critical roles in the immediate defense of disseminated 29. Reference deleted. infection by West Nile encephalitis virus. J. Virol. 77:2578–2586. 30. Calisher CH, et al. 1989. Antigenic relationships between flaviviruses as 54. Diamond MS. 2005. Development of effective therapies against West determined by cross-neutralization tests with polyclonal antisera. J. Gen. Nile virus infection. Expert Rev. Anti Infect. Ther. 3:931–944. Virol. 70:37–43. 55. Douglas MW, Kesson AM, King NJ. 1994. CTL recognition of West 31. Calvo E, Ribeiro JM. 2006. A novel secreted endonuclease from Culex Nile virus-infected fibroblasts is cell cycle dependent and is associated quinquefasciatus salivary glands. J. Exp. Biol. 209:2651–2659. with virus-induced increases in class I MHC antigen expression. Immu- 32. Campbell CL, et al. 2008. Aedes aegypti uses RNA interference in de- nology 82:561–570. fense against Sindbis virus infection. BMC Microbiol. 8:47. doi:10.1186/ 56. Edwards JF, Higgs S, Beaty BJ. 1998. Mosquito feeding-induced en- 1471-2180-8-47. hancement of Cache Valley virus (Bunyaviridae) infection in mice. J. 33. Campbell GL, Marfin AA, Lanciotti RS, Gubler DJ. 2002. West Nile Med. Entomol. 35:261–265. virus. Lancet Infect. Dis. 2:519–529. 57. Elshuber S, Allison SL, Heinz FX, Mandl CW. 2003. Cleavage of 34. Carlson J, Suchman E, Buchatsky L. 2006. Densoviruses for control and protein prM is necessary for infection of BHK-21 cells by tick-borne genetic manipulation of mosquitoes. Adv. Virus Res. 68:361–392. encephalitis virus. J. Gen. Virol. 84:183–191. 35. Casrouge A, et al. 2011. Evidence for an antagonist form of the chemo- 58. Farajollahi A, Nelder MP. 2009. Changes in Aedes albopictus (Diptera: kine CXCL10 in patients chronically infected with HCV. J. Clin. Invest. Culicidae) populations in New Jersey and implications for arbovirus 121:308–317. transmission. J. Med. Entomol. 46:1220–1224. 35a.Centers for Disease Control and Prevention. 2002. Laboratory- 59. Franz AW, et al. 2006. Engineering RNA interference-based resistance acquired West Nile virus infections—United States, 2002. MMWR to dengue virus type 2 in genetically modified Aedes aegypti. Proc. Natl. Morb. Mortal. Wkly. Rep. 51:1133–1135. Acad. Sci. U. S. A. 103:4198–4203.

October 2012 Volume 25 Number 4 cmr.asm.org 643 Colpitts et al.

60. Fredericksen BL, Gale M, Jr. 2006. West Nile virus evades activation of 89. Kato N, et al. 2008. Evaluation of the function of a type I peritrophic interferon regulatory factor 3 through RIG-I-dependent and -indepen- matrix as a physical barrier for midgut epithelium invasion by mosquito- dent pathways without antagonizing host defense signaling. J. Virol. 80: borne pathogens in Aedes aegypti. Vector Borne Zoonotic Dis. 8:701– 2913–2923. 712. 61. Fredericksen BL, Keller BC, Fornek J, Katze MG, Gale M, Jr. 2008. 90. Keene KM, et al. 2004. RNA interference acts as a natural antiviral Establishment and maintenance of the innate antiviral response to West response to O’nyong-nyong virus (Alphavirus; Togaviridae) infection of Nile Virus involves both RIG-I and MDA5 signaling through IPS-1. J. Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 101:17240–17245. Virol. 82:609–616. 91. Kent R, Juliusson L, Weissmann M, Evans S, Komar N. 2009. Seasonal 62. Fredericksen BL, Smith M, Katze MG, Shi PY, Gale M, Jr. 2004. The blood-feeding behavior of Culex tarsalis (Diptera: Culicidae) in Weld host response to West Nile Virus infection limits viral spread through the County, Colorado, 2007. J. Med. Entomol. 46:380–390. activation of the interferon regulatory factor 3 pathway. J. Virol. 78: 92. Khromykh AA, Meka H, Guyatt KJ, Westaway EG. 2001. Essential role 7737–7747. of cyclization sequences in flavivirus RNA replication. J. Virol. 75:6719– 63. Friebe P, Harris E. 2010. Interplay of RNA elements in the dengue virus 6728. Downloaded from 5= and 3= ends required for viral RNA replication. J. Virol. 84:6103–6118. 93. Kilpatrick AM. 2011. Globalization, land use, and the invasion of West 64. Fu G, et al. 2007. Female-specific insect lethality engineered using alter- Nile virus. Science 334:323–327. native splicing. Nat. Biotechnol. 25:353–357. 94. Kilpatrick AM, Daszak P, Jones MJ, Marra PP, Kramer LD. 2006. Host 65. Fu G, et al. 2010. Female-specific flightless phenotype for mosquito heterogeneity dominates West Nile virus transmission. Proc. Biol. Sci. control. Proc. Natl. Acad. Sci. U. S. A. 107:4550–4554. 273:2327–2333. 66. Gibney KB, et al. 2011. West Nile virus RNA not detected in urine of 40 95. Kilpatrick AM, et al. 2005. West Nile virus risk assessment and the people tested 6 years after acute West Nile virus disease. J. Infect. Dis. bridge vector paradigm. Emerg. Infect. Dis. 11:425–429. 203:344–347. 96. Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Daszak P. 2006. West 67. Gilfoy FD, Mason PW. 2007. West Nile virus-induced interferon pro- Nile virus epidemics in North America are driven by shifts in mosquito duction is mediated by the double-stranded RNA-dependent protein feeding behavior. PLoS Biol. 4:e82. doi:10.1371/journal.pbio.0040082. http://cmr.asm.org/ kinase PKR. J. Virol. 81:11148–11158. 97. King NJ, et al. 2007. Immunopathology of flavivirus infections. Immu- 68. Girard YA, Popov V, Wen J, Han V, Higgs S. 2005. Ultrastructural nol. Cell Biol. 85:33–42. study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus 97a.Klein RS, et al. 2005. Neuronal CXCL10 directs CD8ϩ T-cell recruit- (Diptera: Culicidae). J. Med. Entomol. 42:429–444. ment and control of West Nile virus encephalitis. J. Virol. 79:11457– 69. Glass WG, et al. 2006. CCR5 deficiency increases risk of symptomatic 11466. West Nile virus infection. J. Exp. Med. 203:35–40. 98. Kokoza V, et al. 2000. Engineering blood meal-activated systemic im- 70. Goodell JR, Puig-Basagoiti F, Forshey BM, Shi PY, Ferguson DM. munity in the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. 2006. Identification of compounds with anti-West Nile virus activity. J. U. S. A. 97:9144–9149. Med. Chem. 49:2127–2137. 99. Komar N, et al. 2003. Experimental infection of North American birds 71. Gould HL, et al. 2005. Protective and therapeutic capacity of human with the New York 1999 strain of West Nile virus. Emerg. Infect. Dis. on September 5, 2013 by UNIVERSITAT ZURICH single chain Fv-Fc fusion proteins against West Nile virus. J. Virol. 79: 9:311–322. 14606–14613. 100. Kong KF, et al. 2008. Dysregulation of TLR3 impairs the innate immune 72. Gould LH, Fikrig E. 2004. West Nile virus: a growing concern? J. Clin. response to West Nile virus in the elderly. J. Virol. 82:7613–7623. Invest. 113:1102–1107. 101. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB. 1998. 73. Granwehr BP, et al. 2004. West Nile virus: where are we now? Lancet Phylogeny of the genus Flavivirus. J. Virol. 72:73–83. Infect. Dis. 4:547–556. 102. Kurane I, Janus J, Ennis FA. 1992. Dengue virus infection of human 74. Gu B, et al. 2006. Discovery of small molecule inhibitors of West Nile skin fibroblasts in vitro production of IFN-beta, IL-6 and GM-CSF. Arch. virus using a high-throughput sub-genomic replicon screen. Antiviral Virol. 124:21–30. Res. 70:39–50. 103. Kusne S, Smilack J. 2005. Transmission of West Nile virus by organ 75. Gubler DJ. 2007. The continuing spread of West Nile virus in the west- transplantation. Liver Transpl. 11:239–241. ern hemisphere. Clin. Infect. Dis. 45:1039–1046. 104. Lanciotti RS, et al. 1999. Origin of the West Nile virus responsible for an 76. Reference deleted. outbreak of encephalitis in the northeastern United States. Science 286: 77. Guo X, et al. 2009. Inhibition of neutrophil function by two tick salivary 2333–2337. proteins. Infect. Immun. 77:2320–2329. 105. Lanteri MC, et al. 2008. Comprehensive analysis of West Nile virus- 78. Hamdan A, et al. 2002. Possible benefit of intravenous immunoglobulin specific T cell responses in humans. J. Infect. Dis. 197:1296–1306. therapy in a lung transplant recipient with West Nile virus encephalitis. 106. Reference deleted. Transpl. Infect. Dis. 4:160–162. 107. Lanteri MC, et al. 2011. Association between HLA class I and class II 79. Hamer GL, et al. 2008. Culex pipiens (Diptera: Culicidae): a bridge alleles and the outcome of West Nile virus infection: an exploratory vector of West Nile virus to humans. J. Med. Entomol. 45:125–128. study. PLoS One 6:e22948. doi:10.1371/journal.pone.0022948. 80. Hamer GL, et al. 2009. Host selection by Culex pipiens mosquitoes and 108. Lanteri MC, et al. 2009. Tregs control the development of symptomatic West Nile virus amplification. Am. J. Trop. Med. Hyg. 80:268–278. West Nile virus infection in humans and mice. J. Clin. Invest. 119:3266– 81. Hayes EB, O’Leary DR. 2004. West Nile virus infection: a pediatric 3277. perspective. Pediatrics 113:1375–1381. 109. Lazear HM, Pinto AK, Vogt MR, Gale M, Jr, Diamond MS. 2011. Beta 82. Hoffmann AA, et al. 2011. Successful establishment of Wolbachia in interferon controls West Nile virus infection and pathogenesis in mice. J. Aedes populations to suppress dengue transmission. Nature 476:454– Virol. 85:7186–7194. 457. 110. Lee E, Hall RA, Lobigs M. 2004. Common E protein determinants for 83. Holick J, Kyle A, Ferraro W, Delaney RR, Iwaseczko M. 2002. Discov- attenuation of glycosaminoglycan-binding variants of Japanese enceph- ery of Aedes albopictus infected with west nile virus in southeastern alitis and West Nile viruses. J. Virol. 78:8271–8280. Pennsylvania. J. Am. Mosq. Control Assoc. 18:131. 111. Li J, et al. 2003. Asymmetric flaccid paralysis: a neuromuscular presen- 84. Hudson A, Bowman L, Orr CW. 1960. Effects of absence of saliva on tation of West Nile virus infection. Ann. Neurol. 53:703–710. blood feeding by mosquitoes. Science 131:1730–1731. 112. Lim JK, et al. 2009. Genetic variation in OAS1 is a risk factor for initial 85. Iwamoto M, et al. 2003. Transmission of West Nile virus from an organ infection with West Nile virus in man. PLoS Pathog. 5:e1000321. doi: donor to four transplant recipients. N. Engl. J. Med. 348:2196–2203. 10.1371/journal.ppat.1000321. 86. Jarman RV, Morgan PN, Duffy CE. 1968. Persistence of West Nile virus 113. Lim JK, et al. 2008. Genetic deficiency of chemokine receptor CCR5 is a in L-929 mouse fibroblasts. Proc. Soc. Exp. Biol. Med. 129:633–637. strong risk factor for symptomatic West Nile virus infection: a meta- 87. Kajaste-Rudnitski A, et al. 2006. The 2=,5=-oligoadenylate synthetase 1b analysis of 4 cohorts in the US epidemic. J. Infect. Dis. 197:262–265. is a potent inhibitor of West Nile virus replication inside infected cells. J. 114. Lim JK, et al. 2010. CCR5 deficiency is a risk factor for early clinical Biol. Chem. 281:4624–4637. manifestations of West Nile virus infection but not for viral transmission. 88. Kalil AC, et al. 2005. Use of interferon-alpha in patients with West Nile J. Infect. Dis. 201:178–185. encephalitis: report of 2 cases. Clin. Infect. Dis. 40:764–766. 114a.Lim JK, et al. 2011. Chemokine receptor Ccr2 is critical for monocyte

644 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

accumulation and survival in West Nile virus encephalitis. J. Immunol. 141. Murray K, et al. 2010. Persistent infection with West Nile virus years 186:471–478. after initial infection. J. Infect. Dis. 201:2–4. 115. Lim PY, Behr MJ, Chadwick CM, Shi PY, Bernard KA. 2011. Kerati- 142. Reference deleted. nocytes are cell targets of West Nile virus in vivo. J. Virol. 85:5197–5201. 143. Murray KO, Walker C, Gould E. 2011. The virology, epidemiology, and 116. Lin CW, et al. 2008. Interferon antagonist function of Japanese enceph- clinical impact of West Nile virus: a decade of advancements in research alitis virus NS4A and its interaction with DEAD-box RNA helicase since its introduction into the Western Hemisphere. Epidemiol. Infect. DDX42. Virus Res. 137:49–55. 139:807–817. 117. Lindenbach BD, Rice CM. 2003. Molecular biology of flaviviruses. Adv. 144. Reference deleted. Virus Res. 59:23–61. 145. Nash D, et al. 2001. The outbreak of West Nile virus infection in the New 118. Lindenbach DR, CM. 2001. Flaviviridae: the viruses and their replica- York City area in 1999. N. Engl. J. Med. 344:1807–1814. tion, p 991–1041. In Knipe DM, Howley PM (ed), Fields virology, 4th ed. 146. Oliphant T, et al. 2005. Development of a humanized monoclonal Lippincott Williams and Wilkins, Philadelphia, PA. antibody with therapeutic potential against West Nile virus. Nat. Med. 119. Lindsey NP, et al. 2007. West Nile virus activity—United States, 2006. 11:522–530. Downloaded from JAMA 298:619–621. 147. Olson KE, et al. 2002. Developing arbovirus resistance in mosquitoes. 120. Lindsey NP, Sejvar JJ, Bode AV, Pape WJ, Campbell GL. 2012. Delayed Insect Biochem. Mol. Biol. 32:1333–1343. mortality in a cohort of persons hospitalized with West Nile virus disease 148. Olson KE, et al. 1996. Genetically engineered resistance to dengue-2 in Colorado in 2003. Vector Borne Zoonotic Dis. 12:230–235. virus transmission in mosquitoes. Science 272:884–886. 121. Liu WJ, Chen HB, Wang XJ, Huang H, Khromykh AA. 2004. Analysis 149. Papa A, Danis K, Athanasiadou A, Delianidou M, Panagiotopoulos T. of adaptive mutations in Kunjin virus replicon RNA reveals a novel role 2011. Persistence of West Nile virus immunoglobulin M antibodies, for the flavivirus nonstructural protein NS2A in inhibition of beta inter- Greece. J. Med. Virol. 83:1857–1860. feron promoter-driven transcription. J. Virol. 78:12225–12235. 150. Parsons R, et al. 2008. The memory T cell response to West Nile virus in 122. Liu WJ, et al. 2006. A single amino acid substitution in the West Nile symptomatic humans following natural infection is not influenced by age virus nonstructural protein NS2A disables its ability to inhibit alpha/beta and is dominated by a restricted set of CD8ϩ T cell epitopes. J. Immunol. http://cmr.asm.org/ interferon induction and attenuates virus virulence in mice. J. Virol. 181:1563–1572. 80:2396–2404. 151. Pesko K, Mores CN. 2009. Effect of sequential exposure on infection and 123. Loeb M, Eskandarian S, Rupp M. 2011. Genetic variants and suscepti- dissemination rates for West Nile and St. Louis encephalitis viruses in bility to neurological complications following West Nile virus infection. Culex quinquefasciatus. Vector Borne Zoonotic Dis. 9:281–286. J. Infect. Dis. 204:1031–1037. 152. Petersen LR, Marfin AA. 2002. West Nile virus: a primer for the clini- 124. Luplertlop N, et al. 2011. Induction of a peptide with activity against a cian. Ann. Intern. Med. 137:173–179. broad spectrum of pathogens in the Aedes aegypti salivary gland, follow- 153. Reference deleted. ing infection with dengue virus. PLoS Pathog. 7:e1001252. doi:10.1371/ 154. Piazza P, et al. 2010. Surface phenotype and functionality of WNV journal.ppat.1001252. specific T cells differ with age and disease severity. PLoS One 5:e15343. 125. Marfin AA, Gubler DJ. 2001. West Nile encephalitis: an emerging dis- doi:10.1371/journal.pone.0015343. on September 5, 2013 by UNIVERSITAT ZURICH ease in the United States. Clin. Infect. Dis. 33:1713–1719. 155. Pinto AK, et al. 2011. A temporal role of type I interferon signaling in 126. McGee CE, et al. 2010. Infection, dissemination, and transmission of a CD8ϩ T cell maturation during acute West Nile virus infection. PLoS West Nile virus green fluorescent protein infectious clone by Culex pipi- Pathog. 7:e1002407. doi:10.1371/journal.ppat.1002407. ens quinquefasciatus mosquitoes. Vector Borne Zoonotic Dis. 10:267– 156. Pogodina VV, et al. 1983. Study on West Nile virus persistence in 274. monkeys. Arch. Virol. 75:71–86. 127. McMeniman CJ, et al. 2009. Stable introduction of a life-shortening 157. Prince HE, Lape-Nixon M, Yeh C, Tobler LH, Busch MP. 2008. Wolbachia infection into the mosquito Aedes aegypti. Science 323:141– Persistence of antibodies to West Nile virus nonstructural protein 5. J. 144. Clin. Virol. 43:102–106. 128. Medlock J, Luz PM, Struchiner CJ, Galvani AP. 2009. The impact of 158. Puig-Basagoiti F, et al. 2006. Triaryl pyrazoline compound inhibits transgenic mosquitoes on dengue virulence to humans and mosquitoes. flavivirus RNA replication. Antimicrob. Agents Chemother. 50:1320– Am. Nat. 174:565–577. 1329. 129. Mehlhop E, Diamond MS. 2006. Protective immune responses against 159. Qian F, et al. 2011. Impaired interferon signaling in dendritic cells from West Nile virus are primed by distinct complement activation pathways. older donors infected in vitro with West Nile virus. J. Infect. Dis. 203: J. Exp. Med. 203:1371–1381. 1415–1424. 130. Mehlhop E, et al. 2009. Complement protein C1q reduces the stoichi- 160. Ramamoorthi N, et al. 2005. The Lyme disease agent exploits a tick ometric threshold for antibody-mediated neutralization of West Nile protein to infect the mammalian host. Nature 436:573–577. virus. Cell Host Microbe 6:381–391. 161. Ramirez JL, Dimopoulos G. 2010. The Toll immune signaling pathway 131. Mehlhop E, et al. 2005. Complement activation is required for induction control conserved anti-dengue defenses across diverse Ae. aegypti strains of a protective antibody response against West Nile virus infection. J. and against multiple dengue virus serotypes. Dev. Comp. Immunol. 34: Virol. 79:7466–7477. 625–629. 132. Reference deleted. 162. Rappole JH, Derrickson SR, Hubalek Z. 2000. Migratory birds and 133. Reference deleted. spread of West Nile virus in the Western Hemisphere. Emerg. Infect. Dis. 134. Modis Y, Ogata S, Clements D, Harrison SC. 2004. Structure of the 6:319–328. dengue virus envelope protein after membrane fusion. Nature 427:313– 163. Reisen WK, Fang Y, Martinez VM. 2005. Avian host and mosquito 319. (Diptera: Culicidae) vector competence determine the efficiency of West 135. Moesker B, Rodenhuis-Zybert IA, Meijerhof T, Wilschut J, Smit JM. Nile and St. Louis encephalitis virus transmission. J. Med. Entomol. 42: 2010. Characterization of the functional requirements of West Nile virus 367–375. membrane fusion. J. Gen. Virol. 91:389–393. 164. Reisen WK, Fang Y, Martinez VM. 2006. Vector competence of Culiseta 136. Molaei G, Andreadis TG, Armstrong PM, Anderson JF, Vossbrinck incidens and Culex thriambus for West Nile virus. J. Am. Mosq. Control CR. 2006. Host feeding patterns of Culex mosquitoes and West Nile virus Assoc. 22:662–665. transmission, northeastern United States. Emerg. Infect. Dis. 12:468– 165. Rezepova AI, Kuz’mina SV, Kulikova KS, Unanov SS. 1971. Cultiva- 474. tion of various arboviruses in new transplantable lines of mouse fibro- 137. Reference deleted. blasts. Vopr. Virusol. 16:704–707. 138. Molina-Cruz A, et al. 2005. Effect of mosquito midgut trypsin activity 166. Ribeiro JM. 2000. Blood-feeding in mosquitoes: probing time and sali- on dengue-2 virus infection and dissemination in Aedes aegypti. Am. J. vary gland anti-haemostatic activities in representatives of three genera Trop. Med. Hyg. 72:631–637. (Aedes, Anopheles, Culex). Med. Vet. Entomol. 14:142–148. 139. Munoz-Jordan JL, et al. 2005. Inhibition of alpha/beta interferon sig- 167. Ribeiro JM, et al. 2007. An annotated catalogue of salivary gland tran- naling by the NS4B protein of flaviviruses. J. Virol. 79:8004–8013. scripts in the adult female mosquito, Aedes aegypti. BMC Genomics 8:6. 140. Murray K, et al. 2006. Risk factors for encephalitis and death from West doi:10.1186/1471-2164-8-6. Nile virus infection. Epidemiol. Infect. 134:1325–1332. 168. Ribeiro JM, Charlab R, Rowton ED, Cupp EW. 2000. Simulium vitta-

October 2012 Volume 25 Number 4 cmr.asm.org 645 Colpitts et al.

tum (Diptera: Simuliidae) and Lutzomyia longipalpis (Diptera: Psy- 193. Sessions OM, et al. 2009. Discovery of insect and human dengue virus chodidae) salivary gland hyaluronidase activity. J. Med. Entomol. 37: host factors. Nature 458:1047–1050. 743–747. 194. Shao L, Devenport M, Jacobs-Lorena M. 2001. The peritrophic matrix 169. Ribeiro JM, Charlab R, Valenzuela JG. 2001. The salivary adenosine of hematophagous insects. Arch. Insect Biochem. Physiol. 47:119–125. deaminase activity of the mosquitoes Culex quinquefasciatus and Aedes 195. Shen J, Devery JM, King NJ. 1995. Early induction of interferon- aegypti. J. Exp. Biol. 204:2001–2010. independent virus-specific ICAM-1 (CD54) expression by flavivirus in 170. Ribeiro JM, Francischetti IM. 2001. Platelet-activating-factor-hydrolyzing quiescent but not proliferating fibroblasts—implications for virus-host phospholipase C in the salivary glands and saliva of the mosquito Culex interactions. Virology 208:437–449. quinquefasciatus. J. Exp. Biol. 204:3887–3894. 196. Shi PY, Brinton MA, Veal JM, Zhong YY, Wilson WD. 1996. Evidence 171. Ribeiro JM, Rossignol PA, Spielman A. 1985. Aedes aegypti: model for for the existence of a pseudoknot structure at the 3= terminus of the blood finding strategy and prediction of parasite manipulation. Exp. flavivirus genomic RNA. Biochemistry 35:4222–4230. Parasitol. 60:118–132. 197. Shrestha B, Diamond MS. 2004. Role of CD8ϩ T cells in control of West 172. Ribeiro JM, Rossignol PA, Spielman A. 1984. Role of mosquito saliva in Nile virus infection. J. Virol. 78:8312–8321. Downloaded from blood vessel location. J. Exp. Biol. 108:1–7. 197a.Shrestha B, Pinto AK, Green S, Bosch I, Diamond MS. 2012. CD8ϩ T 173. Ribeiro JM, Valenzuela JG. 2003. The salivary purine nucleosidase of cells use TRAIL to restrict West Nile virus pathogenesis by controlling the mosquito, Aedes aegypti. Insect Biochem. Mol. Biol. 33:13–22. infection in neurons. J. Virol. 86:8937–8948. 174. Rice CM. 1996. Flaviviradae: the viruses and their replication, p 931–959. 197b.Shrestha B, et al. 2006. Gamma interferon plays a crucial early antiviral In Fields BN, Knipe DM, Howley PM (ed), Fields virology, 3rd ed. Lip- role in protection against West Nile virus infection. J. Virol. 80:5338– pincott-Raven, Philadelphia, PA. 5348. 175. Richards SL, Anderson SL, Lord CC, Smartt CT, Tabachnick WJ. 2012. 198. Shrestha B, Zhang B, Purtha WE, Klein RS, Diamond MS. 2008. Relationships between infection, dissemination, and transmission of Tumor necrosis factor alpha protects against lethal West Nile virus in- West Nile virus RNA in Culex pipiens quinquefasciatus (Diptera: Culic-

fection by promoting trafficking of mononuclear leukocytes into the cen- http://cmr.asm.org/ idae). J. Med. Entomol. 49:132–142. tral nervous system. J. Virol. 82:8956–8964. 176. Rios JJ, et al. 2010. OAS1 polymorphisms are associated with suscepti- 199. Siddharthan V, et al. 2009. Persistent West Nile virus associated with a bility to West Nile encephalitis in horses. PLoS One 5:e10537. doi: neurological sequela in hamsters identified by motor unit number esti- 10.1371/journal.pone.0010537. mation. J. Virol. 83:4251–4261. 177. Rios M, et al. 2006. Monocytes-macrophages are a potential target in 200. Sim S, Dimopoulos G. 2010. Dengue virus inhibits immune responses in human infection with West Nile virus through blood transfusion. Trans- Aedes aegypti cells. PLoS One 5:e10678. doi:10.1371/journal.pone.0010678. fusion 46:659–667. 200a.Sitati E, McCandless EE, Klein RS, Diamond MS. 2007. CD40-CD40 178. Rocha AC, Braga EM, Araujo MS, Franklin BS, Pimenta PF. 2004. ligand interactions promote trafficking of CD8ϩ T cells into the brain Effect of the Aedes fluviatilis saliva on the development of Plasmodium and protection against West Nile virus encephalitis. J. Virol. 81:9801– gallinaceum infection in Gallus (gallus) domesticus. Mem. Inst. Oswaldo

9811. on September 5, 2013 by UNIVERSITAT ZURICH Cruz 99:709–715. 200b.Sitati EM, Diamond MS. 2006. CD4ϩ T-cell responses are required for 179. Rodenhuis-Zybert IA, et al. 2010. Immature dengue virus: a veiled clearance of West Nile virus from the central nervous system. J. Virol. pathogen? PLoS Pathog. 6:e1000718. doi:10.1371/journal.ppat.1000718. 80:12060–12069. 180. Roehrig JT, et al. 2003. Persistence of virus-reactive serum immuno- 201. Smartt CT, Richards SL, Anderson SL, Erickson JS. 2009. West Nile globulin m antibody in confirmed West Nile virus encephalitis cases. virus infection alters midgut gene expression in Culex pipiens quinque- Emerg. Infect. Dis. 9:376–379. fasciatus Say (Diptera: Culicidae). Am. J. Trop. Med. Hyg. 81:258–263. 181. Samuel MA, Diamond MS. 2005. Alpha/beta interferon protects against 202. Souza-Neto JA, Sim S, Dimopoulos G. 2009. An evolutionary con- lethal West Nile virus infection by restricting cellular tropism and en- served function of the JAK-STAT pathway in anti-dengue defense. Proc. hancing neuronal survival. J. Virol. 79:13350–13361. Natl. Acad. Sci. U. S. A. 106:17841–17846. 182. Samuel MA, et al. 2006. PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread 203. Stracker TH, Thompson S, Grossman GL, Riehle MA, Brown MR. in the periphery and replication in neurons. J. Virol. 80:7009–7019. 2002. Characterization of the AeaHP gene and its expression in the mos- 183. Sangiambut S, et al. 2008. Multiple regions in dengue virus capsid quito Aedes aegypti (Diptera: Culicidae). J. Med. Entomol. 39:331–342. protein contribute to nuclear localization during virus infection. J. Gen. 204. Styer LM, Bernard KA, Kramer LD. 2006. Enhanced early West Nile Virol. 89:1254–1264. virus infection in young chickens infected by mosquito bite: effect of viral 184. Sardelis MR, Turell MJ, O’Guinn ML, Andre RG, Roberts DR. 2002. dose. Am. J. Trop. Med. Hyg. 75:337–345. Vector competence of three North American strains of Aedes albopictus 205. Styer LM, et al. 2007. Mosquitoes inoculate high doses of West Nile virus for West Nile virus. J. Am. Mosq. Control Assoc. 18:284–289. as they probe and feed on live hosts. PLoS Pathog. 3:1262–1270. doi: 185. Scherbik SV, Stockman BM, Brinton MA. 2007. Differential expression 10.1371/journal.ppat.0030132. of interferon (IFN) regulatory factors and IFN-stimulated genes at early 206. Styer LM, et al. 2011. Mosquito saliva causes enhancement of West Nile times after West Nile virus infection of mouse embryo fibroblasts. J. virus infection in mice. J. Virol. 85:1517–1527. Virol. 81:12005–12018. 207. Sultana H, et al. 2009. Fusion loop peptide of the West Nile virus 186. Schneider BS, Higgs S. 2008. The enhancement of arbovirus transmis- envelope protein is essential for pathogenesis and is recognized by a sion and disease by mosquito saliva is associated with modulation of the therapeutic cross-reactive human monoclonal antibody. J. Immunol. host immune response. Trans. R. Soc. Trop. Med. Hyg. 102:400–408. 183:650–660. 187. Schneider BS, et al. 2007. Prior exposure to uninfected mosquitoes 208. Suthar MS, et al. 2010. IPS-1 is essential for the control of West Nile enhances mortality in naturally-transmitted West Nile virus infection. virus infection and immunity. PLoS Pathog. 6:e1000757. doi:10.1371/ journal.ppat.1000757. PLoS One 2:e1171. doi:10.1371/journal.pone.0001171. ϩ 188. Schneider BS, et al. 2010. Aedes aegypti saliva alters leukocyte recruitment 208a.Suthar MS, et al. 2012. The RIG-I-like receptor LGP2 controls CD8 T and cytokine signaling by antigen-presenting cells during West Nile virus cell survival and fitness. Immunity 37:235–248. infection. PLoS One 5:e11704. doi:10.1371/journal.pone.0011704. 209. Szretter KJ, et al. 2010. The innate immune adaptor molecule MyD88 189. Schneider BS, et al. 2006. Potentiation of West Nile encephalitis by restricts West Nile virus replication and spread in neurons of the central mosquito feeding. Viral Immunol. 19:74–82. nervous system. J. Virol. 84:12125–12138. 190. Schneider BS, Soong L, Zeidner NS, Higgs S. 2004. Aedes aegypti 210. Szretter KJ, et al. 2009. The immune adaptor molecule SARM modu- salivary gland extracts modulate anti-viral and TH1/TH2 cytokine re- lates tumor necrosis factor alpha production and microglia activation in sponses to sindbis virus infection. Viral Immunol. 17:565–573. the brainstem and restricts West Nile Virus pathogenesis. J. Virol. 83: 191. Sejvar JJ, et al. 2003. Neurologic manifestations and outcome of West 9329–9338. Nile virus infection. JAMA 290:511–515. 211. Tassaneetrithep B, et al. 2003. DC-SIGN (CD209) mediates dengue 192. Sejvar JJ, Lindsey NP, Campbell GL. 2011. Primary causes of death in virus infection of human dendritic cells. J. Exp. Med. 197:823–829. reported cases of fatal West Nile Fever, United States, 2002-2006. Vector 212. Tempelis CH, Francy DB, Hayes RO, Lofy MF. 1967. Variations in Borne Zoonotic Dis. 11:161–164. feeding patterns of seven culicine mosquitoes on vertebrate hosts in

646 cmr.asm.org Clinical Microbiology Reviews WNV: Biology, Transmission, and Human Infection

Weld and Larimer Counties, Colorado. Am. J. Trop. Med. Hyg. 16: 226. Wang P, et al. 2010. Caspase-12 controls West Nile virus infection via 111–119. the viral RNA receptor RIG-I. Nat. Immunol. 11:912–919. 213. Tesh RB, et al. 2005. Persistent West Nile virus infection in the golden 227. Wang P, et al. 2008. Matrix metalloproteinase 9 facilitates West Nile hamster: studies on its mechanism and possible implications for other virus entry into the brain. J. Virol. 82:8978–8985. flavivirus infections. J. Infect. Dis. 192:287–295. 228. Wang S, et al. 2008. Drak2 contributes to West Nile virus entry into the 214. Thomas DD, Donnelly CA, Wood RJ, Alphey LS. 2000. Insect popu- brain and lethal encephalitis. J. Immunol. 181:2084–2091. lation control using a dominant, repressible, lethal genetic system. Sci- 229. Wang T, et al. 2003. IFN-gamma-producing gamma delta T cells help ence 287:2474–2476. control murine West Nile virus infection. J. Immunol. 171:2524–2531. 215. Thoma-Uszynski S, et al. 2001. Induction of direct antimicrobial activ- 230. Wang T, et al. 2004. Toll-like receptor 3 mediates West Nile virus entry ity through mammalian Toll-like receptors. Science 291:1544–1547. into the brain causing lethal encephalitis. Nat. Med. 10:1366–1373. 216. Tiawsirisup S, Platt KB, Evans RB, Rowley WA. 2005. A comparision 231. Wasserman HA, Singh S, Champagne DE. 2004. Saliva of the Yellow of West Nile Virus transmission by Ochlerotatus trivittatus (COQ.), Fever mosquito, Aedes aegypti, modulates murine lymphocyte function. Downloaded from Culex pipiens (L.), and Aedes albopictus (Skuse). Vector Borne Zoonotic Parasite Immunol. 26:295–306. Dis. 5:40–47. 232. Weiss D, et al. 2001. Clinical findings of West Nile virus infection in 217. Tobler LH, et al. 2008. Interferon and interferon-induced chemokine hospitalized patients, New York and New Jersey, 2000. Emerg. Infect. expression is associated with control of acute viremia in West Nile virus- Dis. 7:654–658. infected blood donors. J. Infect. Dis. 198:979–983. 233. Welte T, et al. 2009. Toll-like receptor 7-induced immune response 218. Town T, et al. 2009. Toll-like receptor 7 mitigates lethal West Nile to cutaneous West Nile virus infection. J. Gen. Virol. 90:2660–2668. encephalitis via interleukin 23-dependent immune cell infiltration and 234. Westaway EG, Mackenzie JM, Khromykh AA. 2002. Replication and homing. Immunity 30:242–253. gene function in Kunjin virus. Curr. Top. Microbiol. Immunol. 267:323– 219. Unlu I, Mackay AJ, Roy A, Yates MM, Foil LD. 2010. Evidence of 351. vertical transmission of West Nile virus in field-collected mosquitoes. J. Vector Ecol. 35:95–99. 235. Whiteman MC, et al. 2010. Development and characterization of non- http://cmr.asm.org/ 220. Vaidyanathan R, Scott TW. 2006. Apoptosis in mosquito midgut epi- glycosylated E and NS1 mutant viruses as a potential candidate vaccine thelia associated with West Nile virus infection. Apoptosis 11:1643–1651. for West Nile virus. Vaccine 28:1075–1083. 221. Vanlandingham DL, et al. 2007. Relative susceptibilties of South Texas 236. Yakub I, et al. 2005. Single nucleotide polymorphisms in genes for mosquitoes to infection with West Nile virus. Am. J. Trop. Med. Hyg. 2=-5=-oligoadenylate synthetase and RNase L inpatients hospitalized with 77:925–928. West Nile virus infection. J. Infect. Dis. 192:1741–1748. 222. Vitek CJ, Richards SL, Mores CN, Day JF, Lord CC. 2008. Arbovirus 236a.Zhang B, Chan YK, Lu B, Diamond MS, Klein RS. 2008. CXCR3 transmission by Culex nigripalpus in Florida, 2005. J. Med. Entomol. mediates region-specific antiviral T cell trafficking within the central 45:483–493. nervous system during West Nile virus encephalitis. J. Immunol. 180: 223. Volfova V, Hostomska J, Cerny M, Votypka J, Volf P. 2008. Hyaluron- 2641–2649.

idase of bloodsucking insects and its enhancing effect on leishmania in- 237. Zhang Y, Kaufmann B, Chipman PR, Kuhn RJ, Rossmann MG. 2007. on September 5, 2013 by UNIVERSITAT ZURICH fection in mice. PLoS Negl. Trop. Dis. 2:e294. Structure of immature West Nile virus. J. Virol. 81:6141–6145. 224. Wanasen N, Nussenzveig RH, Champagne DE, Soong L, Higgs S. 238. Zou S, Foster GA, Dodd RY, Petersen LR, Stramer SL. 2010. West Nile 2004. Differential modulation of murine host immune response by sali- fever characteristics among viremic persons identiﬁed through blood vary gland extracts from the mosquitoes Aedes aegypti and Culex quin- donor screening. J. Infect. Dis. 202:1354–1361. quefasciatus. Med. Vet. Entomol. 18:191–199. 239. Zybert IA, van der Ende-Metselaar H, Wilschut J, Smit JM. 2008. 225. Wang P. IL-22 signaling contributes to West Nile encephalitis pathogen- Functional importance of dengue virus maturation: infectious properties esis. PLoS One, in press. of immature virions. J. Gen. Virol. 89:3047–3051.

Tonya M. Colpitts received a B.A. from the Michael J. Conway received a B.S. from North- University of Hawaii and a Ph.D. in microbiol- ern Michigan University and a Ph.D. in micro- ogy and immunology from the University of biology and immunology in the laboratory of Texas Medical Branch, where she showed that Professor Craig Meyers at the Pennsylvania the cellular endocytic machinery is highly evo- State University College of Medicine, where he lutionarily conserved between mosquitoes and studied differentiation-dependent mechanisms mammals and that alphaviruses require func- of the human papillomavirus life cycle, includ- tional endocytosis and low pH for entry into ing capsid assembly and maturation. He cur- both cell types. She is currently a postdoctoral rently is a postdoctoral research fellow in the associate in the laboratory of Professor Erol laboratory of Professor Erol Fikrig in the Infec- Fikrig in the Section of Infectious Diseases at tious Diseases Section of the Department of In- Yale University School of Medicine. Her research focuses on the exploration ternal Medicine at Yale University School of Medicine. His current research of the interactions between mosquitoes and flaviviruses, the identification of interests involve vector-virus-host interactions that occur as disease vectors human host factors that bind flaviviral proteins, and the examination of how deposit salivary components and pathogens into the host. flavivirus infection affects proteins and pathways of human cells. She is also Continued next page researching the interactions of flavivirus capsid protein with nuclear and cytoplasmic proteins as well as the role of capsid in the nucleus of the cell during infection.

October 2012 Volume 25 Number 4 cmr.asm.org 647 Colpitts et al.

Ruth R. Montgomery received a B.A. from the Erol Fikrig received a B.A. in chemistry from University of Pennsylvania and a Ph.D. from Cornell University and an M.D. from Cornell Rockefeller University, where she studied with University Medical College. Dr. Fikrig did a res- Zanvil Cohn and Carl Nathan. After postdoc- idency in internal medicine at Vanderbilt Uni- toral work on macrophage endocytosis with Ira versity School of Medicine and was a fellow in Mellman, she remained at Yale, where she is infectious diseases and immunobiology at Yale now Associate Professor of Medicine. The focus University School of Medicine. He is currently a of her lab is human innate immunity, speciﬁ- Professor of Medicine, Microbial Pathogenesis, cally the interaction of macrophages, neutro- and Epidemiology and Public Health, a Walde- phils, and dendritic cells with pathogens such as mar Von Zedtwitz Professor of Medicine, Chief West Nile virus and the agent of Lyme disease, of Infectious Diseases, and an investigator with Borrelia burgdorferi, including elucidating effects of vector saliva on phago- the Howard Hughes Medical Institute. He currently leads a research group Downloaded from cyte function. In studies of the pathogenesis of West Nile virus, the Mont- studying the immunopathogenesis of arthropod-borne diseases. Lyme dis- gomery lab has described inhibition of macrophage function, an unexpected ease, human granulocytic anaplasmosis, and West Nile encephalitis are areas biphasic role for PMNs in infection, and effects of aging on innate immunity, of particular interest. Studies are directed at understanding the interactions including dysregulation of TLR3 responses in macrophages and reduced between pathogen, host, and vector that result in virulence and transmission responses of dendritic cells to infection with West Nile virus. and the molecular basis of disease in animal models and patient populations. http://cmr.asm.org/ on September 5, 2013 by UNIVERSITAT ZURICH

648 cmr.asm.org Clinical Microbiology Reviews The Contribution of Culex pipiens Complex Mosquitoes to Transmission and Persistence of West Nile Virus in North America Author(s): Theodore G. Andreadis Source: Journal of the American Mosquito Control Association, 28(4s):137-151. 2012. Published By: The American Mosquito Control Association DOI: http://dx.doi.org/10.2987/8756-971X-28.4s.137 URL: http://www.bioone.org/doi/full/10.2987/8756-971X-28.4s.137

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Copyright E 2012 by The American Mosquito Control Association, Inc.

THE CONTRIBUTION OF CULEX PIPIENS COMPLEX MOSQUITOES TO TRANSMISSION AND PERSISTENCE OF WEST NILE VIRUS IN NORTH AMERICA

THEODORE G. ANDREADIS Center for Vector Biology & Zoonotic Diseases, The Connecticut Agricultural Experiment Station, 123 Huntington Street, New Haven, CT 06511

ABSTRACT. Mosquitoes within the Culex pipiens complex have been implicated as major vectors of West Nile virus (WNV) in North America due to their seasonal abundance, vector competence and high field infection rates. However, the role of Cx. p. pipiens complex mosquitoes in enzootic amplification of WNV among avian hosts and epidemic transmission to humans varies throughout its geographical distribution. In the northeastern United States, Cx. p. pipiens is recognized as the primary enzootic vector responsible for amplification of virus among wild bird populations. However, because this mosquito is strongly ornithophilic, its role in transmission to humans appears to be more limited in this region. In the north central and Mid-Atlantic States by contrast, Cx. p. pipiens shows an increased affinity for human hosts and has been incriminated as a key bridge vector. In southern regions of the United States, Culex p. quinquefasciatus are more opportunistic feeders, and are thought to be principal enzootic and epidemic vectors. In western regions of the United States where Culex tarsalis predominates, especially in rural areas, Cx. p. pipiens and Cx. p. quinquefasciatus play roles that are more limited and are recognized as secondary vectors. In the southwestern United States Cx. p. quinquefasciatus also appears to be the predominant vector in urban habitats, but only a secondary vector in more rural environs. The direct involvement of Cx. p. pipiens form molestus in WNV transmission is largely unknown, but human-biting Cx. p. pipiens are more likely to have a probability of genetic ancestry with Cx. p. pipiens form molestus. The detection of WNV from overwintering populations of diapausing Cx. p. pipiens and non-diapausing Cx. p. quinquefaciatus and their role in local overwintering of WNV are addressed.

KEY WORDS Culex pipiens, Culex quinquefasciatus, West Nile virus, transmission, overwintering

INTRODUCTION movements of migratory birds (Peterson et al. 2003) and infected mosquito vectors (Venkate- The detection and presumed introduction of san and Rasgon 2010); and (3) broad variety West Nile virus (WNV) into the United States and widespread distribution of reservoir com- during the summer of 1999 (Anderson et al. petent avian hosts (Komar 2003, Kilpatrick et 1999, Lanciotti et al. 1999) was a seminal event al. 2007) and mosquito vectors (Turell et al. in realizing the potential threat of introduction 2005). However, it is also quite likely that WNV and subsequent establishment of an exotic may have never become established in North vector-borne disease in the western hemisphere. America where it not for the vectorial capacity Within four years after its initial detection in and intimate involvement of urban Culex New York City, this exotic virus, which was mosquito vectors within the pipiens complex. thought to have been introduced from the Mosquitoes within the Culex pipiens complex Middle East (Lanciotti et al. 1999), rapidly are recognized as major vectors of WNV in North swept across the continental United States, America due to their vector competence, high field moved north into Canada and southward into infection rates, local abundance, and close asso- the Caribbean Islands and Central America to ciation in time and space with virus foci and become the preeminent arboviral disease in human cases. However, the role Cx. pipiens North America. To date, WNV has caused over complex mosquitoes play in enzootic amplifica- 30,000 cases of human disease and more than tion of WNV among avian hosts and epidemic 1,000 fatalities in the United States alone (CDC transmission to humans appears to vary widely 2010a, 2010b, 2011), and has clearly become a throughout its geographical distribution. This permanent part of the North American land- review will examine regional differences in the scape causing seasonal epidemics. This unprec- role of Culex p. pipiens L., Cx. p. quinquefaciatus edented expansion and establishment of WNV Say and their hybrids in transmission, local in North America has been attributed to a overwintering and long-term persistence of number of factors including: (1) the emergence WNV in the United States based on national of a virus strain with greater virulence (Brault surveillance data compiled by the CDC ArboNet et al. 2004, 2007), transmission efficiency since 1999 and our current knowledge of their (Moudy et al. 2007) and epidemic poten- population biology and feeding behavior. The tial (Davis et al. 2005); (2) the long-range contribution of underground populations of Cx.

137 138 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4 pipiens form molestus Forskal to the epidemiology of WNV in urban settings will also be explored. % 5 REGIONAL ANALYSIS OF MOSQUITOES

Northeastern United States No. pools WNV

A summary of mosquito pools that have tested Southwest positive for WNV from different regions of the US from 1999 to 2010 (CDC ArboNet) is shown No. in Table 1. Within the northeastern United species States, where the first isolations were made in 1999 from Culex p. pipiens and Aedes vexans 0.1 - - - 0.1 3 25 0.3 % , (Meigen) (Anderson et al. 1999, Nasci et al. , 2001b), WNV has been identified from 33 species of mosquitoes representing eight different genera. However, over 96% of the positive pools have 4 No. pools WNV

been obtained from Culex mosquitoes, among West which 66% were from Cx. p. pipiens, 27.8% from Culex restuans Theobald, 6.1% from Culex

salinarius Coquillett, 0.3% from Culex erraticus cies

(Dyar and Knab), and , 0.1% from Culex No. spe- territans Walker (Fig. 1). The preponderance of WNV positive pools obtained from field-collected 0.1 4 50 0.3 - - - 0.1------0.10.1------1 2 % , Cx. p. pipiens and to a lesser degree, Cx. restuans , , , clearly incriminate these two species as the most important vectors of WNV in the northeast. This 3 conclusion is supported by their local abundance No. pools in virus foci and high minimum field infection WNV rates revealed from mosquito surveillance con- Midwest ducted in Connecticut (Andreadis et al. 2001,

2004, Anderson et al. 2004, 2006, Andreadis and No. Armstrong 2007), Delaware (Gingrich et al. species 2010), New York City (Kulasekera 2001) and 0.1------New York State (Bernard et al. 2001, White et al. 0.1------0.10.1 1 1 4 6 % , 2001, Ebel et al. 2005, Lukacik et al. 2006). Local , , , regional populations of both species also have 2 been shown to be moderately efficient vectors of WNV in the laboratory (Turell et al. 2000, 2001, No. pools 2005; Sardelis et al. 2001; Ebel et al. 2005). WNV

The ornithophilic feeding behavior of popula- Southeast tions of Cx. p. pipiens from the northeastern

United States is well-established (Crans 1964, No. Means 1968, Spielman 1971, Tempelis 1975, species Magnarelli 1977, Apperson et al. 2002, 2004,

Molaei et al. 2006) and clearly support a major 0.10.1 1 1 1 2 % role for this mosquito in transmission of WNV to , ,

birds throughout the region (Table 2). Culex p. 1 pipiens involvement in both early and late season enzootic transmission is largely based upon the No. pools detection of WNV in July when populations of WNV

Cx. p. pipiens are typically increasing and the Northeast 3 185 1.1 2 60 0.4 2 7 55 15,6831 96.1 32 35 8 0.2 0.2 14,697 4 96.2 1 6 59 25 27,028 0.4 97.9 0.2 4 8 1 13,902 83 30 98.9 0.3 0.1 10 4 7,582 1 97.3 17 1 0.1 2 9 0.1 preponderance of virus positive pools found in 12 316 12 0.1 3 43 0.3 2 10 --- 1 2 --- 1 4 14 363 2.2 12 386 2.5 14 435 1.6 6 79 0.6 9 177 2.3 August and September, when virus activity and No. Cx. p. pipiens populations are at their height species (Andreadis et al. 2001, 2004, Anderson et al. 2004, 2006, Andreadis and Armstrong 2007). Additional support for early season initiation of enzootic transmission by Cx. p. pipiens, comes Table 1. Generic summary of West Nile virus positive mosquito pools reported to CDC ArboNet from different regions of the United States, 1999–2010. Mosquito

from the detection of WNV from overwintering CT, DC, DE, MA,AL, MD, AR, ME, FL, NJ, GA,IA, NY, KY, IL, PA, LA, IN, RI, MS, KS,CA, VT NC, MI, CO, (1999–2010). SC, MN, ID, TN, MO, MT,AZ, VA, ND, NV, NM, WV NE, OR, OK, (2001–2010). OH, UT, TX SD, WA, (2002–2010). WI WY (2001–2010). (2002–2010). 1 2 3 4 5 Culiseta Deinocerites Anopheles Aedes Coquillettidia Culex Mansonia females collected from hibernacula in New York Orthopodomyia Psorophora Uranotaenia CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 139

Fig. 1. Comparative proportion of West Nile virus positive mosquito pools obtained from field-collected Culex species reported to CDC ArboNet from different regions of the United States, 1999–2010. 140 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4

Table 2. Role of Culex p. pipiens complex mosquitoes in enzootic and epidemic transmission of WNV in different geographic regions of the US based on the prevalence of virus detection among Culex mosquitoes and host feeding behavior. Vectorial capacity for Feeding behavior transmission % WNV+ pools Region among Culex Bird Mammal Enzootic Epidemic Cx. p. pipiens Northeast 66.0 ++++ + high low Southeast 18.6 +++ ++ moderate low Midwest 33.6 +++ ++ moderate moderate West 18.5 ++++ + moderate low Cx. p. quinquefasciatus Southeast 64.6 +++ ++ high moderate West 28.7 +++ ++ moderate moderate Southwest 82.1 +++ ++ high high

City, New Jersey and Pennsylvania (Nasci et al. and biting behavior, Kilpatrick et al. (2005) 2001a, Farajollahi et al 2005, Bugbee and Forte estimated that local populations of Cx. p. pipiens 2004, Andreadis et al. 2010), and documentation and Cx. restuans from New Jersey and New York of vertical transmission of the virus by resident might be responsible for up to 80% of human populations in the laboratory (Dohm et al. 2002, infections in that region. In an investigation of Anderson et al. 2008). The important role of Cx. populations from the mid-Atlantic region (Mary- p. pipiens in amplification of WNV in wild bird land and Washington DC), Kilpatrick et al. (2006) populations is further supported by the identifi- similarly implicated Cx. p. pipiens as a major cation of avian communal roosts as amplification epidemic vector based on a late-summer shift in foci in urban centers in the Northeast during the feeding behavior from avian to mammalian hosts. transmission season (Diuk-Wasser at al. 2010), The involvement of Cx. p. pipiens as an epidemic and the detection of significantly greater numbers as well as epizootic vector thus appears probable, of WNV-infected Cx. pipiens in traps placed in especially in densely populated urban areas where the tree canopy when compared to similar traps this species predominates. However, its contribu- placed on the ground (Anderson et al. 2004, tion to epidemic transmission varies greatly, Andreadis and Armstrong 2007). depending on regional differences in host feeding While the role that Cx. pipiens plays in enzootic patterns (Table 2). transmission of WNV among wild bird popula- In addition to Cx. p. pipiens, it is appropriate to tions is unequivocal, its involvement in epidemic comment on the role of two other notable species transmission to humans in the northeastern of Culex mosquitoes that also have been incrim- United States appears to vary widely depending inated as vectors of WNV in the Northeast, Cx. on the location. The majority of investigations on restuans and Cx. salinarius. The abundance of feeding patterns of this species clearly indicate Cx. restuans in June and July, early season that populations from Connecticut (Magnarelli detection of WNV (Andreadis et al. 2001, 2004, 1977, Molaei et al. 2006), Massachusetts (Spielman Andreadis and Armstrong 2007) and high vecto- 1971), New Jersey (Crans 1964) and New York rial capacity (Ebel et al. 2005), support the (Means 1968, Tempelis 1975, Apperson et al. 2002, supposition that this mosquito plays an impor- 2004), predominately feed on birds and rarely feed tant role as an enzootic vector involved in early on humans. However, Apperson et al. (2004) amplification of WNV among wild birds in the identified mammalian-derived blood meals in northeastern US. In addition to being the most 38% of blooded Cx. p. pipiens,11% of which were abundant Culex species at this time of the year, it human-derived, collected from natural and man- is widely distributed throughout the region and it made resting sites in suburban areas of New occurs in both urban and rural environs (Ebel Jersey, while Gingrich and Williams (2005) et al. 2005). This conclusion is fully consistent reported a high percentage (69%) of mammali- with its well-documented ornithophilic feeding an-derived blood meals (albeit no human) from preferences (Means 1968, Magnarelli 1977, Ap- populations in Delaware. Although apparently person et al. 2002, 2004, Molaei et al. 2006). rare, human derived blood meals have been However, there are several reports in the litera- occasionally identified (, 1%)inCx. p. pipiens ture (Hayes 1961, Murphey et al. 1967, Means populations from urban sites in Connecticut as 1968, 1987) indicating that although Cx. restuans well (Molaei et al. 2006). Using a risk-assessment prefers feeding on birds, females from this region model that combined data on mosquito abun- will bite humans on occasion, and a human- dance, infection prevalence, vector competence, derived blood meal has been identified from CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 141 blooded females collected in suburban New mosquitoes in 10 genera (Table 1). However, as Jersey (Apperson et al. 2004). These findings in the Northeast, the overwhelming majority of taken in concert with the multiple isolations virus positive pools (96%) have been made from of WNV obtained from this species in August mosquitoes in the genus Culex (8 species). Among and September (Andreadis et al. 2001, 2004, the Culex mosquitoes, members of the Cx. p. Anderson et al. 2004, Andreadis and Armstrong pipiens complex have accounted for over 88% of 2007) do not preclude its involvement as a bridge the WNV positive pools reported to ArboNet, vector to humans. However, because of its and based on total numbers and infection rates, generally lower abundance at this time of the Culex p. quinquefasciatus, a common and mod- year, virus transmission to humans is likely to be erately efficient vector (Sardelis et al. 2001, Turell relatively rare. et al. 2005, Richards et al. 2007), appears to be Culex salinarius is among the most frequently the predominant vector species (Rutledge et al. captured Culex species in coastal regions of the 2003; Godsey et al. 2005a, 2005b; Gibbs et al. northeastern United States where a large majority 2006; Lindsey et al. 2008) (Fig. 1). of human cases occur, and is locally abundant in The role that members of the Cx. p. pipiens August and September when virus activity is at its complex play in enzootic/epizootic transmission height (Andreadis et al. 2001, 2004; Kulasekera among birds and epidemic transmission to et al. 2001; Anderson et al. 2004; Gingrich and humans appears to vary throughout the South- Casillas 2004; Andreadis and Armstrong 2007; east as it does in the Northeast (Table 2). In a Rochlin et al. 2008). In contrast to Cx. p. pipiens series of detailed studies in the southern portion and Cx. restuans, Cx. salinarius is a well- of the Cx. p. pipiens/quinquefaciatus hybrid zone recognized generalist that feeds indiscriminately in Shelby County, Tennessee, Savage et al. (2006, on both birds and mammals and readily bites 2007, 2008) found that members of the Cx. humans (Crans 1964, Murphey et al. 1967, Means pipiens complex accounted for 97% of all WNV 1987). Studies by Apperson et al. (2002, 2004) positive mosquitoes with no significant differenc- with local populations from New Jersey and New es in infection rates among members within the York reaffirmed the wide-ranging feeding habits complex. They also reported significantly higher reported in these prior investigations; with iden- infection rates in urban sites (Memphis) associ- tification of mammal to bird feeding ratios of 4:1 ated with larger populations of Cx. p. pipiens in blooded females collected from a WNV focus in complex mosquitoes and human cases, reaffirm- Queens, New York in 2000, and 3:1 in blooded ing their importance in these foci. An analysis of females collected from WNV endemic peri-urban feeding preferences again showed no differences areas in New Jersey in 2001 with 8.6% of the mammalian blood meals identified as human- among members of the complex, which fed derived. In an analysis of local populations from predominantly upon avian hosts (73%), support- Connecticut, Molaei et al. (2006) similarly found ing their primary role as enzootic vectors. that Cx. salinarius readily feeds on both birds However, a substantial number of mammalian- (36%) and mammals (53%) including humans, derived blood meals (14%), including humans and further detected mixed blood meals in 11% of (1%) were also identified. Despite the compara- these females, a necessary condition for transmis- tively low rate of human feeding, Savage et al. sion to humans. The frequent isolations of WNV (2007) concluded that the very high rates of WNV from this species in late August and September infection in Cx. p. pipiens complex mosquitoes (Andreadis et al. 2001, Bernard et al. 2001, combined with the extremely high mosquito Kulasekera et al. 2001, White et al. 2001, population levels in this region, supported a role Anderson et al. 2004) when the majority of human for members within the complex in transmission cases were reported, in union with its abundance to humans. The involvement of Cx. restuans as a at this time of the year, broad feeding habits, and principal enzootic vector to birds and occasional demonstrated vector competence (Sardelis et al. vector to humans was also noted. 2001) which equals that of Cx.p.pipiens Studies conducted in more rural areas in the (Anderson et al. 2012), make Cx. salinarius a Tennessee Valley by contrast, indicate a reduced likely bridge vector to humans, horses and other role for Cx. p. pipiens/Cx. quinquefasciatus, where mammals in northeastern United States. the most commonly infected species were Culex erraticus and Cx. salinarius (Cupp et al. 2007). Although the vector competence of Cx. erraticus Southeastern United States for WNV has not been evaluated, the authors of The first WNV positive mosquito pools report- this investigation felt its abundance, wide distri- ed from the southeastern United States were in bution, and strong ornithophilic feeding behavior 2001 (Blackmore et al. 2003, Rutledge et al. 2003, (Hassan et al. 2003), make it a potentially Godsey et al. 2005a), two years following the important enzootic vector in this region of the initial discovery in New York City. Since then, southeastern United States. They also noted that WNV has been detected in 34 species of Cx. salinarius likely played a role as an important 142 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4 bridge vector as suspected in the northeastern This species has since become the most commonly United States. reported WNV-positive mosquito in rural loca- In surveillance studies in East Baton Rouge tions of the Midwest, making up more than one- Parish, Louisiana, where a large number of third of all Culex pools reported to the CDC human cases occurred from 2002 to 2004, over ArboNet through 2010 (Fig. 1). In certain regions 87% of all WNV positive pools were obtained of the midwest, such as Grand Forks, North from Cx. p. quinquefaciatus (Godsey et al. 2005b, Dakota, for example, it is regarded as the most Palmisano et al. 2005, Gleiser et al. 2007, Mackay important vector of WNV, serving as both the et al. 2008). Also underscored in those investiga- enzootic and bridge vector to humans and horses tions was the abundance and widespread distri- (Bell et al. 2005). bution of Cx. p. quinquefaciatus in urban areas Members of the Cx. p. pipiens complex, by and peak temporal association between the onset contrast, appear to be the predominant vectors in of human disease and mosquito WNV infection more densely populated urban and suburban rates. These findings and the demonstrated environments in the Midwest (Gu et al. 2006, feeding of local populations of Cx. p. quinquefa- Hamer et al. 2008a, 2009, Harrison et al. 2009) ciatus on human hosts at a relatively high rate accounting for over 56% of the WNV-positive (7% to 15% of all blood meals) (Niebylski and pools reported through 2010 to the CDC Meek 1992, Mackay et al. 2010), clearly incrim- ArboNet (Fig. 1). Their role as primary enzootic inate this species as the most important vector for vectors involved in amplification of WNV among both enzootic amplification and transmission of wild bird populations in these settings is well WNV to humans in southern Louisiana. recognized (Hamer et al. 2008b, 2009) and widely Among non-Cx. pipiens complex mosquitoes, acknowledged throughout the region (Table 2). Culex nigripalpus Theobald has been implicated The most compelling evidence supporting their as a potentially important vector in some regions role in transmission of WNV to humans comes of the southern United States where it is locally from a series of investigations conducted in an abundant (Godsey et al. 2005a). Although the endemic transmission area in metropolitan Chi- overall number of WNV positive pools reported cago, Illinois, where Hamer et al. (2008a, 2009) from Cx. nigripalpus have been comparatively documented an unusually high rate of human few (1.8% of all Culex) (Fig. 1), this moderately feeding by Cx. pipiens (16% of total blood meals competent vector species (Sardelis et al. 2001) is examined). Based on the: 1.) relatively high rate believed to be one of the more important vectors of feeding on humans, 2.) high prevalence of in Florida, where transmission patterns have been WNV infection in local Cx. p. pipiens populations sporadic and largely focal with rare epidemics (12 per 1,000), 3.) identification of a WNV- (Blackmore et al. 2003, Rutledge et al. 2003, positive female Cx. p. pipiens with a human blood Hribar et al. 2004). Culex nigripalpus is an meal, and 4.) low rate of WNV infection in non- opportunistic species (Edman 1974) that feeds Culex mosquitoes (1 per 1000), the authors on avian hosts during the winter and spring, and concluded that Cx. p. pipiens likely serves as both then purportedly shifts to mammalian hosts, the enzootic and epidemic vector in this metro- including humans during the summer and fall politan area. Microsatellite analysis of popula- (Edman and Taylor 1968). These factors, coupled tions of Cx. p. pipiens from Chicago suggested with its abundance, suggest that Cx. nigripalpus that the probability of genetic ancestry from Cx. likely serves as an enzootic as well as epidemic p. pipiens form molestus may have predisposed vector in this region. It has been further suggested these mosquitoes to readily feed on mammals, that in south Florida drought brings Cx. nigri- although the genetic mechanisms are not known palpus and wild birds into close contact facilitat- (Huang et al. 2009). ing epizootic WNV amplification and generating The degree to which Cx. p. pipiens contribute infection rates necessary to support high levels of to human transmission in other metropolitan WNV transmission (Shaman et al. 2005). districts in the Midwest is less well known and may be markedly reduced. Mosquito and arbovirus surveys conducted in semi-urban regions of Midwestern United States southeastern Kansas in 2007 (Harrison et al. The expansion of WNV into the midwestern 2009) detected high WNV infection rates in Cx. p. United States in 2001 was followed by extensive pipiens (26 per 1,000) that would be typically epidemics of human disease throughout the region associated with an elevated risk of human in 2002 and 2003 (7,067 human cases) (Hayes et al. infection. However, only a single human case 2005). Associated with this was the incrimination was subsequently documented. of Culex tarsalis Coquillett, a highly competent vector (Goddard et al. 2002; Turell et al. 2002, Western United States 2005) and opportunistic feeder that prefers avian hosts, but will readily attack humans (Hayes et al. Mosquito and arbovirus surveillance conduct- 1973, Tempelis 1975, Reisen and Reeves 1990). ed in the western United States since 2002 have CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 143 resulted in the detection of WNV from eight comes from investigations during a severe human species of Culex (Table 1). The most frequently outbreak that occurred in these counties during reported WNV-positive species has been Cx. 2005 (Elnaimen et al. 2008). Culex p. pipiens was tarsalis, which made up nearly one-half (49.0%) the most abundant urban vector collected in CO2- of the positive pools, followed by Cx. p. baited traps placed in residential areas where the quinquefasciatus (28.7%), Cx. p. pipiens (18.5%), epidemic occurred, accounting for 66.8% of all Culex erythrothorax Dyar (2.3%), Culex stigma- Culex mosquitoes. Culex p. pipiens also made up tosoma Dyar (1.3%), Cx. restuans (0.1%), Culex 68.3% of the WNV-infected pools followed by thriambus Dyar (0.1%), and Cx. territans Cx. tarsalis (28.8%), and had an infection rate (, 0.1%) (Fig. 1). that was more than double that detected in Cx. Much attention has focused on Cx. tarsalis, tarsalis. These findings led the authors to which is considered the primary enzootic and conclude that Cx. p. pipiens was the primary epidemic vector of WNV throughout much of the vector likely involved in human transmission. western region, especially in rural areas. This is However, the supposition that Cx. p. pipiens largely due to its high vector efficiency (Goddard functions as a bridge vector in these residential et al. 2002, Turell et al. 2002, Reisen et al. 2005, settings could not be corroborated in a subse- Anderson et al. 2012), widespread abundance quent analysis of blood meals from field-caught (Bolling et al. 2009, Reisen and Reeves 1990, Cx. p. pipiens collected in 2007 and 2008 from Winters et al. 2008), high natural infection rate urban/suburban centers in the same two counties, (Bolling et al. 2007) and propensity of local where . 99% of the blood meals were determined populations to feed on birds and mammals, to be of avian origin and not a single incident of including humans (Kent et al. 2009, Thiemann human feeding was detected (Montgomery et al. et al. 2011). Strong support for this view comes 2011). from studies in northeastern Colorado conducted In the rural lower Coachella Valley of southern from 2003 to 2007, where the abundance of Cx. California, Cx. tarsalis is viewed as the primary tarsalis and vector index for WNV-infected enzootic vector responsible for maintenance and females were strongly associated with the large amplification of WNV (Reisen et al. 2004, 2008b, number of human disease cases that occurred Lothrop et al. 2008). This is based on the frequent during that period (Bolling et al. 2007, 2009). detection of virus in this species and its overall Similarly, Gujral et al. (2007) reported higher abundance, which generally mirrors the temporal vector indices for WNV transmission among local and spatial distribution of enzootic transmission populations of Cx. tarsalis than Cx. p. pipiens in throughout the region. Culex p. quinquefasciatus, two adjacent cites in northern Colorado (Love- by contrast, is considered the primary enzootic land and Fort Collins) that had severe outbreaks andbridgevectortohumansinthemore of human disease in 2003. Bowden et al. (2011) urbanized Upper Valley (Reisen et al. 2004, further demonstrated that the incidence of human 2008b, Lothrop et al. 2008), due to its abundance WNV disease in the northwest was positively in peridomestic habitats and diverse feeding associated with agricultural land covers (grass- habits that include humans (Reisen et al. 1990, land, crops, herbaceous wetland) and not urban Reisen and Reeves 1990). It is also thought to be land covers as observed in other regions of the involved in most tangential transmission of WNV country where Cx. pipiens complex mosquitoes to humans in peridomestic environs in Kern predominate. This analysis is consistent with the County, where rapid spring amplification was preferred breeding sites for Cx. tarsalis, which associated with early season increases in WNV include natural ground pools and ditches with infection incidence in Cx. p. quinquefasiatus emergent vegetation, open grassland and fresh- (Reisen at al. 2009). water pools associated with agricultural sources Host-feeding patterns and WNV infection rates (Reisen and Reeves 1990). in mosquitoes collected from urbanized centers in A more prominent role for Cx. p. pipiens neighboring Orange, Riverside and San Bernar- complex mosquitoes in the ecology and epizoot- dino Counties, equally implicate Cx. p. quinque- iology/epidemiology of WNV transmission in the fasciatus as the primary vector of WNV in this western United States comes from studies in region of southern California as well (Molaei California where WNV was first isolated from a et al. 2010). This mosquito was among the most pool of Cx. tarsalis collected from Imperial commonly trapped species and the main source of County near the Mexican border during July WNV over a two-year period (2006–2008), 2003 (Reisen et al. 2004). In urban/suburban representing nearly 80% of all WNV-positive areas of Sacramento and Yolo counties located in mosquito pools, and blood meal analysis revealed the north central regions of the state, Cx. p. opportunistic feeding on a diversity of competent pipiens appears to function primarily as a WNV- avian (88.4%) and mammalian (11.6%) hosts amplifying enzootic vector (Montgomery et al. including humans (1.9%), further indicating its 2011), but has also been incriminated as an involvement in enzootic as well as epidemic epidemic vector as well. Support for the latter transmission (Table 2). 144 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4

Mosquito and arbovirus surveillance conduct- (2003–2006) further demonstrated a strong posed in Los Angeles County, California, from 2003 itive correlation between WNV-positive Cx. to 2008 that included major human epidemics, quinquefasciatus pools, WNV-positive blue jays similarly identified Cx. p. quinquefasciatus as the and the incidence of monthly human cases most abundant species, and on the basis of (Dennett et al. 2007). The role of this mosquito infection incidence, the species most frequently in both enzootic and epidemic transmission was involved in enzootic and epidemic transmission in explicitly revealed in two host-feeding studies urban Los Angeles (Kwan et al. 2010). Also (Dennett et al. 2007, Molaei et al. 2007) which identified were Cx. tarsalis and Cx. stigmatosoma, showed local populations were very opportunis- the latter a highly competent vector for WNV tic, exhibiting considerable variation in blood- (Goddard et al. 2002, Reisen et al. 2008a) that feeding behavior that included: 1.) A variety of feeds almost exclusively on birds (Reisen et al. competent avian hosts (42% and 39% of total 1990, Molaei et al. 2010). However, both species blood meals), 2.) Several mammals (58% and were significantly less abundant than Cx. p. 53% of total blood meals), and 3.) Humans (0.7% quinquefasciatus (4.1% of total Culex collection), and 23% of mammalian blood meals) (Table 2). were not consistently found infected throughout Molaei et al. (2007) further identified mixed avian the 8-year period, and accounted for only 7.2% of and mammalian blood in 8% of the blood meals the WNV positive pools identified in 2004 when from Cx. p. quinquefasciatus reaffirming its the greatest number of human cases (n 5 168) potential role as a likely bridge vector. were recorded. Culex p. quinquefasciatus also appears to be the Other species of Culex found naturally infected predominant vector in urban habitats in Denton with WNV in southern California include Cx. and surrounding counties in the Dallas-Ft. Worth erythrothorax, a widely distributed species that metropolitan area located in north central Texas develops in permanent and semipermanent (Bolling et al. 2005), while in more rural Lubbock marshes supporting dense tule and cattail stands County in northwestern Texas, Cx. tarsalis (Reisen and Reeves 1990). Culex erythrothorax is constitutes the great majority of WNV positive a competent vector for WNV (Goddard et al. mosquitoes (Bradford et al. 2005). 2002) and opportunistic feeder (Reisen and Studies in the Rio Grande Valley of New Reeves 1990, Molaei et al. 2010) that may serve Mexico, which include the Albuquerque metro- as an occasional bridge vector to humans. politan area, provide evidence to suggest separate However, this mosquito feeds rather infrequently enzootic and epidemic cycles of WNV transmis- on competent avian hosts (Molaei et al. 2010) and sion that involve different species of Culex exhibits substantially lower WNV infection rates mosquitoes. Culex tarsalis appears to be involved in comparison to all other Culex vectors (Kwan in early season amplification of WNV in wild et al. 2010, Molaei et al. 2010) indicating that it is avian hosts, especially in rural areas of the Valley, not likely to be a significant vector of WNV in whereas Cx. salinarius and Cx. p. quinquefasciatus this region. are the two species most likely involved in epidemic transmission to humans in more urban locales. According to this scenario, the virus Southwestern United States builds through multiple amplification cycles Within the southwestern United States, WNV involving avian hosts and Cx. tarsalis and has been detected in 10 different species of Culex eventually spreads throughout the metropolitan mosquitoes (Table 1), but the overwhelming area to populations of Cx. salinarius and Cx. p. majority of virus positive pools reported to quinquefasiatus, which are locally abundant and CDC ArboNet have been from Cx. p. quinque- exhibit comparable WNV infection rates (DiMenna fasciatus (82.1% of all Culex) (Fig. 1). In Harris et al. 2006, 2007). County, Texas, which includes the Houston In more semiarid areas of Don˜a Ana County, metropolitan area where WNV was first detected New Mexico, Cx. tarsalis is reported to be the in June 2002, Cx. p. quinquefasciatus is the primary vector of WNV based on the frequency dominant Culex species, and based on its and preponderance of WNV-positive pools iden- abundance, feeding habits, and high WNV tified from this species (Pitzer et al. 2009). infection rate, is considered the principal vector However, because it is most abundant in sparsely of WNV in the area (Lillibridge et al. 2004, populated riparian and agricultural areas, its Dennett et al. 2007, Molaei et al. 2007). During involvement in transmission to humans appears the 2002 epidemic, when 105 human cases were minimal. Culex p. quinquefasciatus, on the other reported throughout the metropolitan area, it was hand, is reportedly a secondary vector, based on the only species to test positive for the virus, with the detection of substantially fewer WNV-posi- nearly 14% of 69,490 pools WNV-positive and a tive pools from this species, but may be respon- minimum field infection rate of 3.3 per 1,000 sible for most WNV transmission in urbanized (Lillibridge et al. 2004). Surveillance activities areas where 82% of the positive pools for this conducted in the same area in subsequent years species were collected. CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 145

Table 3. Detection of West Nile virus in overwintering populations of Culex p. pipiens in the US. No. virus + pools Location Year No. mosquitoes Isolation PCR Reference New York 2000 2,360 1 2 Nasci et al. 2001a New Jersey 2001–03 1,324 - 1 Farajollahi et al. 2005 Pennsylvania 2003 501 - 1 Bugbee & Forte 2004 Colorado 2003–04 8,017 - - Bolling et al. 2007 New York 2006–09 3,240 1 - Andreadis et al. 2010

CONTRIBUTION OF CULEX PIPIENS micropyle at the time of fertilization rather than COMPLEX MOSQUITOES TO OVERWIN- infecting developing eggs in the ovary, a more TERING AND PERSISTENCE OF WEST efficient mechanism observed with bunyaviruses. NILE VIRUS Nevertheless, WNV has also been isolated from field-collected males, nulliparous females, and The role that Cx. p. pipiens complex mosqui- adults reared from field-collected larvae and toes play in overwintering and long-term persis- WNV-infected females (Anderson and Main tence of WNV has been the subject of several 2006, Anderson et al. 2006, Reisen et al. 2006, investigations and has been recently reviewed McAbbe et al. 2008) reaffirming its occurrence in (Kramer and Ebel 2003, Reisen and Brault 2007, natural populations. Unequivocal evidence that Kramer et al. 2008). In cool temperate regions vertically infected female Cx. p. pipiens that enter where transmission ceases during the winter diapause in the fall are able to initiate infection the months, WNV has been detected in hibernating following spring comes from an investigation by Cx. p. pipiens on several occasions (Nasci et al. Anderson and Main (2006), who documented 2001a, Bugbee and Forte 2004, Farajollahi et al. horizontal transmission of WNV by a vertically 2005, Andreadis et al. 2010), and this species is infected female that had been in diapause for more thought to serve as a natural overwintering host than 5K months. Based on an estimated infection responsible for amplifying transmission of the rate of ,0.05 infected females/1000, these authors virus in the spring. This view is consisitent with concluded that in temperate climates, transge- epidemiological data documenting the annual nerational transmission of WNV by Cx. p. pipiens reemergence of WNV from the same geographic is an important means of enabling the virus to locales (Andreadis et al. 2004; Reisen et al. 2006, persist during the winter and amplify in the spring. 2008b, 2009; Bolling et al. 2007), and molecular An alternative mechanism wherein older pre- evidence of year-to-year persistence of similar hibernating females that had previously acquired viral subclades from foci in the northeastern an infectious blood meal enter hibernacula in the (Armstrong et al. 2011) and midwestern United fall and survive the winter to initiate infection in States (Amore et al. 2010). However, the preva- the spring, albeit rare, remains plausible. Parous lence of viral infection in the overwintering female Cx. p. pipiens from the northeastern population of Cx. p. pipiens appears to be quite United States are known to enter hibernacula in low (Table 3), and the manner in which pre- the fall, and despite significant mortality during hibernating females become infected with WNV the winter months, some individuals survive to in the fall before entering hibernacula is not emerge in the spring (Jumars et al. 1969, entirely clear. Andreadis et al. 2010). However, it is unknown It is widely acknowledged that above ground whether the parous state of these females is due to populations of Culex p. pipiens overwinter in blood feeding and oviposition prior to entering natural and man-made shelters as non-blood fed, the hibernaculum or autogenous egg production. nulliparous, inseminated females (Service 1969, It is generally presumed that diapausing popula- Hayes 1973, Slaff and Crans 1977, Sulaiman and tions of Cx. p. pipiens from northern latitudes are Service 1983, Jaenson 1987, Onyeka and Boreham anautogenous and must acquire a blood meal to 1987, Vinogradova 2000). Since the majority of produce eggs. Autogenous populations have been females that enter diapause do not blood feed identified in North America (Richards 1941, (Eldridge 1987, Mitchell 1988), infection of these Wray 1946, Rozeboom 1951, Spielman 1964, females must occur through vertical transmission 1971, Kent et al. 2007, Huang et al. 2008, Mutebi of the virus. Vertical transmission of WNV by Cx. and Savage 2009) but only among non-diapaus- p. pipiens has been demonstrated in the laboratory ing Cx. p. pipiens form molestus that are confined but appears to be relatively inefficient (Dohm et to enclosed spaces in urban subterranean habitats al. 2002, Goddard et al. 2003, Anderson et al. such as sewer systems and flooded basements. 2008). According to Rosen (1987), flaviviruses Populations of these two physiological biotypes seem to enter the fully formed egg through the are for the most part reproductively isolated due 146 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4 to differences in their breeding sites (Rozeboom detected in Cx. p. quinquefasciatus mosquitoes and Gilford 1954, Spielman 1964, 2001) and collected as larvae from southern Louisiana based on comparative microsatellite analyses are (Unlu et al. 2010). genetically distinct entities (Kent et al. 2007, It is also noteworthy that WNV-positive pools Huang et al. 2008). However, evidence of have been identified from winter-resting and early molestus genetic ancestry among a small portion season Cx. erraticus females in Alabama, well of the aboveground population (Fonseca et al. before significant numbers of this species became 2004;Kent et al. 2007; Kilpatrick et al. 2007; active, adding further evidence that overwintering Huang et al. 2008, 2009), and the documentation mosquitoes in this region maintain virus between of occasional episodes of interbreeding where transmission seasons (Cupp et al. 2007). WNV sympatric populations coexist in the northeastern has also been detected in overwintering larvae of United States (Spielman 1971, 2001), suggest Cx. erythrothorax collected in late October from some level of gene flow and possible hybridiza- Utah demonstrating vertical transmission in this tion between the two biotypes (Kent et al. 2007). mosquito species, and suggesting that vertical The degree to which hybridization occurs where transmission may similarly contribute to WNV populations of these two biotypes are sympatric overwintering in this region (Philips and Christensen in nature and whether specific genes for autogeny 2006). are expressed in above ground populations are intriguing questions that remain to be explored. ACKNOWLEDGMENTS Autogeny has been reported in above ground populations of Cx. p. pipiens from southern I wish to sincerely thank Jennifer Lehman, Europe (Gomes et al. 2009) and the Middle East Division of Vector-Borne Diseases, National (Nudelman et al. 1988) but not from North Center for Infectious Diseases, Centers for America. The role of Cx. p. pipiens form molestus Disease Control and Prevention, Fort Collins, in seasonal transmission and persistence of WNV CO, for providing access to the national ArboNet in North America is entirely unknown. It has also data base. I also thank Philip Armstrong, Louis been suggested that Cx. salinarius may have a role Magnarelli and Goudarz Molaei for their helpful in maintaining WNV in the northeastern United comments on the manuscript. States due to its ability to vertically and horizontally transmit WNV similarly to Cx. p. pipiens (Anderson et al. 2012). REFERENCES CITED In southern regions of the United States where Amore G, Bertolotti L, Hamer GL, Kitron UD, Walker low levels of WNV activity in birds and ED, Ruiz MO, Brawn JD, Goldberg TL. 2010. mosquitoes have been detected during the winter Multi-year evolutionary dynamics of West Nile virus months (Tesh et al. 2004, Reisen et al. 2006), it in suburban Chicago, USA, 2005–2007. Philos has been suggested that the virus may persist Trans R Soc Lond B Biol Sci 365:1871–1878. through continued transmission involving Cx. p. Anderson JF, Andreadis TG, Main AJ, Ferrandino FJ, quinquefasciatus (Reisen and Brault 2007, Richards Vossbrinck CR. 2006. West Nile virus from female et al. 2007). Unlike Cx. p. pipiens, Cx. p. and male mosquitoes (Diptera: Culicidae) in subterranean, ground, and canopy habitats in Connecticut. quinquefasciatus does not enter a true diapause J Med Entomol 43:1010–1019. but rather overwinters in quiescence (Reisen et al. Anderson JF, Andreadis TG, Main MJ, Kline DL. 1986). Adults become inactive during cold 2004. Prevalence of West Nile virus in tree canopy- periods, resting under buildings and in storm inhabiting Culex pipiens and associated mosquitoes. drains and sewers (Tesh et al. 2004), but during Am J Trop Med Hyg 71:112–119. warm periods becomes active again and renew Anderson JF, Andreadis TG, Vossbrinck CR, Tirrell S, blood feeding. According to Tesh et al. (2004), Wakem EM, French RA, Garmendia AE, Van this intermittent host-seeking activity throughout Kruiningen HJ. 1999. Isolation of West Nile virus the winter likely accounts for continued low-level from mosquitoes, crows, and a Cooper’s hawk in Connecticut. Science 286:2331–2333. WNV transmission among resident avian popu- Anderson JF, Main AJ. 2006. Importance of vertical lations in the western Gulf region of Louisiana and horizontal transmission of West Nile virus by and Texas and is the principal mechanism by Culex pipiens in the northeastern United States. which the virus overwinters. Continued WNV J Infect Dis 194:1577–1579. transmission was similarly detected in southern Anderson JF, Main AJ, Cheng G, Ferrandino FJ, California by the recovery of viral RNA from Fikrig E. 2012. Horizontal and vertical transmission dead American crows. This observation in of West Nile virus genotype NY99 by Culex salinarius concert with the isolation of WNV from male and genotypes NY99 and WN02 by Culex tarsalis. Am J Trop Med Hyg 86:134–139. and adults emerging from field-collected imma- Anderson JF, Main AJ, Delroux K, Fikrig E. 2008. ture Cx. p. quinquefasciatus, indicated that Extrinsic incubation periods for horizontal and vertical transmission may be possible during mild vertical transmission of West Nile virus by Culex winter conditions in southern California (Reisen pipiens pipiens (Diptera : Culicidae). J Med Entomol et al. 2006). West Nile virus has also been 45:445–451. CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 147

Andreadis TG, Armstrong PM. 2007. A two-year front range during a period of intense West Nile virus evaluation of elevated canopy trapping for Culex activity. J Am Mosq Control Assoc 23:37–46. mosquitoes and West Nile virus in an operational Bowden SE, Magori K, Drake JM. 2011. Regional surveillance program in the northeastern United differences in the association between land cover and States. J Am Mosq Control Assoc 23:137–148. West Nile virus disease incidence in humans in the Andreadis TG, Armstrong PA, Bajwa WJ. 2010. United States. Am J Trop Med Hyg 84:234–238. Studies on hibernating populations of Culex pipiens Bradford CM, Nascarella MA, Burns TH, Montford (Diptera: Culicidae) from a West Nile virus endemic JR, Marsland EJ, Pepper CB, Presley SM. 2005. First focus in New York City: parity rates and isolation of report of West Nile virus in mosquitoes from West Nile virus. J Am Mosq Control Assoc Lubbock County, Texas. J Am Mosq Control Assoc 26:257–264. 21:102–105. Andreadis TG, Anderson JF, Vossbrinck CR. 2001. Brault AC, Huanh CYH, Langevin SA, Kinney RM, Mosquito surveillance for West Nile virus in Con- Bowen RA, Ramey WA, Panella NA, Holmes EA, necticut, 2000: Isolation from Culex pipiens, Cx. Powers AM, Miller BR. 2007. A single positively restuans, Cx. salinarius, and Culiseta melanura. selected West Nile viral mutation confers increased Emerg Infect Dis 7:670–674. virogenesis in American crows. Nature 39:1162–1166. Andreadis TG, Anderson JF, Vossbrinck CR, Main AJ. Brault AC, Langevin SA, Bowen RA, Panella NA, 2004. Epidemiology of West Nile virus in Connecti- Biggerstaff BJ, Miller BR, Komar N. 2004. Differ- cut: a five-year analysis of mosquito data 1999–2003. ential virulence of West Nile virus strains for Vector-Borne Zoonotic Dis 4:360–378. American crows. Emerg Infect Dis 10:2161–2168. Apperson CS, Harrison BA, Unnasch TR, Hassan HK, Bugbee LM, Forte LR. 2004. The discovery of West Irby WS, Savage HM, Aspen SE, Watson DW, Nile virus in overwintering Culex pipiens (Diptera: Rueda LM, Engber BR, Nasci RS. 2002. Host- Culicidae) mosquitoes in Lehigh County, Pennsylva- feeding habits of Culex and other mosquitoes nia. J Am Mosq Control Assoc 20:326–327. (Diptera: Culicidae) in the borough of Queens in CDC [Centers for Disease Control and Prevention]. New York City, with characters and techniques for 2010a. Surveillance for human West Nile virus the identification of Culex mosquitoes. J Med En- disease – United States, 1999–2008. Morb Mortal tomol 39:777–785. Weekly Rep 59, SS-2:1–18. Apperson CS, Hassan HK, Harrison BA, Savage HM, CDC[Centers for Disease Control and Prevention]. Aspen SE, Farajollahi A, Crans W, Daniels TJ, Falco 2010b. West Nile virus activity – United States, RC, Benedict M, Anderson M, McMillen L, Un- 2009. Morb Mortal Weekly Rep 59:769–772. nasch TR. 2004. Host feeding patterns of established CDC [Centers for Disease Control and Prevention]. and potential mosquito vectors of West Nile virus in 2011. West Nile virus disease and other arboviral the eastern United States. Vector-Borne Zoonotic Dis Morb Mortal Weekly 4:71–82. diseases – United States, 2010. Rep 60:1009–1013. Armstrong PM, Vossbrinck CR, Andreadis TG, Anderson JF, Pesko KN, Newman RM, Lennon Crans WJ. 1964. Continued host preference studies with NJ, Birren BW, Ebel GD, Henn MR. 2011. New Jersey mosquitoes, 1963. Proc 51st Ann Meet NJ Molecular Evolution of West Nile virus in a northern Mosq Exterm Assoc, 50–58. temperate region: Connecticut, USA 1999–2008. Cupp EW, Hassan HK, Yue X, Oldland WK, Lilley Virology 417:203–210. BM, Unnasch TR. 2007. West Nile virus infection in Bell JA, Mickelson NJ, Vaughan JA. 2005. West Nile mosquitoes in the mid-south USA. J Med Entomol virus in host-seeking mosquitoes within a residential 44:117–125. neighborhood in Grand Forks, North Dakota. Davis CT, Ebel GD, Lanciotti RS, Brault AC, Guzman Vector-Borne Zoonotic Dis 4:373–382. H, Siirin M, Lambert A, Parsons RE, Beasley DWC, Bernard KA, Maffei JG, Jones SA, Kauffman EB, Ebel Novak RJ, Elizondo-Quiroga D, Green EN, Young GD, Dupuis IIAP, Ngo KA, Nicholas DC, Young DS, Stark LM, Drebot MA, Artsob H, Tesh RB, DM, Shi PY, Kulasekera VL, Eidson M, White DJ, Kramer LD, Barrett ADT. 2005. Phylogenetic Stone WB, NY State Surveillance Team, Kramer LD. analysis of North American West Nile virus isolates, 2001. West Nile virus infection in birds and 2001–2004: evidence for the emergence of a dominant mosquitoes, New York State, 2000. Emerg Infect genotype. Virology 342:252–265. Dis 7:679–685. Dennett JA, Bala A, Wuithiranyagool T, Randle Y, Blackmore CGM, Stark LM, Jeter WC, Oliveri RL, Sargent C, Guzman H, Siirin M, Hassan HK, Reya- Brooks RG, Conti LA, Wiersma ST. 2003. Surveil- Nava M, Unnasch TR, Tesh R, Parsons RE, Bueno lance results from the first West Nile virus transmis- R Jr. 2007. Associations between two mosquito sion season in Florida, 2001. Am J Trop Med Hyg populations and West Nile virus in Harris County, 69:141–150. Texas, 2003–06. J Am Mosq Control Assoc 23: Bolling BG, Barker CM, Moore CG, Pape WJ, Eisen L. 264–275. 2009. Seasonal patterns for entomological risk for DiMenna MA, Bueno R Jr, Parmenter RR, Norris DE, exposure to Culex vectors and West Nile virus in Sheyka JM, Molina JL, LaBeau EM, Hatton ES, relation to human disease cases in northeastern Glass GE. 2006. Emergence of West Nile virus in Colorado. J Med Entomol 46:1519–1531. mosquito (Diptera:Culicidae) communities of the Bolling BG, Kennedy JH, Zimmerman EG. 2005. New Mexico Rio Grande Valley. J Med Entomol Seasonal dynamics of four potential West Nile vector 43:594–599. species in north-central Texas. JVectorEcol DiMenna MA, Bueno R Jr, Parmenter RR, Norris DE, 30:186–194. Sheyka JM, Molina JL, LaBeau EM, Hatton ES, Bolling BG, Moore CG, Anderson SL, Blair CD, Beaty Roberts CM, Glass GE. 2007. Urban habitat for BJ. 2007. Entomological studies along the Colorado West Nile virus surveillance in mosquitoes in 148 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4

Albuquerque, New Mexico. J Am Mosq Control Goddard LB, Roth AE, Reisen WK, Scott TW. 2002. Assoc 23:153–160. Vector competence of California mosquitoes for Diuk-Wasser MA, Molaei G, Simpson JE, Folsom- West Nile virus. Emerg Infect Dis 8:1385–1391. O’Keefe CM, Armstrong PM, Andreadis TG. 2010. Goddard LB, Roth AE, Reisen WK, Scott TW. 2003. Avian communal roosts as amplification foci for Vertical transmission of West Nile virus by three West Nile virus in urban areas in northeastern United California Culex (Diptera: Culicidae) species. J Med States. Am J Trop Med Hyg 82:337–343. Entomol 40:743–746. Dohm DJ, Sardelis MR, Turell MJ. 2002. Experimental Godsey MS Jr, Blackmore MS, Panella NA, Burkhalter vertical transmission of West Nile virus by Culex K, Gottfried K, Halsey LA, Rutledge R, Langevin pipiens (Diptera: Culicidae). J Med Entomol 39: SA, Gates R, Lamonte KM, Lambert A, Lanciotti 640–644. RS, Blackmore CGM, Loyless T, Stark L, Oliveri R, Ebel GD, Rochlin I, Longacker J, Kramer LD. 2005. Conti L, Komar N. 2005a. West Nile virus epizoot- Culex restuans (Diptera: Culicidae) relative abun- iology in the Southeastern United States, 2001. dance and vector competence for West Nile virus. Vector-Borne Zoonotic Dis 5:82–89. J Med Entomol 42:838–843. Godsey MS Jr, Nasci R, Savage HM, Aspen S, King R, Edman JD. 1974. Host-feeding patterns of Florida Powers AM, Burkhalter K, Colton L, Charnetzky D, mosquitoes III. Culex (Culex)andCulex (Neoculex). Lasater S, Taylor V, Palmisano CT. 2005b. West Nile J Med Entomol 11:95–104. virus-infected mosquitoes, Louisiana, 2002. Emerg Edman JD, Taylor DJ. 1968. Culex nigripalpus: Infect Dis 11:1399–1404. seasonal shift in the bird-mammal feeding ratio in a Gomes B, Sousa CA, Novo MT, Freitas FB, Alves R, mosquito vector of human encephalitis. Science Corte-Real AR, Salgueiro P, Donnelly MJ, Almeida 161:67–68. APG, Pinto J. 2009. Asymmetric introgression Eldridge BF. 1987. Diapause and related phenomena in between sympatric molestus and pipiens forms of Culex mosquitoes: their relation to arbovirus disease Culex pipiens (Diptera: Culicidae) in Comporta ecology. In: Harris KF, ed. Current topics in vector region, Portugal. BMC Evol Biol 9:262. doi:10.1186/ research. Volume 4. New York: Springer-Verlag. 1471-2148-9-262. p 1–28. Gu W, Lampman R, Krasavin N, Berry R, Novak R. Elnaiem D-EA, Kelley K, Wright S, Laffey R, 2006. Spatio-temporal analyses of West Nile virus Yoshimura G, Reed M, Goodman G, Thiemann T, transmission in Culex mosquitoes in northern Illinois, Reimer L, Reisen WK, Brown D. 2008. Impact of USA, 2004. Vector-Borne Zoonotic Dis 6:91–98. aerial spraying of pyrethrin insecticide on Culex Gujral IB, Zielinski-Gutierrez EC, LeBailly A, Nasci R. pipiens and Culex tarsalis (Diptera: Culicidae) abun- 2007. Behavioral risks for West Nile virus disease, dance and West Nile virus infection rates in an urban/ northern Colorado, 2003. Emerg Infect Dis 13:419– suburban area of Sacramento County, California. 425. J Med Entomol 45:751–757. Hamer GL, Kitron UR, Brawn JD, Loss SR, Ruiz MO, Farajollahi A, Crans WJ, Bryant P, Wolf B, Burkhalter Goldberg TL, Walker ED. 2008a. Culex pipiens KR, Godsey MS, Aspen SE, Nasci RS. 2005. (Diptera: Culicidae): a bridge vector of West Nile Detection of West Nile viral RNA from an overwin- virus to humans. J Med Entomol 45:125–128. tering pool of Culex pipiens pipiens (Diptera: Hamer GL, Kitron UR, Goldberg TL, Brawn JD, Loss Culicidae) in New Jersey, 2003. J Med Entomol SR, Ruiz MO, Hayes DB, Walker ED. 2009. Host 42:490–494. selection by Culex pipiens mosquitoes and West Nile Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, virus amplification. Am J Trop Med Hyg 80:268–278. Schaffner F, Mogi M, Fleishcher R, Wilkerson RC. Hamer GL, Walker ED, Brawn JD, Loss SR, Ruiz MO, 2004. Emerging vectors in the Culex pipiens complex. Goldberg TL, Schotthoefer AM, Brown WM, Science 303:1535–1538. Wheller E, Kitron UR. 2008b. Rapid amplification Gibbs SEJ, Wimberly MC, Madden M, Masour J, of West Nile: the role of hatch-year birds. Vector- Yabsley MJ, Stallknecht DE. 2006. Factors affecting Borne Zoonotic Dis 8:57–67. the geographic distribution of West Nile virus in Harrison BA, Whitt PB, Roberts LF, Lehman JA, Georgia, USA: 2002–2004. Vector-Borne Zoonotic Lindsey NP, Nasci RS, Hansen GR. 2009. Rapid Dis 6:73–81. assessment of mosquitoes and arbovirus activity after Gingrich JB, Casillas L. 2004. Selected mosquito flood in southeastern Kansas, 2007. JAmMosq vectors of West Nile virus: comparison of their Control Assoc 25:265–271. ecological dynamics in four woodland and marsh Hassan HK, Cupp EW, Hill GE, Katholi CR, Klinger habitats. J Am Mosq Control Assoc 20:138–145. K, Unnasch TR. 2003. Avian host preference by Gingrich JB, Williams GM. 2005. Host-feeding patterns vectors of eastern equine encephalomyelitis virus. of suspected West Nile virus mosquito vectors in Am J Trop Med Hyg 69:641–647. Delaware, 2001–2002. J Am Mosq Control Assoc Hayes EB, Komar N, Nasci RS, Montgomery SP, 21:194–200. O’Leary DR, Campbell GL. 2005. Epidemiology and Gingrich JB, O’Conner LL, Meredith WH, Pesek JD, transmission of West Nile virus disease. Emerg Infect Shriver WG. 2010. Epidemiology of West Nile virus: Dis 11:1167–1173. a silent epiornitic in northern Delaware in 2007 Hayes J. 1973. Overwintering Culex pipiens pipiens in without associated human cases. J Am Mosq Control the Ohio-Mississippi River basin 1962–1967. Mosq Assoc 26:274–286. News 33:424–428. Gleiser RM, Mackay AJ, Roy A, Yates MM, Vaeth Hayes RO. 1961. Host preferences of Culiseta melanura RH, Faget GM, Folsom AE, Augustine WF Jr, Wells and allied mosquitoes. Mosq News, 179–187. RA, Perich MJ. 2007. West Nile virus surveillance in Hayes RO, Tempelis CH, Hess AD, Reeves WC. 1973. East Baton Rouge Parish, Louisiana. JAmMosq Mosquito host preference studies in Hale County, Control Assoc 26:29–36. Texas. Am J Trop Med Hyg 22:270–277. CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 149

Hribar LJ, Stark LM, Stoner RL, Demay DJ, Nordholt Lillibridge KM, Parsons RE, Randle Y, Travassos Da AL, Hemmen MJ, Vlach JJ, Fussell EM. 2004. Rosa APA, Guzman H, Siirin M, Wuithiranyagool Isolation of West Nile virus from mosquitoes T, Hailey C, Higgs S, Bala A, Pascua R, Meyer T, (Diptera: Culicidae) in the Florida Keys, Monroe Vanlanding DL, Tesh R. 2004. The 2002 introduction County, Florida. Caribb J Sci 40:362–367. of West Nile virus into Harris County, Texas, an area Huang S, Hamer GL, Molaei G, Walker ED, Goldberg historically endemic for St. Louis encephalitis. TL, Kitron UD, Andreadis TG. 2009. Genetic Am J Trop Med Hyg 70:676–681. variation associated with mammalian feeding in Lindsey NP, Kuhn S, Campbell GL, Hayes RO. 2008. Culex pipiens from a West Nile virus epidemic region West Nile virus neuroinvasive disease incidence in the in Chicago, Illinois. Vector-Borne Zoonotic Dis United States, 2002–2006. Vector-Borne Zoonotic Dis 9:637–642. 8:35–39. Huang S, Molaei G, Andreadis TG. 2008. Genetic Lothrop HD, Lothrop BB, Gomsi DE, Reisen WK. insights into the population structure of Culex pipiens 2008. Intensive early season adulticide applications (Diptera: Culicidae) in the northeastern United States decrease arbovirus transmission throughout the by using microsatellite analysis. Am J Trop Med Hyg Coachella Valley, Riverside County, California. 79:518–527. Vector-Borne Zoonotic Dis 8:475–489. Jaenson TGT. 1987. Overwintering of Culex mosqui- Lukacik G, Anand M, Shusas EJ, Howard JJ, Oliver J, toes in Sweden and their potential as reservoirs of Chen H, Backenson PB, Kauffman EB, Bernard KA, human pathogens. Med Vet Entomol 1:151–156. Kramer LD, White DJ. 2006. West Nile virus Jumars PA, Murphey FJ, Lake RW. 1969. Can blood- surveillance in mosquitoes in New York State, fed Culex pipiens L. overwinter? Proc N J Mosq 2000–2004. J Am Mosq Control Assoc 22:264–271. Exterm Assoc 56:219–225. Mackay AJ, Kramer WL, Meece JK, Brumfield RT, Kent RJ, Harrington LC, Norris DE. 2007. Genetic Foil LD. 2010. Host feeding patterns of Culex differences between Culex pipiens f. molestus and mosquitoes (Diptera: Culicidae) in East Baton Rouge Culex pipiens pipiens (Diptera: Culicidae) in New Parish, Louisiana. J Med Entomol 47:238–248. York. J Med Entomol 44:50–59. Mackay AJ, Roy A, Yates MM, Foil LD. 2008. West Kent R, Juliusson L, Weissmann M, Evans S, Komar Nile virus detection in mosquitoes in East Baton N. 2009. Seasonal blood-feeding behavior of Culex Rouge Parish, Louisiana, from November 2002 to tarsalis (Diptera: Culicidae) in Weld County, Colo- October 2004. J Am Mosq Control Assoc 24:28–35. rado, 2007. J Med Entomol 46:380–390. Magnarelli LA. 1977. Host feeding patterns of Con- Kilpatrick AM, Kramer LD, Campbell SR, Alleyne necticut mosquitoes. Am J Trop Med Hyg 26:547–52. EO, Dobson AP, Daszak P. 2005. West Nile virus McAbee RD, Green EN, Holeman J, Christiansen J, risk assessment and the bridge vector paradigm. Frye N, Dealey K, Mulligan IIIFS, Brault AC, Emerg Infect Dis 11:425–429. Cornel AJ. 2008. Identification of Culex pipiens Kilpatrick AM, Kramer LD, Jones MJ, Marra P, complex mosquitoes in a hybrid zone of West Nile Daszak P. 2006. West Nile virus epidemics in North virus transmission in Fresno, California. Am J Trop America are driven by shifts in mosquito feeding Med Hyg 78:303–310. behavior. PLOS Biol 4:606–610. Kilpatrick AM, Kramer LD, Jones MJ, Marra PP, Means RG. 1968. Host preferences of mosquitoes Daszak P, Fonseca DM. 2007. Genetic influences on (Diptera: Culicidae) in Suffolk County, New York. mosquito feeding behavior and the emergence of Ann Entomol Soc Am 61:116–120. zoonotic pathogens. Am J Trop Med Hyg Means RG. 1987. Mosquitoes of New York. Part II. 77:667–671. Genera of Culicidae other than Aedes occurring in Komar N. 2003. West Nile virus: epidemiology and New York. NY State Mus Bull 430b:1–180. ecology in North America. Adv Virus Res 61:185–234. Mitchell CJ. 1988. Occurrence, biology, and physiology Kramer LD, Ebel GD. 2003. Dynamics of flavivirus of diapause in overwintering mosquitoes. In: Monath infection in mosquitoes. Adv Virus Res 60:187–232. TP, ed. The arboviruses: epidemiology and ecology. Kramer LD, Styer LM, Ebel GD. 2008. A global Volume 1. Boca Raton, Florida: CRC Press, Inc. perspective on the epidemiology of West Nile virus. p 191–192. Annu Rev Entomol 53:61–81. Molaei G, Andreadis TG, Armstrong PM, Anderson Kulasekera VL, Kramer L, Nasci RS, Mostashari F, JF, Vossbrinck CR. 2006. Host feeding patterns of Cherry B, Trock SC, Glaser C. 2001. West Nile virus Culex mosquitoes and West Nile virus transmission, infection in mosquitoes, birds, horses and humans, northeastern United States. Emerg Infect Dis Staten Island, New York, 2000. Emerg Infect Dis 12:468–474. 2001; 7:722–725. Molaei G, Andreadis TG, Armstrong PM, Bueno R Jr, Kwan JL, Kluh S, Madon MB, Reisen WK. 2010. West Dennett JA, Real SV, Sargent C, Bala A, Randle Y, Nile virus emergence and persistence in Los Angeles, Guzman H, Travassos da Rosa A, Wuithiranyagool California, 2003–2008. Am J Trop Med Hyg T, Tesh RB. 2007. Host feeding pattern of Culex 83:400–412. quinquefasciatus (Diptera:Culicidae) and its role in Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker transmission of West Nile virus in Harris County, M, Steele K, Crise B, Volpe KE, Crabtree MB, Texas. Am J Trop Med Hyg 77:73–81. Scherret JH, Hall RA, MacKenzie JS, Cropp CB, Molaei G, Cummings RF, Su T, Armstrong PM, Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Williams GA, Cheng ML, Webb JP, Andreadis TG. Banet C, Weissman J, Komar N, Savage HM, Stone 2010. Vector-host interactions governing epidemiol- W, McNamara T, Gubler DJ. 1999. Origin of the ogy of West Nile virus in southern California. West Nile virus responsible for an outbreak of Am J Trop Med Hyg 83:1269–1282. encephalitis in the Northeastern United States. Montgomery MJ, Thiemann T, Macedo P, Brown DA, Science 286:2333–2337. Scott TW. 2011. Blood-feeding patterns of the Culex 150 CULEX PIPIENS COMPLEX SYMPOSIUM VOL.28,SUPPLEMENT TO NO.4

pipiens complex in Sacramento and Yolo Counties, Reisen WK, Fang Y, Martinez VM. 2005. Avian host California. J Med Entomol 48:398–404. and mosquito (Diptera: Culicidae) vector competence Moudy RM, Meola MA, Morin LL, Ebel GD, Kramer determine the efficiency of West Nile virus and St. LD. 2007. A newly emergent genotype of West Nile Louis encephalitis virus transmission. J Med Entomol virus is transmitted earlier and more efficiently by 42:367–375. Culex mosquitoes. Am J Trop Med Hyg 77:365–370. Reisen WK, Lothrop H, Chiles R, Madon M, Cossen Murphey FJ, Burbutis PP, Bray DF. 1967. Bionomics C, Woods L, Husted S, Kramer V, Edman J. 2004. of Culex salinarius Coquillett. II. Host acceptance West Nile virus in California. Emerg Infect Dis and feeding by adult females of C. salinarius and 10:1369–1378. other mosquito species. Mosq News 27:366–374. Reisen WK, Lothrop HD, Wheeler SS, Kennsington M, Mutebi JP, Savage HM. 2009. Discovery of Culex Gutierrez A, Fang Y, Garcia S, Lothrop B. 2008b. pipiens pipiens form molestus in Chicago. JAmMosq Persistent West Nile virus transmission and the Control Assoc 25:500–503. apparent displacement of St. Louis encephalitis virus Nasci RS, Savage HM, White DJ, Miller RJ, Cropp in southeastern California, 2003–2006. J Med En- CB, Godsey MS, Kerst AJ, Bennett P, Gottfried K, tomol 45:494–508. Lanciotti RS. 2001a. West Nile virus in overwintering Reisen WK, Meyer RP, Milby MM. 1986. Overwinter- Culex mosquitoes, New York City, 2000. Emerg ing studies on Culex tarsalis (Diptera: Culicidae) in Infect Dis 7:742–744. Kern County, California: temporal changes in Nasci RS, White DJ, Stirling H, Oliver J, Daniels TJ, abundance and reproductive status with comparative Falco RC, Campbell S, Crans WJ, Savage HM, observations on C. quinquefasciatus (Diptera: Culic- Lanciotti RS, Moore CG, Godsey MS, Gottfried K, idae). Ann Entomol Soc Am 79:677–685. Mitchell CJ. 2001b. West Nile virus isolates from Reisen WK, Meyer RP, Tempelis CH, Spoehel JJ. 1990. mosquitoes in New York and New Jersey, 1999. Mosquito abundance and bionomics in residential Emerg Infect Dis 7:626–630. communities in Orange and Los Angeles Counties, Niebylski ML, Meek CL. 1992. Blood-feeding of Culex California. J Med Entomol 27:356–367. mosquitoes in an urban environment. JAmMosq Reisen WK, Reeves WC. 1990. Bionomics and ecology Control Assoc 8:173–177. of Culex tarsalis and other potential mosquito vector Nudelman S, Galun R, Kitron U, Spielman A. 1988. species. In: WC. Reeves., ed. Epidemiology and Physiological characteristics of Culex pipiens popula- Control of Mosquito-borne Arboviruses in California, tions in the Middle East. Med Vet Entomol 1943–1987. Sacramento, CA: California Mosquito 2:161–169. and Vector Control Association, Inc. p 254–329. Onyeka JOA, Boreham PFL. 1987. Population studies, Richards AG. 1941. A stenogamic autogenous strain of physiological state and mortality factors of overwin- Culex pipiens L. in North America (Diptera: Culic- tering adult populations of females of Culex pipiens idae). Entomol News 52:211–216. L. (Diptera: Culicidae). Bull Entomol Res 77:99–112. Richards SL, Mores CN, Lord CC, Tabachnick WJ. Palmisano CT, Taylor V, Caillouet K, Byrd B, Wesson 2007. Impact of extrinsic incubation temperature and DM. 2005. Impact of West Nile virus-outbreak upon virus exposure on vector competence of Culex pipiens St. Tammany Parish mosquito abatement district. quinquefaciatus Say (Diptera: Culicidae) for West J Am Mosq Control Assoc 21:33–38. Nile virus. Vector-Borne Zoonotic Dis 7:629–636. Peterson AT, Vieglais DA, Andreasen JK. 2003. Rochlin I, Dempsey ME, Campbell SR, Ninivaggi DV. Migratory birds modeled as critical transport agents 2008. Salt marsh as Culex salinarius larval habitat in for West Nile virus in North America. Vector-Borne coastal New York. J Am Mosq Control Assoc 24:359– Zoonotic Dis 3:27–37. 367. Phillips RA, Christensen K. 2006. Field-caught Culex Rosen L. 1987. Overwintering mechanisms of mosqui- erythrothorax larvae found naturally infected with to-borne arboviruses in temperate climates. Am J Trop West Nile virus in Grand County, Utah. JAmMosq Med Hyg 37:69S–76S. Control Assoc 22:561–562. Rozeboom LE. 1951. The Culex pipiens complex in Pitzer JB, Byford RL, Vuong HB, Steiner RL, Creamer North America. Trans R Entomol Soc Lond 102:343– RJ, Caccamise DF. 2009. Potential vectors of West 353. Nile virus in a semiarid environment: Don˜a Ana Rozeboom LE, Gilford BN. 1954. Sexual isolation County, New Mexico. J Med Entomol 46:1474–1482. between populations of the Culex pipiens complex in Reisen WK, Barker CM, Fang Y, Martinez VM. 2008a. North America. J Parasitol 3:237–244. Does variation in Culex (Diptera: Culicidae) vector Rutledge CR, Day JF, Lord CC, Stark LM, Tabach- competence enable outbreaks of West Nile virus in nick WJ. 2003. West Nile virus infection rates in California? J Med Entomol 45:1126–1138. Culex nigripalpus (Diptera: Culicidae) do not reflect Reisen WK, Brault AC. 2007. West Nile virus in North transmission rates in Florida. J Med Entomol America: perspectives on epidemiology and interven- 40:253–258. tion. Pest Manag Sci 63:641–646. Sardelis MR, Turell MJ, Dohm DJ, O’Guinn ML. Reisen WK, Carrol BD, Takahashi R, Fang Y, Garcia 2001. Vector competence of selected North American S, Martinez VM, Quiring R. 2009. Repeated West Culex and Coquillettidia mosquitoes for West Nile Nile virus epidemic transmission in Kern County, virus. Emerg Infect Dis 7:1018–1022. California, 2004–2007. J Med Entomol 46:139–157. Savage HM, Aggarwal D, Apperson CS, Katholi CR, Reisen WK, Fang Y, Lothrop HD, Martinez VM, Gorden E, Hassan HK, Anderson M, Charnetzky D, Wilson J, O’Conner P, Carney R, Cahoon-Young B, McMillen L, Unnasch EA, Unnasch TR. 2007. Host Shafii M, Brault AC. 2006. Overwintering of West choice and West Nile virus infection rates in blood- Nile virus in southern California. J Med Entomol fed mosquitoes, including members of the Culex 43:344–355. pipiens complex, from Memphis and Shelby County, CULEX PIPIENS MOSQUITOES AND WEST NILE VIRUS 151

Tennessee, 2002–2003. Vector-Borne Zoonotic Dis region, Texas and Louisiana. Emerg Infect Dis 7:365–386. 10:1649–1652. Savage HM, Anderson M, Gorden E, McMillen L, Thiemann TC, Wheeler SS, Barker CM, Reisen WK. Colton L, Charnetzky D, Delorey M, Aspen S, 2011. Mosquito host selection varies seasonally with Burkhalter K, Biggerstaff BJ, Godsey M. 2006. host availability and mosquito density. PLos Negl Oviposition activity patterns and West Nile virus Trop Dis 5:e1452. doi:10.1371/journal.pntd.0001452. infection rates for members of the Culex pipiens Turell MJ, Dohm DJ, Sardelis R, O’Guinn ML, complex at different habitat types within the hybrid Andreadis TG, Blow JA. 2005. An update on the zone, Shelby County, TN, 2002 (Diptera:Culicidae). potential of North American mosquitoes (Diptera: J Med Entomol 43:1227–1238. Culicidae) to transmit West Nile virus. JMed Savage HM, Anderson M, Gorden E, McMillen L, Entomol 42:57–62. Colton L, Delorey M, Sutherland G, Aspen S, Turell M, O’Guinn M, Dohm DJ, Jones JW. 2001. Charnetzky D, Burkhalter K, Godsey M. 2008. Vector competence of North American mosquitoes Host-seeking heights, host-seeking activity patterns, (Diptera:Culicidae) for West Nile virus. JMed and West Nile virus infection rates for members of Entomol 38:130–134. the Culex pipiens complex at different habitat types Turell M, O’Guinn M, Dohm DJ, Webb JP Jr, Sardelis within the hybrid zone, Shelby County, TN, 2002 MR. 2002. Vector competence of Culex tarsalis from (Diptera:Culicidae). J Med Entomol 45:276–288. Orange County, California for West Nile virus. Service MW. 1969. Observations on the ecology of some Vector-Borne Zoonotic Dis 2:193–196. British mosquitoes. Bul. Entomol Res 59:161–194. Turell M, O’Guinn M, Oliver J. 2000. Potential for New Shaman J, Day JF, Stieglitz M. 2005. Drought-induced York mosquitoes to transmit West Nile virus. amplification and epidemic transmission of West Nile Am J Trop Med Hyg 62:413–414. virus in southern Florida. J Med Entomol 42:134– Unlu I, Mackay AJ, Roy A, Yates MM, Foil LD. 2010. 141. Evidence of vertical transmission of West Nile virus Slaff ME, Crans WJ. 1977. Parous rates of overwinter- in field-collected mosquitoes. J Vector Ecol 35:95–99. ing Culex pipiens pipiens in New Jersey. Mosq News 37:11–14. Venkatesan M, Rasgon JL. 2010. Population genetic Spielman A. 1964. Studies on autogeny in Culex pipiens data suggest a role for mosquito-mediated dispersal populations in nature. I. Reproductive isolation of West Nile virus across the western United States. between autogenous and anautogenous populations. Mol Ecol 19:1573–1584. Am J Hyg 80:175–183. Vinogradova EB. 2000. Culex pipiens pipiens mosquitoes: Spielman A. 1971. Studies on autogeny in natural taxonomy, distribution, ecology, physiology, genetics, populations of Culex pipiens II. Seasonal abundance applied importance and control. Sofia, Moscow: of autogenous and anautogenous populations. JMed Pensoft. Entomol 8:555–561. White DJ, Kramer LD, Backenson PB, Lukacik GL, Spielman A. 2001. Structure and seasonality of nearctic Johnson G, Oliver J, Howard JJ, Means RG, Eidson Culex pipiens populations. In: White DJ, Morse DL, M, Gotham I, Kulasekera V, Campbell S, the eds. West Nile virus detection, surveillance, and Arbovirus Research Laboratory, the Statewide West control. Ann NY Acad Sci 951:220–234. Nile Virus Response Teams. 2001. Mosquito surveil- Sulaiman S, Service MW. 1983. Studies on hibernating lance and polymerase chain reaction detection of populations of Culex pipiens L. in southern and West Nile virus, New York State. Emerg Infect Dis northern England. J Nat Hist 17:849–857. 4:643–649. Tempelis CH. 1975. Host-feeding patterns of mosqui- Winters AM, Bolling BG, Beaty BJ, Blair CD, Eisen toes, with a review of advances in analysis of blood RJ, Meyer AM, Pape WJ, Moore CG, Eisen L. 2008. meals by serology. J Med Entomol 11:635–653. Combining mosquito vector and human disease data Tesh RB, Parsons R, Siirin M, Randle Y, Sargent C, for improved assessment of spatial West Nile virus Guzman H, Wuithiranyagool T, Higgs S, Vanlan- disease risk. Am J Trop Med Hyg 78:654–665. dingham DL, Bala AA, Haas K, Zerinque B. 2004. Wray FC. 1946. Six generations of Culex pipiens Year-round West Nile virus activity, Gulf Coast without a blood meal. Mosq News 6:71–72. Viruses 2012, 4, 3812-3830; doi:10.3390/v4123812 OPEN ACCESS viruses ISSN 1999-4915 www.mdpi.com/journal/viruses Review Immune Responses to West Nile Virus Infection in the Central Nervous System

Hyelim Cho 1 and Michael S. Diamond 1,2,3,*

1 Departments of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA; E-Mail: [email protected] 2 Departments of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA 3 Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-314-362-2842; Fax: +1-314-362-9230.

Received: 20 November 2012; in revised form: 7 December 2012 / Accepted: 10 December 2012 / Published: 17 December 2012

Abstract: West Nile virus (WNV) continues to cause outbreaks of severe neuroinvasive disease in humans and other vertebrate animals in the United States, Europe, and other regions of the world. This review discusses our understanding of the interactions between virus and host that occur in the central nervous system (CNS), the outcome of which can be protection, viral pathogenesis, or immunopathogenesis. We will focus on defining the current state of knowledge of WNV entry, tropism, and host immune response in the CNS, all of which affect the balance between injury and successful clearance.

Keywords: flavivirus; innate immunity; adaptive immunity; pathogenesis; immunopathogenesis; neuron; brain

1. Introduction

West Nile virus (WNV) is a mosquito borne, neurotropic, positive-stranded, enveloped RNA virus in the Flaviviridae family. WNV is related genetically to other viruses that cause severe visceral and central nervous system (CNS) diseases in humans including dengue (DENV), yellow fever (YFV), Japanese encephalitis (JEV), and tick-borne encephalitis (TBEV) viruses. WNV is maintained in an Viruses 2012, 4 3813 enzootic cycle between mosquitoes and birds, but also infects and causes disease in vertebrate animals including horses and humans. WNV is transmitted primarily by Culex species mosquitoes and the virus amplifies in bird reservoirs, with humans and horses largely considered as dead-end hosts [1]. Although human cases occur primarily after mosquito inoculation, infection after blood transfusion, organ transplantation, and intrauterine transmission has been reported [1]. At present, there are no vaccines or therapeutic agents approved for humans against WNV. WNV was first isolated in 1937 in Uganda from a woman with an undiagnosed febrile illness [2], and historically, has caused outbreaks of a relatively mild febrile illness in regions of Africa, the Middle East, Asia, and Australia [3]. In the 1990’s, the epidemiology of infection changed. New outbreaks in Eastern Europe were associated with higher rates of severe neurological disease [4]. In 1999, WNV entered North America, and caused seven human fatalities in the New York City area as well as a large number of avian and equine deaths. Since then, it has spread to all 48 of the lower continental United States as well as to parts of Canada, Mexico, the Caribbean, and South America. While the majority of human infections are asymptomatic, WNV can cause a severe febrile illness and neuroinvasive syndrome characterized by meningitis, encephalitis, and/or acute flaccid paralysis [5–7]. Persistent movement disorders, cognitive dysfunction, and long-term disability all occur after West Nile neuroinvasive disease. West Nile poliomyelitis-like disease results in limb weakness or paralysis. Patients show markedly decreased motor responses in the paretic limbs, preserved sensory responses, and widespread asymmetric muscle denervation without evidence of demyelination or myopathy [8]. Thus, the neurological and functional disability associated with WNV infection represents a considerable source of morbidity in surviving patients long after the acute illness [9–13]. In the United States alone between 1999 and 2012, ~36,000 cases and ~1,500 deaths have been confirmed. The risk of severe WNV infection in humans is greatest in the elderly and immunocompromised [14,15]. Two studies have estimated a 20-fold increased risk of neuroinvasive disease and death in those over 50 years of age [14,16]. Beyond age, a limited number of host genetic factors have been linked with susceptibility to WNV infection. A deficiency of the chemokine receptor CCR5 increases the risk of symptomatic WNV infection, as a higher incidence (4.2%) of loss-of-function CCR5Δ32 homozygotes was observed in symptomatic WNV infection cohorts compared to that in the general population (1.0%) [17]. A nonsense mutation in the gene encoding 2'-5'-oligoadenylate synthetase/L1 (OAS) isoform is associated with WNV susceptibility in laboratory mice [18]. Correspondingly, a hypomorphic allele of the human ortholog OAS1 is associated with both symptomatic and asymptomatic WNV infection [19]. Finally, an association of single nucleotide polymorphisms (SNP) between symptomatic and asymptomatic WNV infections and IRF3 and Mx1 innate immune response and effector genes has been reported [20]; thus, genetic variation in the interferon (IFN) response pathway appears to correlate with the risk of symptomatic WNV infection in humans. In this review, we will summarize our understanding of the host-virus interface in the CNS and how this determines WNV disease pathogenesis and clinical outcome.

2. Virology and Pathogenesis

Although cellular receptors have not yet been identified definitively, studies suggest that WNV enters cells by endocytosis and fusion with the early endosome [21,22]. Following fusion between the

Viruses 2012, 4 3814 viral and endosomal membranes, the nucleocapsid is released into the cytoplasm and 11 kilobase viral genomic RNA associates with endoplasmic reticulum (ER) membranes. The single open reading frame is translated into a polyprotein and enzymatically processed into three structural proteins (capsid (C), pre-membrane (prM)/membrane (M), and envelope (E)) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Negative strand viral RNA then is synthesized and serves as a template for positive strand RNA synthesis [23]. Positive strand RNA is packaged in progeny virions, which bud into the ER to form enveloped immature virions. A maturation step, cleavage of the prM protein to the membrane M protein, occurs in the trans Golgi network by furin-like proteases [24–26] and results in a reorganization of E proteins on the virus surface into a homodimeric array [27]; these virions are secreted into the extracellular space by exocytosis. Following mosquito inoculation into the skin, it is believed that WNV replicates within epidermal keratinocytes and Langerhans cells [28,29]. Migratory Langerhans dendritic cells enter afferent lymphatics and travel to draining lymph nodes [28]. Here, infection and the risk of dissemination are countered by the rapid development of an early immune response including type I and II IFN production and the effector functions of innate immune cells ( cells, NK cells, neutrophils, macrophages, and IgM-secreting B cells) [30–34]. Virus produced in the lymph node can enter circulation via the efferent lymphatic system and thoracic duct, and viremia allows spread to secondary lymphoid and visceral organs including the spleen and kidney [35,36]. In peripheral tissues, infection is restricted by innate and adaptive immune responses including serum IgM [37], IFN-/ [38], IFN- [32,39], cytolytic CD8+ T cells [39–41], and cell-intrinsic IRF-3-dependent [30,42] antiviral responses.

3. WNV-Induced Pathology in the CNS

WNV causes encephalitis in several vertebrate species by virtue of its ability to infect and injure neurons through direct (viral-induced) and indirect (immune response induced or bystander) mechanisms [43]. Pathologic observations in humans are limited by the small number of autopsy studies on individuals succumbing to WNV infection. In these few reports, gross macroscopic examination of the brain and spinal cord did not reveal any overt pathology [5]. Microscopic examination of the brain in humans and other animals reveals histological changes that are consistent with the clinical disease [5,36]. This includes neuronal cell death, activation of resident microglia and infiltrating macrophages, perivascular and parenchymal accumulation of CD4+ and CD8+ T cells and CD138+ plasma cells, and formation of microglial nodules. These lesions, which can be patchy in distribution, occur in the brainstem, cerebral cortex, the hippocampus, thalamus, and cerebellum [5]. Cellular infiltrates in the meninges also can be present. In some cases, destruction of vascular structures with focal hemorrhage occurs, suggestive of a vasculitis; this may be associated with local compromise of the blood-brain barrier (BBB) [44,45]. Immunohistochemical analysis confirms that WNV antigen is present primarily in neurons from multiple regions of the brain, although other cells (e.g., CD11b+ myeloid cells and possibly astrocytes) may be infected but to lesser degrees [46,47]. In the spinal cord, an intense inflammatory infiltrate around large and small blood vessels is observed with large numbers of microglia in the ventral horn. Anterior horn motor neurons are targeted by WNV [8,48], and studies suggest that axonal transport from peripheral neurons can

Viruses 2012, 4 3815 mediate WNV entry into the spinal cord and induce acute flaccid paralysis [49]. Studies in hamsters reveal that limb paralysis and tremors are directly associated with infection and injury of anterior horn motor neurons in the lumbar section of the spinal cord [50].

4. Neuroinvasion

To establish infection in neurons of the brain, WNV first must cross the BBB (Figure 1). The BBB is composed of endothelial cells, astrocyte foot processes, and pericytes (PCs) and impedes the entry of macromolecules and pathogens from the blood into the brain. The tight junctions between endothelial cells form a diffusion barrier and pose obstacles for pathogens to enter the brain and to infect vulnerable and largely non-renewable neurons [51]. The mechanism by which WNV and other encephalitic flaviviruses cross the BBB remains uncertain. Crossing of the BBB likely occurs through a hematogenous route, as high levels of viremia correlate with greater and more rapid WNV entry into the CNS [52,53]. Intravascular levels of pro-inflammatory cytokines, which are produced during peripheral immune responses, also may modulate WNV entry into the CNS. WNV infection in peripheral tissues induces Toll-like receptor (TLR)-3-mediated secretion of pro-inflammatory cytokines, including IL-6 and TNF-α [44]. Secreted TNF-α can modulate BBB permeability by altering endothelial cell tight junctions, which may allow WNV to cross the BBB and infect neurons [44,54,55]. Semaphorin 7A upregulation after WNV infection also is linked to increased TNF-α production. Mice lacking Semaphorin 7A showed reduced TNF-α levels in serum, less BBB permeability, and reduced viral entry into the brain. [56]. Activation of matrix metalloproteinases also may enhance the flux of WNV by degrading the extracellular matrix of the BBB [57]. In BBB model studies in vitro, treatment with inhibitors of matrix metalloproteinases prevented the disruption of tight junction integrity associated with WNV infection [58]. Beyond compromise of the BBB, in some cases, WNV may penetrate into the CNS through additional mechanisms. Peripheral neurons are susceptible to infection by WNV [59,60]; retrograde axonal transport can bring WNV into the CNS, where transneuronal spread can occur. In contrast to some viruses (e.g., rabies [61]), neuron-to-neuron spread of WNV requires axonal release of viral particles [49]. Other possible entry mechanisms for WNV include (i) infection or passive transport through choroid plexus epithelial cells [62], (ii) a “Trojan horse” mechanism in which the virus is transported by infected immune cells (e.g., neutrophils [34] or CD4+ or CD8+ T cells [63]) that cross the BBB [64], (iii) infection of olfactory neurons and rostral spread from the olfactory bulb [65], or (iv) direct infection of brain microvascular endothelial cells [66]. The precise mechanism of WNV entry into the CNS in humans requires further study, and may differ depending on the route of infection and the pathogenicity of the WNV strain [67].

Viruses 2012, 4 3816

Figure 1. Mechanism of neuroinvasion of West Nile virus (WNV). WNV may enter the central nervous system (CNS) via multiple mechanisms including axonal retrograde transport along peripheral neurons into the spinal cord or hematogenous transport across the blood-brain barrier (BBB). Spinal cord entry is believed to result in interneuron spread to motor neuron cell bodies within the anterior horn of the spinal cord and lead to flaccid paralysis. The possible routes of virus entry across the BBB include (a) “Trojan horse” model; intracellular transport within macrophages or neutrophils, (b) loss of integrity of the BBB; cytokine-mediated (TNF-α, MIF) or matrix metalloproteinases disruption of tight junctions and basement membranes; (c) direct infection of brain microvascular endothelial cells with basolateral spread of the virus; (d) infection of choroid plexus epithelial cells; or (e) direct infection of olfactory neurons adjacent to the cribriform plate.

5. Neuronal Injury

WNV infection of neurons can result in caspase 3-dependent apoptosis, which likely contributes to CNS dysfunction and pathogenesis of severe disease. While no significant difference in peripheral or CNS tissue viral burden was observed in WNV-infected caspase 3−/− mice, these animals were more resistant to lethal WNV infection due to reduced neuronal cell death in the cerebral cortex, brain stem, and cerebellum [68]. Consistent with this, ectopic expression of WNV NS2B-NS3 non-structural proteins activates caspase 3 and induces apoptosis in neuroblastoma cell lines [69], and primary

Viruses 2012, 4 3817 neurons and neuroblastoma cells undergo apoptosis after WNV infection [48,68,70]. Cellular stress pathways including cAMP response element-binding transcription factor homologous protein (CHOP)-dependent apoptotic pathway also likely contribute to WNV-induced neuronal damage [71]. WNV infection may trigger apoptosis by activating non-caspase proteases, such as calpains and cathepsins [72,73]. Finally, WNV infection can induce non-apoptotic pathways of cell death. Cell necrosis can occur, as characterized by extensive cell swelling and loss of membrane integrity likely due to the extensive budding of WNV progeny virions into the ER [74]. In addition to injury imposed directly by WNV infection, neurons may undergo cell death or injury due to bystander damage caused by cytotoxic factors released by neuronal and non-neuronal cells. Neurons that are dying secondary to viral infection or immune-targeted death may release inflammatory molecules (e.g., Cxcl10 L-1β, IL-6, IL-8, and TNF-α) [75,76] with potentially toxic effects on uninfected neurons resulting in irreversible neuronal loss and atrophy. Analogously, glial cells, which are not primary targets of direct WNV infection, can become activated and release excitotoxic amino acids (e.g., glutamic and aspartic acids) and pro-inflammatory cytokines that contribute to the pathogenesis of neurological diseases by virus infections [77,78]. For example, TNF-α and IL1-β released by activated glial cells have direct roles in promoting bystander damage to neurons [79]. Elevated reactive oxygen species secreted by infected or activated microglial cells also may result in oxidative damage to neurons [80].

6. CNS Immune Responses to WNV

Upon entry in the CNS, WNV spreads rapidly between different subtypes of neurons in distinct regions [81]. As neurons are largely non-renewable, controlled immune responses must limit spread and eliminate virus while minimizing neuronal damage [82]. A delay or absence of such responses in genetically deficient mice or immunosuppressed humans results in rapid dissemination, neuronal injury, with an increased risk of mortality. Recent work in animal models has shown that both innate and cellular immune response in the CNS orchestrate control of WNV spread, which ultimately limits the number of neurons targeted for infection or the amount of virus any given infected neurons will produce.

6.1. CNS Innate Immunity

Nucleic acid intermediates of RNA virus replication are recognized by pathogen recognition receptors (PRR) such as TLR and RIG-I like receptors (RLR), which promote an antiviral state by activating IRF-3 and IRF-7-mediated transcriptional programs and type I IFN responses. The importance of these pathways for controlling WNV infection is highlighted by studies in mice that are genetically deficient for key components in this pathway: Type I IFN receptor-knockout mice (Ifnar−/−), Ifnb−/−, Mavs−/−, Tlr3−/−, Tlr7−/−, and Myd88−/− mice all show enhanced viral replication in the CNS and mortality after WNV infection [38,47,83–85]. Irf3−/− neurons showed reduced induction of antiviral defense genes including Rig-I, Mda5, and Ifit1, as well as blunted IFNα/β production [42]. In Irf7−/− neurons, IFN-α production was blunted, which resulted in increased WNV infection [86]. Together, these studies suggest that IRF-3 and IRF-7-dependent transcriptional programs are crucial for protective IFN response in neurons.

Viruses 2012, 4 3818

Stat1-dependent signaling pathways in part, determine the susceptibility of specific neuronal subtypes to WNV infection in the brain. IFN-α/β and Stat1-dependent transcription of IFN-stimulated genes (ISGs) inhibited WNV replication in neurons in vitro and in vivo. Rsad2 (also known as viperin), PKR, and RNase L are induced in neurons of the CNS and restrict WNV infection in vivo [87,88].

6.2. Inflammatory Responses

Neurons in the CNS are immunologically active and initiate inflammatory responses by producing chemokines that recruit immune cells (Figure 2). Infection of neurons by WNV induces expression of the T cell chemoattractant Cxcl10, which promotes trafficking of WNV-specific CD8+ T cells via binding to its cognate receptor Cxcr3 [89,90]. Enhanced expression of Ccl3 (MIP-1α), Ccl4 (MIP-1β), Ccl5 (RANTES) by WNV infection leads to Ccr5-dependent trafficking of CD4+ and CD8+ T cells, NK cells, and macrophages. Deletion or truncation of Ccr5 in mice leads to enhanced viral burden and increased mortality [91,92], and appears to be associated with more severe disease in humans [17]. Trafficking of monocytes into the brain, as precursors of macrophages and possibly microglia, can contribute to CNS injury [93] or survival after WNV infection [94], depending on the virulence of the infecting WNV strain. In mice, deletion of Ccr2, a chemokine receptor on inflammatory monocytes, leads to increased mortality after infection by virulent North American WNV strains, and this is associated with reduced monocyte accumulation in the brain [94]. Study with Il22−/− mice demonstrate that reduced levels of Cxcr2, a chemokine receptor mediating neutrophil migration, correlate with decreased viral loads in the CNS [95], suggesting that entry of WNV-infected neutrophils may contribute to pathogenesis. In Tlr7−/− mice, CD45+ leukocytes and CD11b+ macrophages failed to home to WNV-infected neurons due to blunted IL-23 responses, suggesting Tlr7 reduces WNV infection in part, by enhancing IL-23-dependent immune cell infiltration and homing into the brain [85].

6.3. Cellular Immunity

Studies in mice suggest that T cell-mediated immunity is an essential aspect of immune mediated protection from virulent strains of WNV. The lack of a functional CD4+ and CD8+ T cell response results in inefficient clearance of WNV infection from neurons of the brain [39,40,96]. Nonetheless, an over-exuberant CD8+ T cell-mediated response can lead to injury and or death of infected or uninfected neurons. In mice, within a few days of CNS infection, inflammatory cytokines and chemokines produced by resident cells of the CNS attract antigen-specific CD8+ T cells into the CNS [40,89]. In addition, CD40-CD40L and TNF-TNF-receptor interactions promote CD8+ T cell migration across brain microvascular endothelial cells, likely by increasing expression of adhesion molecules and modulating the integrity of tight junctions [97,98].

Viruses 2012, 4 3819

Figure 2. Leukocyte trafficking into the CNS after WNV. Upon WNV infection of neurons, virus-mediated upregulation of Cxcl10 recruits virus-specific CD8+ T cells via interactions with Cxcr3. Expression of Ccl3, Ccl4, and Ccl5 by other neuronal cells recruits Ccr5-expressing leukocytes. Monocytes and lymphocytes entering the perivascular spaces may be retained initially via Cxcr4 binding Cxcl12 [120]. Leukocyte egress from perivascular spaces requires IL-1β, TNF-, and CD40 interactions, which likely upregulates adhesion molecules including ICAM-1 and VCAM-1 [121,123,124].

CD8+ T cells control WNV infection in the CNS via multiple mechanisms (Figure 3) including the production of antiviral cytokines (e.g., IFN-) or by triggering cell death of target cells through perforin, Fas-Fas ligand, or TRAIL-dependent pathways. Infected neurons up-regulate MHC class I molecules and thus, can be targeted by cytotoxic T cells [99]. Perforin−/− mice showed higher viral burden in CNS and increased mortality after WNV infection [39], as well as a failure to clear WNV resulting in persistent CNS infection. Perforin-mediated control of infected neurons occurs through the granzyme-dependent granule exocytosis pathway, which results in apoptosis of infected neurons in vitro and in vivo [100–102]. Fas ligand (FasL) deficient mice also showed increased susceptibility to lethal WNV infection [103]. Interactions between Fas on infected neurons and FasL on CD8+ T cells leads to programmed cell death of neurons through the activation of a death domain and a caspase apoptosis cascade [102,104,105]. CD8+ T cells also use tumor necrosis factor-related

Viruses 2012, 4 3820 apoptosis-inducing ligand (TRAIL; also known as CD253) to restrict WNV pathogenesis by controlling infection in neurons. TRAIL binding to the death receptor DR5 on neurons activates a caspase-dependent apoptosis cascade [41]. Consistent with results establishing a protective effect of effector CD8+ T cells in mice, humans with impaired T cell immunity have a greater risk of CNS infection with WNV [106]. Although T cell responses are important for viral clearance, they can cause irrevocable damage to the host. Under certain conditions, infection of mice lacking CD8+ T cells with an attenuated lineage 2 WNV (Sarafend) strain resulted in decreased morbidity and mortality compared to wild type mice [107]. Consistent with this, depletion of CD8+ T cells in mice infected with an attenuated genetic variant of a North American WNV strain resulted in prolonged survival [108]. Thus, depending on the virological and immunological context, CD8+ T cells either can protect against or contribute to WNV neurological disease.

Figure 3. Mechanisms of CD8+ T cell clearance in the CNS. CD8+ T cells control WNV infection in the CNS through multiple mechanisms. Infected neurons upregulate surface expression of MHC class I molecules. Antigen-specific CD8+ T cells recognize infected neurons via class I MHC and processed viral peptides and trigger cell death of target cells through perforin, Fas-Fas ligand, or TRAIL-dependent pathways. Perforin-mediated control of infected neurons occurs through the granzyme-dependent granule exocytosis pathway, which results in apoptosis of infected neuron. Interactions between Fas on infected neurons and FasL on CD8+ T cells leads to programmed cell death of neurons through caspase-dependent pathways. CD8+ T cells also utilize TRAIL to restrict WNV infection in neurons. TRAIL binds to DR5 on neurons, which can have a direct antiviral effect against flaviviruses [125] or result in targeted apoptosis. Activated CD8+ T cell also produce IFN-γ, which can induce genes with antiviral effect.

Viruses 2012, 4 3821

7. Viral Persistence in the CNS

Although still controversial, persistent WNV infection and inflammation in the CNS of vertebrate animals has been reported in mice, monkeys, and hamsters [109–113]. These results are consistent with earlier studies in animals and humans showing flavivirus persistence after infection with Saint Louis encephalitis, tick-borne encephalitis, and louping ill viruses [114–117] . In monkeys, the duration of WNV persistence was at least 5.5 months, with infectious virus isolated from the cerebellum and cerebral subcortical ganglia. Virus recovered more than two months after initial infection from these monkeys retained neurovirulence [111]. In hamsters, WNV persistence has been described up to 86 days after initial infection, and this was associated with long term neurological sequelae [112,113]. In mice, infectious WNV was detected in the brains up to 4 months in 12% of mice and viral RNA persisted up to 6 months after infection [110]. Consistent with this, virus-specific B and T cell immune responses persisted in the brains of mice for at least 4 months after infection [109]. Although viral persistence in the CNS has not been documented in humans, chronic WNV infection in the kidney has been reported in some patient cohorts [118,119].

8. Summary and Future Perspectives

WNV continues to spread and cause neurological disease and thus, remains a public health concern in the United States and other countries. Research into the viral and host factors that determine the pathogenesis and outcome of WNV infection is crucial for development of new therapeutic and vaccines strategies. A more complete understanding of the mechanisms of immunopathogenesis in the CNS could facilitate the development tailored anti-inflammatory agents that minimize neuronal damage without preventing clearance. As examples, treatment with the Cxcr4 antagonist AMD3100 enhanced CD8+ T cell trafficking into the parenchyma of CNS and improved survival after WNV encephalitis [120], whereas blockade of migration of nitric oxide-producing inflammatory macrophage using anti-very late antigen (VLA)-4 integrin antibody prolonged survival after WNV encephalitis [121]. Combining such types of immunomodulatory agents with small molecule or antibody-based antiviral molecules [122] that target viral replication or tropism might be a way to maximize viral clearance and minimize neuropathogenesis after WNV infection.

Conflict of Interest

H. Cho declares no conflict of interest. M. Diamond is a consultant for MacroGenics and Elan Pharmaceuticals.

Acknowledgements

NIH grants U54 AI081680 (Pacific Northwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research), U19 AI083019, and R01 AI074973 supported this work.

Viruses 2012, 4 3822

References

1. Hayes, E.B.; Komar, N.; Nasci, R.S.; Montgomery, S.P.; O’Leary, D.R.; Campbell, G.L. Epidemiology and transmission dynamics of West Nile virus disease. Emerging Infect. Dis. 2005, 11, 1167–1173. 2. Smithburn, K.C.; Hughes, T.P.; Burke, A.W.; Paul, J.H. A Neurotropic virus isolated from the blood of a native of Uganda. Am. J. Trop Med. Hyg. 1940, 1, 471–492. 3. Kleiboeker, S.B. West Nile Virus. In Encyclopedia of Environmental Health; Nriagu, J.O., Ed.; Elsevier: Burlington, Canada, 2011; pp. 761–768. 4. Hubálek, Z.; Halouzka, J. West nile fever—A reemerging mosquito-borne viral disease in Europe. Emerging Infect. Dis. 1999, 5, 643–650. 5. Omalu, B.I.; Shakir, A.A.; Wang, G.; Lipkin, W.I.; Wiley, C.A. Fatal fulminant pan-meningo-polioencephalitis due to West Nile virus. Brain Pathol. 2003, 13, 465–472. 6. Armah, H.B.; Wang, G.; Omalu, B.I.; Tesh, R.B.; Gyure, K.A.; Chute, D.J.; Smith, R.D.; Dulai, P.; Vinters, H.V.; Kleinschmidt-DeMasters, B.K.; Wiley, C.A. Systemic distribution of West Nile virus infection: Postmortem immunohistochemical study of six cases. Brain Pathol. 2007, 17, 354–362. 7. Busch, M.P.; Wright, D.J.; Custer, B.; Tobler, L.H.; Stramer, S.L.; Kleinman, S.H.; Prince, H.E.; Bianco, C.; Foster, G.; Petersen, L.R.; et al. West nile virus infections projected from blood donor screening data, United States, 2003. Emerg. Infect. Dis. 2006, 12, 395–402. 8. Leis, A.A.; Fratkin, J.; Stokic, D.S.; Harrington, T.; Webb, R.M.; Slavinski, S.A. West Nile poliomyelitis. Lancet Infect. Dis. 2003, 3, 9–10. 9. Sejvar, J.J.; Haddad, M.B.; Tierney, B.C.; Campbell, G.L.; Marfin, A.A.; Van Gerpen, J.A.; Fleischauer, A.; Leis, A.A.; Stokic, D.S.; Petersen, L.R. Neurologic manifestations and outcome of West Nile virus infection. JAMA 2003, 290, 511–515. 10. Sejvar, J.J.; Marfin, A.A. Manifestations of West Nile neuroinvasive disease. Rev. Med. Virol. 2006, 16, 209–224. 11. Sejvar, J.J.; Bode, A.V.; Marfin, A.A.; Campbell, G.L.; Pape, J.; Biggerstaff, B.J.; Petersen, L.R. West Nile Virus-associated flaccid paralysis outcome. Emerging Infect. Dis. 2006, 12, 514–516. 12. Sejvar, J.J. The long-term outcomes of human West Nile virus infection. Clin. Infect. Dis. 2007, 44, 1617–1624. 13. Sejvar, J.J.; Davis, L.E.; Szabados, E.; Jackson, A.C. Delayed-onset and recurrent limb weakness associated with West Nile virus infection. J. Neurovirol. 2010, 16, 93–100. 14. Nash, D.; Mostashari, F.; Fine, A.; Miller, J.; O’Leary, D.; Murray, K.; Huang, A.; Rosenberg, A.; Greenberg, A.; Sherman, M.; et al. The outbreak of West Nile virus infection in the New York City area in 1999. N. Engl. J. Med. 2001, 344, 1807–1814. 15. Chowers, M.Y.; Lang, R.; Nassar, F.; Ben-David, D.; Giladi, M.; Rubinshtein, E.; Itzhaki, A.; Mishal, J.; Siegman-Igra, Y.; Kitzes, R.; et al. Clinical characteristics of the West Nile fever outbreak, Israel, 2000. Emerging Infect. Dis. 2001, 7, 675–678. 16. Huhn, G.D.; Austin, C.; Langkop, C.; Kelly, K.; Lucht, R.; Lampman, R.; Novak, R.; Haramis, L.; Boker, R.; Smith, S.; et al. The emergence of west nile virus during a large outbreak in Illinois in 2002. Am. J. Trop. Med. Hyg. 2005, 72, 768–776.

Viruses 2012, 4 3823

17. Glass, W.G.; McDermott, D.H.; Lim, J.K.; Lekhong, S.; Yu, S.F.; Frank, W.A.; Pape, J.; Cheshier, R.C.; Murphy, P.M. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 2006, 203, 35–40. 18. Mashimo, T.; Lucas, M.; Simon-Chazottes, D.; Frenkiel, M.-P.; Montagutelli, X.; Ceccaldi, P.-E.; Deubel, V.; Guenet, J.-L.; Despres, P. A nonsense mutation in the gene encoding 2’-5’-oligoadenylate synthetase/L1 isoform is associated with West Nile virus susceptibility in laboratory mice. Proc. Natl. Acad. Sci. USA 2002, 99, 11311–11316. 19. Lim, J.K.; Lisco, A.; McDermott, D.H.; Huynh, L.; Ward, J.M.; Johnson, B.; Johnson, H.; Pape, J.; Foster, G.A.; Krysztof, D.; et al. Genetic variation in OAS1 is a risk factor for initial infection with West Nile virus in man. PLoS Pathog. 2009, 5, e1000321. 20. Bigham, A.W.; Buckingham, K.J.; Husain, S.; Emond, M.J.; Bofferding, K.M.; Gildersleeve, H.; Rutherford, A.; Astakhova, N.M.; Perelygin, A.A.; Busch, M.P.; et al. Host genetic risk factors for West Nile Virus infection and disease progression. PLoS ONE 2011, 6, e24745. 21. Chambers, T.J.; Hahn, C.S.; Galler, R.; Rice, C.M. Flavivirus genome organization, expression, and replication. Annu. Rev. Microbiol. 1990, 44, 649–688. 22. Brinton, M.A. The molecular biology of West Nile Virus: A new invader of the western hemisphere. Annu. Rev. Microbiol. 2002, 56, 371–402. 23. Mackenzie, J.M.; Westaway, E.G. Assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J. Virol. 2001, 75, 10787–10799. 24. Elshuber, S.; Allison, S.L.; Heinz, F.X.; Mandl, C.W. Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J. Gen. Virol. 2003, 84, 183–191. 25. Guirakhoo, F.; Bolin, R.A.; Roehrig, J.T. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology 1992, 191, 921–931. 26. Stadler, K.; Allison, S.L.; Schalich, J.; Heinz, F.X. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 1997, 71, 8475–8481. 27. Mukhopadhyay, S.; Kim, B.-S.; Chipman, P.R.; Rossmann, M.G.; Kuhn, R.J. Structure of West Nile virus. Science 2003, 302, 248. 28. Byrne, S.N.; Halliday, G.M.; Johnston, L.J.; King, N.J. Interleukin-1beta but not tumor necrosis factor is involved in West Nile virus-induced Langerhans cell migration from the skin in C57BL/6 mice. J. Invest. Dermatol. 2001, 117, 702–709. 29. Lim, P.-Y.; Behr, M.J.; Chadwick, C.M.; Shi, P.-Y.; Bernard, K.A. Keratinocytes are cell targets of West Nile Virus in vivo. J. Virol. 2011, 85, 5197–5201. 30. Bourne, N.; Scholle, F.; Silva, M.C.; Rossi, S.L.; Dewsbury, N.; Judy, B.; De Aguiar, J.B.; Leon, M.A.; Estes, D.M.; Fayzulin, R.; Mason, P.W. Early production of type I interferon during West Nile virus infection: Role for lymphoid tissues in IRF3-independent interferon production. J. Virol. 2007, 81, 9100–9108. 31. Purtha, W.E.; Chachu, K.A.; Virgin, H.W., 4th; Diamond, M.S. Early B-cell activation after West Nile virus infection requires alpha/beta interferon but not antigen receptor signaling. J. Virol. 2008, 82, 10964–10974.

Viruses 2012, 4 3824

32. Wang, T.; Scully, E.; Yin, Z.; Kim, J.H.; Wang, S.; Yan, J.; Mamula, M.; Anderson, J.F.; Craft, J.; Fikrig, E. IFN-γ-Producing γδ T cells help control murine West Nile Virus infection. J. Immunol. 2003, 171, 2524–2531. 33. Vargin, V.V.; Semenov, B.F. Changes of natural killer cell activity in different mouse lines by acute and asymptomatic flavivirus infections. Acta. Virol. 1986, 30, 303–308. 34. Bai, F.; Kong, K.-F.; Dai, J.; Qian, F.; Zhang, L.; Brown, C.R.; Fikrig, E.; Montgomery, R.R. A paradoxical role for neutrophils in the pathogenesis of West Nile virus. J. Infect. Dis. 2010, 202, 1804–1812. 35. Diamond, M.S.; Shrestha, B.; Marri, A.; Mahan, D.; Engle, M. B cells and antibody play critical roles in the immediate defense of disseminated infection by West Nile encephalitis virus. J. Virol. 2003, 77, 2578–2586. 36. Eldadah, A.H.; Nathanson, N. Pathogenesis of West Nile Virus encepahlitis in mice and rats. II. Virus multiplication, evolution of immunofluorescence, and development of histological lesions in the brain. Am. J. Epidemiol. 1967, 86, 776–790. 37. Diamond, M.S.; Sitati, E.M.; Friend, L.D.; Higgs, S.; Shrestha, B.; Engle, M. A critical role for induced IgM in the protection against West Nile virus infection. J. Exp. Med. 2003, 198, 1853–1862. 38. Samuel, M.A.; Diamond, M.S. Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival. J. Virol. 2005, 79, 13350–13361. 39. Shrestha, B.; Samuel, M.A.; Diamond, M.S. CD8+ T cells require perforin to clear West Nile Virus from infected neurons. J. Virol. 2006, 80, 119–129. 40. Shrestha, B.; Diamond, M.S. Role of CD8+ T cells in control of west Nile Virus infection. J. Virol. 2004, 78, 8312–8321. 41. Shrestha, B.; Pinto, A.K.; Green, S.; Bosch, I.; Diamond, M.S. CD8+ T cells use TRAIL to restrict West Nile virus pathogenesis by controlling infection in neurons. J. Virol. 2012. 86, 8937–8948. 42. Daffis, S.; Samuel, M.A.; Keller, B.C.; Gale, M.; Diamond, M.S. Cell-Specific IRF-3 responses protect against west Nile Virus infection by interferon-dependent and -independent mechanisms. PLoS Pathog. 2007, 3, e106. 43. Chambers, T.J.; Diamond, M.S. Pathogenesis of flavivirus encephalitis. Adv. Virus Res. 2003, 60, 273–342. 44. Wang, T.; Town, T.; Alexopoulou, L.; Anderson, J.F.; Fikrig, E.; Flavell, R.A. Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis. Nat. Med. 2004, 10, 1366–1373. 45. Diamond, M.S.; Klein, R.S. West Nile virus: Crossing the blood-brain barrier. Nat. Med. 2004, 10, 1294–1295. 46. Diniz, J.A.P.; Da Rosa, A.P.A.T.; Guzman, H.; Xu, F.; Xiao, S.-Y.; Popov, V.L.; Vasconcelos, P.F.C.; Tesh, R.B. West Nile virus infection of primary mouse neuronal and neuroglial cells: The role of astrocytes in chronic infection. Am. J. Trop. Med. Hyg. 2006, 75, 691–696.

Viruses 2012, 4 3825

47. Daffis, S.; Samuel, M.A.; Suthar, M.S.; Gale, M., Jr; Diamond, M.S. Toll-like receptor 3 has a protective role against West Nile virus infection. J. Virol. 2008, 82, 10349–10358. 48. Shrestha, B.; Gottlieb, D.; Diamond, M.S. Infection and injury of neurons by West Nile encephalitis virus. J. Virol. 2003, 77, 13203–13213. 49. Samuel, M.A.; Wang, H.; Siddharthan, V.; Morrey, J.D.; Diamond, M.S. Axonal transport mediates West Nile virus entry into the central nervous system and induces acute flaccid paralysis. Proc. Natl. Acad. Sci. USA 2007, 104, 17140–17145. 50. Morrey, J.D.; Siddharthan, V.; Wang, H.; Hall, J.O.; Skirpstunas, R.T.; Olsen, A.L.; Nordstrom, J.L.; Koenig, S.; Johnson, S.; Diamond, M.S. West Nile virus–induced acute flaccid paralysis is prevented by monoclonal antibody treatment when administered after infection of spinal cord neurons. J. Neurovirol. 2008, 14, 152–163. 51. Ballabh, P.; Braun, A.; Nedergaard, M. The blood–brain barrier: An overview: Structure, regulation, and clinical implications. Neurobiol. Dis. 2004, 16, 1–13. 52. Johnson, R.T.; Mims, C.A. Pathogenesis of Viral infections of the nervous system. New Engl. J. Med. 1968, 278, 84–92. 53. Johnson, R.T.; Mims, C.A. Pathogenesis of viral infections of the nervous system. New Engl. J. Med. 1968, 278, 23–30. 54. de Vries, H.E.; Blom-Roosemalen, M.C.M.; van Oosten, M.; de Boer, A.G.; van Berkel, T.J. C.; Breimer, D.D.; Kuiper, J. The influence of cytokines on the integrity of the blood-brain barrier in vitro. J. Neuroimmunol. 1996, 64, 37–43. 55. Fiala, M.; Looney, D.J.; Stins, M.; Way, D.D.; Zhang, L.; Gan, X.; Chiappelli, F.; Schweitzer, E.S.; Shapshak, P.; Weinand, M.; et al. TNF-alpha opens a paracellular route for HIV-1 invasion across the blood-brain barrier. Mol. Med. 1997, 3, 553–564. 56. Sultana, H.; Neelakanta, G.; Foellmer, H.G.; Montgomery, R.R.; Anderson, J.F.; Koski, R.A.; Medzhitov, R.M.; Fikrig, E. Semaphorin 7A contributes to West Nile virus pathogenesis through TGF-β1/Smad6 signaling. J. Immunol. 2012, 189, 3150–3158. 57. Wang, P.; Dai, J.; Bai, F.; Kong, K.-F.; Wong, S.J.; Montgomery, R.R.; Madri, J.A.; Fikrig, E. Matrix metalloproteinase 9 facilitates West Nile virus entry into the brain. J. Virol. 2008, 82, 8978–8985. 58. Verma, S.; Kumar, M.; Gurjav, U.; Lum, S.; Nerurkar, V.R. Reversal of West Nile virus-induced blood–brain barrier disruption and tight junction proteins degradation by matrix metalloproteinases inhibitor. Virology 2010, 397, 130–138. 59. Hunsperger, E.A.; Roehrig, J.T. Temporal analyses of the neuropathogenesis of a West Nile virus infection in mice. J. Neurovirol. 2006, 12, 129–139. 60. Monath, T.P.; Cropp, C.B.; Harrison, A.K. Mode of entry of a neurotropic arbovirus into the central nervous system. Reinvestigation of an old controversy. Lab. Invest. 1983, 48, 399–410. 61. Dietzschold, B.; Schnell, M.; Koprowski, H. Pathogenesis of rabies. Curr. Top. Microbiol. Immunol. 2005, 292, 45–56. 62. Kramer-Hämmerle, S.; Rothenaigner, I.; Wolff, H.; Bell, J.E.; Brack-Werner, R. Cells of the central nervous system as targets and reservoirs of the human immunodeficiency virus. Virus Res. 2005, 111, 194–213.

Viruses 2012, 4 3826

63. Wang, S.; Welte, T.; McGargill, M.; Town, T.; Thompson, J.; Anderson, J.F.; Flavell, R.A.; Fikrig, E.; Hedrick, S.M.; Wang, T. Drak2 contributes to West Nile virus entry into the brain and lethal encephalitis. J. Immunol. 2008, 181, 2084–2091. 64. Garcia-Tapia, D.; Loiacono, C.M.; Kleiboeker, S.B. Replication of West Nile virus in equine peripheral blood mononuclear cells. Vet. Immunol. Immunopathol. 2006, 110, 229–244. 65. Brown, A.N.; Kent, K.A.; Bennett, C.J.; Bernard, K.A. Tissue tropism and neuroinvasion of West Nile virus do not differ for two mouse strains with different survival rates. Virology 2007, 368, 422. 66. Verma, S.; Lo, Y.; Chapagain, M.; Lum, S.; Kumar, M.; Gurjav, U.; Luo, H.; Nakatsuka, A.; Nerurkar, V.R. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier. Virology 2009, 385, 425–433. 67. Beasley, D.W.C.; Li, L.; Suderman, M.T.; Barrett, A.D.T. Mouse neuroinvasive phenotype of West Nile virus strains varies depending upon virus genotype. Virology 2002, 296, 17–23. 68. Samuel, M.A.; Morrey, J.D.; Diamond, M.S. Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile Virus encephalitis. J. Virol. 2007, 81, 2614–2623. 69. Ramanathan, M.P.; Chambers, J.A.; Pankhong, P.; Chattergoon, M.; Attatippaholkun, W.; Dang, K.; Shah, N.; Weiner, D.B. Host cell killing by the West Nile Virus NS2B–NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway. Virology 2006, 345, 56–72. 70. del Carmen Parquet, M.; Kumatori, A.; Hasebe, F.; Morita, K.; Igarashi, A. West Nile virus-induced bax-dependent apoptosis. FEBS Letters 2001, 500, 17–24. 71. Medigeshi, G.R.; Lancaster, A.M.; Hirsch, A.J.; Briese, T.; Lipkin, W.I.; DeFilippis, V.; Früh, K.; Mason, P.W.; Nikolich-Zugich, J.; Nelson, J.A. West Nile Virus infection activates the unfolded protein response, leading to CHOP induction and apoptosis. J. Virol. 2007, 81, 10849–10860. 72. Hail, N.; Carter, B.; Konopleva, M.; Andreeff, M. Apoptosis effector mechanisms: A requiem performed in different keys. Apoptosis 2006, 11, 889–904. 73. Kroemer, G.; Martin, S.J. Caspase-independent cell death. Nat. Med. 2005, 11, 725–730. 74. Chu, J.J.H.; Ng, M.L. The mechanism of cell death during West Nile virus infection is dependent on initial infectious dose. J. Gen. Virol. 2003, 84, 3305–3314. 75. Kumar, M.; Verma, S.; Nerurkar, V.R. Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death. J. Neuroinflammation 2010, 7, 73. 76. Zhang, B.; Patel, J.; Croyle, M.; Diamond, M.S.; Klein, R.S. TNF-alpha-dependent regulation of CXCR3 expression modulates neuronal survival during West Nile virus encephalitis. J. Neuroimmunol. 2010, 224, 28–38. 77. Bradl, M.; Hohlfeld, R. Molecular pathogenesis of neuroinflammation. J. Neurol. Neurosurg. Psychiatry 2003, 74, 1364–1370. 78. Power, C.; Johnson, R.T. Neuroimmune and neurovirological aspects of human immunodeficiency virus infection. Adv. Virus Res. 2001, 56, 389–433.

Viruses 2012, 4 3827

79. Chen, C.-J.; Ou, Y.-C.; Lin, S.-Y.; Raung, S.-L.; Liao, S.-L.; Lai, C.-Y.; Chen, S.-Y.; Chen, J.-H. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. J. Gen. Virol. 2010, 91, 1028–1037. 80. Schachtele, S.; Hu, S.; Little, M.; Lokensgard, J. Herpes simplex virus induces neural oxidative damage via microglial cell Toll-like receptor-2. J. Neuroimmunol. 2010, 7, 35. 81. McGavern, D.B.; Kang, S.S. Illuminating viral infections in the nervous system. Nat. Rev. Immunol. 2011, 11, 318–329. 82. Griffin, D.E. Immune responses to RNA-virus infections of the CNS. Nat. Rev. Immunol. 2003, 3, 493–502. 83. Lazear, H.M.; Pinto, A.K.; Vogt, M.R.; Gale, M., Jr; Diamond, M.S. Beta interferon controls West Nile virus infection and pathogenesis in mice. J. Virol. 2011, 85, 7186–7194. 84. Szretter, K.J.; Daffis, S.; Patel, J.; Suthar, M.S.; Klein, R.S.; Gale, M., Jr; Diamond, M.S. The innate immune adaptor molecule MyD88 restricts West Nile virus replication and spread in neurons of the central nervous system. J. Virol. 2010, 84, 12125–12138. 85. Town, T.; Bai, F.; Wang, T.; Kaplan, A.T.; Qian, F.; Montgomery, R.R.; Anderson, J.F.; Flavell, R.A.; Fikrig, E. Toll-like receptor 7 mitigates lethal West Nile encephalitis via interleukin 23-dependent immune cell infiltration and homing. Immunity 2009, 30, 242–253. 86. Daffis, S.; Samuel, M.A.; Suthar, M.S.; Keller, B.C.; Gale, M., Jr; Diamond, M.S. Interferon regulatory factor IRF-7 induces the antiviral alpha interferon response and protects against lethal West Nile virus infection. J. Virol. 2008, 82, 8465–8475. 87. Szretter, K.J.; Brien, J.D.; Thackray, L.B.; Virgin, H.W.; Cresswell, P.; Diamond, M.S. The interferon-inducible gene viperin restricts West Nile virus pathogenesis. J. Virol. 2011, 85, 11557–11566. 88. Samuel, M.A.; Whitby, K.; Keller, B.C.; Marri, A.; Barchet, W.; Williams, B.R.G.; Silverman, R.H.; Gale, M., Jr; Diamond, M.S. PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread in the periphery and replication in neurons. J. Virol. 2006, 80, 7009–7019. 89. Klein, R.S.; Lin, E.; Zhang, B.; Luster, A.D.; Tollett, J.; Samuel, M.A.; Engle, M.; Diamond, M.S. Neuronal CXCL10 directs CD8+ T-cell recruitment and control of West Nile virus encephalitis. J. Virol. 2005, 79, 11457–11466. 90. Zhang, B.; Chan, Y.K.; Lu, B.; Diamond, M.S.; Klein, R.S. CXCR3 mediates region-specific antiviral T cell trafficking within the central nervous system during West Nile virus encephalitis. J. Immunol. 2008, 180, 2641–2649. 91. Glass, W.G.; Lim, J.K.; Cholera, R.; Pletnev, A.G.; Gao, J.-L.; Murphy, P.M. Chemokine receptor CCR5 promotes leukocyte trafficking to the brain and survival in West Nile virus infection. J. Exp. Med. 2005, 202, 1087–1098. 92. Shirato, K.; Kimura, T.; Mizutani, T.; Kariwa, H.; Takashima, I. Different chemokine expression in lethal and non-lethal murine West Nile virus infection. J. Med. Virol. 2004, 74, 507–513. 93. Getts, D.R.; Terry, R.L.; Getts, M.T.; Müller, M.; Rana, S.; Shrestha, B.; Radford, J.; Rooijen, N.V.; Campbell, I.L.; King, N.J. C. Ly6c+ “nflammatory monocytes” are microglial precursors recruited in a pathogenic manner in West Nile virus encephalitis. J. Exp. Med. 2008, 205, 2319–2337.

Viruses 2012, 4 3828

94. Lim, J.K.; Obara, C.J.; Rivollier, A.; Pletnev, A.G.; Kelsall, B.L.; Murphy, P.M. Chemokine Receptor Ccr2 is critical for monocyte accumulation and survival in west nile virus encephalitis. J. Immunol. 2011, 186, 471–478. 95. Wang, P.; Bai, F.; Zenewicz, L.A.; Dai, J.; Gate, D.; Cheng, G.; Yang, L.; Qian, F.; Yuan, X.; Montgomery, R.R.; Flavell, R.A.; Town, T.; Fikrig, E. IL-22 Signaling contributes to west nile encephalitis pathogenesis. PLoS ONE 2012, 7, e44153. 96. Sitati, E.M.; Diamond, M.S. CD4+ T-Cell responses are required for clearance of west Nile virus from the central nervous system. J. Virol. 2006, 80, 12060–12069. 97. Sitati, E.; McCandless, E.E.; Klein, R.S.; Diamond, M.S. CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile Virus encephalitis. J. Virol. 2007, 81, 9801–9811. 98. Shrestha, B.; Zhang, B.; Purtha, W.E.; Klein, R.S.; Diamond, M.S. Tumor necrosis factor alpha protects against lethal West Nile Virus infection by promoting trafficking of mononuclear leukocytes into the central nervous system. J. Virol. 2008, 82, 8956–8964. 99. Chevalier, G.; Suberbielle, E.; Monnet, C.; Duplan, V.; Martin-Blondel, G.; Farrugia, F.; Le Masson, G.; Liblau, R.; Gonzalez-Dunia, D. Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection. PLoS Pathog. 2011, 7, e1002393. 100. Harty, J.T.; Badovinac, V.P. Influence of effector molecules on the CD8+ T cell response to infection. Curr. Opin. Immunol. 2002, 14, 360–365. 101. Russell, J.H.; Ley, T.J. Lymphocyte-Mediated Cytotoxicity. Annu. Rev. Immunol. 2002, 20, 323–370. 102. Shresta, S.; Pham, C.T.; Thomas, D.A.; Graubert, T.A.; Ley, T.J. How do cytotoxic lymphocytes kill their targets? Curr. Opin. Immunol. 1998, 10, 581–587. 103. Shrestha, B.; Diamond, M.S. Fas ligand interactions contribute to CD8+ T-cell-mediated control of West Nile Virus infection in the central nervous system. J. Virol. 2007, 81, 11749–11757. 104. Krzyżowska, M.; Cymerys, J.; Winnicka, A.; Niemiałtowski, M. Involvement of fas and fasL in ectromelia virus-induced apoptosis in mouse brain. Virus Res. 2006, 115, 141–149. 105. Nagata, S. Apoptosis by death factor. Cell 1997, 88, 355–365. 106. Pruitt, A.A. Central nervous system infections in cancer patients. Semin. Neurol. 2004, 24, 435–452. 107. Wang, Y.; Lobigs, M.; Lee, E.; Müllbacher, A. CD8+ T cells mediate recovery and immunopathology in West Nile virus encephalitis. J. Virol. 2003, 77, 13323–13334. 108. Szretter, K.J.; Daniels, B.P.; Cho, H.; Gainey, M.D.; Yokoyama, W.M.; Gale, M., Jr; Virgin, H.W.; Klein, R.S.; Sen, G.C.; Diamond, M.S. 2’-O methylation of the viral mRNA cap by West Nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo. PLoS Pathog. 2012, 8, e1002698. 109. Stewart, B.S.; Demarest, V.L.; Wong, S.J.; Green, S.; Bernard, K.A. Persistence of virus-specific immune responses in the central nervous system of mice after West Nile virus infection. BMC Immunol. 2011, 12, 6. 110. Appler, K.K.; Brown, A.N.; Stewart, B.S.; Behr, M.J.; Demarest, V.L.; Wong, S.J.; Bernard, K.A. Persistence of West Nile Virus in the central nervous system and periphery of mice. PLoS ONE 2010, 5, e10649.

Viruses 2012, 4 3829

111. Slavin, H.B. Persistence of the virus of St. Louis encephalitis in the central nervous system of mice for over five months. J. Bacteriol. 1943, 46, 113–116. 112. Pogodina, V.V.; Bochkova, N.G.; Levina, L.S. Persistence of tick-borne encephalitis virus in monkeys. VII. Some features of the immune response. Acta. Virol. 1984, 28, 407–415. 113. Pogodina, V.V.; Frolova, M.P.; Malenko, G.V.; Fokina, G.I.; Levina, L.S.; Mamonenko, L.L.; Koreshkova, G.V.; Ralf, N.M. Persistence of tick-borne encephalitis virus in monkeys. I. Features of experimental infection. Acta. Virol. 1981, 25, 337–343. 114. Zlotnik, I.; Carter, G.B.; Grant, D.P. The persistence of louping ill virus in immunosuppressed guinea-pigs. Br. J. Exp. Pathol. 1971, 52, 395–407. 115. Pogodina, V.V.; Frolova, M.P.; Malenko, G.V.; Fokina, G.I.; Koreshkova, G.V.; Kiseleva, L.L.; Bochkova, N.G.; Ralph, N.M. Study on West Nile virus persistence in monkeys. Arch. Virol. 1983, 75, 71–86. 116. Xiao, S.Y.; Guzman, H.; Zhang, H.; Travassos da Rosa, A.P.; Tesh, R.B. West Nile virus infection in the golden hamster (Mesocricetus auratus): A model for West Nile encephalitis. Emerging Infect. Dis. 2001, 7, 714–721. 117. Siddharthan, V.; Wang, H.; Motter, N.E.; Hall, J.O.; Skinner, R.D.; Skirpstunas, R.T.; Morrey, J.D. Persistent West Nile Virus associated with a neurological sequela in hamsters identified by motor unit number estimation. J. Virol. 2009, 83, 4251–4261. 118. Nolan, M.S.; Podoll, A.S.; Hause, A.M.; Akers, K.M.; Finkel, K.W.; Murray, K.O. Prevalence of chronic kidney disease and progression of disease over time among patients enrolled in the houston West Nile Virus cohort. PLoS ONE 2012, 7, e40374. 119. Murray, K.; Walker, C.; Herrington, E.; Lewis, J.A.; McCormick, J.; Beasley, D.W.C.; Tesh, R.B.; Fisher-Hoch, S. Persistent infection with West Nile virus years after initial infection. J. Infect. Dis. 2010, 201, 2–4. 120. McCandless, E.E.; Zhang, B.; Diamond, M.S.; Klein, R.S. CXCR4 antagonism increases T cell trafficking in the central nervous system and improves survival from West Nile virus encephalitis. PNAS 2008, 105, 11270–11275. 121. Getts, D.R.; Terry, R.L.; Getts, M.T.; Müller, M.; Rana, S.; Deffrasnes, C.; Ashhurst, T.M.; Radford, J.; Hofer, M.; Thomas, S.; et al. Targeted blockade in lethal West Nile virus encephalitis indicates a crucial role for very late antigen (VLA)-4-dependent recruitment of nitric oxide-producing macrophages. J. Neuroimmunol. 2012, 9, 246. 122. Diamond, M.S. Progress on the development of therapeutics against West Nile virus. Antivir. Res. 2009, 83, 214–227. 123. Dai, J.; Wang, P.; Bai, F.; Town, T.; Fikrig, E. ICAM-1 Participates in the entry of West Nile virus into the central nervous system. J. Virol. 2008, 82, 4164–4168.

Viruses 2012, 4 3830

124. Shen, J.; T-To, S.S.; Schrieber, L.; King, N.J. Early e-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines. J. Virol. 1997, 71, 9323–9332. 125. Warke, R.V.; Martin, K.J.; Giaya, K.; Shaw, S.K.; Rothman, A.L.; Bosch, I. TRAIL Is a novel antiviral protein against Dengue virus. J. Virol. 2008, 82, 555–564.

© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Arch Virol (2013) 158:735–752 DOI 10.1007/s00705-012-1516-3

BRIEF REVIEW

West Nile virus associations in wild mammals: a synthesis

J. Jeffrey Root

Received: 20 June 2012 / Accepted: 15 September 2012 / Published online: 2 December 2012 Ó Springer-Verlag (outside the USA) 2012

Abstract Exposures to West Nile virus (WNV) have widely distributed in parts the Old World, such as Africa, been documented in a variety of wild mammals in both the the Middle East, Asia, southern Europe, and elsewhere New and Old Worlds. This review tabulates at least 100 [114]. During the summer of 1999, WNV was first detected mammal species with evidence of WNV exposure. Many in the Western Hemisphere in the northeastern U.S. [80]. of these exposures were detected in free-ranging mammals, The virus spread rapidly across the continental U.S. [69] while several were noted in captive individuals. In addition and expanded its range north into Canada by 2001 [24] and to exposures, this review discusses experimental infections southwardly into the Caribbean basin and Mexico between in terms of the potential for reservoir competence of select 2001 and 2002 [52]. The virus is thought to have reached wild mammal species. Overall, few experimental infections the South American continent by 2004 [52]. Although have been conducted on wild mammals. As such, the role WNV activity is strictly limited to lineage 1 viruses in the of most wild mammals as potential amplifying hosts for New World, lineage 2 viruses occur in various parts of the WNV is, to date, uncertain. In most instances, experimental Old World [5, 78], and additional lineages have been infections of wild mammals with WNV have resulted in no proposed [78]. or low-level viremia. Some recent studies have indicated The natural enzootic cycle of WNV is thought to occur that certain species of tree squirrels (Sciurus spp.), eastern largely among birds and mosquito vectors [56]. Mammals chipmunks (Tamias striatus), and eastern cottontail rabbits have generally been presumed to be dead-end hosts (Sylvilagus floridanus) develop viremia sufficient for because they typically produce short-duration viremia that infecting some mosquito species. Certain mammalian is below the threshold for infecting most mosquito species species, such as tree squirrels, mesopredators, and deer [7]. For example, horses are known to be commonly have been suggested as useful species for WNV surveil- exposed to WNV, which can be associated with morbidity lance. In this review article, the information pertaining to and mortality; however, their low viremia of short duration wild mammal associations with WNV is synthesized. during experimental infections suggest that they are unlikely amplifying hosts [12]. Nonetheless, many reports have suggested that several species of wild mammals are com- Introduction monly exposed to WNV, occasionally with high associated seroprevalence rates. Some recent experimental studies The first isolation of West Nile virus (WNV; family Fla- have documented viremia of [ 105.0 pfu/mL in select viviridae, genus Flavivirus) was documented in Omogo in species of mammals. This level has been commonly used the West Nile District of Uganda from an adult woman as a threshold suggesting that a minimum level is required [103]. Subsequently, the virus has been described to be to infect select mosquito species through blood meals [51]. Overall, the general consensus on the trivial roles mammals play in the epidemiology of WNV may be due to a lack of & J. Jeffrey Root ( ) inquiry rather than a lack of importance [66]. US Department of Agriculture, National Wildlife Research Center, 4101 La Porte Ave, Fort Collins, CO 80521, USA The objective of this paper was to provide a compre- e-mail: [email protected] hensive review of WNV activity in wild mammals. In 123 736 J. Jeffrey Root addition, the potential roles of wild mammals in the ecol- antibody prevalence rates of nearly 50 % have been ogy of WNV are discussed. Due to space limitations, reported for fox squirrels and eastern gray squirrels [91]. Kunjin virus and the Australian continent are not focused Additional WNV infections have been reported from on in this paper. For the purposes of this review, natural ‘‘squirrels’’ in Arizona, Kansas, and Wyoming [70, 71]. exposures to WNV are defined as detections of antibodies, However, the species identifications were not presented for virus, viral RNA, or other forms of detection. When suit- these animals. Overall, WNV exposures in tree squirrels able/possible, common and scientific names have been have been presented from at least thirteen states and the updated to reflect modern taxonomy following Mammal District of Columbia in the contiguous U.S. Additional Species of the World [112]. As reviewed elsewhere, caution WNV exposures from a non-tree squirrel comes from the must be used when interpreting serologic results from European ground squirrel (Spermophilus citellus), in which locations where multiple group B arboviruses exist [38]. antibodies were detected in nine specimens from Austria Many of the older studies cited in this paper used a single [102]. assay for antibody detection, some of which are prone to The reasons why tree squirrels appear to be more cross-react with other flaviviruses. As such, some antibody commonly exposed to WNV when compared to other detections reviewed in this paper are presented for com- sympatric mammal species is undetermined; however, pleteness, but it is acknowledged that some of these reports aspects of their behavioral ecology have been suggested to could represent false positive cross-reactivity. increase their chance of exposure to mosquito vectors [91]. For example, the activity and feeding behavior of Culex pipiens complex mosquitoes in tree canopies in Tennessee Natural exposures of wild mammals to West Nile virus [96] suggest potential increased exposure to tree squirrels. Tree squirrels have been proposed as useful tools for The data presented below represent a synthesis of natural monitoring WNV activity [76, 91], as these animals pro- exposures of wild mammals to WNV. For the sake of vide localized evidence of WNV activity [76]. However, completeness, reports associated with captive wildlife are when antibodies are used to monitor WNV activity, the age also presented in key situations, as zoos and similar facil- structure of populations will need to be accounted for [91], ities have been proposed as potential sentinel sites for young animals will need to be utilized [33], or a longitu- emerging pathogens [61]. dinal approach, such as mark-recapture sampling, may be required [93] to overcome bias generated by studying WNV in rodents animals potentially exposed during previous years.

WNV exposures have been detected in diverse wild rodent Chipmunks species from multiple regions. Rodents represent a taxonomic group with one of the highest number of reported WNV exposures have been documented in eastern chip- species exposed. Some of the earliest observations of WNV munks (Tamias striatus) on two occasions in New York exposures in wild mammals were obtained from rodents and Maryland, USA [33, 63]. However, a multi-state study during the 1960s. Many of the tests used for rodent anti- including New York, Pennsylvania, and Ohio failed to body assessments, especially older accounts, are prone to detect WNV antibodies in any of the [ 30 eastern chip- cross-reactivity and, therefore, should be interpreted with munks sampled [91]. It has been suggested that limited caution. A summary of natural WNV exposures of rodents mosquito exposures or the potential for lethal WNV is presented in Table 1. infections in eastern chipmunks may account for the low seroprevalence observed in this species [33]. Due to their Squirrels small size, dead chipmunks are less likely to be recovered during surveillance efforts when compared to larger tree Dead and sick tree squirrels have become ubiquitous signs squirrels. of WNV activity in some regions of the U.S. [39, 50, 76]. Exposures have been detected in fox squirrels (Sciurus Rats niger)[8, 39, 50, 76, 91–93], eastern gray squirrels (Sciu- rus carolinensis)[21, 33, 39, 55, 91], western gray squir- Exposures of WNV to Old World rat species are fairly rels (Sciurus griseus)[76], and a red squirrel (Tamiasciurus diverse, with many exposures detected in Rattus spp. For hudsonicus)[91]. Some of these exposures have been example, antibodies to WNV were detected in brown rats detected in dead or moribund squirrels, while many others (Rattus norvegicus) and/or roof rats (R. rattus) in Pakistan, have come from healthy squirrels that have developed Israel, Austria, Tunisia, central Africa, and Madagascar antibodies to WNV following their exposures. High [2, 13, 16, 19, 30, 37, 102], from R. rattus alexandrius in 123 West Nile virus associations in wild mammals 737

Table 1 Natural exposures of wild rodents to West Nile virus Common name Scientiﬁc name Detection type Location Reference

Fox squirrel Sciurus niger Antigen; viral RNA MI, USA [50] Antigen; viral RNA IL, USA [39] Antibodies CO, OH, USA [91] Antibodies CO, USA [92] Antibodies; viral RNA CO, USA [93] Viral RNA CA, USA [76] Antibodies IA, USA [8] Eastern gray squirrel Sciurus carolinensis Not speciﬁed NY, USA [55, 63] Antigen; viral RNA IL, USA [39] Antibodies NY, PA, USA [91] Antibodies LA, USA [21] Antibodies MD, D of C, USA [33] Western gray squirrel Sciurus griseus Antibodies CA, ID, USA [76] Red squirrel Tamiasciurus hudsonicus Antibodies NY, USA [91] European ground squirrel Spermophilus citellusa Antibodies Austria [102] Eastern chipmunk Tamias striatus Not speciﬁed NY, USA [63] Antibodies MD, USA [33] Hispid cotton rat Sigmodon hispidus Antibodies LA, USA [21] Roof rat and subspecies Rattus rattus Antibodies Pakistan [37] Antibodies LA, USA [21] Antibodies Madagascar [30] Antibodies Tunisia [13] Antibodies Central Africa [16] R. rattus frugivorus Antibodies Egypt [1] R. rattus alexandrius Antibodies Israel [2] Brown rat Rattus norvegicus Antibodies Israel [2] Antibodies Egypt [1] Antibodies Pakistan [19] Antibodies Austria [102] Antibodies MD, D of C, USA [33] Rat Rattus sp. Antibodies LA, USA [91] African arvicanthis Arvicanthis niloticus Virus Nigeria [49] Antibodiesb Kenya [43] Common dasymys Dasymys incomtus Antibodiesb Kenya [43] Kaiser’s aethomys Aethomys kaiseri Antibodiesb Kenya [43] Rusty-bellied brush-furred rat Lophuromys sikapusic Antibodiesb Kenya [43] Black-tailed thallomys Thallomys nigricauda Antibodiesb Kenya [43] Common metad Millardia meltada Antibodies Pakistan [19] Guenther’s vole Microtus guentherid Antibodies Israel [2] Bank vole Myodes glareoluse Virus Hungary [73] Antibodies Austria [102] Antibodies Italy [59] Common vole Microtus arvalis Antibodies Romania [25] Meadow jumping mouse Zapus hudsonius Antibodies NY, USA [91] Peromyscus mice Peromyscus spp. Antibodies NY, OH, USA [91] White-footed mouse Peromyscus leucopus Antibodies MD, D of C, USA [33] House mouse Mus musculus Antibodies CO, USA [91] Antibodies D of C, USA [33]

123 738 J. Jeffrey Root

Table 1 continued Common name Scientiﬁc name Detection type Location Reference

Western Mediterranean mouse Mus spretus Antibodies Spain [14] Antibodies Morocco [15] Unidentified mouse Mus sp. Antigen Guinea [53] Antibodies Tunisia [13] Northeast African spiny mouse Acomys cahirinusf Antibodies Egypt [1] Eastern spiny mouse Acomys dimidiatusf Antibodiesg Egypt [99] Field mouse Apodemus sp. Antibodies Austria [102] Long-tailed field mouse Apodemus sylvaticus Antibodies Tunisia [13] Maghreb garden dormouse Eliomys munbyanush Antibodies Tunisia [13] Unidentified praomys Praomys sp. Antibodies Central Africa [16] Wagner’s dipodil Dipodillus dasyurus Antibodiesg Egypt [99] Greater Egyptian gerbil Gerbillus pyramidum Antibodies Israel [2] Unidentified gerbil Gerbillus sp. Antibodies Tunisia [13] Indian gerbil Tatera indica Antibodies Pakistan [19] Indian desert jird Meriones hurrianae Antibodies Pakistan [19] Bushy-tailed jird Sekeetamys calurus Antibodiesg Egypt [99] Common hamster Cricetus cricetus Antibodies Austria [102] Common gundi Ctenodactylus gundi Antibodies Tunisia [13] Woodchuck Marmota monax Antibodies MD, USA [33] ‘‘Rodents’’ Not reported Antibodies Hungary [72] Not reported NY, USA [68] a Listed in original paper as Citellus citellus b Note: Sera were generally insufficient to conduct confirmatory tests c Reported as Lophuromys sikapusi, but Mammal Species of the World indicates that eastern distribution limits are unresolved [112] d Reported in original paper as Microtus guntheri e Reported in original papers as Clethrionomys glareolus f Reported in original papers as Acomys cahrines cahirinus and Acomys cahirinus dimidatus for references [1] and [99], respectively g Note: Antibodies were determined by HI tests and were of low titer. The authors concluded that the small number of rodents with antibodies and the low HI titers are insignificant h Reported in original paper as Eliomys tunetae

Israel [2], and R. rattus frugivorus and brown rats in Egypt Louisiana [91]. In addition, antibody-positive hispid cotton [1]. In addition, virus has been isolated from African arv- rats (Sigmodon hispidus) were sampled in Louisiana [21]. icanthis (aka grass rat; Arvicanthis niloticus) collected from a Sudan woodland vegetative zone in Nigeria [49], Mice and exposures have been detected in the common metad (Millardia meltada), also known as the soft-furred rat, Reported exposures of Old World mice to WNV are lim- collected in Pakistan [19]. A serosurvey in Kenya detected ited. WNV antibody detections have been reported in the WNV antibodies in African arvicanthis, common dasymys western Mediterranean mouse (Mus spretus) in Spain [14] (Dasymys incomtus), Kaiser’s aethomys (Aethomys kai- and Morocco [15], in a field mouse (Apodemus sp.) from seri), a rusty-bellied brush-furred rat (Lophuromys sika- Austria [102], and in a long-tailed field mouse (Apodemus pusi), and a black-tailed thallomys (Thallomys nigricauda); sylvaticus), Maghreb garden dormice (Eliomys munby- however, sera were generally insufficient to conduct con- anus), and unidentified Mus sp. in Tunisia [13], in a firmatory tests for these species [43]. In the New World, northeast African spiny mouse (Acomys cahirinus) and an exposures have been primarily limited to Old World rat eastern spiny mouse (Acomys dimidiatus) from Egypt species of the genus Rattus, with antibodies detected in [1, 99], and in unidentified praomys (Praomys sp.) from brown rats from Maryland and Washington DC [33], roof central Africa [16]. Further, viral antigen has recently been rats from Louisiana [21], and Rattus species from reported in an unidentified Mus sp. mouse from Guinea

123 West Nile virus associations in wild mammals 739

[53]. Similar to the Old World, reported WNV exposures in Wyoming, with a high antibody prevalence rate of 63 % of mice found in the New World are limited. Antibodies to [6], thereby suggesting that striped skunks are commonly WNV have been detected in Peromyscus spp. in the eastern exposed to WNV in this area. An additional 90 striped U.S., house mice in the central and eastern U.S., and a skunk sera tested from Arizona, California, Louisiana, and meadow jumping mouse (Zapus hudsonius) in the eastern Texas did not yield evidence of WNV antibodies [6]. U.S. [33, 91]. Canids Other rodents Only limited WNV exposures have been detected in wild Additional WNV exposures, mostly in the Old World, have canids, although this may be due to lack of sampling rather been occasionally documented in other rodent species. than a lack of exposures. Antibodies to WNV have been These included WNV exposures in an Indian gerbil (Tatera detected in coyotes (Canis latrans) during two different indica) and Indian desert jird (Meriones hurrianae) from studies in Wisconsin, with an antibody prevalence rate of Pakistan [19], a bushy-tailed jird (Sekeetamys calurus) and 27 % during a 2003-2004 sampling period [22], 0 % during a a Wagner’s dipodil (Dipodillus dasyurus) from Egypt [99], 2004-2005 sampling period, and increasing to 10 % during a a greater Egyptian gerbil (Gerbillus pyramidum) and 2005-2006 sampling period [23]. An additional example of a Guenther’s vole (Microtus guentheri) from Israel [2], a canid exposed to WNV comes from the red fox (Vulpes vul- bank vole (Myodes glareolus) and ‘‘rodents’’ from Hungary pes), as a single red fox was documented to be antibody [72, 73], additional bank voles from Italy and Austria [59, positive in Wisconsin [22]. Other wild canids, such as the gray 102], and from a common vole (Microtus arvalis) collected fox (Urocyon cinereoargenteus), have been tested for WNV in Romania [25]. In addition, WNV antibodies have been exposure but none were found to have been exposed [6]. detected in the sera of common hamsters (Cricetus cricetus) from Austria [102], and in a common gundi (Cteno- Raccoons dactylus gundi) and an unidentified gerbil (Gerbillus sp.) from Tunisia [13]. Additional rodent WNV exposures were Raccoons (Procyon lotor) have been commonly shown to reported in a woodchuck (Marmota monax) sampled in be exposed to WNV in many regions of the U.S. Feral Maryland [33] and in ‘‘rodents’’ from New York [68]. populations in the Old World may have a similar fate. Overall, only limited testing of rodents for WNV has been Raccoon infections with WNV were first reported from conducted, particularly in the New World, when compared New York during 2000 [68]. Subsequently, antibody to birds and domestic animals. detections in raccoons were reported in 2005 from Penn- sylvania [91] and Louisiana [21]. Additional antibody WNV in wild carnivores and mesocarnivores detections have been documented in raccoons from Wis- consin, Louisiana, Wyoming, Maryland, Washington DC, Detections of WNV antibodies in wild carnivores and and Iowa [6, 8, 22, 23, 33]. mesocarnivores have become fairly common over the last decade in North America. Some species have been afflicted Bears with severe disease, primarily in captive situations, following WNV infection, while disease in other species has Antibodies to WNV have been documented from a small not been routinely reported. The potential roles of various percentage of American black bear (Ursus americanus) peridomestic mesocarnivores in WNV amplification cycles sera collected from New Jersey [27]. In addition, WNV have been proposed as important questions [23], likely due antibodies have been detected in brown bear (Ursus arctos) to the additional public-health burden these species could sera collected from Croatia [62]. foster if they are reservoir competent. Further, mesopredators have been proposed as potentially useful sentinels Virginia opossum for monitoring WNV activity in delineated areas [6]. A summary of natural WNV exposures of carnivores and The Virginia opossum (Didelphis virginiana) is a marsu- mesocarnivores is presented in Table 2. pial and is therefore not a member of the mammalian order Carnivora. However, it is considered a North American Striped skunks mesocarnivore. Virginia opossums have exhibited widespread WNV exposures. Antibodies to WNV have been The first detection of WNV exposure in a striped skunk detected in this species from New York, Ohio, Pennsyl- (Mephitis mephitis) was reported from Connecticut [63]. vania, Louisiana, Wisconsin, Texas, Wyoming, Maryland, Additional exposures were reported in this species sampled Washington DC, and Iowa [6, 8, 21–23, 33, 91]. The 123 740 J. Jeffrey Root

Table 2 Natural exposures of Common name Scientific name Detection type Location Reference wild and zoo members of the order Carnivora to West Nile Striped skunk Mephitis mephitis Not specified CT, USA [63] virus Antibodies WY, USA [6] Raccoon Procyon lotor Antibodies PA, USA [91] Antibodies WI, USA [22] Antibodies LA, USA [21] Antibodies LA, WY, USA [6] Antibodies MD, D of C, USA [33] Antibodies WI, USA [23] Antibodies IA, USA [8] Not reported NY, USA [68] Red panda Ailurus fulgens Antibodies NY, USA [61]* Brown bear Ursus arctos Antibodies Multiple, Croatia [62] American black bear Ursus americanus Antibodies NJ, USA [27] Polar bear Ursus maritimus Antibodies, Viral RNA Toronto, Canada [26]* Wolf Canis sp. IHC, Viral RNA IL, USA [60]* Canis lupus IHC, Viral RNA Que´bec, Canada [57]* Red fox Vulpes vulpes Antibodies WI, USA [22] Coyote Canis latrans Antibodies Yucatan State, Mexico [29]* * = animal living in a zoo or captive outdoor animal facility Antibodies WI, USA [22] a Reported in original paper as Antibodies WI, USA [23] Panthera uncia Jaguar Panthera onca Antibodies Yucatan State, Mexico [29]* b Scientific names were not Snow leopard Uncia unciaa Antibodies NY, USA [61]* listed in the original document. Cougar Puma concolorb Antibodies US (not specified) [48]* These names are assumed to be Tiger Panthera tigrisb Antibodies US (not specified) [48]* correct based on the common b names listed in the original Lion Panthera leo Antibodies US (not specified) [48]* document. Blood samples were Civet Not reported Antibodies Ethiopia [3] collected from private Harbor seal Phoca vitulina Not reported NJ, USA [85]* collections peridomestic nature of this species in some situations may of WNV exposure has been reported in big brown bats make it a useful species for monitoring WNV activity. (Eptesicus fuscus) from New York and Illinois [11, 55, 63], in the little brown myotis (Myotis lucifugus) in New York, Other carnivores Maryland, and New Jersey [33, 55, 63, 81], a northern myotis (Myotis septentrionalis) sampled in New Jersey Antibodies to WNV have been detected in an unidentified [81], and in Mexican free-tailed bats (Tadarida brasilien- civet from Ethiopia [3]. This appears to be one of the first sis) collected in Louisiana [20]. In the Old World, anti- published accounts of WNV exposure in a wild carnivore. bodies were detected in Egyptian rousettes (Rousettus aegyptiacus) from Israel and Uganda [2, 101], unidentified rousette(s) (Rousettus sp.) from central Africa [16], mala- WNV in other wild mammalian species gasy flying foxes (Pteropus rufus) from Madagascar [30], Angolan soft-furred fruit bats (Lissonycteris angolensis) Several additional wild mammalian species have shown collected in Kenya [101], dusky pipistrelles (Pipistrellus evidence of WNV exposure. Some of these represent a hesperidus) from Tunisia [13], and unidentified free-tailed single example or a few examples of exposures in a par- bats (Tadarida sp.) from central Africa [16]. Antibodies to ticular taxonomic group. A summary of natural WNV WNV were described from three additional bat species in exposures in these species is presented in Tables 3, 4, 5. Uganda, which included little free-tailed bats (Chaerephon pumilus), Angola free-tailed bats (Mops condylurus), and a Chiropterans single African straw-colored fruit bat (Eidolon helvum) [98, 101]. Further, WNV was isolated from a Leschenault’s Reports of WNV infections in chiropterans have been rousette (Rousettus leschenaultii) in India [79]. Additional widely documented in the New and Old Worlds. Evidence WNV exposure accounts comes from unidentified ‘‘bats’’ 123 West Nile virus associations in wild mammals 741

Table 3 Natural exposures of Common name Scientific name Detection type Location Reference bats to West Nile virus Big brown bat Eptesicus fuscus Not specified NY, USA [55, 63] Antibodies IL, USA [11] Little brown myotis Myotis lucifugus Not specified NY, USA [55, 63] Antibodies MD, USA [33] Antibodies NJ, USA [81] Northern myotis Myotis septentrionalis Antibodies NJ, USA [81] Mexican free-tailed bat Tadarida brasiliensis Antibodies LA, USA [20] Unidentified free-tailed bat Tadarida sp. Antibodies Central Africa [16] Egyptian rousette Rousettus aegyptiacusa Antibodies Israel [2] Antibodies Uganda [101] Leschenault’s rousette Rousettus leschenaultiib Virus India [79] a Reported in original paper as Unidentified rousette Rousettus sp. Antibodies Central Africa [16] Russettus aegypticus [2] Malagasy flying fox Pteropus rufus Antibodies Madagascar [30] b Reported in original paper as Little free-tailed bat Chaerephon pumilusc Antibodies Uganda [98] Rousettus leschenaulti Angola free-tailed bat Mops condylurusc Antibodies Uganda [98] c Reported in original paper as African straw-colored fruit bat Eidolon helvum Antibodies Uganda [98, 101] Tadarida pumila and Tadarida Angolan soft-furred fruit bat Lissonycteris angolensis Antibodies Kenya [101] condylura Dusky pipistrelle Pipistrellus hesperidusd Antibodies Tunisia [13] d Reported in original paper as Pipistrellus kuhli. Mammal ‘‘Bat’’ Not reported Antibodies Egypt [106] Species of the World [112] Not reported WI, USA [70] indicates that Pipistrellus kuhlii Virus India [46] does not include African populations and is referred to as Antibodies Egypt [46] hesperidus. The same reference Antibodies Ethiopia [3] also indicates that there has Antibodies Central Africa [16] been some discussion about the Not reported NY, USA [68] correct spelling of kuhlii [112] in New York, Wisconsin, India, Egypt, central Africa, and Lagomorphs Ethiopia [3, 16, 46, 68, 70, 106]. The antibody detection in four of 48 unidentified bats from Egypt [106] appears to be Detections of WNV antibodies have been published for one of the first published accounts of WNV exposure in a lagomorph species, primarily in the Old World, with wild mammal. detections occurring in European rabbits (Oryctolagus cuniculus) in France [58] and presumably in Austria (e.g., Soricomorphs Oryctolagus sp.) [102]. WNV antibodies have been reported in European hares (Lepus syriacus), presumably The literature on WNV in soricomorphs is scant. However, now considered to be Lepus europaeus, from Israel [2] and antibodies were detected in Asian house shrews (Suncus from the Czech Republic [44, 45]. An unidentified rabbit murinus) from India [47], white-toothed house shrews and hare yielded evidence of WNV antibodies in Greece (Crocidura russula) in Spain [14], and a Zaphir’s shrew [54], and WNV infections were confirmed in three (Crocidura zaphiri) in Ethiopia [3]. Several African giant unidentified rabbits from New York [68]. In addition, a shrews (Crocidura olivieri), described in the original paper black-tailed jackrabbit (Lepus californicus) in California as C. occidentalis, had antibodies reactive with WNV; tested positive for WNV [76]. however, sera were generally insufficient to conduct confirmatory tests on this species [43]. Additional accounts of Artiodactylids WNV antibodies in unidentified shrews (Crocidura sp.) were described in central Africa [16]. Antibodies reactive West Nile virus activity has been detected in a variety of with WNV in a non-shrew member of the order Sorico- artiodactylids. In the U.S., WNV exposures have been morpha, the Roman mole (Talpa romana), have been reported in white-tailed deer (Odocoileus virginianus)in reported from Italy [59]. Other workers have unsuccess- many regions, with antibodies detected in New Jersey and fully attempted to detect WNV antibodies in shrew sera Iowa [28, 95], and viral RNA detected in Georgia from a from the New World [91]. three-year-old male with a history of signs of disease, such

123 742 J. Jeffrey Root

Table 4 Natural exposures of nonhuman primates to West Nile virus Common name Scientiﬁc name Detection type Location Reference

Ring-tailed lemur Lemur catta Antibodies NY, USA [61]* Antibodies Madagascar [30, 104] Milne-Edward’s sportive lemur Lepilemur edwardsi Antibodies Madagascar [30, 89] Brown lemur Eulemur fulvusa Antibodies Madagascar [30] Weasel lemur Lepilemur mustelinusb Antibodies Madagascar [30] Verreaux’s sifaka Propithecus verreauxi Antibodies Madagascar [30] Coquerel’s sifaka Propithecus coquereli Antibodies Madagascar [17] Barbary macaque Macaca sylvanus Virus isolation, viral RNA, antibodies Toronto, Canada [74]* Baboon Papio cynocephalus anubisc Antibodies Toronto, Canada [74]* Papio spp. Antibodies LA, USA [87]* Japanese macaque Macaca fuscata Antibodies Toronto, Canada [74]* Rhesus monkey Macaca mulatta Antibodies LA, USA [87]* Southern pig-tailed macaque Macaca nemestrina Antibodies LA, USA [87]* Antibodies LA, USA [42]* Common chimpanzee Pan troglodytes Antibodies Congo [75] Antibodies Central Africa [16]** Unidentified monkey Cercopithecus sp. Antibodies South Africa [46] Antibodies Central Africa [16]** Greater spot-nosed monkey Cercopithecus nictitans Antibodies Central Africa [16] Patas monkey Erythrocebus patas Antibodies Central Africa [16]** Sooty mangabey Cercocebus atys Antibodies GA, USA [18]* Unknown mangabey Cercocebus sp. Antibodies Central Africa [16]** Potto Perodicticus potto Antibodies Central Africa [16] Prince Demidoff’s bushbaby Galago demidoffd Antibodies Central Africa [16] * = animal living in a zoo or captive outdoor animal facility ** = assumed to be captive but translation/article is unclear a Reported in original paper as Lemur fulvus b Reported in original paper as Lepilemur mustellinus c Listed in original paper as olive baboons (Papio cynocephalus anubis). Mammal Species of the World [112] recognizes the olive baboon (Papio anubis) and the yellow baboon (Papio cynocephalus) as different species d Reported in original paper as Galago demidoffi as ataxia and tremors [67]. Thus, deer have been proposed Perrissodactylaids as a useful serosurveillance animal for monitoring WNV activity [95]. A greater number of artiodactylid species Antibodies to WNV have been detected in sera from feral with WNV exposures have been reported from the Old horses (Equus caballus) from Nevada during the last dec- World. For example, WNV antibodies have been detected ade, with nearly 1,400 horses sampled during the multi- in European roe (Capreolus capreolus), fallow deer (Dama year study period [31]. A single animal was antibody dama), red deer (Cervus elaphus), and red sheep (Ovis positive during 2004, but none were positive during aries) hunted in Moravia, Czech Republic [44, 45]. In 2005-2006 [31]. This trend changed during the latter part of addition, a dated account of antibodies in an unidentified the decade of collection, as during 2008 and 2009, antibody ‘‘antelope’’ in central Africa was published over four prevalence rates were 19 and 7.2 %, respectively [31]. decades ago [16]. Antibodies have been detected in wild boar/feral Nonhuman primates swine (Sus scrofa) in both hemispheres. These exposures were documented in Florida, Georgia, and Texas in the Published accounts of wild nonhuman primate exposures to U.S. [32], and in Moravia in the Czech Republic [36, 44, WNV are uncommon and have been primarily associated with 45]. Madagascar. Antibodies were detected in a Milne-Edward’s

123 West Nile virus associations in wild mammals 743

Table 5 Natural exposures of other wild and zoo mammals to West Nile virus Common name Scientiﬁc name Detection type Location Reference

Asian house shrew Suncus murinus Antibodies India [47] Greater white-toothed shrew Crocidura russula Antibodies Spain [14] Zaphir’s shrew Crocidura zaphiri Antibodies Ethiopia [3] African giant shrew Crocidura olivieria Antibodiesb Kenya [43] Unidentified shrew Crocidura sp. Antibodies Central Africa [16] Roman mole Talpa romana Antibodies Italy [59] White-tailed deer Odocoileus virginianus Antibodies NJ, USA [28] Antibodies IA, USA [77]* Viral RNA GA, USA [67] Antibodies IA, USA [95] European roe Capreolus capreolus Antibodies Moravia, Czech Republic [44] Antibodies Moravia, Czech Republic [45] Fallow deer Dama dama Antibodies Moravia, Czech Republic [44] Antibodies Moravia, Czech Republic [45] Red deer Cervus elaphus Antibodies Moravia, Czech Republic [45] Reindeer Rangifer tarandus IHC, Viral RNA, antibodies IA, USA [77]* Unidentified antelope Not reproted Antibodies Central Africa [16] Mountain goat Oreamnos americanus Multiple NE, WY, USA [86]* Red sheep Ovis ariesc Antibodies Moravia, Czech Republic [45] Feral horse Equus caballus Antibodies NV, USA [31] Indian rhinoceros Rhinoceros unicornis Antibodies NY, USA [61]* Asian elephant Elephas maximus Antibodies NY, USA [61]* Antibodiesd FL, USA [48]* Unidentified hyrax Dendrohyrax sp. Antibodies Central Africa [16] European rabbit Oryctolagus cuniculus Antibodies France [58] Presumably as above Oryctolagus sp. Antibodies Austria [102] ‘‘Rabbit’’e Not reported Antibodies Greece [54] Not reported NY, USA [68] European hare Lepus europaeusf Antibodies Israel [2] ‘‘Hare’’e Not reported Antibodies Moravia, Czech Republic [44] Antibodies Moravia, Czech Republic [45] Antibodies Greece [54] Black-tailed jackrabbit Lepus californicus Not reported CA, USA [76] Wild boar/feral swine Sus scrofa Antibodies Moravia, Czech Republic [44] Antibodies Moravia, Czech Republic [45] Antibodies South Moravia [36] Antibodies FL, GA, TX, USA [32] Buru babirusa Babyrousa babyrussag Antibodies NY, USA [61]* Killer whale Orcinus orca Viral RNA TX, USA [105]* Bottlenose dolphin Tursiops truncatus Antibodies FL, USA [97]

123 744 J. Jeffrey Root

Table 5 continued Common name Scientiﬁc name Detection type Location Reference

Virginia opossum Didelphis virginiana Antibodies NY, OH, PA, USA [91] Antibodies WI, USA [22] Antibodies LA, USA [21] Antibodies LA, TX, WY, USA [6] Antibodies MD, D of C, USA [33] Antibodies WI, USA [23] Antibodies IA, USA [8] * = animal living in a zoo or captive outdoor animal facility a The original paper indicates this animal is the white toothed shrew (Crocidura occidentalis). However, Mammal Species of the World [112] suggests that this is a synonym of the African giant shrew (Crocidura olivieri) b Sera were generally insufficient to conduct confirmatory tests c Reported in original paper as mouflon (Ovis musimon) d Scientific names were not listed in the original document. This name is assumed to be correct based on the common names given in the original document. Blood samples were collected from captive collections in Florida e It is unclear from the reference if these animals were captive or wild f Reported in original paper as Lepus syriacus g Reported in original paper as Babyrousa babyrousa sportive lemur (Lepilemur edwardsi) in Madagascar [30, Primarily, these exposures have been described from New 89]. In addition, a high percentage of ring-tailed lemurs York, Louisiana, Canada, and Mexico, but a few additional (Lemur catta) were antibody positive in a more recent exposures have been reported elsewhere, with some inter- study in Madagascar, with 94-100 % of animals testing esting cases reported from some marine zoological parks. positive by two different assays [104]. In contrast, in an Data pertaining to WNV associations with captive wildlife earlier account from Madagascar in which 377 individual are summarized in Tables 2, 4 and 5 (denoted by ‘‘*’’). lemurs and sifakas from multiple species, including the ring-tailed lemur, were tested, an overall antibody preva- New York lence of approximately 1.9 % was found [30]. Thus, antibodies have also been detected in the weasel lemur Following the 1999 introduction of WNV into the New (Lepilemur mustelinus), brown lemur (Eulemur fulvus), and World, potential WNV activity was noted in the animal Verreaux’s sifaka (Propithecus verreauxi), all at very low collection of the Bronx Zoo/Wildlife Conservation Park as prevalence levels [30]. Several older accounts of seropos- early as August 1999 [61]. A subsequent serosurvey of the itive nonhuman primates have included common chim- animals in the park led to the detection of WNV antibodies panzees (Pan troglodytes), unidentified monkeys in multiple mammals, such as a buru babirusa (Babyrousa (Cercopithecus sp.), Prince Demidoff’s bushbaby (Galago babyrussa), an Indian rhinoceros (Rhinoceros unicornis), demidoff), the potto (Perodicticus potto), and the greater two ring-tailed lemurs, two Asian elephants (Elephas spot-nosed monkey (Cercopithecus nictitans) from the maximus), two snow leopards (Uncia uncia), and a red African continent [16, 46, 75], with antibody prevalence panda (Ailurus fulgens)[61]. The authors suggested that rates of up to 51 % reported for chimpanzees [75]. the much higher seroprevalence that they observed in birds, as compared to mammals, was likely related to vector host Hyracoidea preferences [61].

Antibodies reactive with WNV were detected in an uniden- Nonhuman primates in the U.S. and Canada tiﬁed hyrax (Dendrohyrax sp.) from central Africa [16]. Following a human epidemic of WNV in southern Loui- siana, 1,692 serum samples were tested from nonhuman WNV in wildlife in captive situations primates housed in an outdoor breeding facility [87]. Antibodies were detected in baboons (Papio spp.), rhesus Exposures of mammalian wildlife to WNV in captive sit- monkeys (Macaca mulatta), and southern pig-tailed uations have been reported on multiple occasions. macaques (Macaca nemestrina), with prevalence rates of

123 West Nile virus associations in wild mammals 745

51.4, 39.4, and 20.3 %, respectively [87]. Of interest, 10-day illness, a 12-year-old harbor seal (Phoca vitulina) antibodies to WNV have been determined to persist in died from a WNV infection at a New Jersey State Aquar- southern pig-tailed macaques for up to 36 months [42]. ium [85]. In addition, a WNV infection associated with Additional antibody detections were reported in sooty nonsuppurative encephalitis was confirmed through RT- mangabeys (Cercocebus atys) from a nonhuman primate PCR and sequencing in a killer whale (Orcinus orca) from facility in Georgia at a low seroprevalence rate of 6.6 % a marine park in Texas [105]. A small percentage of [ 100 [18]. None of the 45 rhesus monkeys tested from the same wild-caught bottlenose dolphins (Tursiops truncatus) from Georgia facility had WNV antibodies [18]. At the Toronto the Indian River Lagoon in Florida tested positive for Zoo in Toronto, Canada, a neurologically ill barbary WNV antibodies [97]. Additional marine mammal infec- macaque (Macaca sylvanus) was diagnosed with a WNV tions have presumably been reported for harbor seals and infection [74]. Subsequently, thirty-three nonhuman pri- monk seals (Monachus schauinslandi) in conference pro- mates from the zoo were tested for WNV antibodies, with ceedings, which have been reviewed elsewhere [48]. one of seven baboons (Papio cynocephalus anubis), two of 16 Japanese macaques (Macaca fuscata), and zero of 10 additional barbary macaques testing positive by at least one Experimental infections assay [74]. Relatively few experimental infection studies have been Anecdotal reports of other captive wildlife conducted on non-domesticated mammals since the discovery of WNV. Some of the recent studies were likely Infections and/or exposures of WNV have been reported motivated by seroprevalence rates and disease observed in from a variety of other captive wildlife from multiple select mammalian species (e.g., Sciurus spp.), while others regions of North America. For example, WNV was were likely motivated by the potential risks to human detected in mountain goats (Oreamnos americanus) from health stemming from the synanthropic nature of select Nebraska and Wyoming, which presumably died from their mammals. These studies have provided valuable informa- infections [86]. Severe morbidity and a fatal WNV infection associated with the potential role of wild mammals in tion in reindeer (Rangifer tarandus) have been reported the ecology of WNV. from a captive facility in Iowa [77], and a fatal WNV Which wild mammal species, if any, have the potential infection associated with encephalitis and myocarditis was for reservoir competence for WNV and which species detected in a three-month-old wolf pup (Canis sp.), pre- possess the natural history attributes and behavioral ecol- sumably Canis lupus, from a private collection in Illinois ogy to be commonly exposed to natural vectors of this [60]. An additional case of WNV in a wolf (C. lupus), virus are important questions. It has been suggested that associated with severe renal lymphoplasmacytic vasculitis, viremia of approximately 105.0 pfu/mL is sufficient to was described from a four-month-old captive pup in Que´- infect select mosquito species and subsequently make a bec [57]. Antibodies to WNV were detected from an vertebrate reservoir competent [51]. However, lower-level asymptomatic coyote and a jaguar (Panthera onca) from viremia has been suggested to be sufficient for infecting the Meridia Zoo, Yucatan State, Mexico [29]. Additional some mosquito species at low efficiency [4]. As such, some accounts of WNV antibodies have been reported from authors have indicated a range of competence for avian captive big cats of the genus Panthera and a cougar (Puma species, with 102 to 105,105 to 108, and 109 to 1012 pfu/mL concolor), all of which were apparently exhibited animals of serum representing a low or absent, moderate, and high moved to various regions of the U.S., and from elephants competence level, respectively [9]. A similar range of associated with captive collections in Florida [48]. A few competence may be applicable to mammals. At present, no additional WNV exposures have been described in pre- mammals have been determined to develop viremia suffi- sumably captive non-human primates from central Africa, cient to warrant their inclusion in the high competence with antibody detections in a mangabey (Cercocebus sp.), a level (Table 6). However, a limited number of species have patas monkey (Erythrocebus patas), an unidentified mon- been assessed to belong to the moderate competence level, key (Cercopithecus sp.), and a common chimpanzee (Pan while many have been assessed to be incompetent troglodytes)[16]. These animals, along with others tested (Table 6). in this reference, often yielded antibodies to multiple viruses tested in a single individual. As such, cross-reac- Tree squirrels tivity may be present in some of these results. Infections with WNV have not been limited to terrestrial The average peak viremia from all published experimental mammals, as WNV infections have been documented in infections with fox squirrels is approximatley105.7 pfu/mL both pinnipeds and cetaceans. For example, following a (Table 6). In addition, a much higher viremia (108.0 pfu/mL) 123 746 J. Jeffrey Root

Table 6 Experimental infections of wild mammals with West Nile virus Common name Scientiﬁc name Exposure method Maximum viremia Reference

Fox squirrel Sciurus niger Subcutaneous inoculation 104.98 pfu/mL [92] Fox squirrel Sciurus niger Intramuscular inoculation 106.1 pfu/mL [83] Mosquito bite 105.3 pfu/mL Fox squirrel Sciurus niger Oral exposure 105.6 pfu/mL [109] Eastern grey squirrel Sciurus carolinensis Subcutaneous inoculation 105.5 pfu/mL [34] Eastern chipmunk Tamias striatus Intramuscular inoculation 107.8 pfu/mL [82] 5.8 a Eastern cottontail Sylvilagus ﬂoridanus Subcutaneous inoculation 10 CID50s/mL [108] Mosquito bite ‘‘Gerbil’’ Not reported Not reported Not reported [46] Not reported Arvicanthis sp. Not reported Not reported [46] African white-tailed rat Mystromys Intracardiac/Intraperitoneal inoculation None detected [64] albicaudatus African arvicanthis Arvicanthis niloticus Intracardiac/Intraperitoneal inoculation None detected [64] Natal mastomys Mastomys natalensis Intracardiac/Intraperitoneal inoculation None detected [64] Red veld aethomys Aethomys Intracardiac/Intraperitoneal inoculation 101.5 (mouse innoc.) [64] chrysophilus Southern African vlei rat Otomys irroratus Intracardiac/Intraperitoneal inoculation None detected [64] Xeric four-striped grass rat Rhabdomys pumilio Intracardiac/Intraperitoneal inoculation None detected [64] Raccoon Procyon lotor Subcutaneous inoculation 104.6 pfu/mL [94] 3.8 Rhesus monkey Macaca mulatta Subcutaneous/intrathalamical inoculations 10 LD50/mL [84]

Intradermal inoculation B100 TCID50/mL [88] Intracerebral, intranasal, and intravenous Not reported [103]b inoculationsa Subcutaneous inoculation 102.0/0.02 ml serum (mouse [40] innoc). Subcutaneous and intravenous inoculation No live virus recoveredc [111] Crab-eating macaque Macaca fascicularis Subcutaneous inoculation No live virus recoveredc [111] Bonnet macaque Macaca radiata Intranasal inoculation ‘‘Low grade’’ [35] d 3.0 Lemur Eulemur spp. Subcutaneous inoculation 10 LD50/mL [90] Hamadryas baboon Papio hamadryas Intradermal inoculation 105–106 copies/mLe [113] Grivet Chlorocebus Intracerebral inoculation Not reported [103] aethiopsf ‘‘Monkey’’ Not reported Aerosol Not reported [46] Big brown bat Eptesicus fuscus Subcutaneous inoculation 180 pfu/mL [20] Mexican free-tailed bat Tadarida brasiliensis Subcutaneous inoculation None detected [20] African straw-colored fruit Eidolon helvum Intraperitoneal inoculation None detected [100] bat Egyptian rousette Rousettus Intraperitoneal inoculation Trace [100] aegyptiacus ‘‘Hedgehog’’ Not reported Intracerebral inoculation Not reported [103] a It is unclear if this titer is associated with a subcutaneous inoculation or mosquito bite b Note: Animals were reported as ‘‘rhesus monkeys’’ in the original article with no corresponding scientiﬁc name. Therefore, these animals are assumed to represent Macaca mulatta c No live virus recovered, but PCR-based viremia described as ‘‘discrete and short-lived’’ in M. mulatta and undetectable in two M. fascicularis that developed fever. M. fascicularis were themectomized and/or CD8 T-cell depleted d Note: Reported as Lemur fulvus fulvus and L. fulvus albifrons in original paper. These are now likely represented by Eulemur fulvus and E. albifrons. However, no distinction is made in the original paper as to which species was experimentally infected with WNV e Viremia range reported by authors is based on quantitative real-time PCR assay f Reported as African monkey (Cercopithecus ethiops centralis) in original paper. Mammal Species of the World [112] suggests that Chlorocebus aethiops has been used a synonym of Cercopithecus aethiops

123 West Nile virus associations in wild mammals 747 has been detected in a naturally infected fox squirrel [76]. especially if one considers how often several of these An experimental infection study has also been conducted species are observed by the general public. However, the on eastern grey squirrels, with a maximum viremia detec- lack of disease noted during experimental infection studies ted of 105.5 pfu/mL [34]. High seroprevalence rates have [108] suggests that natural infections may go unnoticed by also been reported for these two squirrel species, with the public. As such, lagomorphs may be exposed to WNV overall seroprevalence from multiple states and study sites more frequently than has been reported previously. of nearly 50 % [91]. Thus, these tree squirrel species are commonly exposed to WNV and develop viremia of a Baboons moderate level of competence that is sufficient for infecting some mosquito vectors. In addition, based on the detection Peak PCR-based viremia titers of 105 to 106 copies/mL of viral RNA in select tissues long after the clearance of have been reported for baboons (Papio hamadryas)at4 viremia (e.g., 29 DPI), fox squirrels have been suggested as DPI of an experimental infection with WNV [113]. Con- having the potential to be persistently infected [83]. sidering that most reported viremias in nonhuman primates have been low (Table 6), this result is somewhat surprising. Eastern chipmunks Other species Experimentally infected eastern chipmunks yielded moderately high viremia, with up to 107.8 pfu/mL detected [82]. Other species such as rhesus monkeys, big brown bats, However, a lower seroprevalence rate was noted when Mexican free-tailed bats, Egyptian rousettes, African eastern chipmunks were compared to other mammalian straw-colored fruit bats, lemurs (Eulemur spp.), and rac- species tested from Maryland [33]. In addition, no anti- coons did not yield significant viremias during experi- body-positive eastern chipmunks were detected in three mental infections [20, 84, 88, 90, 94, 100]. Of interest, two states where this species was sampled, even though these of three experimentally infected African straw-colored fruit animals were sympatric with antibody-positive tree squir- bats had antibodies in post-inoculation sera with no viremia rels [91]. This low seroprevalence suggests that eastern detected at any time after infection [100]. In addition, one chipmunks may develop fatal WNV infections or are not of six African rodent species tested, the red veld aethomys commonly exposed to appropriate mosquito vectors [33]. (Aethomys chrysophilus), yielded evidence of low-level During experimental infections, no signs of illness were WNV viremia [64]. Maximum viremias during experi- observed in any chipmunk during 1-8 DPI; however, mental infections were not reported for several other potential signs of WNV disease were observed during 9-11 mammalian species (Table 6). As such, their potential DPI, with neurologic symptoms and lethargy as the most contribution to WNV mosquito cycles is not discussed. common signs of disease observed [82]. Most of these With few exceptions, most wild mammals experimentally animals were euthanized for humane reasons prior to death infected with WNV to date have developed low- (many) or so the lethality of WNV infection was not determined with moderate- (few) level viremia. certainty. However, the severity of the disease described [82], along with the additive effects of predation avoidance and the need to forage suggest that WNV infection can Conclusions certainly be lethal for eastern chipmunks. This may represent a reason why chipmunks have been uncommonly It is clear that WNV has the potential to infect a great reported as antibody positive in the literature [33]. diversity of wild mammalian species in most regions of the world. This review tabulates at least 100 wild mammal Eastern cottontails species (including those captive in outdoor enclosures) with some evidence of natural exposure, and there are likely Eastern cottontails (Sylvilagus floridanus) experimentally several other species exposed to WNV that have not been infected with WNV developed a maximum viremia of 105.8 published or were not discovered during this review. Some

CID50s/mL with no signs of disease detected [108]. The species appear to be exposed at much greater frequencies literature on natural WNV infections in rabbits and hares is than others, which may involve one or more facets of their scant; however, antibodies have been reported from rabbits behavioral ecology. In addition, these exposures are likely and hares in Europe and Israel [2, 44, 45, 58]. In addition, a inﬂuenced by a diversity of factors, such as age, urbaniza- black-tailed jackrabbit was exposed to WNV in U.S. [76], tion, date (e.g., timing within an annual WNV cycle), and thereby suggesting that leporids are exposed to WNV in vector feeding preferences [33]. In some instances, some both hemispheres of the world. The small number of doc- mammal species may produce highly localized information umented WNV exposures in lagomorphs is surprising, associated with WNV activity [76]. 123 748 J. Jeffrey Root

The capacity of most wild mammals to be reservoir the virus were noted [84]. Others have noted that experi- competent for WNV remains undermined, as this would mentally infected golden hamsters (Mesocricetus auratus) require many more experimental infection studies or ser- yielded persistent WNV shedding in urine for up to endipitous viremic wild-caught animals to ascertain. 8 months, during which changes were reported in the virus However, studies conducted during the last decade have [107]. Additional studies are warranted to assess any role clearly shown that some mammalian species produce long-term or persistent infections play in non-traditional viremia that is sufficient for infecting some mosquito WNV cycles [109], as ingestion of virus-laden urine and species, although none as of yet have been shown to pro- predator-prey transmission have been speculated as duce the high-level viremia (e.g., [ 108 pfu/mL) that have potential transmission scenarios [110]. Notably, predator- been reported for select avian species [51]. However, some prey transmission has been documented in domestic cats recent work suggests that mammals may warrant further fed WNV-infected mice [4], and oral transmission has been scrutiny. First, viremia for fox squirrels and eastern chip- reported in a variety of vertebrates [51, 109]. munks has been reported up to 108 and 107.8 pfu/mL, Nonviremic transmission of WNV between mosquitoes respectively [76, 82]. Second, a naturally exposed fox has been described from mosquitoes co-feeding on labo- squirrel still had a viremia of 105.7 pfu/mL three days after ratory mice, suggesting that a large number of vertebrates a viremia of 108.0 pfu/mL, thereby suggesting that this could potentially play a role in mosquito infections [41]. If animal maintained a viremia [ 105.0 for 4 days [76]. this commonly occurs in wild mammals in natural settings, Because experimental infections have only been conducted many species could play a role in WNV epidemiology. on a handful of species, it remains to be determined if other Due to their potential for site fidelity, non-migratory wild mammals develop even higher viremia than has been habits, and ease of observation, select mammals have been reported previously. Thus far, based on a small number of proposed as good sentinel animals for WNV in local situ- experimental infections, select members of the rodent ations [76, 91]. For example, dead and moribund tree family Sciuridae and a single lagomorph species have been squirrels have been successfully used for WNV surveil- the only wild mammalian species yielding strong evidence lance, with the dynamics of WNV infections in tree of having the viremic capacity to make reasonable contri- squirrels reflecting that of dead birds [76]. In addition, butions to WNV mosquito cycles. However, experimental serology of mammals can also be used for the surveillance data associated with a recent study on baboons [113] of WNV in certain situations [33, 91, 93, 95]. indicate that this species might warrant more attention. Disease caused by WNV infection in wild mammals has Viral shedding, although less likely than more tradi- varied widely, both at the inter- and intraspecific levels. For tional mechanisms associated with mosquito cycles, may example, signs of disease were uncommon in experimental have the potential to perpetuate some WNV activity. For infections of tree squirrels [34, 83, 92]; however, WNV- example, positive oral swabs, fecal samples, and/or urine infected tree squirrels with severe disease have been samples have been detected in tree squirrels during commonly reported in natural settings from multiple experimental infection studies [34, 83, 92]. These obser- regions [39, 50, 76]. The reason for this discrepancy is vations, along with the demonstration of successful oral unclear. However, experimental studies could show vary- WNV transmission in fox squirrels [109] and successful ing results from natural infections and from other experi- predator-prey transmission in a domestic mammal [4], mental studies for several reasons. For example, crows suggest that viral shedding and other alternative routes of (Corvus brachyrhynchos) experimentally infected with transmission should not be completely discounted as a North American and Old World strains of WNV had higher potential transmission mechanism of this virus among viremia titers and death rates when infected with the former mammals. [10]. In addition, others have noted different responses in The role of persistent WNV infections in the epidemi- birds experimentally infected with Saint Louis encephalitis ology of this virus in mammals is undermined. However, virus when exposed to a virus of varying doses and passage some reports suggest that WNV may be present in certain history [65]. species long after their viremia has cleared. For example, Age may play a role in the severity of infections in some WNV was isolated from urine and oral swabs of fox mammal species. For example, many WNV-positive tree squirrels up to 17 and 22 DPI, respectively, and WNV squirrels in California were juvenile animals in 2005; RNA was detected in kidney tissue up to 29 DPI [83]. In a however, the authors were unable to assess if young ani- second study based on oral exposure of WNV, viral RNA mals were more susceptible to severe WNV infections than was detected in select tissues (i.e., salivary gland and/or adults or if their observation was due to the timing of late kidney) from multiple squirrels during 65-72 DPI [109]. litters of young animals [76]. Additional juvenile mammals Virus was detected in select organs of some experimentally have been reported with severe disease associated with infected rhesus macaques [ 160 DPI; however, changes in WNV infection. Of interest, two wolf pups, ages 3 and 123 West Nile virus associations in wild mammals 749

4-months-old, succumbed to WNV infection during the last Nile strains for American crows. Emerg Infect Dis 10:2161– decade [57, 60]. In contrast, severe disease in older 2168 11. Bunde JM, Heske EJ, Mateus-Pinilla NE, Hofmann JE, Novak mammals has also been reported, as acute neurologic dis- RJ (2006) A survey for West Nile virus in bats from Illinois. ease associated with WNV infection has been described in J Wildl Dis 42:455–458 a 25-year-old barbary macaque [74]. Overall, data related 12. Bunning ML, Bowen RA, Bruce Cropp C, Sullivan KG, Davis to age effects of disease associated with WNV infection in BS, Komar N, Godsey MS, Baker D, Hettler DL, Holmes DA, Biggerstaff BJ, Mitchell CJ (2002) Experimental infection of nonhuman mammals is inadequate and could be an horses with West Nile virus. Emerg Infect Dis 8:380–386 important topic for future study. 13. Chastel C, Rogues G, Beaucournu-Saguez F (1977) Enqueˆte As has been suggested previously, this review indicates se´ro-e´pide´miologique mixte arbovirus-areńavirus chez les petits that most wild mammals will likely only play a minor role mammife`res de Tunisie. Bull Soc Pathol Exot Filiales 70:471– 479 in WNV epidemiology. At present, no mammals have been 14. Chastel C, Launay H, Rogues G, Beaucournu JC (1980) Infec- shown to yield the high-level viremia noted in some avian tions a` arbovirus en Espagne: enqueˆte se´rologique chez les petits species. However, the scant number of experimental mammife`res. Bull Soc Pathol Exot Filiales 73:384–390 infection studies that have been conducted to date on wild 15. Chastel C, Launay H, Bailly Choumara H (1982) Infections a` arbovirus au Maroc: sondage se´rologique chez les petis mam- mammals suggests that this topic has not been rigorously mife`res du nord du pays. Bull Soc Pathol Exot Filiales evaluated. As such, more experimental infection studies are 75:466–475 warranted in key mammalian species, especially studies 16. Chippaux-Hyppolite C, Chippaux A (1969) Contribution a` pertaining to ubiquitous peridomestic mammals. Based on l’e´tude d’un re´servoir de virus animal dans le cycle de certains arbovirus en Centrafrique. I. Etude immunologique chez divers experimental evidence from the New World, Old World animaux domestiques et sauvages. Bull Soc Pathol Exot Filiales tree squirrels in WNV endemic areas would be an excellent 62:1034–1045 choice for future evaluations of the reservoir competence 17. Clerc Y, Rodhain F, Digoutte JP, Albignac R, Coulanges P of wild mammals. (1982) Le programme exploratoire arbovirus de l’Institut Pas- teur de Madagascar: bilan 1976–1980. Arch Inst Pasteur Mad- agascar 49:65–78 Acknowledgments I thank K. Bentler and R. McLean for excellent 18. Cohen JK, Kilpatrick AM, Stroud FC, Paul K, Wolf F, Else JG reviews of an earlier version of this manuscript, and A. Lavelle for (2007) Seroprevalence of West Nile virus in nonhuman primates assistance in obtaining literature. The opinions and conclusions of this as related to mosquito abundance at two national primate article are those of the author and do not necessarily represent those of research centers. Comp Med 57:115–119 the U.S. Department of Agriculture. 19. Darwish MA, Hoogstraal H, Roberts TJ, Ahmed IP, Omar F (1983) A sero-epidemiological survey for certain arboviruses (Togaviridae) in Pakistan. Trans R Soc Trop Med Hyg References 77:442–445 20. Davis A, Bunning M, Gordy P, Panella N, Blitvich B, Bowen R 1. Abdel-Wahab KS, Imam IZ (1970) Antibodies to arboviruses in (2005) Experimental and natural infection of North American rodent sera. J Egypt Public Health Assoc 45:370–375 bats with West Nile virus. Am J Trop Med Hyg 73:467–469 2. Akov Y, Goldwasser R (1966) Prevalence of antibodies to 21. Dietrich G, Montenieri JA, Panella NA, Langevin S, Lasater SE, arboviruses in various animals in Israel. Bull World Health Klenk K, Kile JC, Komar N (2005) Serologic evidence of West Organ 34:901–909 Nile virus infection in free-ranging mammals, Slidell, Louisiana, 3. Andral L, Bre`sP,Se´rie´ C, Casals J, Panthier R (1968) Etudes sur 2002. Vector Borne Zoonotic Dis 5:288–292 la fie`vre jaune en Ethiopie. 3. Etude se´rologique et virologique 22. Docherty DE, Samuel MD, Nolden CA, Egstad KF, Griffin KM de la faune sylvatique. Bull World Health Organ 38:855–861 (2006) West Nile virus antibody prevalence in wild mammals, 4. Austgen LE, Bowen RA, Bunning ML, Davis BS, Mitchell CJ, Southern Wisconsin. Emerg Infect Dis 12:1982–1984 Chang GJJ (2004) Experimental infection of cats and dogs with 23. Docherty DE, Samuel MD, Egstad KF, Griffin KM, Nolden CA, West Nile virus. Emerg Infect Dis 10:82–86 Karwal L, Ip HS (2009) Short report: changes in West Nile virus 5. Bakonyi T, Ivanics E´ , Erde´lyi K, Ursu K, Ferenczi E, Weiss- seroprevalence and antibody titers among Wisconsin mesopre- enbo¨ck H, Nowotny N (2006) Lineage 1 and 2 strains of dators 2003–2006. Am J Trop Med Hyg 81:177–179 encephalitic West Nile virus, Central Europe. Emerg Infect Dis 24. Drebot MA, Lindsay R, Barker IK, Buck PA, Fearon M, Hunter 12:618–623 F, Sockett P, Artsob H (2003) West Nile virus surveillance and 6. Bentler KT, Hall JS, Root JJ, Klenk K, Schmit B, Blackwell BF, diagnostics: a Canadian perspective. Can J Infect Dis Ramey PC, Clark L (2007) Serologic evidence of West Nile 14:105–114 virus exposure in North American mesopredators. Am J Trop 25. Duca M, Duca E, Buiuc D, Luca V (1989) Studiul seroepi- Med Hyg 76:173–179 demiologic s¸i virologic al prezent¸ei unor arbovirusuri pe teri- 7. Blitvich BJ (2008) Transmission dynamics and changing epi- toriul Moldovei, 1961–1982. Rev Med Chir Soc Med Nat Iasi demiology of West Nile virus. Anim Health Res Rev 9:71–86 93:719–733 8. Blitvich BJ, Juarez LI, Tucker BJ, Rowley WA, Platt KB (2009) 26. Dutton CJ, Quinnell M, Lindsay R, Delay J, Barker IK (2009) Antibodies to West Nile virus in raccoons and other wild peri- Paraparesis in a polar bear (Ursus maritimus) associated with domestic mammals in Iowa. J Wildl Dis 45:1163–1168 West Nile virus infection. J Zoo Wildl Med 40:568–571 9. Bowen RA, Nemeth NM (2007) Experimental infections with 27. Farajollahi A, Panella NA, Carr P, Crans W, Burguess K, Komar West Nile virus. Curr Opin Infect Dis 20:293–297 N (2003) Serologic evidence of West Nile virus infection in 10. Brault AC, Langevin SA, Bowen RA, Panella NA, Biggerstaff black bears (Ursus americanus) from New Jersey. J Wildl Dis BJ, Miller BR, Komar N (2004) Differential virulence of West 39:894–896

123 750 J. Jeffrey Root

28. Farajollahi A, Gates R, Crans W, Komar N (2004) Serologic serological studies on, wild and domestic vertebrates from Kano evidence of West Nile virus and St. Louis encephalitis virus Plain, Kenya. Trans R Soc Trop Med Hyg 71:512–517 infections in white-tailed deer (Odocoileus virginianus) from 44. Jurˇicova´ Z (1992) Protila´tky proti arboviru˚m u lovne´ zveˇre New Jersey, 2001. Vector Borne Zoonotic Dis 4:379–383 odchycene´ na Moraveˇ. Vet Med (Praha) 37:633–636 29. Farfań-Ale JA, Blitvich BJ, Marlenee NL, Loronõ-Pino MA, 45. Jurˇicova´ Z, Huba´lek Z (1999) Serological surveys for arbovi- Puerto-Manzano F, Garcıá-Rejoń JE, Rosado-Paredes EP, Flo- ruses in the game animals of southern Moravia (Czech Repub- res-Flores LF, Ortega-Salazar A, Cha´vez-Medina J, Cremieux- lic). Folia Zool 48:185–189 Grimaldi JC, Correa-Morales F, Hernańdez-Gaona G, Meńdez- 46. Karabatos N (ed) (1985) International catalogue of arboviruses, Galvań JF, Beaty BJ (2006) Antibodies to West Nile virus in including certain other viruses of vertebrates, 3rd edn. American asymptomatic mammals, birds, and reptiles in the Yucatan Society of Tropical Medicine and Hygiene, San Antonio Peninsula of Mexico. Am J Trop Med Hyg 74:908–914 47. Kaul HN, Venkateshan CN, Mishra AC (1976) Serological 30. Fontenille D, Rodhain F, Digoutte JP, Mathiot C, Morvan J, evidence of arbovirus activity in birds and small mammals in Coulanges P (1989) Les cycles de transmission du virus West- Japanese encephalitis affected areas of Bankura District, West Nile a` Madagascar, Oceán Indien. Ann Soc Belg Med Trop Bengal. Indian J Med Res 64:1735–1739 69:233–243 48. Keller M (2005) Development of a competitive inhibition 31. Franson JC, Hofmeister EK, Collins GH, Dusek RJ (2011) Short enzyme-linked immunosorbent assay (CI ELISA) for serosurvey report: Seroprevalence of West Nile virus in feral horses on of wildlife species for West Nile virus emphasizing marine Sheldon National Wildlife Refuge, Nevada, United States. Am J mammals. Veterinary Medicine. University of Florida, p 102 Trop Med Hyg 84:637–640 49. Kemp GE, Causey OR, Setzer HW, Moore DL (1974) Isolation 32. Gibbs SEJ, Marlenee NL, Romines J, Kavanaugh D, Corn JL, of viruses from wild mammals in West Africa, 1966–1970. Stallknecht DE (2006) Antibodies to West Nile virus in feral J Wildl Dis 10:279–293 swine from Florida, Georgia, and Texas, USA. Vector Borne 50. Kiupel M, Simmons HA, Fitzgerald SD, Wise A, Sikarskie JG, Zoonotic Dis 6:261–265 Cooley TM, Hollamby SR, Maes R (2003) West Nile virus 33. Go´mez A, Kilpatrick AM, Kramer LD, Dupuis Ii AP, Maffei JG, infection in eastern fox squirrels (Sciurus niger). Vet Pathol Goetz SJ, Marra PP, Daszak P, Aguirre AA (2008) Land use and 40:703–707 West Nile virus seroprevalence in wild mammals. Emerg Infect 51. Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler Dis 14:962–965 D, Davis B, Bowen R, Bunning M (2003) Experimental infec- 34. Go´mez A, Kramer LD, Dupuis Ii AP, Kilpatrick AM, Davis LJ, tion of North American birds with the New York 1999 strain of Jones MJ, Daszak P, Aguirre AA (2008) Experimental infection West Nile virus. Emerg Infect Dis 9:311–322 of eastern gray squirrels (Sciurus carolinensis) with West Nile 52. Komar N, Clark GG (2006) West Nile virus activity in Latin virus. Am J Trop Med Hyg 79:447–451 America and the Caribbean. Rev Panam Salud Publica 35. Goverdhan MK, Kulkarni AB, Gupta AK, Tupe CD, Rodrigues 19:112–117 JJ (1992) Two-way cross-protection between West Nile and 53. Konstantinov OK, Diallo SM, Inapogi AP, Ba A, Kamara SK Japanese encephalitis viruses in bonnet macaques. Acta Virol (2006) The mammals of Guinea as reservoirs and carriers of 36:277–283 arboviruses. Med Parazitol (Mosk):34–39 36. Halouzka J, Juricova Z, Jankova J, Hubalek Z (2008) Serologic 54. Koptopoulos G, Papadopoulos O (1980) Zooanthroponoses in survey of wild boars for mosquito-borne viruses in South Greece. A serological survey for antibodies to the arboviruses of Moravia (Czech Republic). Vet Med (Praha) 53:266–271 tick-borne encephalitis and West Nile. In: Vesenjak-Hirjan J 37. Hayes CG, Baqar S, Ahmed T, Chowdhry MA, Reisen WK (ed) Arboviruses in the Mediterranean Countries, Zbl Bakt (1982) West Nile virus in Pakistan. 1. Sero-epidemiological Suppl 9. Gustav Fischer Verlag, Stuttgart, pp 185–188 studies in Punjab Province. Trans R Soc Trop Med Hyg 55. Kramer LD, Bernard KA (2001) West Nile virus infection in 76:431–436 birds and mammals. Ann N Y Acad Sci 951:84–93 38. Hayes CG (1988) West Nile Fever. In: Monath TP (ed) The 56. Kramer LD, Styer LM, Ebel GD (2008) A global perspective on Arboviruses: epidemiology and ecology. CRC Press, Boca the epidemiology of West Nile virus. Annu Rev Entomol Raton, pp 59–88 53:61–81 39. Heinz-Taheny KM, Andrews JJ, Kinsel MJ, Pessier AP, Pink- 57. Lanthier I, He´bert M, Tremblay D, Harel J, Dallaire AD, Girard erton ME, Lemberger KY, Novak RJ, Dizikes GJ, Edwards E, C (2004) Natural West Nile virus infection in a captive juvenile Komar N (2004) West Nile virus infection in free-ranging Arctic wolf (Canis lupus). J Vet Diagn Invest 16:326–329 squirrels in Illinois. J Vet Diagn Invest 16:186–190 58. Le Lay-Rogues G, Arthur CP, Vanderwalle P, Hardy E, Chasterl 40. Henderson BE, Cheshire PP, Kirya GB, Lule M (1970) Immu- C (1990) Wild rabbit, Oryctolagus cuniculus L., and arboviruses nologic studies with yellow fever and selected African group B in southeast France. Results of two serologic investigations. Bull arboviruses in rhesus and vervet monkeys. Am J Trop Med Hyg Soc Pathol Exot 83:446–457 19:110–118 59. Le Lay Rogues G, Valle M, Chastel C, Beaucournu JC (1983) 41. Higgs S, Schneider BS, Vanlandingham DL, Klingler KA, Petits mammiferes sauvages et arbovirus en Italie. Bull Soc Gould EA (2005) Nonviremic transmission of West Nile virus. Pathol Exot 76:333–345 Proc Natl Acad Sci USA 102:8871–8874 60. Lichtensteiger CA, Heinz-Taheny K, Osborne TS, Novak RJ, 42. Hukkanen RR, Liggitt HD, Kelley ST, Grant R, Anderson DM, Lewis BA, Firth ML (2003) West Nile virus encephalitis and Hall RA, Tesh RB, DaRosa APT, Bielefeldt-Ohmann H (2006) myocarditis in wolf and dog. Emerg Infect Dis 9:1303–1306 West Nile and St. Louis encephalitis virus antibody serocon- 61. Ludwig GV, Calle PP, Mangiafico JA, Raphael BL, Danner DK, version, prevalence, and persistence in naturally infected pig- Hile JA, Clippinger TL, Smith JF, Cook RA, McNamara T tailed macaques (Macaca nemestrina). Clin Vaccine Immunol (2002) An outbreak of West Nile virus in a New York City 13:711–714 captive wildlife population. Am J Trop Med Hyg 67:67–75 43. Johnson BK, Chanas AC, Shockley P, Squires EJ, Gardner P, 62. Madic´ J, Huber D, Lugovic´ B (1993) Serologic survey for Wallace C, Simpson DIH, Bowen ETW, Platt GS, Way H, selected viral and rickettsial agents of brown bears (Ursus arc- Parsons J, Grainger WE (1977) Arbovirus isolations from, and tos) in Croatia. J Wildl Dis 29:572–576

123 West Nile virus associations in wild mammals 751

63. Marfin AA, Petersen LR, Eidson M, Miller J, Hadler J, Farello 80. Petersen LR, Roehrig JT (2001) West Nile virus: a reemerging C, Werner B, Campbell GL, Layton M, Smith P, Bresnitz E, global pathogen. Emerg Infect Dis 7:611–614 Cartter M, Scaletta J, Obiri G, Bunning M, Craven RC, Roehrig 81. Pilipski JD, Pilipski LM, Risley LS (2004) West Nile virus JT, Julian KG, Hinten SR, Gubler DJ, Hilger T, Jones JE, antibodies in bats from New Jersey and New York. J Wildl Dis Lehman JA, Medlin K, Morris T, Perilla MJ, Sutliff S, Withum 40:335–337 D, Sorhage F, Tan C, Beckett G, Gensheimer K, Greenblatt J, 82. Platt KB, Tucker BJ, Halbur PG, Tiawsirisup S, Blitvich BJ, Montero J, Galbraith P, Tassler P, DeMaria A, Matyas B, Fabiosa FG, Bartholomay LC, Rowley WA (2007) West Nile Timperi R, Bandy U, Breslosky T, Andreadis T, McCarthy T, virus viremia in eastern chipmunks (Tamias striatus) sufficient Backenson B, Hagiwara Y, Kramer L, Morse D, Wallace B, for infecting different mosquitoes. Emerg Infect Dis 13:831–837 White D, Willsey A, Wong S, Cherry B, Fine A, Kellachan J, 83. Platt KB, Tucker BJ, Halbur PG, Blitvich BJ, Fabiosa FG, Kulakasera V, Poshni I, Rankin J, Hathcock L, Wolfe D, Roche Mullin K, Parikh GR, Kitikoon P, Bartholomay LC, Rowley J, Jenkins S, Levy M, MacCormack JN, Gibson J, Blake P, WA (2008) Fox squirrels (Sciurus niger) develop West Nile Kramer S, Lance-Parker S, Conti L, Hopkins RS, Oliveri R, virus viremias sufficient for infecting select mosquito species. Lofgren JP, Woernle CH, Currier M, Slavinski S, Kelso K, Vector Borne Zoonotic Dis 8:225–233 Rawlings J (2001) Widespread West Nile virus activity, eastern 84. Pogodina VV, Frolova MP, Malenko GV (1983) Study on West United States, 2000. Emerg Infect Dis 7:730–735 Nile virus persistence in monkeys. Arch Virol 75:71–86 64. McIntosh BM (1961) Susceptibility of some African wild 85. ProMED-mail (2002) West Nile virus update 2002-USA (28). rodents to infection with various arthropod-borne viruses. Trans ProMED-mail Archive number 20021031.5674 R Soc Trop Med Hyg 55:63–68 86. ProMED-mail (2002) West Nile virus, Caprine and Ovine-USA 65. McLean RG, Mullenix J, Kerschner J, Hamm J (1983) The (NE) (04). ProMED-mail Archive number 20020920.5368 house sparrow (Passer domesticus) as a sentinel for St. Louis 87. Ratterree MS, Travassos da Rosa APA, Bohm RP Jr, Cogswell encephalitis virus. Am J Trop Med Hyg 32:1120–1129 FB, Phillippi KM, Caillouet K, Schwanberger S, Shope RE, 66. McLean RG, Ubico SR, Bourne D, Komar N (2002) West Nile Tesh RB (2003) West Nile virus infection in nonhuman primate virus in livestock and wildlife. Curr Top Microbiol Immunol breeding colony, concurrent with human epidemic, southern 267:271–308 Louisiana. Emerg Infect Dis 9:1388–1394 67. Miller DL, Radi ZA, Baldwin C, Ingram D (2005) Fatal West 88. Ratterree MS, Gutierrez RA, Travassos Da Rosa APA, Dille BJ, Nile virus infection in a white-tailed deer (Odocoileus virgini- Beasley DWC, Bohm RP, Desai SM, Didier PJ, Bikenmeyer anus). J Wildl Dis 41:246–249 LG, Dawson GJ, Leary TP, Schochetman G, Phillippi-Falken- 68. MMWR (2000) Update: West Nile Virus Activity—Eastern stein K, Arroyo J, Barrett ADT, Tesh RB (2004) Experimental United States, 2000. Morb Mortal Wkly Rep 49:1044–1047 infection of rhesus macaques with West Nile virus: level and 69. MMWR (2002) Provisional surveillance summary of the West duration of viremia and kinetics of the antibody response after Nile virus epidemic—United States, January–November 2002. Infection. J Infect Dis 189:669–676 Morb Mortal Wkly Rep 51:1129–1133 89. Rodhain F, Clerc Y, Albignac R, Ricklin B, Ranaivosata J, 70. MMWR (2004) West Nile Virus Activity—United States, Coulanges P (1982) Arboviruses and lemurs in Madagascar: a November 3–8, 2004. Morb Mortal Wkly Rep 53:1050–1051 preliminary note. Trans R Soc Trop Med Hyg 76:227–231 71. MMWR (2006) West Nile Virus Activity—United States, Jan- 90. Rodhain F, Petter JJ, Albignac R (1985) Arboviruses and lemurs in uary 1–August 15, 2006. Morb Mortal Wkly Rep 55:879–880 Madagascar: experimental infection of Lemur fulvus with yellow 72. Molna´r E, Gresı´kova´ M, Kuba´sova T, Kubinyi L, Szabo´ JB fever and West Nile viruses. Am J Trop Med Hyg 34:816–822 (1973) Arboviruses in Hungary. J Hyg Epidemiol Microbiol 91. Root JJ, Hall JS, McLean RG, Marlenee NL, Beaty BJ, Gan- Immunol 17:1–10 sowski J, Clark L (2005) Serologic evidence of exposure of wild 73. Molna´r E, Gulya´s MS, Kubinyi L, Nosek J, Kozuch O, Ernek E, mammals to flaviviruses in the central and eastern United States. Labuda M, Grulich I (1976) Studies on the occurrence of tick- Am J Trop Med Hyg 72:622–630 borne encephalitis in Hungary. Acta Vet Acad Sci Hung 92. Root JJ, Oesterle PT, Nemeth NM, Klenk K, Gould DH, 26:419–437 McLean RG, Clark L, Hall JS (2006) Experimental infection of 74. Ølberg RA, Barker IK, Crawshaw GJ, Bertelsen MF, Drebot fox squirrels (Sciurus niger) with West Nile virus. Am J Trop MA, Andonova M (2004) West Nile virus encephalitis in a Med Hyg 75:697–701 barbary macaque (Macaca sylvanus). Emerg Infect Dis 93. Root JJ, Oesterle PT, Sullivan HJ, Hall JS, Marlenee NL, 10:712–714 McLean RG, Montenieri JA, Clark L (2007) Fox squirrel 75. Osterrieth PM, Deleplanque-Liegeois P (1961) Presence d’ an- (Sciurus niger) associations with West Nile virus. Am J Trop ticorps vis * a*vis des virus transmis par arthropodes chez le Med Hyg 76:782–784 chimpanze (Pan troglodites). Comparaison de leur etat im- 94. Root JJ, Bentler KT, Nemeth NM, Gidlewski T, Spraker TR, munitaire a celui de l’homme. Ann Soc Belg Med Trop 1:63–72 Franklin AB (2010) Experimental infection of raccoons (Pro- 76. Padgett KA, Reisen WK, Kahl-Purcell N, Fang Y, Cahoon- cyon lotor) with West Nile virus. Am J Trop Med Hyg Young B, Carney R, Anderson N, Zucca L, Woods L, Husted S, 83:803–807 Kramer VL (2007) West Nile virus infection in tree squirrels 95. Santaella J, McLean R, Hall JS, Gill JS, Bowen RA, Hadow HH, (Rodentia: Sciuridae) in California, 2004–2005. Am J Trop Med Clark L (2005) West Nile virus serosurveillance in Iowa white- Hyg 76:810–813 tailed deer (1999–2003). Am J Trop Med Hyg 73:1038–1042 77. Palmer MV, Stoffregen WC, Rogers DG, Hamir AN, Richt JA, 96. Savage HM, Anderson M, Gordon E, McMillen L, Colton L, Pedersen DD, Waters WR (2004) West Nile virus infection in Delorey M, Sutherland G, Aspen S, Charnetzky D, Burkhalter reindeer (Rangifer tarandus). J Vet Diagn Invest 16:219–222 K, Godsey M (2008) Host-seeking heights, host-seeking activity 78. Papa A, Bakonyi T, Xanthopoulou K, Va´zquez A, Tenorio A, patterns, and West Nile virus infection rates for members of the Nowotny N (2011) Genetic characterization of West Nile virus Culex pipiens complex at different habitat types within the lineage 2, Greece, 2010. Emerg Infect Dis 17:920–922 hybrid zone, Shelby County, TN, 2002 (Diptera: Culicidae). 79. Paul SD, Rajagopalan PK, Sreenivasan MA (1970) Isolation of J Med Entomol 45:276–288 the West Nile virus from the frugivorous bat, Rousettus lesch- 97. Schaefer AM, Reif JS, Goldstein JD, Ryan CN, Fair PA, Bossart enaulti. Indian J Med Res 58:1169–1171 GD (2009) Serological evidence of exposure to selected viral,

123 752 J. Jeffrey Root

bacterial, and protozoal pathogens in free-ranging atlantic bot- 107. Tesh RB, Siirin M, Guzman H, Travassos Da Rosa APA, Wu X, tlenose dolphins (Tursiops truncatus) from the Indian River Duan T, Lei H, Nunes MR, Xiao SY (2005) Persistent West Nile Lagoon, Florida, and Charleston, South Carolina. Aquat Mamm Virus infection in the golden hamster: studies on its mechanism 35:163–170 and possible implications for other flavivirus infections. J Infect 98. Shepherd RC, Williams MC (1964) Studies on viruses in East Dis 192:287–295 African bats (Chiroptera). 1. Haemagglutination inhibition and 108. Tiawsirisup S, Platt KB, Tucker BJ, Rowley WA (2005) Eastern circulation of arboviruses. Zoonoses Res 3:125–139 cottontail rabbits (Sylvilagus floridanus) develop West Nile 99. Sherif NEDH (2007) Serological evidence of antibodies to virus viremias sufficient for infecting select mosquito species. certain arboviruses in desert rodent sera in Egypt. Egypt J Hosp Vector Borne Zoonotic Dis 5:342–350 Med 28:342–346 109. Tiawsirisup S, Blitvich BJ, Tucker BJ, Halbur PG, Bartholomay 100. Simpson DI, O’Sullivan JP (1968) Studies on arboviruses and LC, Rowley WA, Platt KB (2010) Susceptibility of fox squirrels bats (Chiroptera) in East Africa. I. Experimental infection of (Sciurus niger) to West Nile virus by oral exposure. Vector bats and virus transssion attempts in Aedes (Stegomyia) aegypti Borne Zoonotic Dis 10:207–209 (Linnaeus). Ann Trop Med Parasitol 62:422–431 110. Tonry JH, Xiao SY, Siirin M, Chen H, Travassos Da Rosa APA, 101. Simpson DI, Williams MC, O’Sullivan JP, Cunningham JC, Tesh RB (2005) Persistent shedding of West Nile virus in urine Mutere FA (1968) Studies on arboviruses and bats (Chiroptera) of experimentally infected hamsters. Am J Trop Med Hyg in East Africa. II. Isolation and haemagglutination-inhibition 72:320–324 studies on bats collected in Kenya and throughout Uganda. Ann 111. Wertheimer AM, Uhrlaub JL, Hirsch A, Medigeshi G, Sprague Trop Med Parasitol 62:432–440 J, Legasse A, Wilk J, Wiley CA, Didier P, Tesh RB, Murray 102. Sixl W, Kock M, Withalm H, Stunzner D, Sixl-Voigt B (1989) KO, Axthelm MK, Wong SW, Nikolich-Zˇ ugich J (2010) Serological investigations of small mammals in waste disposal Immune response to the West Nile virus in aged non-human sites in Austria. Geographia Medica Suppl 2:65–68 primates. PLoS ONE 5:e15514 103. Smithburn KC, Hughes TP, Burke AW, Paul JH (1940) A 112. Wilson DE, Reeder DM (2005) Mammal species of the world. A neurotropic virus isolated form the blood of a native of Uganda. taxonomic and geographic reference, 3rd edn. Johns Hopkins Am J Trop Med Hyg 20:471–492 University Press, Baltimore 104. Sondgeroth K, Blitvich B, Blair C, Terwee J, Junge R, Sauther 113. Wolf RF, Papin JF, Hines-Boykin R, Chavez-Suarez M, White M, VandeWoude S (2007) Assessing flavivirus, lentivirus, and GL, Sakalian M, Dittmer DP (2006) Baboon model for West herpesvirus exposure in free-ranging ring-tailed lemurs in Nile virus infection and vaccine evaluation. Virology 355:44–51 southwestern Madagascar. J Wildl Dis 43:40–47 114. Zeller HG, Schuffenecker I (2004) West Nile virus: an overview 105. St. Leger J, Wu G, Anderson M, Dalton L, Nilson E, Wang D of its spread in Europe and the Mediterranean basin in contrast (2011) West Nile virus infection in killer whale, Texas, USA, to its spread in the Americas. Eur J Clin Microbiol Infect Dis 2007. Emerg Infect Dis 17:1531–1533 23:147–156 106. Taylor RM, Work TH, Hurlbut HS, Rizk F (1956) A study of the ecology of West Nile virus in Egypt. Am J Trop Med Hyg 5:579–620

123 Rev. sci. tech. Off. int. Epiz., 2012, 31 (3), 829-844

West Nile virus epidemiology and factors triggering change in its distribution in Europe

S. Pradier (1, 3), S. Lecollinet (3) & A. Leblond (2, 4)

(1) Clinique équine, Ecole nationale vétérinaire d’Alfort, 7 Av. du Général de Gaulle, Maisons-Alfort F-94704, France (2) Institut national de la recherche agronomique, UR346 Epidémiologie animale, Centre de recherche de Clermond-Ferrand/Theix, Saint-Genès-Champanelle F-63122, France (3) Université Paris-Est, Ecole nationale vétérinaire d’Alfort, Unités mixtes de recherche 1161 Virologie, Institut national de la recherche agronomique, Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail, 23 Av. du Général de Gaulle, Maisons-Alfort F-94704, France (4) Département hippique, VetAgro Sup Campus vétérinaire de Lyon, 1 Av. Bourgelat, Marcy-l’Étoile F-69280, France

Summary West Nile virus (WNV) has historically been considered among the least virulent members of the Japanese serogroup complex (family Flaviviridae, genus Flavivirus). The WNV natural cycle involves birds as the main amplifying hosts and several species of mosquito as vectors. Many outbreaks occurred during the past decade, causing severe human encephalitis in the Old World, and the virus has become established in many European countries. Emergence of WNV is difficult to predict and even more difficult to prevent. In this review, the latest information on the epidemiology, transmission dynamics and clinical aspects of WNV is presented, with particular focus on specific factors likely to trigger changes in the distribution of the disease in Europe, such as climate changes and their consequences on the potential vectors of WNV or bird migration routes. The control of some anthropogenic and environmental factors could help prevent extension and re-emergence of WNV epidemics.

Keywords Birds – Encephalitis – Epidemiology – Flavivirus – Horses – Mosquitoes – Vector-borne disease – West Nile virus – Zoonosis.

occurred in urban areas where cellars flooded with sewage- Introduction polluted water were highly productive breeding sites for an effective vector, Culex pipiens (107). Outbreaks on this scale West Nile virus (WNV) is a flavivirus transmitted in natural have also occurred in Israel (114). cycles between birds and mosquitoes, particularly Culex spp. (Fig. 1) (104), and was first isolated in Since 2008, outbreaks have been reported in several 1937 from the blood of a woman suffering from a mild countries of Europe with neuroinvasive human cases febrile illness in the West Nile district of Uganda (99). In potentially associated with equine cases. In Italy between the Old World, many outbreaks causing severe human 2008 and October 2011, 39 human neuroinvasive cases encephalitis were observed during the mid-1990s and associated with two fatalities were observed, together with early 2000s: Algeria 1994 (50 cases, including 8 fatalities), 191 equine cases and 19 fatalities (8, 15, 29, 87). Strains of Morocco 1996 (one fatal case), Romania 1996 (393 cases, WNV isolated in 2008 and 2009 from samples of equids 17 fatalities), Tunisia 1997 (111 cases, 8 fatalities), and birds (one pigeon, three magpies) were genetically very Russia 1999–2001 (361 cases, 40 fatalities), close to the 1998 Tuscan strain, as well as to isolates from Israel 2000 (326 cases, 33 fatalities) and France 2003 Romania, Russia, Senegal and Kenya (67, 94). In Spain (7 cases, 0 fatalities) (10, 23, 70, 79, 95, 106, 107) (Fig. 2). (Andalusia) in 2010 and 2011, two non-fatal human cases In Bucharest (Romania) and Volgograd (Russia) epidemics and 42 equine cases with ten fatalities were reported for the 830 Rev. sci. tech. Off. int. Epiz., 31 (3)

first time in that country (83). In the same period, large has become newly established in several countries and outbreaks occurred in Greece (322 human neuroinvasive there is little likelihood that it can be eliminated. This cases, including 39 fatalities) and Russia (596 human paper discusses factors that could be involved in changes neuroinvasive cases, including 6 fatalities) (83). Other recently observed in the distribution of WNV in Europe, countries such as Albania, Bulgaria, Macedonia, Portugal, with particular reference to risk assessment models. Romania and Turkey were also affected by the disease (83). In Italy, only limited reports of avian mortality have been available until now (15), in contrast to the situation in the United States (USA) since 1999, Israel in 1998 and Disease transmission Hungary in 2004–2005 (10, 31, 64). Some of these European outbreaks were associated with the emergence of and epidemiology lineage 2 viruses never isolated in Europe before 2004 (1, 20, 57, 78). Molecular epidemiology West Nile virus was first detected in the Western West Nile virus is mosquito borne and belongs to the Hemisphere in 1999 in New York City (58). Subsequently, genus Flavivirus, family Flaviviridae (70). European the virus spread across continental USA, leading to epidemics may be initiated by the introduction of variants unparalleled morbidity and mortality rates in humans and from Africa carried north by migratory birds, although equids, then continued its progression northward into introduction from Europe to Africa cannot be ruled out. Canada and southward into the Caribbean Islands and Phylogenetic studies have shown the existence of two main Latin America (55, 115, 116). In the USA, 1,263 fatal cases lineages (9). Lineage 1 includes WNV strains from Africa, and 31,392 reported cases of WNV infection occurred Europe, the Middle East, North America, India and between 1999 and 2011 (19). Currently, WNV is Australia. Until the mid-2000s, lineage 2 was considered to considered endemic throughout much of the Western have low pathogenicity and its distribution was restricted Hemisphere. to African countries. However, lineage 2 was isolated for the first time from birds in Hungary in 2004 and 2005, West Nile disease will most probably continue to be a demonstrating enzootic circulation in this area (31). Both public health concern. Because the virus has the most lineages have since been described in Hungary and the widespread geographical distribution and the largest vector disease reported in wild and domestic birds, and in sheep and host range of all mosquito-borne flaviviruses (116), it and horses (57). More recently, outbreaks caused by

Migratory birds Migratory birds

Introduction Spread Incidental hosts Horses Wild or domestic birds Non strictly ornithophilic mosquitoes Amplification = (Culex sp.) Enzootic cycle between or bridge vectors reservoir hosts and Direct (Aedes sp.) ? transmission ornithophilic mosquitoes Culex sp. (oro-faecal)

Humans Wild or Wild or domestic birds domestic birds

Long-term perpetuation Potential

secondary amplification Other potential Chronic infection Vertical transmission hosts (squirrels, Continual (birds or others) in mosquitoes rabbits, alligators…) transmission

Fig. 1 West Nile virus transmission cycle Rev. sci. tech. Off. int. Epiz., 31 (3) 831

Greece

Countries in red: confirmed human cases of WNV Countries in yellow: WNV antibodies in vertebrates Fig. 2 Geographical distribution of West Nile virus in the Old World lineage 2 have been reported in other European countries Phylogenetic analysis suggests a common evolution such as Russia (78), Romania (97), Greece (109) and Italy between this Spanish strain and lineage 4 virus. (1). Of note is that lineage 2 WNV has also been associated Lineage 3 and 4 strains have not been associated with with fatal neurological cases in horses and humans in natural disease in animals or humans. South Africa, in 2002 and 2009 (14, 111). In summary, a great diversity of WNV strains is encountered in Europe and virulence data on the newly The virus isolated in New York City in 1999 was closely described strains begin to be available (100). related to a strain isolated in Israel a year earlier (58). As a result of the rapid evolution and adaptation of WNV, genetic variants of the NY99 strain have been identified in Texas since 2002. Moreover, these new variants replaced Mosquito (Diptera: Culicidae) transmission the initial one in just a few years (22, 28) as they have a Enzootic cycle selective advantage arising from a shorter extrinsic incubation period in Culex mosquitoes, thus enhancing In Europe and Africa, the main enzootic vectors are their transmission speed and efficacy (68). Subclades of Cx. pipiens, Cx. modestus, Cx. univittatus and Cx. antennatus lineage 1 occur in Europe, America, the Middle East, (5, 41). Infectious mosquitoes carry WNV in their salivary Africa, Asia (lineage 1a) and Australia (lineage 1b), and a glands and are thus able to infect susceptible vertebrate distinct lineage has also been identified in India (lineage 5) hosts during feeding. Virus can be detected in almost all (11) (Fig. 3). Strains isolated in central Europe and tissues of the mosquito, particularly in the brain and Russia were classified as new lineages of WNV ventral nerve cord with its associated ganglia. The infection (lineages 3 and 4 respectively) (2), but their taxonomic of vectors occurs mostly through feeding on viraemic status is currently unclear. In Spain, a putative new lineage hosts; however, transovarial transmission has been shown of WNV was recently detected in pooled Cx. pipiens (110). to occur in a small proportion of infected Cx. pipiens (25). 832 Rev. sci. tech. Off. int. Epiz., 31 (3)

Even at these low transmission rates, transovarial both temperature and humidity (21). After this period, transmission may have an important role in the ecology of mosquitoes can transmit the virus to susceptible hosts. the virus. Maintenance of an outbreak is highly dependent on the After infection, a temperature-dependent extrinsic longevity of the mosquitoes and the speed of development incubation period ensues, during which virus replicates of the virus, both influenced by temperature. An optimum and enters the salivary glands. Typically, this period lasts temperature is probably necessary for maintenance of a for two weeks during warm periods, but is sensitive to WNV outbreak, but this remains to be determined.

100 Italy 2009 (human) GU011992 71 Italy 2008 (magpie) FJ483549 100 France 2004 (magpie) DQ786573 59 Morocco 2003 (horse) AY701413 46 Italy 1998 (horse) AF404757 100 France 2000 (horse) AY268132

100 Morocco 1996 (horse) AY701412 Kenya 1998 (C. univittatus) AY262283 Romania 1996 (C. pipiens) AF260969 100 88 Russia 1999 (human) AF317203 Clade 1a Lineage 1 Tunisia 1997 (human) AY268133 Hungary 2003 (goose) DQ118127 100 Texas 2002 (human) AY289214 100 100 100 Israël 1998 (stork) AF481864 59 New York 1999 (flamingo) AF196835 New York 1999 (human) AF202541 83 100 China 2001 AY490240 100 Egypt 1951 Eg101 (human) AF260968 100 100 India 1968 (fruit bat) EU249803 Kunjin 1980 (C. annulirostris) D00246 Clade 1b 99 India 1980 (human) DQ256376 Lineage 5 Sarafend AY688948 100 South Africa 2000 (human) EF429199 75 100 Hungary 2004 (goshawk) DQ116961 Lineage 2 66 South Africa 1958 H442 (human) EF429 200 95 Uganda 1937 B956 (human) NC 001563 RabV 1997 (C. pipiens) AY765264 Lineage 3 Lineage 4 Russia 1998 LEIV-Kmd88-190 (Dermacentor marginatus) AY277251 Japanese encephalitis virus (JEV) GP78 AF075723 0,05 Fig. 3 Phylogenetic tree of West Nile virus strains, based on their complete genomic sequence GenBank accession numbers are indicated on tree branches for each viral strain The tree was constructed with the MEGA program (Molecular Evolutionary Genetics Analysis) by neighbour-joining with Jukes–Cantor parameter distances (scale bar). Bootstrap confidence level (1,000 replicates) and a confidence probability value based on the standard error test were calculated using MEGA. Japanase encephalitis virus, a close flavivirus, was used as an outgroup to root the phylogenetic tree Rev. sci. tech. Off. int. Epiz., 31 (3) 833

Bridge vectors without any case of West Nile fever in horses or humans during that time (4). Bridge vectors that are distinct from enzootic vector species are not involved in the maintenance of WNV, because of Vertical transmission of WNV in Culex and Aedes their feeding preferences (predominantly mammals), but mosquitoes has been demonstrated experimentally (7) and can be implicated in the epizootic transmission of WNV the virus has been isolated from field-collected larvae of from birds to humans and/or horses. To determine the Cx. univittatus in Kenya (66). Whether these low rates of potential for a mosquito species to be an efficient vector, it vertical transmission provide an effective way to perpetuate is necessary to consider not only its vector competence for the virus remains unclear. WNV under laboratory conditions but also its abundance, host-feeding preference, infection with other viruses with similar transmission cycles and whether WNV has been Birds, natural hosts for West Nile virus isolated from this species under natural conditions Competent birds will sustain an infectious viraemia (52, 108). for 1 to 4 days after exposure, after which time they develop life-long immunity (48). In Europe, bird mortality Population density, host-feeding studies and genetic related to WNV infection is rare; however, in analyses have implicated Cx. pipiens as the most important Israel, Hungary and Spain, several storks, geese and bridge vector in eastern Europe and Russia (36). raptors are reported to have died from WNV infection (3, Anautogenous populations of Cx. pipiens form pipiens, 44, 65). In North America, emergence of WNV in New which feed mostly on birds, could hybridise with York City was revealed by the deaths of thousands of birds autogenous populations of Cx. pipiens form molestus in (102). late summer and then feed on either birds or mammals (101). Increased mammalian feeding by Cx. pipiens late in In laboratory studies, Passeriformes (song birds), the season in temperate climates is a common observation. Charadriiformes (shorebirds), Strigiformes (owls) and Recent genetic studies show that populations of Cx. pipiens Falconiformes (hawks) developed viraemia levels sufficient in the USA are a mix of pipiens-molestus-quinquefasciatus to infect most feeding mosquitoes (105 plaque-forming genotypes and therefore express phenotypic traits units/ml serum for Cx. pipiens, for example) (56). variously (52). Culex pipiens may therefore be involved in Some passerines, including common grackles early-season amplification of WNV in enzootic cycles and (Quiscalus quiscula), various corvids (crows: Corvus serve as bridge vectors when autogenous-anautogenous brachyrhynchos, jays: Cyanocitta cristata, magpies: hybrids become abundant (52, 101). Pica pica), house finches (Carpodacus mexicanus) and house Other potential vectors sparrows (Passer domesticus) could be important amplifying hosts. The American robin (Turdus migratorius) The role of non-mosquito vectors in WNV epizootiology has been identified as an important amplifying host for continues to be explored. West Nile virus has been WNV in Maryland and Washington, DC, whereas the detected in hippoboscid flies in the USA, using a Taqman contribution of corvids to WNV amplification appeared reverse transcription polymerase chain reaction (RT-PCR) relatively unimportant, despite their being largely affected (34), and isolated from soft ticks in Israel (69) and from by the disease (51). In Europe, amplifying hosts have been hard ticks in Russia (63). The potential role of other biting little understood until recently (15, 46). flies (e.g. biting midges, sand flies and black flies) remains to be explored. Other possible transmission Long-term perpetuation and spread cycles for West Nile virus Potential mechanisms of WNV perpetuation within an Other potential amplifying hosts enzootic focus include low-level continuous enzootic transmission, vertical transmission by mosquito vector(s) Humans, horses and most other mammals are incidental and chronic infection in birds (7, 56, 86) (Fig. 1). Equine (dead-end) hosts of WNV because they do not produce epizootics accompanied by sentinel bird seroconversions significant viraemia and do not contribute to the occurred in the Camargue (delta of the River Rhône, transmission cycle (13). The virus has been detected in France) between 2000 and 2004, most probably as the several animals such as bats, cats, dogs, raccoons, rabbits, result of local perpetuation (59). Enzootic activity of WNV mountain goats, reindeer, alpacas, camels, skunks, in the Camargue has also been highlighted through harbour-seals, raptors, amphibians and reptiles, most of isolation of the virus, in 2004, from symptomatic wild them being considered as incidental hosts (42, 61). birds in the vicinity of equine clinical cases (47) and However, squirrels, eastern chipmunks (Tamias striatus), through detection of seropositive birds, including eastern cottontail rabbits (Sylvilagus floridanus), and certain juveniles, in migratory birds between 2005 and 2007, but species of lemur and alligator experience sufficiently high 834 Rev. sci. tech. Off. int. Epiz., 31 (3)

levels of viraemia to infect at least a low proportion of mosquitoes and may be competent amplification hosts for Clinical symptoms WNV (42, 80, 89, 93, 105). Clinical features in humans Non-viraemic transmission About 80% of WNV infections in humans are asymptomatic (77). West Nile fever presents as a minor The possible infection of mosquitoes feeding on influenza-like illness characterised by abrupt onset of non-infected hosts but near other infected mosquitoes has moderate-to-high fever lasting 3 to 5 days (incubation been suggested by some authors (39). The phenomenon of period 3 to 6 days). A maculopapular or roseolar rash (35) cofeeding is well known in ticks (84). Remarkably, appears in approximately 50% of cases, spreading from the 2.3% (n = 87) of Cx. quinquefasciatus cofeeding with a trunk to the extremities and head. Lymphadenopathy, single infected mosquito on the same mouse anorexia, nausea, abdominal pain, diarrhoea, myositis, became infected (39). This mechanism could greatly orchitis and respiratory symptoms are also encountered accelerate the establishment of a WNV outbreak in a given (98). Hepatosplenomegaly, hepatitis, pancreatitis, area, independently of virus multiplication and myocarditis (76) and haemorrhagic fever have been development of viraemia in amplifying hosts. The reported infrequently (73). Occasionally (< 15% of cases), mechanism of transmission during cofeeding should be acute aseptic meningitis or encephalitis occurs (92). determined for other potential vectors of WNV in order to Flaccid paralysis can accompany other syndromes, evaluate its importance in the infection of mosquitoes with generally without sensory loss (96); however, there have this virus. been reports of axonal polyneuropathy in which both sensory and motor neurons appear to be affected (40). A New routes of transmission: non-vector transmission striking feature of WNV encephalitis is disorder of There is strong evidence for non-vector routes of movement, some of these abnormalities being transmission, as in bird-to-bird transmission through the characterised as Parkinsonism (88). Up to a year may be faecal-oral route, or via cloacal shedding or the necessary for convalescence following encephalitis. consumption of infected carrion. Transmission of WNV Myalgia, confusion and lightheadedness may persist even between crows in the same cage and oral transmission from beyond this period and prolonged depression persists in as infected carcasses to crows have been demonstrated (56). many as 31% of patients (72). Direct transmission has also been demonstrated or strongly suspected in farmed alligators, domestic turkeys in Wisconsin and domestic geese in Canada (6, 17, 42). Host risk factors The incidence of neuroinvasive WNV disease and death In humans, the most important area of concern with increases with age. Other risk factors associated with respect to new modes of transmission of WNV is the development of neuroinvasive disease rather than West transfusion of infected blood components, which was Nile fever include male gender, hypertension and diabetes documented in a group of patients, some of whom were mellitus (43). Hypertension and cerebrovascular disease immunocompromised, in the USA in 2002 (75). presumably promote virus entry and replication in the Serological studies conducted in 2000 among blood endothelium of the blood-brain barrier. Diabetes and donors living in the South of France showed significant hypertension are both independent risk factors for prevalence of WNV IgG and IgM antibodies (37). These increased permeability of the blood-brain barrier. cases led to the implementation of comprehensive surveillance of blood donations. Immunocompromised humans are at the greatest risk for disseminated WNV infection. The induction of a specific, Other cases of WNV contamination transplacentally, neutralising IgM response early in the course of WNV through breast milk (38) or through dialysis (18) have infection limits viraemia and dissemination into the central been reported. Infection has also been occupationally nervous system and protects against lethal infection (24). acquired by laboratory workers and veterinarians through percutaneous inoculation and possibly through aerosol exposure, for example during a horse autopsy (16, 112). Clinical features in animals An outbreak of WNV disease among turkey handlers at a turkey farm raised the possibility of aerosol exposure (17). Following WNV infection, approximately 10% of horses Although these alternative routes of transmission are present neurological disorders. Apart from fever (>38.5°C), possible, their importance in the WNV cycle is difficult to clinical signs of WNV in horses almost exclusively reflect establish and measure; indeed, individual cases are difficult lesions of the central nervous system: ataxia in hindlimbs to prove conclusively because almost all people are and/or forelimbs, abnormal behaviour, paresis, paralysis, potentially exposed to mosquito vectors. muscular tremors, myoclonia, lethargy and cranial nerve Rev. sci. tech. Off. int. Epiz., 31 (3) 835

deficits (71). Mortality rates among clinically affected may be positive or negative, depending on the relative horses have been estimated as approximately 40% to 50% contributions of variations in each of the contributory (71). Two articles report results of experimental WNV parameters. For example, it is likely that increasing infection in limited numbers of horses (12 horses and 9 temperature will lead not only to an increase in the vector- equids respectively) and tend to reinforce many field biting rate and speed of virus development in the mosquito observations. Major clinical signs were those of infectious but also to an increase in the vector mortality rate (91). The meningoencephalomyelitis (fever, lethargy, ataxia, transition from low transmission to increased transmission paresis…) but they appeared in only 40% and 8% of due to temperature effects appears to occur over a narrow infected animals respectively (13, 45). Viraemia levels in range of temperatures, indicating that there is usually an infected horses are too low in magnitude and duration to optimum temperature for transmission (53). infect vectors efficiently, confirming that horses are unlikely to serve as amplifying hosts for WNV (13). Drought brings avian hosts and vector mosquitoes into close contact and facilitates the epizootic cycling and Encephalitis and myocarditis have been reported in dogs amplification of arboviruses within these populations (30). (61). Mice and other laboratory rodents are the most In the South of France, a retrospective study of the 2000 thoroughly studied and well-characterised models for WNV outbreak showed that the biting rate of Cx. modestus experimental studies. Following infection with WNV, these was positively correlated with temperature and humidity, animal models develop encephalitis showing many coupled with rainfall and hours of sunshine (62). similarities to the human disease. However, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterised (54). Apart from seropositivity, there is only limited information on the Factors linked to bird populations occurrence of WNV in sheep (49). A case of a 4-year-old Bird movements: migration and dispersion ewe with WNV infection and neurological symptoms has been described in Hungary (50). Reptiles are also During migratory movements, birds may carry pathogens susceptible to the infection. Clinical signs observed in that can be transmitted between species at breeding sites, alligators are anorexia, lethargy, intention tremors, during overwintering and at stopover places where swimming on their sides, spinning in the water, and numerous birds of various species are concentrated. A opisthotonus (42). Death occurred 24 h to 48 h after the good example of the probable introduction of WNV by onset of clinical signs. In Hungary in 2004 a goshawk migratory birds occurred in Israel in 1998 when an (Accipiter gentilis) died after showing central nervous signs outbreak of the virus on goose farms and evidence of due to infection with a lineage 2 strain (3). infection in dead migratory birds were reported (10). Although it is widely accepted that bird migrations are important factors allowing WNV to translocate between Africa and Europe, there is a knowledge gap between what Factors triggering changes is known about bird migrations and full understanding of the phenomena underlying WNV transcontinental in West Nile virus distribution translocations.

Factors linked to climate change First, some aspects of bird physiology and pathology of and mosquito populations WNV infection should be considered. For example, duration of viraemia and the associated transmission risk As early as 1999, Epstein suggested that a mild winter to mosquito vectors does not go beyond 3 to 4 days in followed by dry spring and summer seasons, heat waves in most cases, whereas transcontinental migration from July and storms with rainfall at the beginning of autumn endemic areas in sub-Saharan Africa can easily take 15 to would favour the emergence of mosquito-borne diseases 20 days for a rapid migrant. It could be argued that stress (30). During the WNV outbreak in Israel in 2000, the during migration could lengthen the viraemic period, but minimum temperature was found to be the most important whether a diseased bird can fly for so long is speculative. climatic factor that encouraged earlier appearance of disease (74). The development of the outbreak in Romania Secondly, it is often taken for granted that the direction of in 1996 was comparable (107). Each of these epidemics WNV import is from Africa to Europe, but little evidence appeared after a long heatwave. supports this, there is much more consistent evidence supporting introduction in the opposite direction. For Climate has a key influence on vector abundance, biology example, the only isolation of WNV from birds in obvious and physiology (91) and mosquitoes are very sensitive to migration was described in Israel in a flock of infected weather conditions at many stages of their life cycle. The storks migrating from Europe to Africa (65). Further, the net effect of global climate change on vector-borne diseases transmission periods in Africa and Europe support the 836 Rev. sci. tech. Off. int. Epiz., 31 (3)

view that the virus travels from Europe to Africa more found near the airport (58). Although genetic relationships easily than in the opposite direction. Thus, the between African and European strains of WNV support the transmission period in endemic parts of sub-Saharan existence of a translocation mechanism in which migrating Africa, such as in Senegal, is from October to January, but birds possibly play a role, the details of such a role are not the main body of bird migration occurs from late February yet fully understood. to May. It is therefore almost impossible for any migrating bird to become infected during the migration season. Host diversity and heterogeneity Moreover, in temperate Europe the virus circulates in summer and autumn, just when most migrant birds return Recent ideas suggest that biodiversity could play a major to their winter headquarters in Africa. Clearly, some of role in influencing contact rates between hosts or between them will be infected like the storks cited above. A list of hosts and vectors. However, biodiversity appears to play an birds potentially involved in the introduction, ambiguous role in WNV circulation, with two opposite amplification and spread of WNV in the Camargue was effects. The nature of the contradiction is only apparent, provided recently (48). Birds were classified according to because the effect could depend upon the biodiversity of their migration routes and to other ecological factors such each site. In some instances, an increase in biodiversity as habitat use and abundance. For example, in spring, would result in a rise in WNV transmission. In North passerines and shorebirds could introduce the virus from America, transmission of the virus is dominated by the African continent to Europe. Birds that could extreme heterogeneity in the community of avian host potentially be involved in the spread of WNV from wet to species, with a single species, the American robin (Turdus dry areas at the end of summer are herons (Bubulcus ibis, migratorius), accounting for the majority of WNV- Ardea cinerea), gulls (Larus cachinnans, L. melanocephalus, infectious mosquitoes (51). In southern France (the Var) in L. ridibundus), corvids (Pica pica, Corvus corone, 2003, the two areas with IgM-positive horses were located C. monedula), starlings (Sturnus vulgaris) and small near protected natural zones where biodiversity is greater passerine species. than elsewhere (26). A recent study has investigated the role of migratory birds, In another instance, increased avian diversity in the eastern principally Passeriformes, in the dissemination of WNV in USA was associated with a lower incidence of human the USA (27). In examination of more than 13,000 avian WNV infection (103), illustrating the concept of the blood samples, specific WNV neutralising antibodies were dilution effect and demonstrating that wildlife diversity detected in 254 birds (39 species), and WNV viraemias can help in protecting human populations from infectious were identified in 19 birds, but only during fall migration. disease. Where native vertebrate diversity is high, This study also supports the view that the virus is carried mosquito vectors can feed on a large variety of hosts, southward during autumn migration in the Americas, but mostly poor reservoirs for WNV, resulting in lower not northwards. Migratory birds might have a role in the prevalence of infection. Richness (numbers) of non- spread of WNV in the USA but major contradictions exist: passerine species has been negatively correlated with both progression of WNV in the USA has occurred mainly from mosquito and human infection rates for WNV (32). In east to west, whereas birds migrate from north to south these situations conservation of avian diversity and and the virus has spread at a pace not compatible with biodiversity in general might help in the prevention of migrating birds as principal factors of dispersal (85). WNV epidemics.

Bird roosting behaviour may be a critical component regulating WNV transmission because of the Environmental, anthropogenic crepuscular/nocturnal feeding behaviour of Culex changes and socio-economic patterns mosquitoes. A study of the roosting behaviour of American crows (Corvus brachyrhynchos) and northern cardinals The distribution of both susceptible avian reservoir hosts (Cardinalus cardinalus) showed that crows could spread the and competent mosquito vectors is influenced by virus throughout an area of approximately 20 km², geographic variables such as land use/land cover, elevation, whereas viraemic cardinals would only spread the virus human population density, physiographic region and over an average area of 0.03 km² (113). temperature. Identifying the links between environmental variables and risk of infectious disease is essential for understanding how human-induced environmental Lastly, other mechanisms not related to bird migration changes will affect the dynamics of human and wildlife should be taken into account. The trade in birds and bird diseases. products (legal or illegal) could also account for some of the introductions into Europe. An example of In the Camargue, two clusters of equine WNV cases were intercontinental introduction of WNV, probably identified after the 2004 outbreak, both in wet areas (60) independent of migrating birds, was the outbreak of WNV (Fig. 4). Rice fields, dry bushes, wet sansouire (saltmarsh in New York in 1999, where the first disease clusters were habitat dominated by Salicornia species) and open water Rev. sci. tech. Off. int. Epiz., 31 (3) 837

b) Results of a survey conducted in an equine population in the Camargue, 2003: resultant spatial prevalence-map; white areas indicate a high prevalence of WNV IgG (80%) (except for the white area in the north, which is an artefact due to a border effect)

a) Results of a survey conducted in an equine population in the Camargue, 2003: location of stables according to the presence (in red)/absence (in yellow) of IgG-positive horses

c) 2004 West Nile disease outbreak in horses in the Camargue, France: location of stables with horses testing positive (in red) and negative (in yellow) for WNV IgM and IgG antibodies and analysis of environmental risk factors identified in SPOT-4 satellite imaging Fig. 4 Results of serological, clinical and environmental studies conducted on West Nile virus circulation in horses in the Camargue, France, 2003–2004 838 Rev. sci. tech. Off. int. Epiz., 31 (3)

were the major components of the landscape that were WNV cases identified by surveillance systems. External associated with the presence of cases. In the Var and the validation of these models is therefore required. Camargue, a high level of virus circulation was positively associated with the landscape metrics and the In France, WNV disease is notifiable by veterinarians but interspersion and juxtaposition index, as well as with the the vaccination of horses is as yet very limited; however, a surface covered by heterogenous agricultural areas. Both syndromic surveillance system has allowed collection of variables are indicators of a complex landscape structure clinical cases in horses during consecutive years. A model that may favour the contact rates of competent vectors with was built to generate a prediction map for the areas at risk reservoir hosts (82). of endemic circulation of WNV along the Mediterranean coast (82). Areas with significant probability of high-level In Colorado, WNV infection rates among Culex viral circulation status were located in the Camargue and mosquitoes were found to decline with increasing wetland the Var, and also at an intermediate distance between these cover, suggesting that preservation of large wetland areas zones, near Aix-en-Provence. Equine cases near Perpignan may represent a valuable ecosystem-based approach for in 2006 were also located near areas of high risk. Further controlling WNV outbreaks (33). In the northeastern USA, work is needed to provide external validation of this a study based on analysis of eight years (1999 to 2006) of predictive map, and generalisation to other European human WNV disease surveillance data showed that countries should be a further objective. It should be borne urbanisation is a risk factor for this disease but that the risk in mind that WNV is a very complex pathogen behaving is independent of human population density (12). These completely differently according to the particular results are consistent with our knowledge of vector species conditions at each of the numerous geographical niches in this area. compatible with WNV transmission.

Models of risk prediction Conclusion

Methods aiming at predicting the risk of WNV emergence The spectacular panzootic of WNV in the Americas has are based on statistical or mathematical modelling. The drawn attention to this virus and it has frequently been objective is to identify key parameters that could be suggested that it is also an emerging pathogen in the Old included in routine surveillance systems and enable early World. Recent data suggest an increase in the circulation of warning and implementation of control measures (90). WNV in European countries and the Mediterranean basin. However, it is important to put into perspective the fact that, even if the urban outbreaks in Romania and Russia Vector-based models of prediction are included, fewer than 200 deaths in humans have been recorded over the past decade. Of course, many countries A theoretical framework allowing calculation of the have limited facilities for surveillance and diagnosis, but abundance dynamics of emerging adult mosquitoes from there is certainly no evidence of large-scale epidemics rain-fed ponds has been developed for forecasting the anywhere in the region. What will happen with WNV dynamics of Aedes and Culex populations (81). The lineage 2 in the future remains uncertain. dynamic of vectorial abundance was sensitive to flooding dynamics and depended on the bio-ecology of mosquito Many factors could be involved in the explanation for the species and the topography of breeding sites. recrudescence of WNV in Europe. Temperature is only one of many interacting factors that are likely to influence These models have also taken into account the capacity of transmission. The history of yellow fever, dengue, malaria, flight and dispersion of the vectors (5). For example, chikungunya and other diseases in Europe shows that the Cx. modestus has a small flight distance in open spaces but risk of importation and establishment of exotic pathogens can fly longer distances along vegetation corridors. is a direct result of the revolution in transport technologies and increasing global trade. Globalisation, whether caused by intentional economic activity or by large-scale economic Population-based models disruption, is potentially a far greater challenge to public Instead of focusing on vector populations, other studies health than any future changes in climate. have examined the links between cases in WNV hosts (reservoirs and/or incidental vectors, humans or horses) and the environment in order to build predictive models for the risk of transmission. This approach, mainly Acknowledgements statistical, has the advantage of requiring less information This review is based on an extensive literature study about vector abundance but the disadvantage of needing conducted as part of the V-borne project funded by the Rev. sci. tech. Off. int. Epiz., 31 (3) 839

European Centre for Disease Prevention and control responsibility of the authors and do not necessarily reflect (ECDC): ‘Assessment of the magnitude and impact of the views of the ECDC or the European Commission. vector-borne diseases in Europe’, tender OJ/2007/04/13- PROC/2007/003. The contents of this publication are the

L’épidémiologie du virus West Nile et les facteurs favorisant les changements de sa distribution en Europe

S. Pradier, S. Lecollinet & A. Leblond

Résumé Le virus West Nile a toujours été considéré comme le moins virulent des membres du complexe formé par le sérogroupe du virus de l’encéphalite japonaise (famille Flaviviridae, genre Flavivirus). Le cycle naturel du virus West Nile comprend les oiseaux, qui en sont les principaux hôtes amplificateurs, et plusieurs espèces de moustiques qui jouent le rôle de vecteurs. Plusieurs foyers d’encéphalite humaine sévère ont été enregistrés depuis dix ans dans l’Ancien Monde et le virus est désormais bien établi dans plusieurs pays européens. Il est difficile d’anticiper l’émergence du virus, et encore plus de la prévenir. Les auteurs font le point sur les découvertes les plus récentes relatives à l’épidémiologie, à la dynamique de la transmission et aux aspects cliniques de l’infection par le virus West Nile, en mettant particulièrement l’accent sur les facteurs spécifiques susceptibles de favoriser les changements de distribution de la maladie en Europe, tels que le changement climatique et ses effets sur les vecteurs potentiels du virus ou sur les routes migratoires des oiseaux. La maîtrise de certains facteurs anthropiques ou environnementaux pourrait contribuer à prévenir la propagation et la réémergence d’épidémies dues au virus West Nile.

Mots-clés Chevaux – Encéphalite – Épidémiologie – Flavivirus – Maladie à transmission vectorielle – Moustiques – Oiseaux – Virus West Nile – Zoonose.

Epidemiología del virus West Nile y factores desencadenantes de cambios en su distribución europea

S. Pradier, S. Lecollinet & A. Leblond

Resumen Tradicionalmente se ha considerado que el virus West Nile (VWN) es uno de los menos virulentos del complejo de serogrupos de la encefalitis japonesa (familia Flaviviridae, género Flavivirus). Intervienen en su ciclo natural las aves, que cumplen la función de principal anfitrión amplificador, y diversas especies de mosquitos que actúan de vectores. En el último decenio se han producido muchos brotes causantes de graves casos de encefalitis humana en el Viejo Mundo, y el virus se ha asentado en numerosos países europeos. Resulta difícil 840 Rev. sci. tech. Off. int. Epiz., 31 (3)

predecir, y aún más prevenir, su aparición. Los autores presentan los datos más recientes sobre la epidemiología, dinámica de transmisión y aspectos clínicos del VWN, deteniéndose especialmente en una serie de factores que seguramente pueden inducir cambios en la distribución de la enfermedad en Europa, por ejemplo la modificación del clima y sus efectos sobre los eventuales vectores del VWN o las rutas migratorias de las aves. El control de ciertos factores antropogénicos y ambientales podría ayudar a impedir la extensión y reaparición de epidemias provocadas por el virus West Nile.

Palabras clave Aves – Encefalitis – Enfermedad transmitida por un vector – Epidemiología – Equinos – Flavivirus – Mosquitos – Virus West Nile – Zoonosis.

References

1. Bagnarelli P., Marinelli K., Trotta D., Monachetti A., Tavio M., 8. Barzon L., Pacenti M., Cusinato R., Cattai M., Franchin E., Del Gobbo R., Capobianchi M., Menzo S., Nicoletti L., Pagni S., Martello T., Bressan S., Squarzon L., Cattelan A.M., Magurano F. & Varaldo P. (2011). – Human Pellizzer G., Scotton P., Beltrame A., Gobbi F., Bisoffi Z., case of autochthonous West Nile virus lineage 2 infection Russo F. & Palu G. (2011). – Human cases of West Nile virus in Italy, September 2011. Eurosurveillance, infection in north-eastern Italy, 15 June to 15 November 16 (43), pii: 20002 (accessed on 26 November 2012). 2010. Eurosurveillance, 16 (33), pii: 19949 (accessed on 26 November 2012). 2. Bakonyi T., Hubalek Z., Rudolf I. & Nowotny N. (2005). – Novel flavivirus or new lineage of West Nile virus, central 9. Berthet F.X., Zeller H.G., Drouet M.T., Rauzier J., Digoutte J.P. Europe. Emerg. infect. Dis., 11 (2), 225–231. & Deubel V. (1997). – Extensive nucleotide changes and deletions within the envelope glycoprotein gene of Euro- 3. Bakonyi T., Ivanics E., Erdelyi K., Ursu K., Ferenczi E., African West Nile viruses. J. gen. Virol., 78 (Pt 9), 2293–2297. Weissenbock H. & Nowotny N. (2006). – Lineage 1 and 2 strains of encephalitic West Nile virus, central Europe. Emerg. 10. Bin H., Grossman Z., Pokamunski S., Malkinson M., Weiss L., infect. Dis., 12 (4), 618–623. Duvdevani P., Banet C., Weisman Y., Annis E., Gandaku D., Yahalom V., Hindyieh M., Shulman L. & Mendelson E. 4. Balanca G., Gaidet N., Savini G., Vollot B., Foucart A., (2001). – West Nile fever in Israel 1999–2000: from geese to Reiter P., Boutonnier A., Lelli R. & Monicat F. (2009). – Low humans. Ann. N.Y. Acad. Sci., 951, 127–142. West Nile virus circulation in wild birds in an area of recurring outbreaks in southern France. Vector-borne zoonotic 11. Bondre V.P., Jadi R.S., Mishra A.C., Yergolkar P.N. & Dis., 9 (6), 737–741. Arankalle V.A. (2007). – West Nile virus isolates from India: evidence for a distinct genetic lineage. J. gen. Virol., 5. Balenghien T. (2006). – De l’identification des vecteurs du 88 (Pt 3), 875–884. virus West Nile à la modélisation du risque d’infection dans le sud de la France [Doctoral Thesis]. Université des Sciences 12. Brown H.E., Childs J.E., Diuk-Wasser M.A. & Fish D. (2008). des Technologies et de la Santé, Grenoble, 223 pp. – Ecological factors associated with West Nile virus transmission, northeastern United States. Emerg. infect. Dis., 6. Banet-Noach C., Simanov L. & Malkinson M. (2003). – 14 (10), 1539–1545. Direct (non-vector) transmission of West Nile virus in geese. Avian Pathol., 32 (5), 489–494. 13. Bunning M.L., Bowen R.A., Cropp C.B., Sullivan K.G., Davis B.S., Komar N., Godsey M.S., Baker D., Hettler D.L., 7. Baqar S., Hayes C.G., Murphy J.R. & Watts D.M. (1993). – Holmes D.A., Biggerstaff B.J. & Mitchell C.J. (2002). – Vertical transmission of West Nile virus by Culex and Aedes Experimental infection of horses with West Nile virus. Emerg. species mosquitoes. Am. J. trop. Med. Hyg., 48 (6), 757–762. infect. Dis., 8 (4), 380–386. Rev. sci. tech. Off. int. Epiz., 31 (3) 841

14. Burt F.J., Grobbelaar A.A., Leman P.A., Anthony F.S., 27. Dusek R.J., McLean R.G., Kramer L.D., Ubico S.R., Gibson G.V. & Swanepoel R. (2002). – Phylogenetic Dupuis A.P., Ebel G.D. & Guptill S.C. (2009). – Prevalence of relationships of southern African West Nile virus isolates. West Nile virus in migratory birds during spring and fall Emerg. infect. Dis., 8 (8), 820–826. migration. Am. J. trop. Med. Hyg., 81 (6), 1151–1158.

15. Calistri P., Giovannini A., Savini G., Monaco F., Bonfanti L., 28. Ebel G.D., Carricaburu J., Young D., Bernard K.A. & Ceolin C., Terregino C., Tamba M., Cordioli P. & Lelli R. Kramer L.D. (2004). – Genetic and phenotypic variation of (2009). – West Nile virus transmission in 2008 in north- West Nile virus in New York, 2000–2003. Am. J. trop. Med. eastern Italy. Zoonoses public Hlth, 57 (3), 211–219. Hyg., 71 (4), 493–500.

16. Centers for Disease Control and Prevention (CDC) (2002). – 29. European Centre for Disease Prevention and Control (ECDC) Laboratory-acquired West Nile virus infections – United (2011). – West Nile fever maps. Available at: ecdc.europa. States, 2002. MMWR, 51 (50), 1133–1135. eu/en/healthtopics/west_nile_fever/West-Nile-fever-maps/ Pages/index.aspx (accessed on 26 November 2012). 17. Centers for Disease Control and Prevention (CDC) (2003). – West Nile virus infection among turkey breeder farm workers 30. Epstein P.R. (2001). – West Nile virus and the climate. – Wisconsin, 2002. MMWR, 52 (42), 1017–1019. J. urban Hlth, 78 (2), 367–371.

18. Centers for Disease Control and Prevention (CDC) (2004). – 31. Erdelyi K., Ursu K., Ferenczi E., Szeredi L., Ratz F., Skare J. & Possible dialysis-related West Nile virus transmission – Bakonyi T. (2007). – Clinical and pathologic features of Georgia, 2003. MMWR, 53 (32), 738–739. lineage 2 West Nile virus infections in birds of prey in Hungary. Vector-borne zoonotic Dis., 7 (2), 181–188. 19. Centers for Disease Control and Prevention (CDC) (2011). – Statistics, surveillance and control of West Nile virus. 32. Ezenwa V.O., Godsey M.S., King R.J. & Guptill S.C. (2006). Available at: www.cdc.gov/ncidod/dvbid/westnile/surv& – Avian diversity and West Nile virus: testing associations control_archive.htm (accessed on 26 November 2012). between biodiversity and infectious disease risk. Proc. biol. Sci., 273 (1582), 109–117. 20. Chaskopoulou A., Dovas C., Chaintoutis S., Bouzalas I., Ara G. & Papanastassopoulou M. (2011). – Evidence of 33. Ezenwa V.O., Milheim L.E., Coffey M.F., Godsey M.S., enzootic circulation of West Nile virus (Nea Santa-Greece- King R.J. & Guptill S.C. (2007). – Land cover variation and 2010, lineage 2), Greece, May to July 2011. Eurosurveillance, West Nile virus prevalence: patterns, processes, 16 (31), pii: 19933 (accessed on 26 November 2012). and implications for disease control. Vector-borne zoonotic Dis., 7 (2), 173–180. 21. Cornel A.J., Jupp P.G. & Blackburn N.K. (1993). – Environmental temperature on the vector competence of 34. Farajollahi A., Crans W., Nickerson D., Bryant P. & Wolf B. Culex univittatus (Diptera: Culicidae) for West Nile virus. (2005). – Detection of West Nile virus RNA from the louse fly J. med. Entomol. 30 (2), 449–456. Icosta americana (Diptera: Hippoboscidae). J. Am. Mosq. Control Assoc., 21 (4), 474–476. 22. Davis C., Ebel G., Lanciotti R., Brault A. & Guzman H. (2005). – Phylogenetic analysis of North American West Nile 35. Ferguson D., Gershman K., LeBailly A. & Petersen L. (2005). virus isolates, 2001–2004: evidence for the emergence of a – Characteristics of the rash associated with West Nile virus dominant genotype. Virology, 342 (2), 252–265. fever. Clin. infect. Dis., 41 (8), 1204–1207.

23. Del Giudice P., Schuffenecker I., Vandenbos F., Counillon E. 36. Fyodorova M.V., Savage H.M., Lopatina J.V., Bulgakova T.A., & Zeller H. (2004). – Human West Nile virus, France. Ivanitsky A.V., Platonova O.V. & Platonov A.E. (2006). – Emerg. infect. Dis., 10 (10), 1885–1886. Evaluation of potential West Nile virus vectors in Volgograd region, Russia, 2003 (Diptera: Culicidae): species 24. Diamond M.S., Shrestha B., Mehlhop E., Sitati E. & Engle M. composition, bloodmeal host utilization, and virus infection (2003). – Innate and adaptive immune responses determine rates of mosquitoes. J. med. Entomol., 43 (3), 552–563. protection against disseminated infection by West Nile encephalitis virus. Viral Immunol., 16 (3), 259–278. 37. Gallian P., de Lamballerie X., de Micco P. & Andreu G. (2005). – West Nile virus (WNV): generalities and 25. Dohm D.J., Sardelis M.R. & Turell M.J. (2002). – implications for blood transfusion [Article in French]. Experimental vertical transmission of West Nile virus by Transfus. clin. biol., 12 (1), 11–17. Culex pipiens (Diptera: Culicidae). J. med. Entomol., 39 (4), 640–644. 38. Hayes E. & O’Leary D. (2004). – West Nile virus infection: a pediatric perspective. Pediatrics, 113 (5), 1375–1381. 26. Durand B., Dauphin G., Zeller H., Labie J., Schuffenecker I., Murri S., Moutou F. & Zientara S. (2005). – Serosurvey for 39. Higgs S., Schneider B., Vanlandingham D., Kingler K. & West Nile virus in horses in southern France. Vet. Rec., Gould E. (2005). – Nonviremic transmission of West Nile 157 (22), 711–713. virus. Proc. natl Acad. Sci. USA, 102 (25), 8871–8874. 842 Rev. sci. tech. Off. int. Epiz., 31 (3)

40. Holman R., Monserrate N., Czander E. & Rushing E. 53. Kilpatrick A.M., Meola M.A., Moudy R.M. & Kramer L.D. (2004). – West Nile poliomyelitis. Emerg. infect. Dis., (2008). – Temperature, viral genetics, and the transmission of 10 (3), 547–548. West Nile virus by Culex pipiens mosquitoes. PLoS Pathog., 4 (6), e1000092 (accessed on 26 November 2012). 41. Hubalek Z. & Halouzka J. (1999). – West Nile fever: a reemerging mosquito-borne viral disease in Europe. Emerg. 54. Kimura T., Sasaki M., Okumura M., Kim E. & Sawa H. infect. Dis., 5 (5), 643–650. (2010). – Flavivirus encephalitis: pathological aspects of mouse and other animal models. Vet. Pathol., 42. Jacobson E.R., Ginn P.E., Troutman J.M., Farina L., Stark L., 47 (5), 806–818. Klenk K., Burkhalter K.L. & Komar N. (2005). – West Nile virus infection in farmed American alligators (Alligator 55. Komar N. & Clark G.G. (2006). – West Nile virus activity in mississippiensis) in Florida. J. Wildl. Dis., 41 (1), 96–106. Latin America and the Caribbean. Rev. panam. Salud pública, 19 (2), 112–117. 43. Jean C.M., Honarmand S., Louie J.K. & Glaser C.A. (2007). – Risk factors for West Nile virus neuroinvasive disease, 56. Komar N., Langevin S., Hinten S., Nemeth N., Edwards E., California, 2005. Emerg. infect. Dis., 13 (12), 1918–1920. Hettler D., Davis B., Bowen R. & Bunning M. (2003). – Experimental infection of North American birds with the 44. Jiménez-Clavero M.A., Sotelo E., Fernández-Pinero J., New York 1999 strain of West Nile virus. Emerg. infect. Dis., Llorente F., Blanco J.M., Rodríguez-Ramos J., 9 (3), 311–322. Pérez-Ramírez E. & Höfle U. (2008). – West Nile virus in golden eagles, Spain, 2007. Emerg. infect. Dis., 57. Kutasi O., Bakonyi T., Lecollinet S., Biksi I., Ferenczi E., 14 (9), 1489–1491. Bahuon C., Sardi S., Zientara S. & Szenci O. (2011). – Equine encephalomyelitis outbreak caused by a genetic lineage 2 45. Joubert L., Oudar J., Hannoun C. & Chippaux M. (1971). – West Nile virus in Hungary. J. vet. internal Med., 25 (3), Reproduction expérimentale de la méningo-encéphalomyélite 586–591. du cheval par l’arbovirus West Nile. III. Relations entre la virologie, la sérologie et l’évolution anatomo-clinique. 58. Lanciotti R.S., Roehrig J.T., Deubel V., Smith J., Parker M., Conséquences épidémiologiques et prophylactiques. Bull. Steele K., Crise B., Volpe K.E., Crabtree M.B., Scherret J.H., Acad. vét. Fr., 44 (3), 159–167. Hall R.A., MacKenzie J.S., Cropp C.B., Panigrahy B., Ostlund E., Schmitt B., Malkinson M., Banet C., Weissman J., 46. Jourdain E., Gauthier-Clerc M., Sabatier P., Grege O., Komar N., Savage H.M., Stone W., McNamara T. & Greenland T., Leblond A., Lafaye M. & Zeller H.G. (2008). – Gubler D.J. (1999). – Origin of the West Nile virus Magpies as hosts for West Nile virus, southern France. Emerg. responsible for an outbreak of encephalitis in the infect. Dis., 14 (1), 158–160. northeastern United States. Science, 286 (5448), 2333–2337.

47. Jourdain E., Schuffenecker I., Korimbocus J., Reynard S., 59. Leblond A., Hendrikx P. & Sabatier P. (2007). – West Nile Murri S., Kayser Y., Gauthier-Clerc M., Sabatier P. & virus outbreak detection using syndromic monitoring in Zeller H.G. (2007). – West Nile virus in wild resident birds, horses. Vector-borne zoonotic Dis., 7 (3), 403–410. southern France, 2004. Vector-borne zoonotic Dis., 7 (3), 448–452. 60. Leblond A., Sandoz A., Lefebvre G., Zeller H. & Bicout D.J. (2007). – Remote sensing-based identification of 48. Jourdain E., Toussaint Y., Leblond A., Bicout D.J., Sabatier P. environmental risk factors associated with West Nile disease & Gauthier-Clerc M. (2007). – Bird species potentially in horses in Camargue, France. Prev. vet. Med., 79 (1), 20–31. involved in introduction, amplification, and spread of West Nile virus in a Mediterranean wetland, the Camargue 61. Lichtensteiger C.A., Heinz-Taheny K., Osborne T.S., (southern France). Vector-borne zoonotic Dis., 7 (1), 15–33. Novak R.J., Lewis B.A. & Firth M.L. (2003). – West Nile virus encephalitis and myocarditis in wolf and dog. Emerg. infect. 49. Juricová Z., Mitterpák J., Prokopic J. & Hubálek Z. (1986). – Dis., 9 (10), 1303–1306. Circulation of mosquito-borne viruses in large-scale farms in eastern Slovakia. Folia parasitol., 33 (3), 285–288. 62. Ludwig A., Bicout D.J., Chalvet-Monfray K. & Sabatier P. (2005). – Modélisation de l’agressivité de Culex modestus, 50. Kecskeméti S., Bajmócy E., Bacsadi A., Kiss I. & Bakonyi T. vecteur potentiel de West Nile en Camargue, en fonction de (2007). – Encephalitis due to West Nile virus in a sheep. Vet. données météorologiques. Environ. Risques Santé, 4 (2), 1–5. Rec., 161 (16), 568–569. 63. L’vov D.K., Dzharkenov A.F., L’vov D.N., Aristova V.A., 51. Kilpatrick A.M., Daszak P., Jones M.J., Marra P.P. & Kovtunov A.I., Gromashevskii V.L., Vyshemirskii O.I., Kramer L.D. (2006). – Host heterogeneity dominates West Galkina I.V., Al’khovskii S.V., Samokhvalov E.I., Nile virus transmission. Proc. biol. Sci., 273 (1599), Prilipov A.G., Deriabin P.G., Odolevskii E.I. & 2327–2333. Ibragimov R.M. (2002). – Isolation of the West Nile fever virus from the great cormorant Phalacrorax carbo, the crow 52. Kilpatrick A., Kramer L., Jones M., Marra P. & Daszak P. Corvus corone, and Hyalomma marginatum ticks associated (2006). – West Nile virus epidemics in North America are with them in natural and synanthroic biocenosis in the Volga driven by shifts in mosquito feeding behaviour. PLoS Biol., delta (Astrakhan region, 2001) [Article in Russian]. Vopr. 4 (4), e82 (accessed on 26 November 2012). Virusol., 47 (5), 7–12. Rev. sci. tech. Off. int. Epiz., 31 (3) 843

64. McLean R.G., Ubico S.R., Bourne D. & Komar N. (2002). – 77. Petersen L.R. & Marfin A.A. (2002). – West Nile virus: a West Nile virus in livestock and wildlife. Curr. Top. Microbiol. primer for the clinician. Ann. internal Med., 137 (3), Immunol., 267, 271–308. 173–179.

65. Malkinson M., Banet C., Weisman Y., Pokamunski S., 78. Platonov A., Karan L., Shopenskaia T., Fedorova M., King R., Drouet M.T. & Deubel V. (2002). – Introduction of Koliasnikova N., Rusakova N., Shishkina L., Arshba T., West Nile virus in the Middle East by migrating white storks. Zhuravlev V., Govorukhina M., Valentseva A. & Shipulin G. Emerg. infect. Dis., 8 (4), 392–397. (2011). – Genotyping of West Nile fever virus strains circulating in southern Russia as an epidemiological 66. Miller B.M., Nasci R., Godsey M.S., Savage H.M., investigation method: principles and results [Article in Lutwama J.J., Lanciotti R. & Peters C.J. (2000). – First field Russian]. Zhurnal Mikrobiol. Epidemiol. Immunobiol. (Moskva), evidence for natural vertical transmission of West Nile virus 2, 29–37. in Culex univittatus complex mosquitoes from Rift Valley 79. Platonov A.E., Shipulin G.A., Shipulina O.Y., Province, Kenya. Am. J. trop. Med. Hyg., 62 (2), 240–246. Tyutyunnik E.N., Frolochkina T.I., Lanciotti R.S., Yazyshina S., Platonova O.V., Obukhov I.L., Zhukov A.N., Vengerov Y.Y. 67. Monaco F., Lelli R., Teodori L., Pinoni C., Di Gennaro A., & Pokrovskii V.I. (2001). – Outbreak of West Nile virus Polci A., Calistri P. & Savini G. (2009). – Re-emergence of infection, Volgograd Region, Russia, 1999. Emerg. infect. Dis., West Nile virus in Italy. Zoonoses public Hlth, 57 (7–8), 7 (1), 128–132. 476–486. 80. Platt K.B., Tucker B.J., Halbur P.G., Tiawsirisup S., 68. Moudy R.M., Meola M.A., Morin L.L., Ebel G.D. & Blitvich B.J., Fabiosa F.G., Bartholomay L.C. & Rowley W.A. Kramer L.D. (2007). – A newly emergent genotype of West (2007). – West Nile virus viremia in eastern chipmunks Nile virus is transmitted earlier and more efficiently by Culex (Tamias striatus) sufficient for infecting different mosquitoes. mosquitoes. Am. J. trop. Med. Hyg., 77 (2), 365-370. Emerg. infect. Dis., 13 (6), 831–837.

69. Mumcuoglu K.Y., Banet-Noach C., Malkinson M., Shalom U. 81. Porphyre T., Bicout D.J. & Sabatier P. (2005). – Modelling the & Galun R. (2005). – Argasid ticks as possible vectors of West abundance of mosquito vectors versus flooding dynamics. Nile virus in Israel. Vector-borne zoonotic Dis., 5 (1), 65–71. Ecol. Modell., 183 (2–3), 173–181. 82. Pradier S., Leblond A. & Durand B. (2008). – Land cover, 70. Murgue B., Murri S., Triki H., Deubel V. & Zeller H.G. landscape structure, and West Nile virus circulation in (2001). – West Nile in the Mediterranean basin: 1950–2000. southern France. Vector-borne zoonotic Dis., 8 (2), 253–263. Ann. N.Y. Acad. Sci., 951, 117–126. 83. Promed (2012). – West Nile Alerts. Available at: 71. Murgue B., Murri S., Zientara S., Durand B., Durand J.P. & www.promedmail.org/ (accessed on 26 November 2012). Zeller H. (2001). – West Nile outbreak in horses in southern France, 2000: the return after 35 years. Emerg. infect. Dis., 84. Randolph S., Gern L. & Nuttall P. (1996). – Co-feeding ticks: 7 (4), 692–696. epidemiology significance for tick-borne pathogen transmission. Parasitol. Today, 12 (12), 472–479.

72. Murray K., Resnick M. & Miller V. (2007). – Depression after 85. Rappole J.H. & Hubalek Z. (2003). – Migratory birds and infection with West Nile virus. Emerg. infect. Dis., 13 (3), West Nile virus. J. appl. Microbiol., 94 (Suppl.), 47S–58S. 479–481. 86. Reisen W., Fang Y., Lothrop H., Martínez V., Wilson J., 73. Paddock C.D., Nicholson W.L., Bhatnagar J., Goldsmith C.S., O’Connor P., Carney R., Cahoon-Young B., Shafil M. & Greer P.W., Hayes E.B., Risko J.A., Henderson C., Brault A. (2006). – Overwintering of West Nile virus in Blackmore C.G., Lanciotti R.S., Campbell G.L. & Zaki S.R. southern California. J. med. Entomol., 43 (2), 344–355. (2006). – Fatal hemorrhagic fever caused by West Nile virus in the United States. Clin. infect. Dis., 42 (11), 1527–1535. 87. Rizzo C., Vescio F., Declich S., Finarelli A.C., Macini P., Mattivi A., Rossini G., Piovesan C., Barzon L., Palu G., 74. Paz S. (2006). – The West Nile virus outbreak in Israel (2000) Gobbi F., Macchi L., Pavan A., Magurano F., Ciufolini M.G., from a new perspective: the regional impact of climate Nicoletti L., Salmaso S. & Rezza G. (2009). – West Nile virus change. Int. J. environ. Hlth Res., 16 (1), 1–13. transmission with human cases in Italy, August–September 2009. Eurosurveillance, 14 (40), pii: 19353 (accessed on 75. Pealer L.N., Marfin A.A., Petersen L.R., Lanciotti R.S., 26 November 2012). Page P.L., Stramer S.L., Stobierski M.G., Signs K., 88. Robinson R.L., Shahida S., Madan N., Rao S. & Khardori N. Newman B., Kapoor H., Goodman J.L. & Chamberland M.E. (2003). – Transient parkinsonism in West Nile virus (2003). – Transmission of West Nile virus through blood encephalitis. Am. J. med., 115 (3), 252–253. transfusion in the United States in 2002. N. Engl. J. Med., 349 (13), 1236–1245. 89. Rodhain F., Petter J.J., Albignac R., Coulanges P. & Hannoun C. (1985). – Arboviruses and lemurs in 76. Pergam S., DeLong C., Echevarria L., Scully G. & Goade D. Madagascar: experimental infection of Lemur fulvus with (2006). – Myocarditis in West Nile virus infection. Am. J. trop. yellow fever and West Nile viruses. Am. J. trop. Med. Hyg., Med. Hyg., 75 (6), 1232–1233. 34 (4), 816–822. 844 Rev. sci. tech. Off. int. Epiz., 31 (3)

90. Rogers D. (2006). – Models for vectors and vector-borne 104. Taylor R.M., Hurlbut H.S., Dressler H.R., Spangler E.W. & diseases. Adv. Parasitol., 62, 1–35. Thrasher D. (1953). – Isolation of West Nile virus from Culex mosquitoes. J. Egypt. med. Assoc., 36 (3), 199–208. 91. Rogers D.J. & Randolph S.E. (2006). – Climate change and vector-borne diseases. Adv. Parasitol., 62, 345–381. 105. Tiawsirisup S., Platt K., Tucker B. & Rowley W. (2005). – Eastern cottontail rabbits (Sylvilagus floridanus) develop 92. Roos K.L. (2004). – West Nile encephalitis and myelitis. West Nile virus viraemias sufficient for infecting select Curr. Op. Neurol., 17 (3), 343–346. mosquito species. Vector-borne zoonotic Dis., 5 (1), 342–350.

93. Root J., Oesterle P., Nemeth N., Klenk K. & Gould D. 106. Triki H., Murri S., Le Guenno B., Bahri O., Hili K., (2006). – Experimental infection of fox squirrels (Sciurus Sidhom M. & Dellagi K. (2001). – West Nile viral meningo- niger) with West Nile virus. Am. J. trop. Med. Hyg., 75 (4), encephalitis in Tunisia [Article in French]. Méd. trop., 697–701. 61 (6), 487–490. 107. Tsai T.F., Popovici F., Cernescu C., Campbell G.L. & 94. Savini G., Monaco F., Calistri P. & Lelli R. (2008). – Nedelcu N.I. (1998). – West Nile encephalitis epidemic in Phylogenetic analysis of West Nile virus isolated in Italy in south-eastern Romania. Lancet, 352 (9130), 767–771. 2008. Eurosurveillance, 13 (48), pii: 19048 (accessed on 26 November 2012). 108. Turell M.J., Dohm D.J., Sardelis M.R., Oguinn M.L., Andreadis T.G. & Blow J.A. (2005). – An update on the 95. Schuffenecker I., Peyrefitte C., El Harrak M., Murri S., potential of North American mosquitoes (Diptera: Leblond A. & Zeller H. (2005). – West Nile virus in Culicidae) to transmit West Nile virus. J. med. Entomol., Morocco, 2003. Emerg. infect. Dis., 11 (2), 306–309. 42 (1), 57–62.

96. Sejvar J., Bode A., Marfin A., Campbell G., Ewing D., 109. Valiakos G., Touloudi A., Iacovakis C., Athanasiou L., Mazowiecki M., Pavot P., Schmitt J., Pape J., Biggerstaff B. & Birtsas P., Spyrou V. & Billinis C. (2011). – Molecular Petersen L. (2005). – West Nile virus-associated flaccid detection and phylogenetic analysis of West Nile virus paralysis. Emerg. infect. Dis., 11 (7), 1021–1027. lineage 2 in sedentary wild birds (Eurasian magpie), Greece, 2010. Eurosurveillance, 16 (18), pii: 19862 (accessed on 97. Sirbu A., Ceianu C., Panculescu-Gatej R., Vázquez A., 26 November 2012). Tenorio A., Rebreanu R., Niedrig M., Nicolescu G. & Pistol A. (2011). – Outbreak of West Nile virus infection in humans, 110. Vázquez A., Sánchez-Seco M.P., Ruiz S., Molero F., Romania, July to October 2010. Eurosurveillance, 16 (2), pii: Hernández L., Moreno J., Magallanes A., Tejedor C.G. & 19762 (accessed on 26 November 2012). Tenorio A. (2010). – Putative new lineage of West Nile virus, Spain. Emerg. infect. Dis., 16 (3), 549–552. 98. Smith R.D., Konoplev S., DeCourten-Myers G. & Brown T. (2004). – West Nile virus encephalitis with myositis and 111. Venter M., Human S., Zaayman D., Gerdes G.H., orchitis. Hum. Pathol., 35 (2), 254–258. Williams J., Steyl J., Leman P.A., Paweska J.T., Setzkorn H., Rous G., Murray S., Parker R., Donnellan C. & 99. Smithburn K.C., Hughes T.P., Burke A.W. & Paul J.H. Swanepoel R. (2009). – Lineage 2 West Nile virus as cause (1940). – A neurotropic virus isolated from the blood of a of fatal neurologic disease in horses, South Africa. Emerg. native of Uganda. Am. J. trop. Med. Hyg., 20, 471–492. infect. Dis., 15 (6), 877–884.

100. Sotelo E., Fernández-Pinero J., Llorente F., Aguero M., 112. Venter M., Steyl J., Human S., Weyer J., Zaayman D., Hoefle U., Blanco J.M. & Jiménez-Clavero M.A. (2009). – Blumberg L., Leman P., Paweska J. & Swanepoel R. (2010). Characterization of West Nile virus isolates from Spain: new – Transmission of West Nile virus during horse autopsy. insights into the distinct West Nile virus eco-epidemiology Emerg. infect. Dis., 16 (3), 573–575. in the western Mediterranean. Virology, 395 (2), 289–297. 113. Ward M., Raim A., Yaremich-Hamer S., Lampman R. & Novak R. (2006). – Does the roosting behaviour of birds 101. Spielman A. (2001). – Structure and seasonality of nearctic affect transmission dynamics of West Nile virus? Am. J. trop. Culex pipiens populations. Ann. N.Y. Acad. Sci., Med. Hyg., 75 (2), 350–355. 951, 220–234. 114. Weinberger M. & Pitlik S. (2001). – West Nile fever 102. Steele K.E., Linn M.J., Schoepp R.J., Komar N., outbreak, Israel, 2000: epidemiologic aspects. Emerg. infect. Geisbert T.W., Manduca R.M., Calle P.P., Raphael B.L., Dis., 7 (4), 686–691. Clippinger T.L., Larsen T., Smith J., Lanciotti R.S., Panella N.A. & McNamara T.S. (2000). – Pathology of fatal 115. Weir E. (2000). – West Nile fever heads north. Can. med. West Nile virus infections in native and exotic birds during Assoc. J., 163 (7), 878. the 1999 outbreak in New York City, New York. Vet. Pathol., 37 (3), 208–224. 116. Weissenbock H., Hubálek Z., Bakonyi T. & Nowotny N. (2010). – Zoonotic mosquito-borne flaviviruses: worldwide 103. Swaddle J.P. & Calos S.E. (2008). – Increased avian presence of agents with proven pathogenicity and potential diversity is associated with lower incidence of human West candidates of future emerging diseases. Vet. Microbiol., Nile infection: observation of the dilution effect. PLoS ONE, 140 (3–4), 271–280. 3 (6), e2488 (accessed on 26 November 2012). REVIEW ARTICLE published: 21 March 2012 doi: 10.3389/fneur.2012.00037 Neuromuscular manifestations of West Nile virus infection

A. Arturo Leis* and Dobrivoje S. Stokic

Center for Neuroscience and Neurological Recovery, Methodist Rehabilitation Center, Jackson, MS, USA

Edited by: The most common neuromuscular manifestation of West Nile virus (WNV) infection is a Marianne De Visser, Academic poliomyelitis syndrome with asymmetric paralysis variably involving one (monoparesis) to Medical Centre, Netherlands four limbs (quadriparesis), with or without brainstem involvement and respiratory failure. Reviewed by: Marianne De Visser, Academic This syndrome of acute flaccid paralysis may occur without overt fever or meningoen- Medical Centre, Netherlands cephalitis. Although involvement of anterior horn cells in the spinal cord and motor neurons S. H. Subramony, University of Florida in the brainstem are the major sites of pathology responsible for neuromuscular signs, College of Medicine, USA inflammation also may involve skeletal or cardiac muscle (myositis, myocarditis), motor *Correspondence: axons (polyradiculitis), and peripheral nerves [Guillain–Barré syndrome (GBS), brachial plex- A. Arturo Leis, Methodist Rehabilitation Center and University opathy]. In addition, involvement of spinal sympathetic neurons and ganglia provides an of Mississippi Medical Center, Center explanation for autonomic instability seen in some patients. Many patients also experi- for Neuroscience and Neurological ence prolonged subjective generalized weakness and disabling fatigue. Despite recent Recovery, 1350 East WoodrowWilson, evidence that WNV may persist long-term in the central nervous system or periphery in Suite 2, Jackson, MS 39216, USA. e-mail: [email protected] animals, the evidence in humans is controversial. WNV persistence would be of great concern in immunosuppressed patients or in those with prolonged or recurrent symptoms. Support for the contention thatWNV can lead to autoimmune disease arises from reports of patients presenting with various neuromuscular diseases that presumably involve autoimmune mechanisms (GBS, other demyelinating neuropathies, myasthenia gravis, brachial plexopathies, stiff-person syndrome, and delayed or recurrent symptoms). Although there is no specific treatment or vaccine currently approved in humans, and the standard remains supportive care, drugs that can alter the cascade of immunobiochemical events leading to neuronal death may be potentially useful (high-dose corticosteroids, interferon preparations, and intravenous immune globulin containing WNV-specific antibodies). Human experience with these agents seems promising based on anecdotal reports.

Keywords:West Nile virus, infection, poliomyelitis, fever

INTRODUCTION The viral strain introduced into the United States likely originated West Nile virus (WNV), a mosquito-borne RNA flavivirus and from a strain that was circulating in Israel during 1998 (Lanciotti human neuropathogen, was first isolated from a febrile woman et al., 1999). In the past decade,WNV has spread and is now widely in the West Nile region of Uganda, Africa in 1937 (Smithburn established from Canada to Venezuela (World Health Organiza- et al., 1940). During the 1940s and 1950s, transmission of WNV tion1). In 2011, human cases were reported in Albania, Greece, by mosquitoes was demonstrated, the close antigenic relationship Israel, Italy, Romania, Russia, and Mexico. Hence, the geographic with flaviviruses was described, and neutralizing antibody was range of WNV now includes six of seven continents, including found in many residents of East-Central Africa (for a historical Africa, Asia, Europe, Australia (subtype Kunjin), North America, review see Campbell et al., 2002). The virus also became recog- and South America. nized as a cause of human meningitis or encephalitis in elderly Before 1996, WNV was known to cause high fever, chills, patients during an outbreak in Israel in 1957 (Spigland et al., malaise, headache, backache, arthralgia, myalgias, retro-orbital 1958), although CNS involvement in middle-aged and younger pain, and a maculopapular rash, but neurological symptoms were subjects remained unusual and outcome in the younger age-group uncommon. However, since the New York City outbreak, severe was generally excellent (Weinberger et al., 2001). During the 1960s neurological illness, including encephalitis and meningitis, has to 1970s, birds were identified as a major host, horses were com- been reported much more frequently, together with neuromus- monly infected, and there were large human epidemics in Africa cular manifestations. The diagnosis of WNV infection should be and the Middle East. Europe also experienced its first WNV out- considered in any patient with an unexplained acute febrile or break (Murgue et al., 2001). During the 1980s and 1990s, there neurological illness during the summer months, particularly if were major outbreaks in Africa, Middle East, Europe, and Russia, recently exposed to mosquitoes. In such cases, serum should be although the Romania epidemic in 1996 marked the geographic tested for class M immunoglobulin (IgM) antibody toWNV,which transition of WNV epidemics from rural areas to urban industrial- ized areas (Campbell et al., 2002). In 1999, WNV gained entry into North America in the New York City outbreak (Nash et al., 1999). 1http://www.who.int/mediacentre/factsheets

www.frontiersin.org March 2012 | Volume 3 | Article 37 | 1 Leis and Stokic West Nile virus infection

indicates a recent infection. If there are signs of CNS involvement, syndrome (GBS), motor axonopathy, or severe axonal polyneu- cerebrospinal fluid (CSF) should be analyzed and also tested for ropathy (Nash et al., 1999; Asnis et al., 2000; Sampson et al., 2000). WNV IgM antibody. CSF findings typically show increased leuko- In the 2002 epidemic, more cases of WNV-associated acute flaccid cytes (usually >200 cells/mm3), increased protein, and normal paralysis were also seen across the Southern United States. These glucose. Almost half of WNV meningitis patients may have at patients had asymmetric acute flaccid paralysis, absent deep ten- least 50% neutrophils in their initial CSF specimen (Tyler et al., don reflexes in affected limbs, preserved sensation, bowel, or blad- 2006), followed by a shift to lymphocytosis. Imaging studies in der dysfunction, and respiratory distress. Electrodiagnostic studies WNV infection are frequently normal,although they may be useful in these subjects revealed markedly decreased or absent motor in excluding other etiologies of acute myelomeningoencephalitis. responses in the paretic limbs, preserved sensory responses, and When abnormal, findings are generally non-specific and without widespread asymmetric muscle denervation, without evidence of mass effect. T2-weighted magnetic resonance signal abnormali- demyelination or myopathy. The clinical and electrodiagnostic ties have been reported in brainstem, deep gray structures (basal findings were classic for poliomyelitis and inconsistent with GBS ganglia or thalami), and cerebellum (Petropoulou et al., 2005). or other peripheral nerve disorders. These cases were reported in However, other imaging series have found no definite predilection the Centers for Disease Control Morbidity and Mortality Weekly for any specific area of the brain parenchyma (Ali et al., 2005). Report on September 20, 2002 (Centers for Disease Control and In patients with WNV-associated limb paralysis, abnormal sig- Prevention, 2002), with clinical, laboratory, and electrophysio- nal intensity may be more pronounced in the spinal cord ventral logic guidelines to help physicians discern poliomyelitis from GBS horns with enhancement around the conus medullaris and cauda (Table 1). Subsequently, clinical, laboratory, and neurophysiologic equina (Petropoulou et al., 2005). However, follow-up MRIs may findings suggested that WNV-associated acute flaccid paralysis was show complete resolution of signal abnormalities. a poliomyelitis syndrome with involvement of anterior horn cells Neuromuscular manifestations are now recognized as a of the spinal cord (Glass et al., 2002; Leis et al., 2002; Li et al., prominent feature in patients with WNV neuroinvasive disease 2003; Al-Shekhlee and Katirji, 2004). Soon thereafter pathologic (encephalitis, meningitis). In the 1999 New York City outbreak, confirmation of WNV poliomyelitis (Figures 1A–C) was provided more than 50% of patients with confirmed WNV encephalitis had by several groups (Doron et al., 2003; Jeha et al., 2003; Kelly et al., severe muscle weakness as a cardinal sign (Nash et al.,1999). Weak- 2003; Leis et al., 2003a; Fratkin et al., 2004; Guarner et al., 2004). ness was an apparent risk factor predicting death in patients with Our postmortem examinations of four patients with WNV infec- WNV encephalitis (Nash et al., 1999; Petersen and Marfin, 2002). tion from the 2002 epidemic, who developed muscle weakness and In the 2002 and 2003 WNV epidemics in the United States, neuro- acute respiratory distress, showed that poliomyelitis was the major muscular manifestations were a well-recognized feature associated CNS finding in each case (Leis et al., 2003a; Fratkin et al., 2004). with increased morbidity and mortality (Jeha et al., 2003). In Col- This is in agreement with the neuropathology of experimental orado, the state with the most reported cases of WNV infection or naturally occurring WNV infection in monkeys (Manuelidis, (2,943) and fatalities (63) during the 2003 epidemic, as many as 1956), horses (Cantile et al., 2000, 2001), and birds (Steele et al., 50% of patients with encephalitis had evidence of acute flaccid 2000). In these vertebrates, WNV shows a pronounced tropism for paralysis (Tyler, 2004). gray matter of the spinal cord, causing poliomyelitis. In contrast, peripheral nerves are not commonly involved. WEST NILE VIRUS POLIOMYELITIS Most investigators actively involved in WNV clinical research In the original New York City outbreak, several case series now accept poliomyelitis to be the most common cause of WNV- attributed neuromuscular complications,particularly acute flaccid associated acute flaccid paralysis in humans. Moreover, the CDC paralysis, to peripheral neuronal processes, namely Guillain–Barré now classifies WNV infection into WNV fever or neuroinvasive

Table 1 | Characteristics in West Nile virus-associated poliomyelitis compared with typical Guillain–Barré syndrome.

West Nile poliomyelitis Guillain–Barré syndrome

Timing of onset Acute phase of infection Weeks after acute infection Fever, leukocytosis Present Absent Weakness distribution Asymmetric; monoplegia to quadriplegia Generally symmetric; proximal and distal muscles Sensory symptoms Some myalgias, infrequent numbness, paresthesias, or sensory loss Sensory loss, painful distal paresthesias Bowel and bladder Often involved Rarely involved Encephalopathy Often present Absent CSF proﬁle Pleocytosis, elevated protein No pleocytosis, elevated protein (albuminocyto- logic dissociation) Electrodiagnostic features Anterior horn cell or motor axon loss (reduced/absent CMAPs, Demyelination (marked slowing of conduction preserved SNAPs, asymmetric denervation) velocity, conduction block, temporal dispersion); reduced SNAPs

CSF,cerebrospinal ﬂuid; CMAP,compound muscle action potential; SNAP,sensory nerve action potential.

Frontiers in Neurology | Neuromuscular Diseases March 2012 | Volume 3 | Article 37 | 2 Leis and Stokic West Nile virus infection

disease,with further subdivision of the latter group into encephali- encephalitis, or respiratory distress from involvement of spinal tis, meningitis, and poliomyelitis. Patients with the poliomyelitis motor neurons supplying the phrenic nerves to the diaphragm, presentation commonly have associated signs of meningitis, but acute flaccid paralysis also may occur in the absence of fever or meningoencephalitis (Leis et al., 2003b; Li et al., 2003; Sej- var et al., 2003). Recovery from neurological sequelae of WNV neuroinvasive disease may be slow and incomplete (Davis et al., 2006), with a poorer prognosis for recovery of physical function in patients with acute flaccid paralysis (Johnstone et al., 2011). Our initial four patients in 2002 with acute flaccid paralysis and profound loss of anterior horn cells in paretic limbs based on electrodiagnostic studies (CDC 2002; Leis et al., 2002) remain weak in affected limbs almost a decade after the acute WNV illness. However, each of these patients had needle electromyographic (EMG) evidence of profound denervation in muscles of the most affected limbs with few or no voluntarily recruited motor unit potentials (MUPs) and absent or markedly reduced motor responses with normal sensory responses on nerve conduction studies. Table 2 shows motor and sensory amplitudes in one of these patients, a 50-year-old man with WNV poliomyelitis limited to the right upper limb. Follow-up nerve conduction studies at 3-, 6-, and 12-month continued to show markedly FIGURE 1 | Spectrum of pathological findings in West Nile virus reduced motor responses in proximal muscles with normal sen- poliomyelitis: (A) Chromatolytic neuron with eccentric nucleus and sory responses. Follow-up needle EMG examinations also showed distended cell body. Chromatolysis is usually triggered by damage to the persistent profound denervation in shoulder girdle muscles with cell body or axon. Neuronal recovery through regeneration can occur after only a single voluntarily recruited MUP in deltoid and biceps chromatolysis, but most often it is a precursor of cell death (apoptosis). The event of chromatolysis is also characterized by a prominent migration of the (Figure 2). This patient never regained the ability to abduct or nucleus toward the periphery of the cell and increase in the size of the cell forward elevate the arm past the horizontal position. In contrast, body (hematoxylin–eosin, original magnification ×400); (B) Activated limbs with lesser degrees of anterior horn cell loss, based on the microglial cells surround and ingest a dead neuron (neuronophagia), which is presence of recordable motor responses and preserved innerva- typical of viral infections (hematoxylin–eosin, original magnification ×400); (C) Blood vessel at the interface between ventral horn gray matter and tion on needle EMG, had better recovery of function. Indeed, adjacent white matter surrounded by a dense cuff of chronic inflammatory our observations after performing electrodiagnostic evaluations cells (perivascular inflammation); wm, white matter; gm, gray matter on many WNV patients with varying degrees of acute flaccid (hematoxylin–eosin, original magnification ×100); (D) Cervical sympathetic paralysis suggest that prognosis for recovery of function is greatly ganglia. Microglial nodules are clustered around eosinophilic husks of dying dependent on the degree of motor neuron loss; limbs with absent ganglion cells. Microglial cells are consuming the pyknotic sympathetic neurons (neuronophagia; hematoxylin–eosin, original magnification ×400). motor responses and no voluntary EMG activity have a poorer long-term prognosis while those with relatively preserved motor

Table 2 | Baseline-to-peak amplitudes of motor (m, in mV) and sensory (s, in μV) responses in a 50 year-old man with West Nile poliomyelitis of right upper limb.

Nerve Stimulation Recording Initial 3-month 6-month 12-month Normal site site study follow-up follow-up follow-up values

RLRLRLRL

Median (m) Wrist APB 2.0 11.4 3.8 11.2 6.3 – 8.4 12.7 ≥5.0 Median (s) Wrist Digit II 33.4 35.4 27.2 32.8 38.1 – 34.0 37.3 ≥20.0 Ulnar (m) Wrist ADM 1.6 7. 5 2.1 12.1 3.9 – 5.6 14.0 ≥4.5 Ulnar (s) Wrist Digit V 28.8 27.6 21.8 23.5 30.7 – 29.2 28.0 ≥15.0 Musculocutaneous (m) Erb’s point Biceps 0.2 7. 6 0.2 7. 0 0.6 – 2.0 11.6 ≥4.0 Musculocutaneous (s) Arm Forearm 27.1 32.6 21.4 – 22.0 – 18.6 19.5 ≥10.0 Axillary (m) Erb’s point Deltoid 0.4 5.3 0.2 7. 3 1.4 – 2.8 13.4 ≥4.0 Radial (s) Forearm Dorsum hand 31.9 31.8 39.8 40.3 39.4 – 34.8 44.5 ≥15.0

He initially presented with acute ﬂaccid paralysis and areﬂexia in the right arm, without pain or paresthesias. Sensory examination was normal. Initial electrodiagnostic studies showed markedly reduced motor responses in the monoplegic right limb (particularly in proximal muscles), with normal sensory responses. Note persistently reduced proximal motor responses 1 year after the acute illness. R, right; L, left; APB, abductor pollicis brevis; ADM, abductor digiti minimi; bold data are abnormal.

www.frontiersin.org March 2012 | Volume 3 | Article 37 | 3 Leis and Stokic West Nile virus infection

FIGURE 2 | Electromyographic (EMG) examination in a voluntarily recruited rapidly firing motor unit potential. Similar 50-year-old man with WNV poliomyelitis limited to the right denervation was noted in other shoulder girdle muscles. Clinical upper limb. Needle examination of the biceps muscle at 1 year follow-up 5 years later revealed persistent severe weakness in follow-up showed persistent profound denervation with only a single proximal upper limb muscles. responses and some voluntary activity have a more favorable although confusion may arise when acute flaccid paralysis attrib- outcome. utable to poliomyelitis is limited to one limb (monoparesis; Leis et al., 2003b). In one series that included 10 patients with WNV OTHER SPINAL CORD NEUROPATHOLOGY IN WEST NILE poliomyelitis, 4 patients had monoparesis (Emig and Apple, 2004). VIRUS INFECTION We have also encountered several patients with WNV and asym- Although the anterior horns are the major site of spinal cord metric weakness in the arms or legs who initially were thought to pathology (Kelly et al., 2003; Leis et al., 2003a; Fratkin et al., 2004), have cervical or lumbosacral radiculopathies (Leis et al., 2002; Leis autopsy series show that pathologic changes may extend beyond and Stokic, 2005). the spinal cord gray matter with focal inflammatory changes involving the adjacent white matter (Fratkin et al., 2004). This may WEST NILE VIRUS PERIPHERAL NERVE INVOLVEMENT explain the infrequent occurrence of WNV-associated transverse West Nile virus can involve peripheral nerves although this man- myelitis with clinical involvement of spinal sensory and motor ifestation is much less frequent than originally assumed during pathways (Leis, personal observation). In addition, neuronopha- the 1999 outbreak, when weakness and acute flaccid paralysis gia, neuronal disappearance, and pathologic alterations have been were attributed to GBS or axonal polyneuropathy (Nash et al., described in dorsal root ganglia and sympathetic ganglia (Fratkin 1999; Asnis et al., 2000; Sampson et al., 2000). In 1 series of et al., 2004). Involvement of dorsal root ganglia may explain some 64 patients with WNV infection, 3 patients had mixed axonal of the sensory deficits and reduced sensory nerve action poten- degenerating and demyelinating processes, and 1 had a pure tials on electrodiagnostic testing that occasionally are reported demyelinating neuropathy (Pepperell et al., 2003). In addition, in patients with WNV infection (Doron et al., 2003; Jeha et al., there are case reports of patients with true GBS (Ahmed et al., 2003). However, sensory loss attributed to WNV has not been a 2000; Xu et al., 2003; Sejvar et al., 2004), unilateral brachial prominent clinical finding.Another often overlooked finding is the plexopathy (Almhanna et al., 2003; Sejvar et al., 2004), and bilat- disappearance of neurons in the sympathetic ganglia (Figure 1D), eral diaphragmatic paralysis attributed to loss of motor neurons which offers a plausible explanation for the autonomic instability or motor axons supplying the phrenic nerves (Betensley et al., observed in some patients, including labile vital signs, hypoten- 2004). Lymphocytic infiltration of nerves and occasional degen- sion, and potentially lethal cardiac arrhythmias (Pepperell et al., erating axons also has been described (Li et al., 2003; Smith et al., 2003; Fratkin et al., 2004; Bode et al., 2006). In rodents, WNV can 2004), prompting a suggestion that WNV may reach the CNS lead to autonomic dysfunction by infecting neurons controlling viaperipheralnerves(Smith et al., 2004). Indeed, recent evidence cardiac and gastrointestinal function (Wang et al., 2011). These suggests that axonal transport mediates WNV entry into the CNS data lead to hypothesis that autonomic instability may play a role and induces acute flaccid paralysis (Samuel et al., 2007). How- in the morbidity and mortality of human WNV infection. ever, most autopsy series do not suggest that WNV exhibits a predilection for peripheral nerves. Similarly, WNV infection in WEST NILE VIRUS SPINAL NERVE ROOT INVOLVEMENT monkeys (Manuelidis, 1956), horses (Cantile et al., 2000, 2001), Inflammatory changes in spinal cord gray matter also may extend and birds (Steele et al., 2000) does not commonly involve periph- into the spinal nerve roots to cause a myeloradiculitis (Jeha et al., eral nerves. In addition, it should be recognized that prolonged 2003; Bouffard et al., 2004). Involvement of ventral spinal roots critical illness can be associated with an axonal sensorimotor may contribute to the asymmetric acute flaccid paralysis seen in polyneuropathy, termed critical illness polyneuropathy (Hund, many patients with WNV infection. Magnetic resonance imaging 2001), which can confound the interpretation of WNV-associated findings showing apparent enhancement of ventral nerve roots polyneuropathy. support the concept that anterior radiculopathy should be considered, in addition to anterior horn cell pathology, when assess- NEUROMUSCULAR JUNCTION AND SKELETAL MUSCLE IN ing patients with WNV-associated acute flaccid paralysis (Park WEST NILE VIRUS INFECTION et al., 2003). However, acute isolated radiculopathy as the sole West Nile virus has not been reported to cause a defect in neu- neuromuscular manifestation of WNV infection is not common, romuscular transmission, but we have encountered three patients

Frontiers in Neurology | Neuromuscular Diseases March 2012 | Volume 3 | Article 37 | 4 Leis and Stokic West Nile virus infection

with WNV poliomyelitis who developed classic myasthenia gravis neuromuscular diseases that have a presumed autoimmune mech- with positive acetylcholine receptor antibodies and a marked anism, including GBS (Ahmed et al., 2000; Xu et al., 2003; Sejvar decremental response on repetitive nerve stimulation studies (ini- et al., 2004, 2005), other demyelinating neuropathies (Pepperell tial case reported in Leis and Stokic, 2005). We also are aware et al., 2003; Sumner and Jones, 2008), myasthenia gravis (Leis and of the association of WNV and myasthenia gravis in two other Stokic, 2005), brachial plexopathies (Almhanna et al., 2003; Sej- patients (personal communication). Myopathy also is an uncom- var et al., 2004), and stiff-person syndrome (Hassin-Baer et al., mon manifestation of WNV infection, although there are several 2004). In the latter case, stiff-person syndrome with antibod- reports of rhabdomyolysis with creatine kinase levels as high as ies to glutamic acid decarboxylase developed several weeks after 45,000 U/L (normal <220 U/L; Doron et al., 2003; Jeha et al., WNV fever. CNS vasculitis with acute stroke or loss of vision 2003; Gupta et al., 2008). However, in one of these reports, post- due to WNV infection has also been reported (Kaiser et al., 2003; mortem examination confirmed poliomyelitis with striking loss of Kulstad and Wichter, 2003; Lowe et al., 2005). There is also exper- motor neurons in the anterior horn and brainstem, and only mild imental evidence that WNV causes a significant upregulation of inflammation without necrosis in skeletal muscle (Doron et al., class I and II major histocompatibility complex (MHC) mole- 2003). In another series, muscle biopsies on four patients with cules in Lewis rat Schwann cells (Argall et al., 1991). Moreover, WNV with acute asymmetric paralysis showed scattered necrotic irradiated medium from WNV-infected Schwann cell cultures muscle fibers invaded by macrophages (two patients), normal upregulated class I molecule expression on dissociated Schwann muscle fibers with inflammatory cells surrounding small blood cell cultures. These findings were considered to have implica- vessels (one patient), and a normal muscle biopsy (one patient; Li tions for virally triggered autoimmune disease of nervous tissue. et al., 2003). In one of the patients with scattered necrotic muscle Infection of human embryonic myoblasts by WNV also causes fibers, immunohistochemistry with polyclonal antibodies against a significant upregulation of class I and II MHC expression flaviviruses did not detect WNV on biopsied muscle. These inves- (Bao et al., 1992). MHC class I was increased after exposure to tigators acknowledged that the scattered necrotic muscle fibers virus-inactivated supernatant from infected cells, indicating the were an unlikely explanation for the severe paralysis observed (Li presence of additional factors contributing to the MHC class I et al., 2003). In the most comprehensive series of rhabdomyolysis increase. The investigators concluded that these findings may be in patients with WNV neuroinvasive disease, 9 of 244 hospi- important in establishing a link between viral infection of human talized patients had rhabdomyolysis (median age 70 years, CK cells and induction of inflammatory autoimmune myositis (Bao levels ranged from 1,153 to 42,113 IU; Montgomery et al., 2005). et al., 1992). Other studies have shown that WNV can regu- However, six of nine patients had history of recent falls prior late the cell surface expression of numerous immune recognition to admission. The authors concluded that although the tempo- molecules, resulting in increased recognition by WNV-specific ral relationship of rhabdomyolysis and WNV illness suggested a and allo-specific cytotoxic T cells (Kesson et al., 2002). How- common etiology, these patients presented with complex clinical ever, long-term follow-up of patients with WNV infection should conditions that may have led to development of rhabdomyoly- clarify whether there is an increased incidence of autoimmune sis from other causes (Montgomery et al., 2005). In addition, it diseases. is now commonly recognized that critical illness can be associated with a diffuse myopathy, termed critical illness myopathy, PERSISTENCE OF WEST NILE VIRUS IN THE HUMAN BODY which can cause generalized weakness, respiratory failure, and Acute WNV infection is usually cleared by an effective immune inability to wean from the respirator (Lacomis et al.,2000).Accord- response after several days of viremia. Recent evidence, however, ingly, the role played by direct WNV invasion of muscles and the suggests that WNV may persist long-term in animals (Appler et al., clinical significance of WNV-associated myositis remains to be 2010) and humans (Murray et al., 2010). Immunocompetent mice elucidated. inoculated with WNV showed infectious virus or WNV RNA West Nile virus has also been reported to cause myocarditis in the CNS and periphery for up to 6 months post-inoculation (Braun et al., 2006; Kushawaha et al., 2009) and cardiomyopathy (Appler et al., 2010). Viral persistence occurred in the face of a (Khouzam, 2009), which can predispose to fatal arrhythmia. How- robust antibody response and in the presence of inflammation in ever, cardiac arrhythmias have also been reported to occur across the brain. Furthermore, persistence in the CNS and pathological the spectrum of WNV disease, including in cases of WNV fever evidence of encephalitis were observed even in mice with subclin- not associated with myocarditis (Bode et al., 2006). Since statis- ical infections. In such cases, the CNS immune response appears tics on the incidence of WNV myocarditis as the cause of cardiac to be ineffective in clearing the virus (Stewart et al., 2011). In arrhythmias are lacking, it is possible that autonomic instability humans, WNV RNA was recently demonstrated in 5 (20%) of 25 caused by direct WNV infection of neurons controlling cardiac urine samples collected from convalescent WNV patients diag- function (Fratkin et al., 2004; Wang et al., 2011) may also have nosed with WNV neuroinvasive disease between 2002 and 2007 precipitated cardiac arrhythmias. (Murray et al., 2010). However, attempts to grow the virus from urine of these five patients were not successful. These findings WEST NILE VIRUS AND AUTOIMMUNE DISEASE suggest that in some individuals, WNV RNA may persist for sev- An unresolved issue that may have important neuromuscular eral years after infection. These results may also have implications implications is whether WNV infection can induce autoimmune for some WNV-infected humans, particularly immunosuppressed disease (Leis and Stokic, 2005). Support for this contention patients or those with long-term or recurrent sequelae. Conversely, arises from the numerous reports of WNV patients with various a subsequent study failed to demonstrate WNV RNA in urine

www.frontiersin.org March 2012 | Volume 3 | Article 37 | 5 Leis and Stokic West Nile virus infection

samples collected from an established cohort of 40 persons over during the phase of acute WN illness,with full recovery of function 6 years after initial infection with WNV. Urine from all partic- occurring within several weeks. Although rapid reversal of paraly- ipants tested negative for WNV RNA by reverse-transcription sis has rarely been reported withWNV infection,this phenomenon polymerase chain reaction and transcription-mediated amplifi- is not new, and was first observed in patients with poliomyelitis cation (Gibney et al., 2011). These conflicting findings suggest caused by the poliovirus (Paul, 1971). Jacob von Heine (1799– that prospective studies are needed to determine if and for how 1878) recognized the transitory nature of some attacks of paralysis long WNV may persist in urine or other tissues following acute early in the nineteenth century, and attributed the rapid improve- infection. ment to fluid exudate and edema in the spinal cord that was reabsorbed (Paul, 1971). Therefore, in acute WNV infection, as in SPECTRUM OF NEUROMUSCULAR SYMPTOMS AND SIGNS acute poliovirus infection,reversible paralysis may reflect transient IN WEST NILE VIRUS INFECTION anterior horn cell dysfunction. Two other patients developed exag- In our initial series of 54WNV patients from 2002 to 2006,who had gerated weakness in the distribution of preexisting lower motor extensive electrodiagnostic evaluation and four autopsies, neu- neuron dysfunction after WNV infection. One of these patients romuscular manifestations included: acute flaccid paralysis with developed persistent weakness limited to the same leg that had electrophysiologic or pathologic features of poliomyelitis (n = 23), been symptomatic 14 years earlier from spinal stenosis caused by clinical findings of autonomic instability (cardiac dysrhythmias, a synovial cyst. Another patient had a four-level lumbar laminec- marked fluctuations in blood pressure, gastrointestinal complica- tomy in 2001 for spinal stenosis that caused weakness, pain, and tions including gastroparesis) or pathologic alterations in sym- altered sensation in the right more than left leg. After surgery, pathetic ganglia (n = 8), brainstem dysfunction (n = 4) including symptoms improved although tingling persisted in the right leg. three cases of seventh nerve palsies (two delayed several weeks after After acute WNV illness 11 months later, the patient reported dis- acute illness and one with acute illness), myopathy (n = 3, in two abling fatigue and exaggerated weakness in the same distribution attributed to critical illness myopathy and one case of rhabdomy- as the preexisting weakness. In these patients, the observation that olysis with acute renal failure),new onset myasthenia gravis (n = 3, WNV infection caused exaggerated weakness in the previously one case diagnosed in 2011),diffuse axonal polyneuropathy (n = 2, weak limbs suggests that preexisting dysfunction may predispose one attributed to critical illness polyneuropathy and one to acute anterior horn cells to additional injury during acute WNV infec- polyneuropathy associated with WNV), transverse myelitis with tion. It is speculated that anterior horn cells that have survived involvement of sensory and motor pathways (n = 1), gait apraxia an initial insult, or incorporated too many muscle fibers from (n = 1), and optic nerve involvement (n = 1). In the latter case, denervated motor units beyond the metabolic capability, may be full-field pattern reversal visual evoked potential studies showed especially prone to injury (Leis et al., 2003b). a monocular abnormality that suggested a conduction defect in the visual pathways anterior to the optic chiasm. In nine patients, WEST NILE VIRUS IMMUNITY AND PATHOGENESIS the chief complaint was severe or disabling fatigue without objec- Although postmortem examinations have confirmed WNV tive muscle weakness on clinical examination or abnormalities on poliomyelitis and encephalitis, including patchy infection of neu- neurophysiologic studies. The specific physiologic mechanism for rons in the cerebral cortex, hippocampus, basal ganglia, cerebel- WNV fatigue remains unknown. Aside from the psycho-social fac- lum, and brain stem (Doron et al., 2003; Kelly et al., 2003; Leis tors, residual dysfunction in anterior horn cells and involvement et al., 2003a; Fratkin et al., 2004; Guarner et al., 2004), the pre- of brainstem reticular activating system may play a role. In the cise mechanisms that underlie destruction of neurons remain to post-polio syndrome, poliovirus-induced lesions in the reticular be fully elucidated. The increased risk of severe WNV infection activating system are thought to contribute to the subjective fatigue in immunosuppressed and elderly patients suggests that an intact (Bruno et al., 1994). In patients with WNV infection, prolonged immune system is essential for control of WNV. WNV-specific fatigue is common after the acute illness (Campbell et al., 2002), antibodies are responsible for reducing viremia and preventing affecting nearly two-thirds of patients. At 1 year follow-up, fatigue development of severe disease, while different T-lymphocyte pop- was the most common persistent symptom in patients hospital- ulations play an important role in clearing infection from tissues ized from the 1999 New York outbreak, affecting 67% of patients and preventing viral persistence (Lim et al., 2011). However, the whereas muscle weakness was found in 44% of patients (New pathologic effect of the immune system in WNV neuroinvasive York Department of Health, 2001; Petersen and Marfin, 2002). disease cannot be overlooked. Studies with a neuroadapted strain Thus, physicians should be aware that fatigue and subjective weak- of Sindbis virus, a related RNA neurotropic virus capable of induc- ness may be the major complaint in patients with WNV infection, ing death of spinal motor neurons in rodents resulting in acute particularly in those with WNV fever. flaccid hind-limb paralysis, revealed that many degenerating neu- In addition, we observed two patients that developed severe, rons were not directly infected with the virus (Jackson et al., but reversible, muscle weakness that recovered completely within 1988; Havert et al., 2000; Darman et al., 2004). Rather, bystander weeks. Both patients were hospitalized for their weakness. Weak- neuronal death occurred as a result of a cascade of immuno- ness involved both lower limbs in one patient (paraparesis) and biochemical events in the spinal cord that impaired glutamate one upper limb in the other (monoparesis). Their neurophysi- transport and allowed excess glutamate to accumulate extracel- ologic studies were unremarkable after recovery of function, in lularly around motor neurons. This excess synaptic glutamate is agreement with the clinical examination. The reversible muscle bound by calcium permeable glutamate receptors on post-synaptic weakness likely reflects transient anterior horn cell dysfunction motor neurons, causing the excitotoxic destruction of that neuron,

Frontiers in Neurology | Neuromuscular Diseases March 2012 | Volume 3 | Article 37 | 6 Leis and Stokic West Nile virus infection

whether it harbored the virus or not (Darman et al., 2004). WNV There also has been great interest in passive immunization with neuroinvasive disease is also characterized by increased produc- intravenous immune globulin (IVIG) for the treatment of patients tion of pro-inflammatory cytokines derived from infected cells with acute WNV infection. Immune serum frequently was used (Kumar et al., 2010) and upregulation of other pro-inflammatory in the pre-antibiotic era to treat infectious diseases, and animal genes (van Marle et al., 2007). These cytokines and other pro- data indicate an important role for humoral immunity in con- inflammatory factors are either neurotoxic or attract leukocytes trolling WNV infection (Gea-Banacloche et al., 2004). Given the into the affected area, which further contribute to WNV-induced endemic nature of WNV in the Middle East, IVIG obtained from neurotoxicity. Hence, WNV-induced inflammation is now recog- Israeli blood donors that contains WNV-specific antibodies pro- nized as a major contributor of neuropathogenesis. The concept vided clear-cut protection to animals treated before or shortly after of a pathologic effect of the immune system in WNV neuroin- infectious challenge with WNV (Ben-Nathan et al., 2003). In con- vasive disease provides a framework for the development of anti- trast, IVIG obtained from blood donors from the United States inflammatory drugs as much needed interventions to limit the without WNV-specific antibodies had no similar protective effect cascade of immunobiochemical events leading to neurotoxicity (Ben-Nathan et al., 2003). Several case reports also show efficacy (Kumar et al., 2010). of IVIG containing WNV-specific antibodies in aborting human WNV infection (Hamdan et al.,2002;Agrawal and Peterson,2003), TREATMENT FOR NEUROINVASIVE WEST NILE INFECTION although other case studies found no clear benefit (Haley et al., At present, no specific therapy has been approved for human use 2003). A double-blind placebo-controlled multicenter trial was in WNV infection. However, the merging knowledge about the initiated in 2003 to compare the efficacy of an Israeli IVIG prepa- pathogenesis of WNV infection has direct therapeutic implica- ration that had a high-titer anti-WNV antibody (Omr-IgG-am; tions. Treatment strategies that control the previously described Omrix, Tel Aviv) with that of U.S. IVIG (Polygam; Baxter Health- cascade of events leading to neuronal death may prove beneficial. care Corp.,Deerfield,IL,USA),which lacked detectable anti-WNV Promising therapies include the use of interferon and interferon antibody. However,the study did not enroll enough cases to permit inducers, which have been shown to reduce mortality in mice data analysis in the 2003 epidemic season. Nonetheless, neutral- infected by subcutaneous injection of WNV (Morrey et al., 2004). izing antibody therapeutics show promise in inhibiting WNV A preliminary report described three WNV patients with neuro- infection and preventing acute flaccid paralysis in vivo (Diamond, logic syndromes who showed improvement after receiving inter- 2009), which justifies phase I and II studies using humanized or feron alpha, thereby increasing optimism toward this treatment human monoclonal antibodies. (Sayao et al., 2004). Interferon gamma producing gamma-delta T cells also have been shown to prevent mortality from murine WNV DIFFERENTIAL DIAGNOSIS OF NEUROINVASIVE WEST NILE infection (Wanget al.,2003). Other potential therapies include rib- INFECTION avirin, nucleic acids, RNA interference, antisense oligomers, pep- Our observations and literature review suggest that patients with tides, imino sugars, and mycophenolic acid (for a review, see Dia- WNV infection who have muscle weakness or other neuromus- mond, 2009). These agents act through distinct mechanisms and cular signs and symptoms often are given erroneous diagnoses are moving through various stages of pre-clinical development. and may receive inappropriate, potentially injurious treatments. The role of corticosteroids in WNV neuroinvasive disease is Among patients referred to our rehabilitation center with WNV controversial, with concern that immunosuppressive effects may neuroinvasive disease,initial diagnoses that ultimately proved to be worsen outcome. However, high-dose steroids have been used erroneous included evolving stroke, GBS, myopathy, food poison- to successfully treat a patient with WNV-associated acute flac- ing, endocarditis, sepsis, heat stroke, malingering, gastroenteritis, cid paralysis (Pyrgos and Younus, 2004). In addition, findings drug reaction, spinal cord compression, diabetic amyotrophy, and in 14 patients with acute infection suggest that intravenous dex- myocardial infarction. Diagnostic studies and therapies directed at amethasone may be the reason for shortening the acute phase these erroneous diagnoses are typically ineffective and can produce of WNV meningoencephalitis and hastening patient recovery significant morbidity. Hence, physicians should consider a diag- (Narayanaswami et al., 2004). We also have administered high- nosis of WNV infection in any patient who presents with a febrile dose intravenous steroids to two patients with acute flaccid paral- illness that progresses over several days associated with neurologi- ysis and brainstem involvement, including progressive seventh cal signs or symptoms, especially during the summer months (i.e., nerve palsies, who showed clear improvement in brainstem symp- “summer flu” plus neurological symptoms). Health care providers toms and facial paralysis within 24 h of treatment. However, both also need to be aware that the spectrum of neuromuscular mani- cases were characterized by an atypical temporal pattern of pro- festations may range from a poliomyelitis syndrome in the absence gressive symptoms or new deficits occurring several weeks after of overt fever or meningoencephalitis to subjective weakness and the onset of the acute illness. In most cases, WNV is thought disabling fatigue. This awareness will help to avoid less tenable to be cleared by an effective immune response after only several diagnoses and inappropriate treatment. days of viremia. Accordingly, this relatively delayed progression The differential diagnoses of WNV infection include other of symptoms is more likely to reflect secondary injury from the arbovirus encephalitides (e.g., St. Louis encephalitis virus, Japan- downstream cascade of excitotoxic events and a secondary wave ese encephalitis virus, Eastern, Western, and Venezuelan equine of inflammation. In cases where the temporal course suggests that encephalitis viruses, tick borne encephalitis virus), other viral indirect immune-mediated mechanisms may be contributing to meningoencephalitides (La Crosse virus, Murray Valley virus, neuronal injury,a trial of high-dose corticosteroids seems justified. coxsackievirus, echovirus, enterovirus), bacterial meningitis or

www.frontiersin.org March 2012 | Volume 3 | Article 37 | 7 Leis and Stokic West Nile virus infection

encephalitis (including Lyme disease and Leptospirosis), tick CONCLUDING REMARKS paralysis, and non-infectious conditions that affect brain or spinal Although anterior horns are the major site of spinal cord pathol- cord (e.g., stroke, brain or spinal cord tumors, spinal cord com- ogy, inflammatory changes may also involve muscle fibers, periph- pression). Guillian–Barre syndrome and other immune-mediated eral nerves, spinal roots, and spinal sympathetic neurons and neuropathies are also diagnostic considerations, since in some ganglia, contributing to the wide spectrum of neuromuscular cases WNV poliomyelitis may mimic these disorders (see Table 1). manifestations of WNV infection. Unresolved issues with impor- Poliovirus poliomyelitis should also be considered in the differen- tant implications include whether WNV may persist in the CNS tial diagnosis if the patient resides in or travels to a polio-endemic or periphery, and whether WNV infection may lead to autoim- region. However, in 2011 only four countries (India, Afghanistan, mune disease. Although there is no specific treatment or vaccine Nigeria, and Pakistan) remain polio-endemic (see text footnote 1). approved for human WNV infection, several drugs that can alter In addition, poliovirus infection mainly affects infants or young the cascade of immunobiochemical events leading to neuronal children. death may be potentially useful.

REFERENCES Ben-Nathan, D., Lustig, S., Tam, G., Centers for Disease Control and Pre- after acute West Nile virus disease. J. Agrawal, A. G., and Peterson, L. R. Robinzon, S., Segal, S., and Rager- vention. (2002). Acute flaccid paral- Infect. Dis. 203, 344–347. (2003). Human immunoglobulin as Zisman, B. (2003). Prophylactic and ysis syndrome associated with West Glass, J. D., Samuels, O., and Rich, M. a treatment for WestNile virus infec- therapeutic efficacy of human intra- Nile virus infection- Mississippi M. (2002). Polio myelitis due to West tion. J. Infect. Dis. 188, 1–4. venous immunoglobulin in treating and Louisiana, July-August 2002. Nile virus. N.Engl.J.Med.347, Ahmed, S., Libman, R., Wesson, West Nile virus infection in mice. J. MMWR 51, 825–828. 1280–1281. K., Ahmed, F., and Einberg, K. Infect. Dis. 188, 5–12. Darman, J., Backovic, S., Dike, S., Mara- Guarner, J., Shieh, W. J., Hunter, S., Pad- (2000). Guillain-Barre syndrome: Betensley, A. D., Jaffery, S. H., Collins, gakis, N. J., Krishnan, C., Roth- dock, C. D., Morken, T., Campbell, an unusual presentation of West H., Sripathi, N., and Alabi, F. (2004). stein, J. D., Irani, D. N., and Kerr, G. L., Marfin, A. A., and Zaki, S. R. Nile virus infection. Neurology 55, Bilateral diaphragmatic paralysis D. A. (2004). Viral-induced spinal (2004). Clinicopathologic study and 144–146. and related respiratory complica- motor neuron death is non-cell- laboratory diagnosis of cases with Ali, M., Safriel, Y., Sohi, J., Llave, A., tions in a patient with West Nile autonomous and involves gluta- West Nile virus encephalomyelitis. and Weathers, S. (2005). West Nile virus infection. Thorax 59, 268–269. mate excitotoxicity. J. Neurosci. 34, Hum. Pathol. 35, 983–990. virus infection: MR imaging find- Bode, A. V., Sejvar, J. J., Pape, W. J., 7566–7575. Gupta, M., Ghaffari, M., and Freire, ings in the nervous system. AJNR Campbell, G. L., and Marfin, A. Davis, L. E., DeBiasi, R., Goade, D. E., A. X. (2008). Rhabdomyolysis in a Am. J. Neuroradiol. 26, 289–297. A. (2006). West Nile virus disease: Haaland, K. Y., Harrington, J. A., patient with West Nile encephalitis Almhanna, K., Palanichamy, N., a descriptive study of 228 patients Harnar, J. B., Pergam, S. A., King, and flaccid paralysis. Tenn. Med. 101, Sharma, M., Hobbs, R., and Sil, A. hospitalized in a 4-county region of M. K., DeMasters, B. K., and Tyler, 45–47. (2003). Unilateral brachial plexopa- Colorado in 2003. Clin. Infect. Dis. K. L. (2006). West Nile virus neu- Haley, M., Retter, A. S., Fowler, D., thy associated with West Nile virus 42, 1234–1240. roinvasive disease. Ann. Neurol. 60, Gea-Banacloche, J., and O’Grady, N. meningoencephalitis. Clin. Infect. Bouffard, J. P., Riudavets, M. A., Hol- 286–300. P. (2003). The role for intravenous Dis. 36, 1629–1630. man, R., and Rushing, E. J. (2004). Diamond, M. S. (2009). Progress on the immunoglobulin in the treatment of Al-Shekhlee, A., and Katirji, B. (2004). Neuropathology of the brain and development of therapeutics against West Nile virus encephalitis. Clin. Electrodiagnostic features of acute spinal cord in human West Nile West Nile virus. Antiviral Res. 83, Infect. Dis. 37, e88–e90. paralytic poliomyelitis associated virus infection. Clin. Neuropathol. 214–227. Hamdan, A., Green, P., Mendelson, E., with West Nile virus infection. Mus- 23, 59–61. Doron, S. I., Dashe, J. F., Adelman, Kramer, M. R, Pitlik, S., and Wein- cle Nerve 29, 376–380. Braun, L. E., Tsuchida, T., and Spiegel, L. S., Brown, W. F., Werner, B. G., berger, M. (2002). Possible benefit of Appler, K. K., Brown, A. N., Stewart, B. H. (2006). Meningoencephalitis in and Hadley, S. (2003). Histopatho- intravenous immunoglobulin ther- S., Behr, M. J., Demarest,V.L.,Wong, a child complicated by myocarditis, logically proven poliomyelitis with apy in a lung transplant recipient S. J., and Bernard, K. A. (2010). Per- quadriparesis and respiratory fail- quadriplegia and loss of brainstem with West Nile virus encephalitis. sistence of West Nile virus in the ure. Pediatr. Infect. Dis. J. 25, 853, function due to West Nile infection. Transpl. Infect. Dis. 4, 160–162. central nervous system and periph- 855–856. Clin. Infect. Dis. 37, e74–e77. Hassin-Baer, S., Kirson, E. D., Shul- ery of mice. PLoS ONE 5, e10649. Bruno, R. L., Cohen, J. M., Gal- Emig,M.,and Apple,D. J. (2004). Severe man, L., Buchman, A. S., Bin, H., doi:10.1371/journal.pone.0010649 ski, T., and Frick, N. M. (1994). West Nile virus disease in healthy Hindiyeh, M., Markevich, L., and Argall, K. G., Armati, P. J., King, N. The neuroanatomy of post-polio adults. Clin. Infect. Dis. 38, 289–292. Mendelson, E. (2004). Stiff-person J., and Douglas, M. W. (1991). The fatigue. Arch. Phys. Med. Rehabil. 75, Fratkin, J. D., Leis, A. A., Stokic, D. S., syndrome following West Nile fever. effects of West Nile virus on major 498–504. Slavinski, S. A., and Geiss, R. W. Arch. Neurol. 61, 938–941. histocompatibility complex class I Campbell, G. L., Marfin, A. A., Lan- (2004). Spinal cord neuropathology Havert, M. B., Schofield, B., Griffin, D. and II molecule expression by Lewis ciotti, R. S., and Gubler, D. J. (2002). in human West Nile virus infection. E., and Irani, D. N. (2000). Activa- rat Schwann cells in vitro. J. Neu- West Nile virus. Lancet. Infect. Dis. 2, Arch. Pathol. Lab. Med. 128,533–537. tion of divergent neuronal cell death roimmol. 35, 273–284. 519–529. Gea-Banacloche, J., Johnson, R. T., pathways in different target cell pop- Asnis, D. S., Conetta, R., Teixeira, A. A., Cantile, C., Di Guardo, G., Eleni, C., Bagic, A., Butman, J. A., Murray, P. ulations during neuroadapted Sind- Waldman, G., and Sampson, B. A. and Arispici, M. (2000). Clinical and R., and Agrawal, A. G. (2004). West bis virus infection of mice. J. Virol. (2000). The WestNile virus outbreak neuropathological features of West Nile virus: pathogenesis and ther- 4, 5352–5356. of 1999 in New York City: the flush- Nile virus equine encephalomyelitis apeutic options. Ann. Intern. Med. Hund, E. (2001). Critical illness ing hospital experience. Clin. Infect. in Italy. Equine Vet. J. 32, 31–35. 140, 545–553. polyneuropathy. Curr. Opin. Neurol. Dis. 30, 413–418. Cantile, C., Del Piero, F., Di Guardo, G., Gibney, K. B., Lanciotti, R. S., Sejvar, J. J., 4, 649–653. Bao, S., King, N. J., and Dos Reme- and Arispici, M. (2001). Pathologic Nugent, C. T., Linnen, J. M., Delorey, Jackson, A. C., Moench, T. R., Trapp, dios, C. G. (1992). Flavivirus induces and immunohistochemical findings M. J., Lehman, J. A., Boswell, E. N., B. D., and Griffin, D. E. (1988). MHC antigen on human myoblasts: in naturally occuring West Nile virus Staples, J. E., and Fischer, M. (2011). Basis of neurovirulence in Sind- a model of autoimmune myositis? infection in horses. Vet. Pathol. 38, West nile virus RNA not detected bis virus encephalomyelitis of mice. Muscle Nerve 15, 1271–1277. 414–421. in urine of 40 people tested 6 years Lab. Invest. 58, 503–509.

Frontiers in Neurology | Neuromuscular Diseases March 2012 | Volume 3 | Article 37 | 8 Leis and Stokic West Nile virus infection

Jeha, L. E., Sila, C. A., Lederman, R. J., (2003b). Clinical spectrum of mus- encephalitis: experience with cortico syndrome in Calgary, Alberta: clini- Prayson,R. A.,Isada,C. M.,and Gor- cle weakness in human West Nile steroid therapy. Neurology 62, A404. cal presentations and treatment with don, S. M. (2003). West Nile virus virus infection. Muscle Nerve 28, Nash, D., Mostashari, F., Fine, A., Miller, interferon alpha-2B. Neurology 62, infection. A new acute paralytic ill- 302–308. J.,O’Leary,D.,Murray,K.,Huang,A., A404. ness. Neurology 61, 55–59. Leis, A. A., and Stokic, D. S. (2005). Rosenberg, A., Greenberg, A., Sher- Sejvar, J., Bode, A., Ewing, D., Johnstone, J., Hanna, S. E., Nicolle, L. E., Neuromuscular manifestations of man, M., Wong, S., Layton, M., and Mazowiecki, M., Pavot, P., Marfin, Drebot, M. A., Neupane, B., Mahony, human West Nile virus infection. West Nile Outbreak Response Work- A., and Petersen, L. (2004). The J. B., and Loeb, M. B. (2011). Prog- Curr. Treat. Options Neurol. 7, ing Group. (1999). The outbreak of spectrum of West Nile virus- nosis of West Nile virus associated 15–22. West Nile virus infection in the New associated weakness. Neurology 62, acute flaccid paralysis: a case series. Leis, A. A., Stokic, D. S., Polk, J. L., York City area in 1999. N. Engl. J. A446. J. Med. Case Reports 5, 395. Dostrow, V., and Winkelmann, M. Med. 344, 1807–1814. Sejvar, J. J., Bode, A. V., Marfin, Kaiser, P. K., Lee, M. S., and Martin, D. (2002). Poliomyelitis-like syndrome New York Department of Health. A. A., Campbell, G. L., Ewing, A. (2003). Occlusive vasculitis in a from West Nile virus infection. N. (2001). West Nile virus surveillance, D., Mazowiecki, M., Pavot, P. V., patient with concomitant West Nile Engl.J.Med.347, 1279–1280. and control: an update for health- Schmitt, J., Pape, J., Biggerstaff, virus infection. Am. J. Ophthalmol. Li, J., Loeb, J. A., Shy, M. E., Shah, careprovidersinNewYorkCity.City B. J., and Petersen, L. R. (2005). 136, 928–930. A. K., Tselis, A. C., Kupski, W. J., Health Inf. 20, 1–7. West Nile virus-associated flaccid Kelly, T. W., Prayson, R. A., and Isada, C. and Lewis, R. A. (2003). Asym- Park, M., Hui, J. S., and Bartt, R. paralysis. Emerging Infect. Dis. 11, M. (2003). Spinal cord pathology in metric flaccid paralysis: a neuro E. (2003). Acute anterior radiculi- 1021–1027. West Nile virus infection. N. Engl. J. muscular presentation of West Nile tis associated with West Nile virus Sejvar, J. J., Leis, A. A., Stokic, D. S., Med. 348, 564–566. virus infection. Ann. Neurol. 53, infection. J. Neurol. Neurosurg. Psy- Van Gerpen, J. A., Marfin, A. A., Kesson, A. M., Cheng, Y., and King, 703–710. chiatr. 74, 823–825. Webb, R., Haddad, M. B., Tierney, N. J. (2002). Regulation of immune Lim, S. M., Koraka, P., Osterhaus, A. Paul, J. R. (1971). A History of B. C., Slavinski, S. A., Polk, J. L., recognition molecules by flavivirus, D. M. E., and Martina, B. E. E. Poliomyelitis. New Haven: Yale Uni- Dostrow, V., Winkelmann, M., and West Nile. Viral Immunol. 15, (2011). West Nile virus: immu- versity Press. Petersen, L. R. (2003). Acute flac- 273–283. nity and pathogenesis. Viruses 3, Pepperell, C., Rau, N., Krajden, S., Kern, cid paralysis and West Nile virus Khouzam, R. N. (2009). Significant car- 811–828. R., Humar, A., Mederski, B., Simor, infection. Emerging Infect. Dis. 9, diomyopathy secondary to West Nile Lowe, L. H., Morello, F. P., Jack- A., Low, D. E., McGeer, A., Maz- 788–793. virus infection. South. Med. J. 102, son, M. A., and Lasky, A. (2005). zulli, T., Burton, J., Jaigobin, C., Smith, R. D., Konoplev, S., DeCourten- 527–528. Application of transcranial Doppler Fearon, M., Artsob, H., Drebot, M. Myers, G., and Brown, T. (2004). Kulstad, E. B., and Wichter, M. D. sonography in children with acute A., Halliday, W., and Brunton, J. West Nile virus encephalitis with (2003). West Nile encephalitis pre- neurologic events due to primary (2003). West Nile virus infection myositis and orchitis. Hum. Pathol. senting as a stroke. Ann. Emerg. Med. cerebral and West Nile vasculi- in 2002: morbidity and mortality 35, 254–258. 41, 283. tis. AJNR Am. J. Neuroradiol. 26, among patients admitted to hospital Smithburn, K. C., Hughes, T. P., Burke, Kumar, M., Verma, S., and Nerurkar, 1698–1701. in southcentral Ontario. CMAJ 168, A. W., and Paul, J. H. (1940). A V. R. (2010). Pro-inflammatory Manuelidis, E. E. (1956). Neuropathol- 1399–1405. neurotropic virus isolated from the cytokines derived from West Nile ogy of experimental West Nile Petersen, L. R., and Marfin,A. A. (2002). blood of a native of Uganda. Am. J. virus (WNV)-infected SK-N-SH virus infection in monkeys. J. West Nile virus: a primer for the Trop. Med. 20, 471–492. cells mediate neuroinflammatory Neuropathol. Exp. Neurol. 15, clinician. Ann. Intern. Med. 137, Spigland, I., Jasinska-Klinberg, W., Hof- markers and neuronal death. J. Neu- 448–460. 173–179. shi, E., and Goldblum, N. (1958). roinflammation 31, 7–73. Montgomery, S. P., Chow, C. C., Smith, Petropoulou, K. A., Gordon, S. M., Clinical and laboratory observa- Kushawaha, A., Jadonath, S., and S. W., Marfin, A. A., O’Leary, D. Prayson, R. A., and Ruggierri, P. tions in an outbreak of West Mobarakai, N. (2009). West Nile R., and Campbell, G. L. (2005). M. (2005). West Nile virus menin- Nile fever in Israel. Harefuah 54, virus myocarditis causing a fatal Rhabdomyolysis in patients with goencephalitis: MR imaging find- 275–281. arrhythmia: a case report. Cases J. 2, west nile encephalitis and menin- ings. AJNR Am. J. Neuroradiol. 8, Steele, K. E., Linn, M. J., Schoepp, R. 7147. gitis. Vector Borne Zoonotic Dis. 5, 1986–1995. J., Komar, N., Geisbert, T. W., Man- Lacomis, D., Zochodne, D. W., and Bird, 252–257. Pyrgos, V.,and Younus, F. (2004). High- duca, R. M., Calle, P. P., Raphael, S. J. (2000). Critical illness myopa- Morrey, J. D., Day, C. W., Julander, J. G., dose steroids in the management of B. L., Clippinger, T. L., Larsen, T., thy. Muscle Nerve 23, 1785–1788. Blatt, L. M., Smee, D. F., and Sidwell, acute flaccid paralysis due to West Smith, J., Lanciotti, R. S., Panella, Lanciotti, R. S., Roehrig, J. T.,Deubel,V., R. W. (2004). Effect of interferon- Nile virus infection. Scand. J. Infect. N. A., and McNamara, T. S. (2000). Smith, J., Parker, M., Steele, K., Crise, alpha and interferon-inducers on Dis. 36, 509–512. Pathology of fatal West Nile virus B., Volpe, K. E., Crabtree, M. B., West Nile virus is mouse and ham- Sampson, B. A., Ambrosi, C., Char- infection in native and exotic birds Scherret, J. H., Hall, R. A., MacKen- ster animal models. Antivir. Chem. lot, A., Reiber, K., Veress, J. F., during the 1999 outbreak in New zie, J. S., Cropp, C. B., Panigrahy, B., Chemother. 15, 101–109. and Armbrustmacher, V. (2000). York Cit y, New York. Vet. Pathol. 37, Ostlund, E., Schmitt, B., Malkinson, Murgue, B., Murri, S., Triki, H., Deubel, The pathology of human West Nile 208–224. M., Banet, C., Weissman, J., Komar, V.,and Zeller,H. G. (2001).WestNile virus infection. Hum. Pathol. 31, Stewart, B. S., Demarest, V. L., Wong, N., Savage, H. M., Stone, W., McNa- in the Mediterranean basin: 1950- 527–531. S. J., Green, S., and Bernard, KA. mara, T., and Gubler, D. J. (1999). 2000. Ann. N. Y. Acad. Sci. 951, Samuel, M. A., Wang, H., Siddharthan, (2011). Persistence of virus-specific Origin of the WestNile virus respon- 117–126. V., Morrey, J. D., and Diamond, immune responses in the central sible for an outbreak of encephalitis Murray, K., Walker, C., Herrington, E., M. S. (2007). Axonal transport nervous system of mice after West in the northeastern United States. Lewis, J. A., McCormick, J., Beasley, mediates West Nile virus entry Nile virus infection. BMC Immunol. Science 286, 2333–2337. D. W., Tesh, R. B., and Fisher- into the central nervous system 12, 6. doi:10.1186/1471-2172-12-6 Leis, A. A., Fratkin, J., Stokic, D. S., Hoch, S. (2010). Persistent infec- and induces acute flaccid paralysis. Sumner, N., and Jones, L. (2008). Mul- Harrington, T., Webb, R. M., and tion with West Nile virus years after Proc. Natl. Acad. Sci. U.S.A. 104, tifocal neuropathy associated with Slavinski, S. A. (2003a). West Nile initial infection. J. Infect. Dis. 201, 17140–17145. West Nile virus infection. Neurology poliomyelitis. Lancet. Infect. Dis. 3, 2–4. Sayao, A., Patry, D., Suchowersky, O., 71, 1123. 9–10. Narayanaswami, P., Edwards, L., Hyde, Al-Khathaami, A., and Klassen, B. Tyler, K. L. (2004). West Nile virus Leis, A. A., Stokic, D. S., Webb, R. C., Page, C., and Hastings, N. (2004). A report of seven cases infection in the United States. Arch. M., Slavinski, S. A., and Fratkin, J. E. (2004). West Nile meningitis/ of West Nile virus neurological Neurol. 61, 1190–1195.

www.frontiersin.org March 2012 | Volume 3 | Article 37 | 9 Leis and Stokic West Nile virus infection

Tyler, K. L., Pape, J., Goody, R. J., Corkill, nervous dysfunction in hamsters T. (2001). West Nile fever out- Received: 13 October 2011; accepted: M., and Kleinschmidt-DeMasters, infected with West Nile virus. PLoS break, Israel, 2000: epidemiologic 26 February 2012; published online: 21 B. K. (2006). CSF findings in ONE 6, e19575. doi:10.1371/jour- aspects. Emerging Infect. Dis. 7, March 2012. 250 patients with serologically con- nal.pone.0019575 686–691. Citation: Leis AA and Stokic DS (2012) firmed West Nile virus meningi- Wang, T., Scully, E., Yin, Z., Kim, Xu, M. Y., Talkad, A. V., Karbowska- Neuromuscular manifestations of West tis and encephalitis. Neurology 66, J. H., Wang, S., Yan, J., Mamula, Jankowska, M., Blume, G. M., Nile virus infection. Front. Neur. 3:37. 361–365. M., Anderson, J. F., Craft, J., Somaraju, V., and Slagle, D. C. doi: 10.3389/fneur.2012.00037 van Marle, G., Antony, J., Ostermann, and Fikrig, E. (2003). IFN-gamma- (2003). Guillain-Barre syndrome This article was submitted to Frontiers H., Dunham, C., Hunt, T., Hall- producing gamma delta T cells associated with West Nile virus in Neuromuscular Diseases, a specialty of iday, W, Maingat, F., Urbanowski, help control murine West Nile infection. Neurology 60, A107. Frontiers in Neurology. M. D., Hobman, T., Peeling, J., virus infection. J. Immunol. 171, Copyright © 2012 Leis and Stokic. This is and Power, C. (2007). West Nile 2524–2531. an open-access article distributed under virus-induced neuroinflammation: Weinberger, M., Pitlik, S. D., Gan- Conflict of Interest Statement: The the terms of the Creative Commons Attri- glial infection and capsid protein- dacu, D., Lang, R., Nassar, F., Ben authors declare that the research was bution Non Commercial License, which mediated neurovirulence. J. Virol. David, D., Rubinstein, E., Izthaki, conducted in the absence of any com- permits non-commercial use, distribu- 81, 10933–10949. A., Mishal, J., Kitzes, R., Siegman- mercial or financial relationships that tion, and reproduction in other forums, Wang, H., Siddharthan, V., Hall, J. O., Igra,Y.,Giladi, M., Pick, N., Mendel- could be construed as a potential con- provided the original authors and source and Morrey, J. D. (2011). Autonomic son, E., Bin, H., and Shohat, flict of interest. are credited.

Frontiers in Neurology | Neuromuscular Diseases March 2012 | Volume 3 | Article 37 | 10 De Filette et al. Veterinary Research 2012, 43:16 http://www.veterinaryresearch.org/content/43/1/16 VETERINARY RESEARCH

REVIEW Open Access Recent progress in West Nile virus diagnosis and vaccination Marina De Filette1*, Sebastian Ulbert2, Mike Diamond3 and Niek N Sanders1

Abstract West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.

Table of contents 1. Introduction 1. Introduction West Nile virus (WNV) is a zoonotic mosquito-trans- 2. West Nile virus mitted arbovirus belonging to the genus Flavivirus in 3. Epidemiology the family Flaviviridae. WNV is maintained in a mos- 4. Pathogenesis quito-bird-mosquito transmission cycle [1], whereas 5. Diagnosis humans and horses are considered dead-end hosts. WNV is transmitted primarily by the bite of infected 5.1. Nucleic acid based tests for WNV mosquitoes, themselves acquiring the virus by feeding 5.2. Serologic diagnosis of WNV infections on infected birds. 5.3. WNV antigen detection The West Nile virus has been reported in dead or dying birds of at least 326 species [2]. The clinical outcome of 6. Vaccination infection is variable e.g. chickens and turkeys are resistant to disease while some species are particularly susceptible, 6.1. Licensed West Nile virus vaccines for animals e.g. crows, Carolina chickadees, tufted titmice, blue jays, 6.2. WNV vaccines under development American robins, and eastern bluebirds. 6.3. Clinical trials with West Nile virus vaccines in WNV has a wide geographical range that includes por- humans tions of Europe, Asia, Africa, Australia (Kunjin virus) and North, Central and South America [3]. Migratory birds are 7. Conclusions thought to be primarily responsible for virus dispersal, 8. Competing interests including reintroduction of WNV from endemic areas 9. Authors’ contributions into regions that experience sporadic outbreaks [3]. 10. Acknowledgments In humans, it was first isolated in the West Nile pro- 11. References vince of Uganda in 1937 from the blood of a woman suffering from a mild febrile illness [4]. Until the mid 1990’s, West Nile (WN) disease was considered as a minor risk for humans and horses because it only * Correspondence: [email protected] appeared sporadically. The first cases of West Nile virus 1Laboratory of Gene Therapy, Faculty of Veterinary Sciences, Ghent in its lethal encephalitic form were reported in Algeria University, Heidestraat 19, 9820 Merelbeke, Belgium Full list of author information is available at the end of the article in 1994. Since the first large outbreak in Romania in

© 2012 De Filette et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. De Filette et al. Veterinary Research 2012, 43:16 Page 2 of 15 http://www.veterinaryresearch.org/content/43/1/16

1996, which was characterized by a high number of neu- protease. Flaviviruses replication requires the viral pro- roinvasive cases, and the huge epidemics and equine tein NS5, which is an RNA-dependent RNA polymerase epizootics into North America that followed West Nile [15,16]. An “antisense” negative strand RNA is produced virus introduction in New York city in 1999 [5], WN by this enzyme, which then serves as a template for the associated disease has become a major public health and synthesis of many new copies of the infectious positive veterinary concern. strand RNA genome. WNV assembles on virus-induced membranes derived 2. West Nile virus from the endoplasmic reticulum and buds into the The structure of WNV particles have been elucidated by lumen as immature virions on which E and prM pro- Mukhopadhay et al. [6]. Electron microscopy and image teins form 60 heterotrimeric spikes. Transit of the reconstruction techniques established that WNV is a immature virion through the mildly acidic compart- small spherical icosahedral virus with a 50 nm diameter ments of the trans-Golgi network triggers a rearrange- and a lipid envelope surrounding an icosahedral nucleo- ment of E proteins on the immature virion; the lower capsid consisting of capsid proteins that are associated pH induces a structural transition such that E proteins with the RNA genome (Figure 1a). The sequence of the lie flat as antiparallel dimers on the surface of the virion WNV genome and the function of the different viral [17]. Under acidic conditions, prM remains associated proteins in the viral lifecycle as well as immune evasion with the virion and protrudes from the surface of an have been described in detail elsewhere (Figure 1b) otherwise smooth virus particle. This pH-dependent (reviewed by Ulbert et al. [7]). conformational change increases the susceptibility of During viral entry (Figure 2), the E protein interacts prM for a furin-like serine protease. Cleavage and with one or more cell surface receptor(s). It is not com- release of prM completes the virion maturation process, pletely clear which cellular receptors are involved in and is a required step in the virus lifecycle. WNV binding, however DC-SIGN, alphaVbeta3 integrin Flavivirus nonstructural (NS) proteins modulate the [8] and laminin-binding protein [9] have been reported host antiviral response. This was recently reviewed by as potential receptors. After binding to the cell, the Diamond et al. [18]. Flavivirus NS1 is a versatile nonvirus is taken up via clathrin-mediated endocytosis [10] structural glycoprotein, with intracellular NS1 function- and in the acidified endosome the E protein undergoes ing as an essential cofactor for viral replication [19] and conformational changes resulting in fusion between the antagonist of TLR signaling [20], whereas NS1 at the viral and cellular membranes [11]. After the fusion cell surface and secreted NS1 antagonize complement event the positive-stranded RNA genome is released activation [21,22]. NS2A is involved in the biogenesis of into the cytoplasm of the cell. The viral RNA is trans- virus-induced membranes, which have a vital role in lated into a single polyprotein [12], which is proteolyti- virus assembly [23]. NS2A also inhibits interferon-b pro- cally processed to yield three structural proteins (the moter activation [24]. The flavivirus non-structural pro- envelope protein E; the membrane precursor protein tein NS4B and NS5 block activation of the JAK-STAT1 prM; and the capsid protein C) and seven non-structural signaling pathway [25], which limits production of anti- (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and viral interferon stimulated genes in infected cells. NS5). Whereas the cleavages at the junctions C-prM, prM-E, E-NS1, NS4A-NS4B [13], and likely also NS1- 3. Epidemiology NS2A [14], are performed by the host signal peptidase Birds are the primary vertebrate hosts of WNV and in located within the lumen of the ER, the remaining pep- endemic regions, the virus is maintained in an enzootic tide bonds are cleaved by the virus encoded NS3 cycle between Culex mosquitoes and birds [1]. Mosquito

Figure 1 The structure (a) and 11 kb long RNA genome (b) of West Nile virus. De Filette et al. Veterinary Research 2012, 43:16 Page 3 of 15 http://www.veterinaryresearch.org/content/43/1/16

Figure 2 West Nile virus life cycle. After binding and uptake, the virion envelope fuses with cellular membranes, followed by uncoating of the nucleocapsid and release of the RNA genome into the cytoplasm. The viral genome serves as messenger RNA (mRNA) for translation of all viral proteins and as template during RNA replication. Copies are subsequently packaged within new virus particles which are transported in vesicles to the cell membrane. Reprinted with permission from PNAS 2002, vol. 99 no. 18 11555-11557. Copyright 2002 National Academy of Sciences, U.S.A. species from other genera are also susceptible to infec- However in 2010 the epidemiological situation changed tion. There is now indirect evidence that WNV is trans- with the second largest outbreak of the disease in the ported by migratory birds to the temperate areas of EU occurring in Greece [35]. During this outbreak 262 Europe during spring migration [26-28]. Mammals are cases were confirmed with 197 patients suffering from less important than birds in maintaining transmission West Nile neuroinvasive disease and 33 reported fatal- cycles of the virus as viremia is too low in most of the ities. In the same year 480 cases of West Nile infections mammal species to reinfect mosquitoes. Humans are were detected in Russia and also some cases were regarded as dead-end hosts because the concentration of reported in Romania. virus within the blood is insufficient to infect a feeding In 1999, WNV was detected for the first time in the naïve mosquito [29]. However, in humans, the virus can Western Hemisphere in New York [36]. During this out- spread between individuals by blood transfusion and break 62 human cases of WNV including 7 deaths were organ transplantation [30]. Few reports describe the identified in New York. Despite intense mosquito control possible transmission from a mother to her newborn via measures to minimize human infections, WNV spread the intrauterine [31] route or via breast-feeding [32]. into Canada and the remainder of the lower 48 continen- West Nile virus was first isolated in Uganda in 1937 tal states. WNV became endemic within 10 years of its from the blood of a febrile woman [4]. It is now recog- introduction in North America. So far, in the United nized as the most widespread of the flaviviruses, with States between 1999 and 2010, 30 662 cases were con- geographic distribution in the United States of America, firmed and associated with 1163 deaths [37]. Australia, Africa and Eurasia. The largest outbreak of It has been recently proposed that WNV can be WNVinEuropetodatewasinRomaniain1996when grouped into 7 lineages [38]. Two major genetic lineages 800 clinical cases of neuroinvasive disease were reported of WNV have been well described based on phylogenetic and in 393 cases the presence of WNV was confirmed analysis [39]. Lineage 1 is widespread and contains iso- [33]. A total of 17 deaths were reported during this out- lates from Europe, the USA, the Middle East, India, break. Between 1997 and 2010 in Europe, WNV infec- Africa and Australia. Lineage 1 is further segregated into tions have been observed sporadically in Portugal, Spain, three different clades: 1a, 1b and 1c. WNV-1a is mainly France, Czech Republic, Hungary and Italy and during a found in Europe, North America, Middle East and Africa. severe outbreak in Russia in 1999 (318 cases) [34]. This clade can further be divided in six clusters with De Filette et al. Veterinary Research 2012, 43:16 Page 4 of 15 http://www.veterinaryresearch.org/content/43/1/16

distinct evolutionary histories [40]. WNV-1b contains the has yet been approved for humans, patients infected with Australian Kunjin virus and lineage 1c some Indian iso- WNV have limited treatment options. The primary lates. Lineage 2 contains isolates from Southern Africa course of action is supportive. Small numbers of patients and Madagascar. Since 2004 lineage 2 has also been have received antibody therapy against WNV infection observed in central [41] and Eastern [42] Europe. In 2010 [56] or off-label treatment with IFN-a [57]. it caused outbreaks in Romania [43] and Greece [44] and In mammals, the initial replication of WNV after mos- in 2011 it was detected for the first time in Italy [45]. The quito inoculation is believed to occur in Langerhans den- Greek and Italian strains showed the highest homology to dritic cells. These infected cells migrate to draining Hungarian and South African strains, differing from the lymph nodes resulting in a primary viremia [58] and a Russian lineage 2 strains. This means that at least two subsequent infection of peripheral tissues such as the lineage 2 strains are circulating in Europe causing severe spleen and kidney. Viremia ensues and after spread to disease in humans. In general the lineage 1 viruses are the visceral organs, WNV may cross the blood-brain bar- considered to be more virulent than the lineage 2 viruses. rier (BBB) and enter the central nervous system (CNS). However, animal experiments have demonstrated that The mechanism by which WNV cross the BBB remains highly and less neuroinvasive phenotypes exist in both largely unknown, although tumor necrosis factor alpha lineages. Mutations responsible for increasing virulence (TNF-a)-mediated changes in endothelial cell permeabil- in lineage 2 viruses have previously been described in ity have been proposed to facilitate CNS entry [59]. lineage 1 viruses like the substitution of amino acids at Other models have been proposed like infection of olfac- position 249 for proline in NS3 [46,47]. Also the glycosy- tory neurons and spread to the olfactory bulb [60], direct lation state of the E protein is an important determinant axonal retrograde transport from infected peripheral neu- of pathogenicity [48,49]. Many of the WNV strains rons [61] or transport of the virus by infected immune responsible for more severe outbreaks of WN disease are cells trafficking to the CNS [62]. Neuronal infection is glycosylated at position 154. The lineage 3 is represented associated with degeneration, a loss of cell architecture, by a virus strain that was isolated from mosquitoes in the and cell death. Later in the course of infection, a mono- Czech Republic, designated the Rabensburg virus [50] nuclear cell infiltrate appears diffusely throughout and lineage 4 was isolated from a thick from the Cauca- infected regions, although it is not clear whether these sus [51]. WNV strains from India belonging to a subclus- inflammatory cells eradicate infection or contribute to ter of lineage 1 are sometimes classified as lineage 5 [52]. pathogenesis by destroying infected neurons and releas- The Sarawak Kunjin virus strain is significantly different ing pro-inflammatory cytokines [63]. to the other Kunjin viruses, and therefore re-classification TheprotectiveimmuneresponsetoWNVrequires of this virus as lineage 6 has been proposed. Furthermore, both innate and adaptive immunity. IFN-a and IFN-b the African virus, Koutango, is closely related to the WN are produced during the earliest stages of WNV infec- virus lineages, and could be considered as a seventh tion after host cell recognition of viral RNA. Mice with lineage. a defect in type I IFN signaling are much more sensitive to WNV infection than their wild-type counterparts 4. Pathogenesis [64]. The complement also is required for protection Mosquitoes become infected with WNV after biting a from lethal WNV infection in mice. WNV activates bird with high-level viremia and may then transmit it to complement in vivo, and mice lacking the central com- humans following a blood meal from the host. The plement protein C3 showed enhanced lethality after human incubation period of West Nile virus is 2 to 14 WNV infection [65]. Macrophage uptake of WNV can days [53]. The majority (~75 to 80%) of humans infected control infection through direct viral clearance, with WNV usually have no or very mild symptoms. enhanced antigen presentation, and cytokine and che- Approximately 20% of infected patients develop a febrile mokine secretion [66]. gδ T cells also directly limit illness ("West Nile fever”) with malaise, myalgias, head- WNV infection in early immune responses [67]. As they ache and lymphadenopathy [54]. A small number of the lack classical MHC restriction, they can react with viral symptomatic cases progress to the neuroinvasive form of antigens in the absence of conventional antigen proces- WNV infection [55], which can be characterized by acute sing [68]. Furthermore, the importance of adaptive flaccid paralysis, meningitis, encephalitis and ocular man- immunity has also been demonstrated as passive transfer ifestations. Overall, only 1 in 150 infections results in the of immune monoclonal and polyclonal antibodies pro- most severe and potentially lethal form of the disease, tected mice from lethal WNV infection [69]. IgM is cri- although the relative risk is increased in the elderly or tically important for the control of early WNV infection immunocompromised. Long-term complications (1 year and is detectable approximately 4 to 7 days after infec- or more after infection) are common in patients recover- tion [70]. After four to five days of illness, IgG antibo- ing from severe WNV infection. Since no specific therapy dies are measurable in patients presumably conferring De Filette et al. Veterinary Research 2012, 43:16 Page 5 of 15 http://www.veterinaryresearch.org/content/43/1/16

long-term protection against WNV re-infection [71]. TaqMan assay failed to detect 47% of possible single Cellular immune responses also control WNV infec- nucleotide variations in the probe-binding site. Johnson tions. Cytolytic T cells clear WNV infection by lysing et al. designed a pan-flavivirus RT-PCR utilizing degen- infected cells directly through the delivery of perforin erate primers targeting the NS5 gene to allow the detec- and granzymes A and B [68,72]. CD8+ deficient mice tion of a range of flaviviruses including WNV. This develop persistent WNV infections in the brain and SYBR Green based RT-PCR was able to detect WNV humans with impaired T cell function are at increased however the sensitivity was much lower compared to risk of neuroinvasive WNV infection and poor outcome WNV-specific TaqMan RT-PCR assays [76]. SYBR [73]. Green has been shown to inhibit the PCR reaction to some extent and melt curve analysis is complicated by 5. Diagnosis dye redistribution during melting. Eischeid analyzed the A review of the developments in WNV diagnosis was behavior of other DNA dyes in real-time PCR and published in 2007 by Dauphin et al. [74]. The current found that EvaGreen and SYTO dyes 13, 16, 80 and 82 paper focuses more on the new trends in diagnosis outperformed SYBR Green in real-time PCR [77]. since 2007. The two most popular alternatives to SYBR Green are TaqMan and molecular beacons, both of which use 5.1. Nucleic acid based tests for WNV hybridization probes and rely on fluorescence resonance Because the virus is present at very low levels in human energy transfer (FRET) for quantification. A TaqMan blood and tissues, an in vitro amplification of the qRT-PCR for rapid detection of WNV in human clinical genetic material is used to enhance the detection rate of specimens was first developed by Lanciotti et al. [78]. WNV infections. Several investigators have reported Compared to the traditional RT-PCR assay, this TaqMan real-time PCR-based detection systems for rapid detec- RT-PCR was more sensitive and could detect less than 1 tion of WNV infection in clinical samples (Table 1). PFU of virus whereas the traditional RT-PCR had a These real-time PCR systems rely upon the detection detection limit of 1 PFU of virus [78]. Chao et al. devel- and quantification of a fluorescent reporter. Single-tube, oped a multiplex TaqMan RT-PCR for the simultaneous real-time RT-PCR shows many advantages over end- identification of four different flaviviruses [80], including point RT-PCR because it is more rapid, often more sen- YFV, JEV, WNV and SLEV with a detection limit of sitive, more specific, and minimizes contamination. In respectively 3.5, 2, 10 and 10 PFU/mL. Dyer et al. also addition, real-time RT-PCR is easily standardized and developed a multiplexed TaqMan assay for the detection enable nucleic acid quantification. In the simplest and of SLE, WNV, Dengue and TBE species [88]. Naze et al. least expensive system the reporter is the double-strand developed a multiplex real-time RT-PCR assay for the DNA-specific dye SYBR Green. A disadvantage is that simultaneous detection and quantification of dengue SYBR Green will bind to any double-stranded DNA in virus (DENV) and Chikungunya virus (CHIKV) from the reaction, including non-specific PCR products and plasma samples [81]. In parallel, a real-time RT-PCR primer-dimers. Papin et al. developed a SYBR Green assay was developed for the detection and quantification based assay [75] that could detect 100% of the different of WNV using the same blood extract and identical WNV target region variants in their study, whereas a amplification conditions asforDENVandCHIKV[81]. Jiménez-Clavero et al. developed an improved TaqMan real-timeRT-PCRbyintroducingaminorgroovebinder Table 1 Overview of nucleic acid based assays for WNV ’ detection. (MGB), a 3 -labeling group that in addition to acting as a quencher also increases the binding affinity between the Technique used Reference probe and its target sequence [79]. Two different probes SYBR Green RT-PCR [75,76] were used and all samples were analyzed in parallel with TaqMan RT-PCR [78] the TaqMan RT-PCR described by Lanciotti et al. Both TaqMan-MGB RT-PCR [79] methods performed comparably in terms of sensitivity Multiplex RT-PCR [80,81] and were able to detect all clade 1a isolates, however Molecular beacon RT-PCR [82] Kunjin and lineage 2 isolates were only detected by the RT-PCR/ESI-MS [83] new TaqMan-MGB real-time PCR. RT-PCR/LDR [84] Molecular beacons, like TaqMan probes, also contain RT-PCR/FRET [85] fluorescent and quenching dyes but FRET only occurs when the quenching dye is directly adjacent to the fluor- RT-LAMP [86] escent dye. Lee et al. developed a combined RT-PCR, NASBA [87] using a FAM-labeled molecular beacon probe for WNV Digital PCR Invitrogen detection and a VIC-labeled TaqMan probe for internal De Filette et al. Veterinary Research 2012, 43:16 Page 6 of 15 http://www.veterinaryresearch.org/content/43/1/16

control detection [82]. The assay was highly specific for WNV and demonstrated no reactivity with 15 other viruses. Grant-Klein et al. combined RT-PCR with electrospray ionization mass spectrometry (ESI-MS) to detect tick- and mosquito-born flaviviruses on the Ibis T5000 platform [83]. The Ibis T5000 analyses DNA and determines the base composition (AxGxTxCx) of PCR amplicons by using ESI-MS. For the RT-PCR assay they developed eight primer pairs able to detect a broad range of flaviviruses. WNV was correctly detected by RT-PCR/ESI-MS in blood, serum and urine spiked with WNV demonstrating that these clinical matrices did not inhibit the detection of this virus. Using WNV, the sensitivity of the assay was determined to be approximately 2 PFU/mL. Rondini et al. developed a sensitive assay for the detection of both lineages of WNV by coupling multiplex RT- PCR and ligase detection reaction (LDR) [84]. Multiple PCR primers amplify three distinct regions of the WNV cDNA. Each PCR primer contains between one and three degenerate positions to accommodate minor sequence variation at the primer binding sites. Within each PCR amplicon, LDR primer pairs are designed to identify SNPs (Figure 3). Ligation of the appropriate LDR primers results in fluorescently labeled products of different lengths that are then separated using capillary electrophoresis. The Figure 3 Schematic representation of the ligase detection broad strain coverage was confirmed by testing 34 WNV reaction. PCR products are denatured and common (black) and isolates belonging to lineages 1 and 2. The detection limit discriminating primers (blue and red) annealed. DNA ligase only was very low reaching 0.005 and 0.017 PFU for respec- ligates those duplexes which contain exact matches and thermocycling with DNA ligase amplifies the ligated products. tively the one-step or two-step procedure. Detection of LDR products could also be achieved by hybridization to a universal DNA microarray. these newly developed NASBA assays a diagnosis can be PCR based diagnostic assay requires specialized equip- made within a hour and their sensitivity is similar or ment and well-trained personnel which is difficult to even greater than the sensitivity of their previously devel- obtain in developing countries. A relatively inexpensive oped TaqMan assay. assay for WNV is the loop-mediated isothermal amplifica- A major disadvantage of all real-time PCR techniques is tion (LAMP) assay described by Parida et al. [86]. The that new emerging WNV strains may acquire mutations LAMP assay is based on the principle of autocycling in the PCR-primer binding sites, which render them unde- strand displacement DNA synthesis performed by Bst tectable to currently existing assays. In addition, new (Bacillus stearothermophilus) DNA polymerase and a set WNVisolatesbelongingtoWNVlineage2mayposea of two inner primers and two outer primers. The advan- problem. Indeed, data from an external quality assessment tage of LAMP is that the amplification reaction can be of the molecular diagnostic of West Nile virus showed performed under isothermal conditions between 63°C and that some of the participating laboratories could not detect 65°C, thereby obviating the need for a thermal cycler [89]. lineage 2 viruses by their PCR assays [90]. To avoid that The RT-LAMP assay of Parida et al. [86] demonstrated a lineage 2 viruses are missed by the assay, Linke et al. high degree of specificity for WNV and was more sensitive developed a real-time PCR targeting a conserved region of than the traditional RT-PCR detecting 0.1 PFU of WNV. lineage 1 and 2 WNV [91]. Eiden et al. developed two Another technology that works at isothermal condi- real-time quantitative RT-PCRs for the detection of tions is the nucleic acid sequence-based amplification lineages 1 and 2 WNV strains. The primers and probe (NASBA) assay. Lanciotti et al. developed two NASBA were located either in the 5’-untranslated region or in assays each with a different detection format, i.e. electro- NS2A. Both assays allowed detection of both lineages with chemiluminescence or 6-carboxyfluorescein-labeled high sensitivity [92]. Several groups started to combine virus-specific molecular beacon probes, for the detection detection and genotyping of WNV by real-time PCR. of WNV and Saint Louis encephalitis (SLE) [87]. With Zaayman et al. [85] performed genotyping of WNV strains De Filette et al. Veterinary Research 2012, 43:16 Page 7 of 15 http://www.veterinaryresearch.org/content/43/1/16

by means of dissociation-curve analysis using the fluores- whereas neutralizing antibodies were detected by PRNT cence resonance energy transfer probe technology whereas at day 10. The diagnostic sensitivity was 100% compared Papin et al. used a genome-wide, multiple primer-based to PRNT but the specificity was only 79.5%. Kitai et al. real-time quantitative PCR assay [93]. Considerable efforts established an epitope-blocking ELISA based on NS1 have been put into the development of a third generation that differentiated WNV from JEV infections in horse of PCR, namely digital PCR (dPCR). It involves partition- sera [98]. Since several vaccines under development are ing of a sample into thousands of nanoliter sized droplets based on the prM and E proteins, NS1 based ELISAs will by means of an emulsion droplet generator [94]. Subse- still be able to discriminate between vaccinated and natu- quently, fluorescent labeled probes are added to the dro- rally infected animals. While comparing different assays, plets and PCR is performed on a standard thermal cycler. they observed that neutralizing antibodies were detect- After the PCR the droplets are analyzed by passing them able on day 7 by PRNT, and anti-WNV antibodies were in a single file in front of fluorescence detectors. Subse- observed from day 10 by conventional ELISA and day 12 quently, the outcome of the reaction is determined using a in their blocking ELISA. rigorous statistical analysis. In a multiplex PCR, the reac- Indirect IgG and IgM antibody-capture ELISA (MAC) tion mixture contains varying concentrations of the differ- are used primarily for serological detection of WNV in ent fluorogenic probes of the same color. It is then acute or convalescent serum or CSF samples. However, possible to identify the different probes on the basis of there are limitations to these tests such as the lack of cer- fluorescence intensity. Life Technologies have successfully tain anti-species secondary antibody conjugates and cross- applied their dPCR technology for the detection of WNV. reactivity with other flaviviruses. Alonso-Padilla et al. described an indirect ELISA for detection of anti-WNV 5.2. Serologic diagnosis of WNV infections IgG antibodies based on recombinant insect-cell derived Following exposure to WNV, both IgM and IgG antibo- soluble WNV-E protein [103]. Comparison of this ELISA dies are produced. In most cases, IgM antibodies can be based on recombinant E protein with the ELISA using detected within 4 to 7 days after the initial exposure and inactivated whole virus as antigen showed an equivalence may persist more than one year [95]. In comparison, in sera reactivity, with excellent specificity and sensitivity anti-WNV IgG are reliably detected ~ 8 days after the when compared with the “gold-standard” PRNT technique. onset of symptoms and they have a limited use in the In an attempt to differentiate between St. Louis ence- initial diagnosis of WNV infection [71]. Flavivirus- phalitis virus (SLEV) and WNV, Chang et al. performed infected sera show cross-reactions in serodiagnosis with extensive mutagenesis within the cross-reactive epitopes heterologous flavivirus infections. Therefore, the plaque of the E proteins of the two flaviviruses. Subsequently, reduction neutralization test (PRNT) is still used as the each mutant E protein was presented on the surface of reference assay for specific diagnosis of WNV infection. virus-like particles (VLP) to evaluate their diagnostic However, PRNT is a laborious test and must be carried potential. The assay was validated using human serum out in a biosafety level 3 (BSL-3) facility as viable WNV samples from patients infected with SLEV, WNV or viruses are used in this assay. For high-throughput other flaviviruses. It was found that in the MAC-ELISA screening, different ELISA methods (e.g. indirect IgG, higher specificity was obtained using the VLP containing IgM antibody-capture and blocking ELISA) have been the mutant antigens [104]. developed over the last years [74]. ELISAs have the To further improve the specificity of the WNV ELISAs, advantage of being rapid, reproducible and less expensive fragments of WNV proteins have also been used to dif- than other methods. ferentiate between different flaviviruses. The DIII of the The WNV blocking ELISA measures the ability of anti- E protein is an immunoglobulin-like domain protruding bodies present in sera to block the binding of a monoclo- from the otherwise smooth particle surface. Studies on nal antibody (mAb) to the NS1 protein [96-98], the E- the DIII of the E protein from JEV, DENV and WNV protein [96] or WNV-specific antigens present in cell showed slight differences in their structures, particularly extracts of WNV infected cells [99]. The advantage of the in areas that constitute virus neutralizing epitopes [105]. method is that it is species independent as demonstrated This observation led different groups to study the diag- by Blivitch et al. [96,99-101]. Their blocking ELISA based nostic potential of domain III of the E protein. Beasley et on the E-protein reliably detected flavivirus antibodies in al. demonstrated that an ELISA with WNV-DIII pro- several species of domestic mammals including horses, duced in bacteria could differentiate clearly between anti- cows, pigs and cats [100]. Sotelo et al. developed a block- body responses to WNV and those produced by other ing ELISA with a monoclonal antibody recognizing related flaviviruses, such as SLEV, JEV and Murray Valley domain III of the E glycoprotein [102]. After experimen- encephalitis virus (MVEV) [106]. tal infection of partridges this blocking-ELISA detected Other serological tests have been explored for use in WNV-specific antibodies as early as 3 days post-infection diagnosis of WNV infections. Kitai et al. developed a De Filette et al. Veterinary Research 2012, 43:16 Page 8 of 15 http://www.veterinaryresearch.org/content/43/1/16

complement-dependent cytotoxicity (CDC) assay (Figure 4) can be used for different mammalian species (but not to measure antibodies to the West Nile virus NS1 protein avian due to low binding affinity for protein A/G) within horses [107]. The antigen used for the assay was out the requirement of species specific secondary anti- obtained from a stably transfected cell line that constitu- body conjugates. tively expressed the NS1 protein of the WNV Eg101 strain. After incubation of the cells with heat-inactivated test 5.3. WNV antigen detection ® serum, commercial rabbit complement is added and The VecTest is an antigen panel assay designed by release of lactose dehydrogenase from cells was measured. Medical Analysis Systems to detect WNV, SLE and East- A comparison between a conventional ELISA, the blocking ern Equine Encephalitis (EEE). It uses a detection dip- ELISA, and a virus neutralization test (VNT) revealed that stick coated with specific antibodies. Although it is less this system detected anti-NS1 antibodies at similar time sensitive than the plaque assay in Vero cells or RT-PCR points as the conventional ELISA, but later as the VNT [111], it has the advantage that it gives a result in less and earlier as the blocking ELISA. than 20 minutes and it does not require sophisticated Microsphere-based immunoassays (MIAs) are becom- equipment. ing increasingly popular for the diagnosis of many dis- Two groups developed sensitive antigen capture ELISAs eases. This technology is based on the covalent bonding (ACE) for the detection of secreted NS1. MacDonald et al. of antigen to microspheres. The detecting device is a produced two ACEs for the detection of NS1 in experi- simplified flow cytometer and the procedure can be mentally infected hamsters [112]. In their first ACE a poly- completed in a few hours. Balasuriya et al. developed clonal antiserum was used as detecting antibody, and in four MIAs using recombinant WNV E, NS1, NS3 or their second ACE they used the same monoclonal anti- NS5 proteins for the detection of equine IgG antibodies body for capturing and detecting the NS1 antigen. The in sera of vaccinated or naturally infected horses [108]. first ACE was more specific for recombinant forms of The NS-based MIAs were less sensitive than both NS1, while the latter detected native NS1 at high sensitiv- PRNT and E-MIA. However, the NS1-MIA was able to ity. The detection limit was less than 1 ng/mL. Chung et distinguish between horses vaccinated with the canary- al. developed a similar ACE using two different monoclo- pox virus vaccine and horses that were naturally nals [113]. Their ACE detected as little as 0.5 ng/mL of infected. Johnson et al. produced a human IgM specific soluble NS1 and showed no cross-reactivity with yellow WNV/SLE MIA using recombinant WNV prM and E fever, Dengue and SLE virus NS1. and SLEV antigen extracted from mice brains as detect- A membrane-based electrochemical nanobiosensor ing antigens [109]. that recognizes viral particles or virus E protein was fab- A surface enhanced Raman scattering (SERS) immu- ricated by Nguyen et al. by putting a nanoporous alu- noassay for antibody detection in serum was developed mina membrane over a sensing electrode [114]. IgM by Neng et al. [110]. This assay utilizes gold nanoparti- raised against domain III of protein E was used as the cles coated with the E protein of WNV as the SERS- specific biorecognition probe for WNV particles. This active substrate and protein A/G conjugated with the assay was highly sensitive toward whole WNV particles Raman tag malachite green (MG) as a bi-functional with a detection limit of 2 viral particles per 100 mL, Raman tag/antibody binding reporter. The assay was which is comparable to PCR techniques. This might be validated by incubation of the E protein-coated gold useful for the detection of virus early during infection of nanoparticles with immune serum from rabbits. Subse- the host, i.e. before the onset of antibody production. quent laser interrogation of the sandwiched immuno- complex revealed a SERS signaling response diagnostic 6. Vaccination for MG. Since protein A/G can interact with a range of A review of the trends in vaccine development was pub- mammalian antibody subclasses, this SERS immunoassay lished in 2007 by Dauphin et al. [74]. The current paper focuses more on the evolution in vaccine development since 2007.

6.1. Licensed West Nile virus vaccines for animals Although no human vaccine is available to date, there are currently three WNV vaccines licensed for horses (Table 2). The first licensed vaccine was developed by Fort Dodge Animal Health, which is now subsidiary of Pfizer. It contains a formalin-inactivated, whole West Figure 4 CDC assay as developed by Kitai.Seetextabovefor Nile virus. This vaccine is currently commercialized in further details. ® theUSAunderthetradenameWestNile-Innovator De Filette et al. Veterinary Research 2012, 43:16 Page 9 of 15 http://www.veterinaryresearch.org/content/43/1/16

and is quite effective. Indeed, 12 months after two doses is given two to four weeks after the first. This vaccine ® of West Nile-Innovator 94% of the animals were pro- has recently been discontinued by Pfizer. A Flavivirus tected against viremia after challenge. In a safety trial of chimera vaccine for horses (PreveNile, Intervet) contain- the vaccine, less than 5% of the horses showed adverse ing West Nile virus pre-membrane (prM) and envelope responses to vaccination [115]. Another killed virus vac- (E) genes (from the NY99 strain) in a backbone of yel- ® cine (Vetera WNV vaccine) developed by Boehringer low fever (YF17D vaccine virus), was granted a full Ingelheim Vetmedica was also licensed by the United license by USDA in 2006. However, in 2010 it was States Department of Agriculture (USDA). A third com- recalled from the market after the observation of mercialized WNV vaccine in the United States for adverse effects like acute anaphylaxis, colics, respiratory ® horses is Recombitek Equine West Nile Virus Vaccine distress and even death in horses. (Merial, now Sanofi Aventis), which is a chimeric recombinant canarypoxvirus vaccine [116]. This vaccine 6.2. WNV vaccines under development expresses the prM and E genes derived from a 1999 A recombinant influenza virus expressing domain III of New York isolate of West Nile virus (WNV). All of the the WNV E protein has been evaluated as a WNV vac- vaccinated horses developed neutralizing antibodies cine candidate in a mouse model [121]. The WNV DIII against WNV and showed significantly fewer clinical was cloned in the N-terminal region of the influenza signs of WNV disease upon challenge [117]. Also an virus neuraminidase destroying the functional activity of inactivated form of this chimeric vaccine has been the influenza protein. Subcutaneous immunization of licensed by the USDA. In 2005, a WNV DNA plasmid- mice with the vaccine, FLU-NA-DIII, resulted in higher based vaccine was licensed in the United States by Fort virus-neutralizing and WNV-specific IgG ELISA titers Dodge Animal Health/Pfizer under the trade name of than intranasal administration. In addition, cellular DIII- ® West Nile-Innovator DNA. The vaccine contains an specific responses as determined by IFN-g ELISPOT unformulated plasmid DNA encoding the prM and E assay were also stronger in the subcutaneously immu- protein of WNV and MetaStim™ as adjuvant. The vac- nized group. After subcutaneous challenge with WNV, cine is administered intramuscularly and a second dose higher morbidity as assessed by loss of body weight was

Table 2 Overview of the different commercialized and candidate West Nile vaccines. Name Viral antigen(s) State of development Reference West Nile-Innovator Whole virus Commercialized for horses [63] (Pfizer) WNV prM-E in Commercialized for horses RecombiTek (Merial) canarypox virus West Nile-Innovator Plasmid DNA prM/E Licensed for horses [23] DNA PreveNile WNV prM-E in yellow fever backbone Commercialized for horses (Intervet) (recalled in 2010) Vetera West Nile Killed virus Commercialized for horses vaccine (Boehringer Ingelheim) ChimeriVax Yellow fever PrM-E substituted by WNV Phase II human clinical trial [5,8] (Sanofi) prM-E WN-DEN4 WNV prM-E in dengue-4 backbone Phase II human clinical trial [72] VRC303 Plasmid encoding WNV prM and E Phase I human clinical trial [41] (NIAID/Vical) STF2Δ.EIII S. typhimurium flagellin fused to E domain Evaluated in mice [55] III rWNV-ET Truncated protein E Evaluated in mice and horses [19,42] SRIP prM-E VLPs Evaluated in mice and horses [12] RepliVAX WN Single-cycle West Nile virus Evaluated in mice [118], hamsters [119], non-human primates [120] Plasmid encoding E domain III fused to P28 Evaluated in mice [22] DIII-C-AP205 E domain III coupled to bacteriophage Evaluated in mice [87] AP205 FLU-NA-DIII E domain III inserted into NA of influenza Evaluated in mice [54] CAdVax-WNVII C, preM, E and NS1 expressed in adenovirus Evaluated in mice [81] De Filette et al. Veterinary Research 2012, 43:16 Page 10 of 15 http://www.veterinaryresearch.org/content/43/1/16

observed in the intranasal immunized group. Survival Interestingly, cross-neutralizing IgG against JEV also rates were 100% and 75% in mice immunized with FLU- were produced. Mice vaccinated with rDIII and chal- NA-DIII via the subcutaneous or intranasal route, lenged with either WNV or JEV were protected against respectively. morbidity as determined monitoring the body weight. Schepp-Berglind et al. created an adenoviral vaccine However, the survival rates were lower (80% to WNV vector (CAdVax-WNVII) expressing four WNV pro- and 60% to JEV) compared to mice vaccinated with BPL teins, C, prM, E and NS1. Although these proteins origi- inactivated WNV (100% to WNV and 80% to JEV). nated from a lineage II virus strain, serum samples McDonald et al. fused bacterial flagellin to the domain III collected after vaccination of mice with CAdVax-WNVII of the WNV envelope protein (STF2Δ.EIII) providing the contained antibodies that neutralized lineage I and II fusion protein the ability to engage the TLR5 receptor. viruses. In vaccinated mice T cell activity against WNV Mammalian hosts detect the conserved domain on flagel- antigens was observed in splenocytes after re-stimulation lin monomers through TLR5, which triggers proinflam- in vitro with WNV infected target cells [122]. matory and adaptive immune responses. Mice injected The emergence of pathogenic lineage 2 strains in Eur- either subcutaneously or intraperitoneally with the flagel- ope raised the question whether the existing WNV vac- lin-DIII fusion protein produced significant levels of anti- cines, mainly based on lineage 1 strains, can also protect WNVEIgGasdeterminedbyELISA.Inaddition,sera against the new circulating lineage 2 strains of WNV. from vaccinated mice had neutralizing antibody titers > ® Minke et al. demonstrated that Recombitek Equine 1/40. In a mouse model, > 90% survival was observed in West Nile, that expresses the prM/E genes of lineage 1 animals that were immunized with STF2Δ.EIII [127]. strain in a recombinant canarypox virus, could protect Spohn et al. also have used recombinantly expressed horses against a contemporary neurovirulent lineage 2 domain III of the WNV E protein as an immunogen. WNV isolate [123]. Finally, Yamshchikov et al. reported This group chemically coupled the DIII protein to VLP that an attenuated non-epidemic West Nile virus strain derived from bacteriophage AP205. This conjugate vac- of lineage 2 can be used as an effective vaccine against a cine DIII-C-AP205 was more immunogenic in mice than virulent epidemic strain of lineage 1 in mice [124]. a mixture of corresponding amounts of free DIII and its Another strategy that has been evaluated is vaccination carrier AP205. Neutralizing antibodies could be detected with purified viral proteins. Although these vaccines proin 75% of the mice after one injection with the DIII-C- tect against disease in animal models, multiple injections AP205 vaccine while all animals scored positive after and/or strong adjuvants were required to reach accepta- three injections. The latter group was also fully protected ble efficacy. Demento et al. [125] formulated recombi- against a lethal challenge with the virus [128]. nant E protein onto poly(lactic-co-glycolic acid) (PLGA) Finally, a fourth strategy uses DNA vaccination as a nanoparticles that contained CpG oligonucleotides at platform for WNV vaccination. Davis et al. were the first their surface. Activation of dendritic cells as determined to demonstrate that plasmid DNA encoding the WNV by the secretion of IL-6 and IL-12 was stronger when the membrane (M) and envelope (E) proteins injected intra- CpG-modified E protein-loaded nanoparticles were used muscularly in mice and horses provided protection compared with E protein adsorbed to Alhydrogel. C3H/ against a WNV challenge [129]. DNA vaccination HeN mice immunized with encapsulated E protein in resulted in both a humoral response as well as a strong CpG modified nanoparticles or with E protein adsorbed Th1 response. This study paved the way for the licensing to alhydrogel elicited equivalent titers of IgG however, of the first DNA vaccine for animal use, i.e. West Nile- ® the isotype profiles were very different. Only CpG-modi- Innovator DNA. Later on, other administration routes fied particles loaded with E protein raised high IgG2a and carrier mediated delivery of the WNV DNA vaccines and IgG2b titers. Lymphocytes from mice vaccinated have been exploited. Zhao et al. showed that inoculation with encapsulated E protein in CpG modified nanoparti- of plasmid (mixed with colloidal gold) via intravenous cles produced higher levels of IFN-g and IL-2 in vitro and intradermal injection elicited stronger and more sus- after re-stimulation with E protein compare to lympho- tained neutralizing immune responses than intramuscu- cytes from Alhydrogel-rWNV-ET immunized mice. lar or intraperitoneal injection [130]. Prow et al. used a CD8+CD44+ T cells from mice vaccinated with CpG/ nanopatch with a microneedle array to deliver a West rWNV E nanoparticles had a larger KLRG1+CD127- Nile virus DNA vaccine that was complexed to poly(ethy- population, a subset of terminally differentiated effector lenimine) [131]. This simple needle-free technique cells. resulted into an effective vaccine delivery with a cuta- Martina et al. produced domain III protein of the E neous expression of encoded proteins within 24 h [131]. proteinofWNV(rDIII)andcompareditwithab-pro- Dunn et al. evaluated DNA vaccines with derivatives of piolactone (BPL) inactivated WNV vaccine [126]. Neutra- the WNV E gene (full length, truncated E or DIII region) lizing antibodies against WNV were detected in all mice. conjugated to the P28 region of the complement protein De Filette et al. Veterinary Research 2012, 43:16 Page 11 of 15 http://www.veterinaryresearch.org/content/43/1/16

C3d. Mice were vaccinated three times either intramus- The CD8+ T cells produced TNF-a and IFN-g after sti- + cularly or by the gene gun route. The latter resulted in mulation with the NS4B2488 peptide. Significant CD4 T higher IgG titers against WNV DIII. Gene gun DNA vac- cell responses were also detected against peptides cination induced primarily a Th2 response (characterized NS31616,E641,E431 and NS32066 that peaked on day 13. + by IgG1 antibodies) whereas intramuscular administra- Specific cytolysis of NS32066 pulsed cells by CD4 spleno- tion resulted more in a Th1 response (characterized by cytes was observed by day 6 and peaked on day 8. CD4+ IgG2 antibodies). Eighty percent of the mice vaccinated T cells produced predominantly IFN-g and no IL-4 fol- by gene gun with DIII-DNA survived a lethal WNV chal- lowing restimulation with peptide in vitro. Memory CD4 lenge with little weight loss, while no mice survived in + and CD8+ T cells were detected 8 months post immu- the intramuscularly vaccinated group. However, the sur- nization [134]. vival rates after IM administration increased to 60% by conjugating P28 to DIII [132]. At the moment, all the 6.3. Clinical trials with West Nile virus vaccines in humans commercially available DNA vaccines against any patho- At present, there are no FDA-approved vaccines for gen contain unformulated DNA. Chang et al. developed a human use but several clinical trials are ongoing. In plasmid DNA (pDNA) that after transfection gives rise to 2005, Acambis (Sanofi-Pasteur) successfully completed a single-round infectious particles (SRIPs) based on WNV. Phase I clinical trial with its live-attenuated Chimeri- Flavivirus RNAs that contain large deletions in the capsid Vax-WN. ChimeriVax-West Nile (Acambis, Sanofi-Pas- gene cannot produce infectious virions but retain the teur) utilizes the attenuated YFV vaccine strain (17D) to ability to replicate their RNA backbone and express prM build a live chimeric virus that consists of the prM and and E proteins. After transfection, the plasmid DNA gen- E proteins of WNV in the context of the YFV capsid erates two different mRNAs: one encoding capsid protein and non-structural proteins [135]. ChimeriVax-West and the other for the prM, the E, non-structural proteins Nile is the most advanced vaccine in development. In and a truncated capsid. Only the latter RNA molecule thefirstpartofaPhaseIItrialinhealthyadults18-40 can replicate and become incorporated in the SRIPs. years of age, a single dose of ChimeriVax-West Nile Cells transfected with SRIPs will produce subviral parti- raised neutralizing antibodies 28 days after vaccination cles that contain the prM and E-proteins but lack virus [135]. The second part of the Phase II trial determined genomic material. This plasmid DNA vaccine was deliv- safety and tolerance in healthy individuals over 41 years ered using a gene-gun with DNA-coated gold particles. of age. Seroconversion was achieved at day 28 by more Vaccination of mice with plasmid DNA encoding SRIPs than 96% of the healthy adults in both age groups. elicited higher overall and neutralizing antibody titers Another chimeric vaccine (WN-DEN4) that uses attenu- than after vaccination with a plasmid encoding prM-E ated dengue virus as a backbone for prM-E genes of [133]. After intraperitoneal challenge, all mice vaccinated WNV [136] is being evaluated in a Phase II human trial with SRIPs were protected against morbidity whereas at the John Hopkins School of Public Health in adults some mice in the prM-E DNA plasmid group developed 18-50 years of age. The Vaccine Research Center (VRC) signs of disease although all mice survived infection. The at the National Institute of Allergy and Infectious Dis- SRIP vaccine also was able to induce virus-neutralizing eases (NIAID) has, in collaboration with Vical, devel- antibodies in horses. A similar strategy was followed by oped a DNA plasmid-based vaccine. In 2005, the VRC Mason et al. They produced RepliVAX WN, a live-atte- initiated a successful Phase I clinical trial demonstrating nuated virus in which the gene encoding the capsid pro- its safety, tolerability and ability to induce neutralizing tein was deleted from the WNV genome. Vaccination antibodies. Subsequently, a second-generation DNA vac- with RepliVAX WN induced protective immunity in cine using an improved vector was evaluated in a Phase mice [118], hamsters [119] and non-human primates I clinical trial. Naked plasmid DNA was administered [120]. Nelson et al. characterized the nature of the via needle-free intramuscular injection on days 0, 28 immune response to RepliVAX in mice [134]. They and 56 with at least 21 days between injections. The found that the number of B cells secreting IgG specific plasmid in this vaccine is incapable of replicating in ani- for NS1 peaked at day 8 and was dose-dependent. Long- mal cells and does generate infectious virions. The vac- term presence of NS1-specific plasma cells through 8 cine was well tolerated without serious adverse events. months was observed. The IgG subclass of the induced All individuals that completed the 3-dose vaccination antibodies was predominantly IgG2 while only little IgG1 schedule developed neutralizing antibodies [137]. The was produced. Immunized mice mounted a strong CD8+ majority of the subjects developed a CD4+ response + T cell response against NS4B2488 and E347 that peaked at rather than a CD8 response as assessed by intracellular day 6. The cytotoxic activity of these cells was confirmed cytokine staining. Vaccine-induced T cell responses by analysis of the killing of peptide-pulsed target cells. were mainly directed against WNV E protein. De Filette et al. Veterinary Research 2012, 43:16 Page 12 of 15 http://www.veterinaryresearch.org/content/43/1/16

7. Conclusions 4. Smithburn KCHT, Burke AW, Paul JH: A neurotropic virus isolated from the blood of a native of Uganda. Am J Trop Med Hyg 1940, 20:471-492. West Nile virus remains a serious threat to the public 5. Hayes CG: West Nile virus: Uganda, 1937, to New York City, 1999. Ann N health, especially to very young, elderly and immunocom- Y Acad Sci 2001, 951:25-37. promised individuals. There is currently no antiviral 6. Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ: Structure of West Nile virus. Science 2003, 302:248. treatment to cure WNV infections and only supportive 7. Ulbert S: West nile virus: the complex biology of an emerging pathogen. care can be administered. Ribavirin [138], interferon-a Intervirology 2011, 54:171-184. [57,139] and WNV-specific immunoglobulin [56,140] 8. Bogachek MV, Zaitsev BN, Sekatskii SK, Protopopova EV, Ternovoi VA, Ivanova AV, Kachko AV, Ivanisenko VA, Dietler G, Loktev VB: have all been considered as specific treatments for WNV Characterization of glycoprotein E C-end of West Nile virus and disease, but no rigorously conducted clinical trials have evaluation of its interaction force with alphaVbeta3 integrin as putative been completed. Diagnostic tests have improved consid- cellular receptor. Biochemistry (Mosc) 2010, 75:472-480. 9. Bogachek MV, Protopopova EV, Loktev VB, Zaitsev BN, Favre M, Sekatskii SK, erably and allow a rapid detection of the presence of Dietler G: Immunochemical and single molecule force spectroscopy WNV. Vaccine development against WNV continues to studies of specific interaction between the laminin binding protein and progress. Four WNV vaccines are currently available in the West Nile virus surface glycoprotein E domain II. J Mol Recognit 2008, 21:55-62. the USA for horses. Although several clinical trials in var- 10. Chu JJ, Ng ML: Infectious entry of West Nile virus occurs through a ious phases in humans are ongoing, it will take several clathrin-mediated endocytic pathway. J Virol 2004, 78:10543-10555. years before any vaccine is available. Given the relatively 11. Gollins SW, Porterfield JS: pH-dependent fusion between the flavivirus West Nile and liposomal model membranes. J Gen Virol 1986, 67:157-166. low incidence of WNV neuroinvasive disease in healthy 12. Chambers TJ, Hahn CS, Galler R, Rice CM: Flavivirus genome organization, individuals and the focal occurrence of WNV epidemics expression, and replication. Annu Rev Microbiol 1990, 44:649-688. thus far, vaccination will likely target the groups at higher 13. Nowak T, Farber PM, Wengler G: Analyses of the terminal sequences of West Nile virus structural proteins and of the in vitro translation of risk for WNV neuroinvasive infection. At the moment, these proteins allow the proposal of a complete scheme of the the best way to prevent West Nile virus infection remains proteolytic cleavages involved in their synthesis. Virology 1989, to avoid mosquito bites. 169:365-376. 14. Falgout B, Markoff L: Evidence that flavivirus NS1-NS2A cleavage is mediated by a membrane-bound host protease in the endoplasmic 8. Competing interests reticulum. J Virol 1995, 69:7232-7243. The authors declare that they have no competing 15. Poch O, Sauvaget I, Delarue M, Tordo N: Identification of four conserved motifs among the RNA-dependent polymerase encoding elements. interests. EMBO J 1989, 8:3867-3874. 16. Rice CM, Aebersold R, Teplow DB, Pata J, Bell JR, Vorndam AV, Trent DW, 9. Authors’ contributions Brandriss MW, Schlesinger JJ, Strauss JH: Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses. Virology MDF developed the structural design of the review, 1986, 151:1-9. organized the work and did together with NS the draft- 17. Konishi E, Mason PW: Proper maturation of the Japanese encephalitis ing of the manuscript. SU and MD performed detailed virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol 1993, 67:1672-1675. and critical revisions of the manuscript. All authors read 18. Diamond MS: Progress on the development of therapeutics against West and approved the final manuscript. Nile virus. Antiviral Res 2009, 83:214-227. 19. Lindenbach BD, Rice CM: trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 1997, 71:9608-9617. 10. Acknowledgements 20. Wilson JR, de Sessions PF, Leon MA, Scholle F: West Nile virus This research was funded by the EU FP7 project WINGS (grant n° 261426). nonstructural protein 1 inhibits TLR3 signal transduction. J Virol 2008, 82:8262-8271. Author details 21. Avirutnan P, Fuchs A, Hauhart RE, Somnuke P, Youn S, Diamond MS, 1Laboratory of Gene Therapy, Faculty of Veterinary Sciences, Ghent Atkinson JP: Antagonism of the complement component C4 by flavivirus University, Heidestraat 19, 9820 Merelbeke, Belgium. 2Vaccine Technologies nonstructural protein NS1. J Exp Med 2010, 207:793-806. Unit, Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, 22. Chung KM, Liszewski MK, Nybakken G, Davis AE, Townsend RR, D-04103 Leipzig, Germany. 3Departments of Medicine, Molecular Fremont DH, Atkinson JP, Diamond MS: West Nile virus nonstructural Microbiology and Pathology & Immunology, Washington University School protein NS1 inhibits complement activation by binding the regulatory of Medicine, 660 South Euclid Avenue, Box 8051, St. Louis, Missouri 63110, protein factor H. Proc Natl Acad Sci USA 2006, 103:19111-19116. USA. 23. Leung JY, Pijlman GP, Kondratieva N, Hyde J, Mackenzie JM, Khromykh AA: Role of nonstructural protein NS2A in flavivirus assembly. J Virol 2008, Received: 24 October 2011 Accepted: 1 March 2012 82:4731-4741. Published: 1 March 2012 24. Liu WJ, Chen HB, Wang XJ, Huang H, Khromykh AA: Analysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon References promoter-driven transcription. J Virol 2004, 78:12225-12235. 1. Work TH, Hurlbut HS, Taylor RM: Indigenous wild birds of the Nile Delta 25. Laurent-Rolle M, Boer EF, Lubick KJ, Wolfinbarger JB, Carmody AB, Rockx B, as potential West Nile virus circulating reservoirs. Am J Trop Med Hyg Liu W, Ashour J, Shupert WL, Holbrook MR, Barrett AD, Mason PW, 1955, 4:872-888. Bloom ME, Garcia-Sastre A, Khromykh AA, Best SM: The NS5 protein of the 2. The CDC Database. [http://www.cdc.gov/ncidod/dvbid/westnile/ virulent West Nile virus NY99 strain is a potent antagonist of type I birds&mammals.htm]. interferon-mediated JAK-STAT signaling. J Virol 2010, 84:3503-3515. 3. Mackenzie JS, Gubler DJ, Petersen LR: Emerging flaviviruses: the spread 26. Calistri P, Giovannini A, Hubalek Z, Ionescu A, Monaco F, Savini G, Lelli R: and resurgence of Japanese encephalitis, West Nile and dengue viruses. Epidemiology of west nile in europe and in the mediterranean basin. Nat Med 2004, 10(Suppl 12):98-109. Open Virol J 2010, 4:29-37. De Filette et al. Veterinary Research 2012, 43:16 Page 13 of 15 http://www.veterinaryresearch.org/content/43/1/16

27. Ernek E, Kozuch O, Nosek J, Teplan J, Folk C: Arboviruses in birds captured 51. Lvov DK, Butenko AM, Gromashevsky VL, Kovtunov AI, Prilipov AG, Kinney R, in Slovakia. J Hyg Epidemiol Microbiol Immunol 1977, 21:353-359. Aristova VA, Dzharkenov AF, Samokhvalov EI, Savage HM, Shchelkanov MY, 28. Jourdain E, Olsen B, Lundkvist A, Hubalek Z, Sikutova S, Waldenstrom J, Galkina IV, Deryabin PG, Gubler DJ, Kulikova LN, Alkhovsky SK, Moskvina TM, Karlsson M, Wahlstrom M, Jozan M, Falk KI: Surveillance for West Nile virus Zlobina LV, Sadykova GK, Shatalov AG, Lvov DN, Usachev VE, Voronina AG: in wild birds from northern Europe. Vector Borne Zoonotic Dis 2011, West Nile virus and other zoonotic viruses in Russia: examples of 11:77-79. emerging-reemerging situations. Arch Virol Suppl 2004, 18:85-96. 29. Rossi SL, Ross TM, Evans JD: West Nile virus. Clin Lab Med 2010, 30:47-65. 52. Bondre VP, Jadi RS, Mishra AC, Yergolkar PN, Arankalle VA: West Nile virus 30. Charatan F: Organ transplants and blood transfusions may transmit West isolates from India: evidence for a distinct genetic lineage. J Gen Virol Nile virus. BMJ 2002, 325:566. 2007, 88:875-884. 31. Alpert SG, Fergerson J, Noel LP: Intrauterine West Nile virus: ocular and 53. Petersen LR, Marfin AA: West Nile virus: a primer for the clinician. Ann systemic findings. Am J Ophthalmol 2003, 136:733-735. Intern Med 2002, 137:173-179. 32. Centers for Disease Control and Prevention: Possible West Nile virus 54. Hayes EB, Sejvar JJ, Zaki SR, Lanciotti RS, Bode AV, Campbell GL: Virology, transmission to an infant through breast-feeding–Michigan. JAMA 2002, pathology, and clinical manifestations of West Nile virus disease. Emerg 288:1976-1977. Infect Dis 2005, 11:1174-1179. 33. Tsai TF, Popovici F, Cernescu C, Campbell GL, Nedelcu NI: West Nile 55. Sejvar JJ, Haddad MB, Tierney BC, Campbell GL, Marfin AA, Van Gerpen JA, encephalitis epidemic in southeastern Romania. Lancet 1998, 352:767-771. Fleischauer A, Leis AA, Stokic DS, Petersen LR: Neurologic manifestations 34. Lundstrom JO: Mosquito-borne viruses in western Europe: a review. J and outcome of West Nile virus infection. JAMA 2003, 290:511-515. Vector Ecol 1999, 24:1-39. 56. Shimoni Z, Niven MJ, Pitlick S, Bulvik S: Treatment of West Nile virus 35. Papa A, Danis K, Baka A, Bakas A, Dougas G, Lytras T, Theocharopoulos G, encephalitis with intravenous immunoglobulin. Emerg Infect Dis 2001, Chrysagis D, Vassiliadou E, Kamaria F, Liona A, Mellou K, Saroglou G, 7:759. Panagiotopoulos T: Ongoing outbreak of West Nile virus infections in 57. Kalil AC, Devetten MP, Singh S, Lesiak B, Poage DP, Bargenquast K, Fayad P, humans in Greece, July - August 2010. Euro Surveill 2010, 34:1-5. Freifeld AG: Use of interferon-alpha in patients with West Nile 36. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, Crise B, encephalitis: report of 2 cases. Clin Infect Dis 2005, 40:764-766. Volpe KE, Crabtree MB, Scherret JH, Hall RA, MacKenzie JS, Cropp CB, 58. Johnston LJ, Halliday GM, King NJ: Langerhans cells migrate to local Panigrahy B, Ostlund E, Schmitt B, Malkinson M, Banet C, Weissman J, lymph nodes following cutaneous infection with an arbovirus. J Invest Komar N, Savage HM, Stone W, McNamara T, Gubler DJ: Origin of the West Dermatol 2000, 114:560-568. Nile virus responsible for an outbreak of encephalitis in the 59. Wang T, Town T, Alexopoulou L, Anderson JF, Fikrig E, Flavell RA: Toll-like northeastern United States. Science 1999, 286:2333-2337. receptor 3 mediates West Nile virus entry into the brain causing lethal 37. The CDC Database. [http://www.cdc.gov/ncidod/dvbid/westnile/ encephalitis. Nat Med 2004, 10:1366-1373. surv&control.htm]. 60. Monath TP, Cropp CB, Harrison AK: Mode of entry of a neurotropic 38. Mackenzie JS, Williams DT: The zoonotic flaviviruses of southern, south- arbovirus into the central nervous system. Reinvestigation of an old eastern and eastern Asia, and Australasia: the potential for emergent controversy. Lab Invest 1983, 48:399-410. viruses. Zoonoses Public Health 2009, 56:338-356. 61. Samuel MA, Wang H, Siddharthan V, Morrey JD, Diamond MS: Axonal 39. Kramer LD, Styer LM, Ebel GD: A global perspective on the epidemiology transport mediates West Nile virus entry into the central nervous system of West Nile virus. Annu Rev Entomol 2008, 53:61-81. and induces acute flaccid paralysis. Proc Natl Acad Sci USA 2007, 40. May FJ, Davis CT, Tesh RB, Barrett AD: Phylogeography of West Nile virus: 104:17140-17145. from the cradle of evolution in Africa to Eurasia, Australia, and the 62. Garcia-Tapia D, Loiacono CM, Kleiboeker SB: Replication of West Nile virus Americas. J Virol 2011, 85:2964-2974. in equine peripheral blood mononuclear cells. Vet Immunol 41. Bakonyi T, Ivanics E, Erdelyi K, Ursu K, Ferenczi E, Weissenbock H, Immunopathol 2006, 110:229-244. Nowotny N: Lineage 1 and 2 strains of encephalitic West Nile virus, 63. Lazear HM, Pinto A, Vogt MR, Gale M Jr, Diamond MS: Beta interferon central Europe. Emerg Infect Dis 2006, 12:618-623. controls west nile virus infection and pathogenesis in mice. J Virol 2011, 42. Platonov AE, Fedorova MV, Karan LS, Shopenskaya TA, Platonova OV, 85:7186-7194. Zhuravlev VI: Epidemiology of West Nile infection in Volgograd, Russia, in 64. Samuel MA, Diamond MS: Alpha/beta interferon protects against lethal relation to climate change and mosquito (Diptera: Culicidae) bionomics. West Nile virus infection by restricting cellular tropism and enhancing Parasitol Res 2008, 103(Suppl 1):45-53. neuronal survival. J Virol 2005, 79:13350-13361. 43. Sirbu A, Ceianu CS, Panculescu-Gatej RI, Vazquez A, Tenorio A, Rebreanu R, 65. Mehlhop E, Whitby K, Oliphant T, Marri A, Engle M, Diamond MS: Niedrig M, Nicolescu G, Pistol A: Outbreak of West Nile virus infection in Complement activation is required for induction of a protective humans, Romania, July to October 2010. Euro Surveill 2011, 2:1-5. antibody response against West Nile virus infection. J Virol 2005, 44. Papa A, Xanthopoulou K, Gewehr S, Mourelatos S: Detection of West Nile 79:7466-7477. virus lineage 2 in mosquitoes during a human outbreak in Greece. Clin 66. Ben-Nathan D, Huitinga I, Lustig S, van Rooijen N, Kobiler D: West Nile Microbiol Infect 2011, 17:1176-1180. virus neuroinvasion and encephalitis induced by macrophage depletion 45. Bagnarelli P, Marinelli K, Trotta D, Monachetti A, Tavio M, Del Gobbo R, in mice. Arch Virol 1996, 141:459-469. Capobianchi M, Menzo S, Nicoletti L, Magurano F, Varaldo P: Human case 67. Wang T, Gao Y, Scully E, Davis CT, Anderson JF, Welte T, Ledizet M, Koski R, of autochthonous West Nile virus lineage 2 infection in Italy, September Madri JA, Barrett A, Yin Z, Craft J, Fikrig E: Gamma delta T cells facilitate 2011. Euro Surveill 2011, 43:1-4. adaptive immunity against West Nile virus infection in mice. J Immunol 46. Brault AC, Huang CY, Langevin SA, Kinney RM, Bowen RA, Ramey WN, 2006, 177:1825-1832. Panella NA, Holmes EC, Powers AM, Miller BR: A single positively selected 68. Wang Y, Lobigs M, Lee E, Mullbacher A: Exocytosis and Fas mediated West Nile viral mutation confers increased virogenesis in American cytolytic mechanisms exert protection from West Nile virus induced crows. Nat Genet 2007, 39:1162-1166. encephalitis in mice. Immunol Cell Biol 2004, 82:170-173. 47. Papa A, Bakonyi T, Xanthopoulou K, Vazquez A, Tenorio A, Nowotny N: 69. Diamond MS, Shrestha B, Marri A, Mahan D, Engle M: B cells and antibody Genetic characterization of West Nile virus lineage 2, Greece, 2010. play critical roles in the immediate defense of disseminated infection by Emerg Infect Dis 2011, 17:920-922. West Nile encephalitis virus. J Virol 2003, 77:2578-2586. 48. Beasley DW, Whiteman MC, Zhang S, Huang CY, Schneider BS, Smith DR, 70. Diamond MS, Sitati EM, Friend LD, Higgs S, Shrestha B, Engle M: A critical Gromowski GD, Higgs S, Kinney RM, Barrett AD: Envelope protein role for induced IgM in the protection against West Nile virus infection. glycosylation status influences mouse neuroinvasion phenotype of J Exp Med 2003, 198:1853-1862. genetic lineage 1 West Nile virus strains. J Virol 2005, 79:8339-8347. 71. Tardei G, Ruta S, Chitu V, Rossi C, Tsai TF, Cernescu C: Evaluation of 49. Botha EM, Markotter W, Wolfaardt M, Paweska JT, Swanepoel R, Palacios G, immunoglobulin M (IgM) and IgG enzyme immunoassays in serologic Nel LH, Venter M: Genetic determinants of virulence in pathogenic diagnosis of West Nile Virus infection. J Clin Microbiol 2000, 38:2232-2239. lineage 2 West Nile virus strains. Emerg Infect Dis 2008, 14:222-230. 72. Shrestha B, Samuel MA, Diamond MS: CD8+ T cells require perforin to 50. Bakonyi T, Hubalek Z, Rudolf I, Nowotny N: Novel flavivirus or new lineage clear West Nile virus from infected neurons. J Virol 2006, 80:119-129. of West Nile virus, central Europe. Emerg Infect Dis 2005, 11:225-231. De Filette et al. Veterinary Research 2012, 43:16 Page 14 of 15 http://www.veterinaryresearch.org/content/43/1/16

73. Shrestha B, Diamond MS: Role of CD8+ T cells in control of West Nile 95. Roehrig JT, Nash D, Maldin B, Labowitz A, Martin DA, Lanciotti RS, virus infection. J Virol 2004, 78:8312-8321. Campbell GL: Persistence of virus-reactive serum immunoglobulin m 74. Dauphin G, Zientara S: West Nile virus: recent trends in diagnosis and antibody in confirmed west nile virus encephalitis cases. Emerg Infect Dis vaccine development. Vaccine 2007, 25:5563-5576. 2003, 9:376-379. 75. Papin JF, Vahrson W, Dittmer DP: SYBR green-based real-time quantitative 96. Blitvich BJ, Marlenee NL, Hall RA, Calisher CH, Bowen RA, Roehrig JT, PCR assay for detection of West Nile Virus circumvents false-negative Komar N, Langevin SA, Beaty BJ: Epitope-blocking enzyme-linked results due to strain variability. J Clin Microbiol 2004, 42:1511-1518. immunosorbent assays for the detection of serum antibodies to west 76. Johnson N, Wakeley PR, Mansfield KL, McCracken F, Haxton B, Phipps LP, nile virus in multiple avian species. J Clin Microbiol 2003, 41:1041-1047. Fooks AR: Assessment of a novel real-time pan-flavivirus RT-polymerase 97. Hall RA, Broom AK, Hartnett AC, Howard MJ, Mackenzie JS: chain reaction. Vector Borne Zoonotic Dis 2010, 10:665-671. Immunodominant epitopes on the NS1 protein of MVE and KUN viruses 77. Eischeid AC: SYTO dyes and EvaGreen outperform SYBR Green in real- serve as targets for a blocking ELISA to detect virus-specific antibodies time PCR. BMC Res Notes 2011, 4:263. in sentinel animal serum. J Virol Methods 1995, 51:201-210. 78. Lanciotti RS, Kerst AJ, Nasci RS, Godsey MS, Mitchell CJ, Savage HM, 98. Kitai Y, Shoda M, Kondo T, Konishi E: Epitope-blocking enzyme-linked Komar N, Panella NA, Allen BC, Volpe KE, Davis BS, Roehrig JT: Rapid immunosorbent assay to differentiate west nile virus from Japanese detection of west nile virus from human clinical specimens, field- encephalitis virus infections in equine sera. Clin Vaccine Immunol 2007, collected mosquitoes, and avian samples by a TaqMan reverse 14:1024-1031. transcriptase-PCR assay. J Clin Microbiol 2000, 38:4066-4071. 99. Lorono-Pino MA, Farfan-Ale JA, Blitvich BJ, Beebe JL, Jarman RG, Beaty BJ: 79. Chao DY, Davis BS, Chang GJ: Development of multiplex real-time reverse Evaluation of an epitope-blocking enzyme-linked immunosorbent assay transcriptase PCR assays for detecting eight medically important for the diagnosis of West Nile virus infections in humans. Clin Vaccine flaviviruses in mosquitoes. J Clin Microbiol 2007, 45:584-589. Immunol 2009, 16:749-755. 80. Dyer J, Chisenhall DM, Mores CN: A multiplexed TaqMan assay for the 100. Blitvich BJ, Bowen RA, Marlenee NL, Hall RA, Bunning ML, Beaty BJ: detection of arthropod-borne flaviviruses. J Virol Methods 2007, 145:9-13. Epitope-blocking enzyme-linked immunosorbent assays for detection of 81. Naze F, Le Roux K, Schuffenecker I, Zeller H, Staikowsky F, Grivard P, west nile virus antibodies in domestic mammals. J Clin Microbiol 2003, Michault A, Laurent P: Simultaneous detection and quantitation of 41:2676-2679. Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays 101. Sondgeroth K, Blitvich B, Blair C, Terwee J, Junge R, Sauther M, and dengue virus typing using high resolution melting. J Virol Methods VandeWoude S: Assessing flavivirus, lentivirus, and herpesvirus exposure 2009, 162:1-7. in free-ranging ring-tailed lemurs in southwestern Madagascar. J Wildl 82. Jimenez-Clavero MA, Aguero M, Rojo G, Gomez-Tejedor C: A new Dis 2007, 43:40-47. fluorogenic real-time RT-PCR assay for detection of lineage 1 and 102. Sotelo E, Llorente F, Rebollo B, Camunas A, Venteo A, Gallardo C, Lubisi A, lineage 2 West Nile viruses. J Vet Diagn Invest 2006, 18:459-462. Rodriguez MJ, Sanz AJ, Figuerola J, Jimenez-Clavero MA: Development and 83. Lee DH, Mathew J, Pfahler W, Ma D, Valinsky J, Prince AM, Andrus L: evaluation of a new epitope-blocking ELISA for universal detection of Individual donor nucleic acid amplification testing for detection of West antibodies to West Nile virus. J Virol Methods 2011, 174:35-41. Nile virus. J Clin Microbiol 2005, 43:5111-5116. 103. Alonso-Padilla J, Jimenez de Oya N, Blazquez AB, Loza-Rubio E, 84. Grant-Klein RJ, Baldwin CD, Turell MJ, Rossi CA, Li F, Lovari R, Crowder CD, Escribano JM, Saiz JC, Escribano-Romero E: Evaluation of an enzyme-linked Matthews HE, Rounds MA, Eshoo MW, Blyn LB, Ecker DJ, Sampath R, immunosorbent assay for detection of West Nile virus infection based Whitehouse CA: Rapid identification of vector-borne flaviviruses by mass on a recombinant envelope protein produced in Trichoplusia ni larvae. J spectrometry. Mol Cell Probes 2010, 24:219-228. Virol Methods 2010, 166:37-41. 85. Rondini S, Pingle MR, Das S, Tesh R, Rundell MS, Hom J, Stramer S, Turner K, 104. Roberson JA, Crill WD, Chang GJ: Differentiation of West Nile and St. Rossmann SN, Lanciotti R, Spier EG, Munoz-Jordan J, Larone D, Spitzer E, Louis encephalitis virus infections by use of noninfectious virus-like Barany F, Golightly LM: Development of multiplex PCR-ligase detection particles with reduced cross-reactivity. J Clin Microbiol 2007, 45:3167-3174. reaction assay for detection of West Nile virus. J Clin Microbiol 2008, 105. Volk DE, Beasley DW, Kallick DA, Holbrook MR, Barrett AD, Gorenstein DG: 46:2269-2279. Solution structure and antibody binding studies of the envelope protein 86. Parida M, Posadas G, Inoue S, Hasebe F, Morita K: Real-time reverse domain III from the New York strain of West Nile virus. J Biol Chem 2004, transcription loop-mediated isothermal amplification for rapid detection 279:38755-38761. of West Nile virus. J Clin Microbiol 2004, 42:257-263. 106. Beasley DW, Holbrook MR, Travassos Da Rosa AP, Coffey L, Carrara AS, 87. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Phillippi-Falkenstein K, Bohm RP Jr, Ratterree MS, Lillibridge KM, Ludwig GV, Hase T: Loop-mediated isothermal amplification of DNA. Nucleic Acids Res Estrada-Franco J, Weaver SC, Tesh RB, Shope RE, Barrett AD: Use of a 2000, 28:e63. recombinant envelope protein subunit antigen for specific serological 88. Lanciotti RS, Kerst AJ: Nucleic acid sequence-based amplification assays diagnosis of West Nile virus infection. J Clin Microbiol 2004, 42:2759-2765. for rapid detection of West Nile and St. Louis encephalitis viruses. J Clin 107. Kitai Y, Kondo T, Konishi E: Complement-dependent cytotoxicity assay for Microbiol 2001, 39:4506-4513. differentiating West Nile virus from Japanese encephalitis virus 89. Linke S, Mackay WG, Scott C, Wallace P, Niedrig M: Second external quality infections in horses. Clin Vaccine Immunol 2010, 17:875-878. assessment of the molecular diagnostic of West Nile virus: are there 108. Balasuriya UB, Shi PY, Wong SJ, Demarest VL, Gardner IA, Hullinger PJ, improvements towards the detection of WNV? J Clin Virol 2011, Ferraro GL, Boone JD, De Cino CL, Glaser AL, Renshaw RW, Ledizet M, 52:257-260. Koski RA, MacLachlan NJ: Detection of antibodies to West Nile virus in 90. Linke S, Ellerbrok H, Niedrig M, Nitsche A, Pauli G: Detection of West Nile equine sera using microsphere immunoassay. J Vet Diagn Invest 2006, virus lineages 1 and 2 by real-time PCR. J Virol Methods 2007, 18:392-395. 146:355-358. 109. Johnson AJ, Cheshier RC, Cosentino G, Masri HP, Mock V, Oesterle R, 91. Eiden M, Vina-Rodriguez A, Hoffmann B, Ziegler U, Groschup MH: Two new Lanciotti RS, Martin DA, Panella AJ, Kosoy O, Biggerstaff BJ: Validation of a real-time quantitative reverse transcription polymerase chain reaction microsphere-based immunoassay for detection of anti-West Nile virus assays with unique target sites for the specific and sensitive detection and anti-St. Louis encephalitis virus immunoglobulin M antibodies. Clin of lineages 1 and 2 West Nile virus strains. J Vet Diagn Invest 2010, Vaccine Immunol 2007, 14:1084-1093. 22:748-753. 110. Neng J, Harpster MH, Zhang H, Mecham JO, Wilson WC, Johnson PA: A 92. Zaayman D, Human S, Venter M: A highly sensitive method for the versatile SERS-based immunoassay for immunoglobulin detection using detection and genotyping of West Nile virus by real-time PCR. J Virol antigen-coated gold nanoparticles and malachite green-conjugated Methods 2009, 157:155-160. protein A/G. Biosens Bioelectron 2010, 26:1009-1015. 93. Papin JF, Vahrson W, Larson L, Dittmer DP: Genome-wide real-time PCR 111. Ryan J, Dave K, Emmerich E, Fernandez B, Turell M, Johnson J, Gottfried K, for West Nile virus reduces the false-negative rate and facilitates new Burkhalter K, Kerst A, Hunt A, Wirtz R, Nasci R: Wicking assays for the rapid strain discovery. J Virol Methods 2010, 169:103-111. detection of West Nile and St. Louis encephalitis viral antigens in 94. Pohl G, Shih IeM: Principle and applications of digital PCR. Expert Rev Mol mosquitoes (Diptera: Culicidae). J Med Entomol 2003, 40:95-99. Diagn 2004, 4:41-47. De Filette et al. Veterinary Research 2012, 43:16 Page 15 of 15 http://www.veterinaryresearch.org/content/43/1/16

112. Macdonald J, Tonry J, Hall RA, Williams B, Palacios G, Ashok MS, Jabado O, 129. Dunn MD, Rossi SL, Carter DM, Vogt MR, Mehlhop E, Diamond MS, Ross TM: Clark D, Tesh RB, Briese T, Lipkin WI: NS1 protein secretion during the Enhancement of anti-DIII antibodies by the C3d derivative P28 results in acute phase of West Nile virus infection. J Virol 2005, 79:13924-13933. lower viral titers and augments protection in mice. Virol J 2010, 7:95. 113. Chung KM, Diamond MS: Defining the levels of secreted non-structural 130. Chang DC, Liu WJ, Anraku I, Clark DC, Pollitt CC, Suhrbier A, Hall RA, protein NS1 after West Nile virus infection in cell culture and mice. J Khromykh AA: Single-round infectious particles enhance immunogenicity Med Virol 2008, 80:547-556. of a DNA vaccine against West Nile virus. Nat Biotechnol 2008, 26:571-577. 114. Nguyen BT, Koh G, Lim HS, Chua AJ, Ng MM, Toh CS: Membrane-based 131. Widman DG, Ishikawa T, Fayzulin R, Bourne N, Mason PW: Construction electrochemical nanobiosensor for the detection of virus. Anal Chem and characterization of a second-generation pseudoinfectious West Nile 2009, 81:7226-7234. virus vaccine propagated using a new cultivation system. Vaccine 2008, 115. Ng T, Hathaway D, Jennings N, Champ D, Chiang YW, Chu HJ: Equine 26:2762-2771. vaccine for West Nile virus. Dev Biol (Basel) 2003, 114:221-227. 132. Widman DG, Ishikawa T, Winkelmann ER, Infante E, Bourne N, Mason PW: 116. El Garch H, Minke JM, Rehder J, Richard S, Edlund Toulemonde C, Dinic S, RepliVAX WN, a single-cycle flavivirus vaccine to prevent West Nile Andreoni C, Audonnet JC, Nordgren R, Juillard V: A West Nile virus (WNV) disease, elicits durable protective immunity in hamsters. Vaccine 2009, recombinant canarypox virus vaccine elicits WNV-specific neutralizing 27:5550-5553. antibodies and cell-mediated immune responses in the horse. Vet 133. Widman DG, Ishikawa T, Giavedoni LD, Hodara VL, Garza Mde L, Immunol Immunopathol 2008, 123:230-239. Montalbo JA, Travassos Da Rosa AP, Tesh RB, Patterson JL, Carrion R Jr, 117. Seino KK, Long MT, Gibbs EP, Bowen RA, Beachboard SE, Humphrey PP, Bourne N, Mason PW: Evaluation of RepliVAX WN, a single-cycle flavivirus Dixon MA, Bourgeois MA: Comparative efficacies of three commercially vaccine, in a non-human primate model of West Nile virus infection. Am available vaccines against West Nile Virus (WNV) in a short-duration J Trop Med Hyg 2010, 82:1160-1167. challenge trial involving an equine WNV encephalitis model. Clin Vaccine 134. Nelson MH, Winkelmann E, Ma Y, Xia J, Mason PW, Bourne N, Milligan GN: Immunol 2007, 14:1465-1471. Immunogenicity of RepliVAX WN, a novel single-cycle West Nile virus 118. Martina BE, van den Doel P, Koraka P, van Amerongen G, Spohn G, vaccine. Vaccine 2010, 29:174-182. Haagmans BL, Provacia LB, Osterhaus AD, Rimmelzwaan GF: A 135. Biedenbender R, Bevilacqua J, Gregg AM, Watson M, Dayan G: Phase II, Recombinant Influenza A Virus Expressing Domain III of West Nile Virus randomized, double-blind, placebo-controlled, multicenter study to Induces Protective Immune Responses against Influenza and West Nile investigate the immunogenicity and safety of a West Nile virus vaccine Virus. PLoS One 2011, 6:e18995. in healthy adults. J Infect Dis 2011, 203:75-84. 119. Schepp-Berglind J, Luo M, Wang D, Wicker JA, Raja NU, Hoel BD, 136. Pletnev AG, Swayne DE, Speicher J, Rumyantsev AA, Murphy BR: Chimeric Holman DH, Barrett AD, Dong JY: Complex adenovirus-mediated West Nile/dengue virus vaccine candidate: preclinical evaluation in mice, expression of West Nile virus C, PreM, E, and NS1 proteins induces both geese and monkeys for safety and immunogenicity. Vaccine 2006, humoral and cellular immune responses. Clin Vaccine Immunol 2007, 24:6392-6404. 14:1117-1126. 137. Ledgerwood JE, Pierson TC, Hubka SA, Desai N, Rucker S, Gordon IJ, 120. Minke JM, Siger L, Cupillard L, Powers B, Bakonyi T, Boyum S, Nowotny N, Enama ME, Nelson S, Nason M, Gu W, Bundrant N, Koup RA, Bailer RT, Bowen R: Protection provided by a recombinant ALVAC((R))-WNV Mascola JR, Nabel GJ, Graham BS: A West Nile virus DNA vaccine utilizing vaccine expressing the prM/E genes of a lineage 1 strain of WNV a modified promoter induces neutralizing antibody in younger and against a virulent challenge with a lineage 2 strain. Vaccine 2011, older healthy adults in a phase I clinical trial. J Infect Dis 2011, 29:4608-4612. 203:1396-1404. 121. Yamshchikov G, Borisevich V, Seregin A, Chaporgina E, Mishina M, Mishin V, 138. Loginova S, Borisevich SV, Pashchenko Iu A, Bondarev VP: Ribavirin Kwok CW, Yamshchikov V: An attenuated West Nile prototype virus is prophylaxis and therapy of experimental West Nile fever. Antibiot highly immunogenic and protects against the deadly NY99 strain: a Khimioter 2009, 54:17-20. candidate for live WN vaccine development. Virology 2004, 330:304-312. 139. Sayao AL, Suchowersky O, Al-Khathaami A, Klassen B, Katz NR, Sevick R, 122. Demento SL, Bonafe N, Cui W, Kaech SM, Caplan MJ, Fikrig E, Ledizet M, Tilley P, Fox J, Patry D: Calgary experience with West Nile virus Fahmy TM: TLR9-targeted biodegradable nanoparticles as immunization neurological syndrome during the late summer of 2003. Can J Neurol Sci vectors protect against West Nile encephalitis. J Immunol 2010, 2004, 31:194-203. 185:2989-2997. 140. Hamdan A, Green P, Mendelson E, Kramer MR, Pitlik S, Weinberger M: 123. Martina BE, Koraka P, van den Doel P, van Amerongen G, Rimmelzwaan GF, Possible benefit of intravenous immunoglobulin therapy in a lung Osterhaus AD: Immunization with West Nile virus envelope domain III transplant recipient with West Nile virus encephalitis. Transpl Infect Dis protects mice against lethal infection with homologous and 2002, 4:160-162. heterologous virus. Vaccine 2008, 26:153-157. 124. McDonald WF, Huleatt JW, Foellmer HG, Hewitt D, Tang J, Desai P, Price A, doi:10.1186/1297-9716-43-16 Jacobs A, Takahashi VN, Huang Y, Nakaar V, Alexopoulou L, Fikrig E, Cite this article as: De Filette et al.: Recent progress in West Nile virus Powell TJ: A West Nile virus recombinant protein vaccine that diagnosis and vaccination. Veterinary Research 2012 43:16. coactivates innate and adaptive immunity. J Infect Dis 2007, 195:1607-1617. 125. Spohn G, Jennings GT, Martina BE, Keller I, Beck M, Pumpens P, Osterhaus AD, Bachmann MF: A VLP-based vaccine targeting domain III of the West Nile virus E protein protects from lethal infection in mice. Virol J 2010, 7:146. 126. Davis BS, Chang GJ, Cropp B, Roehrig JT, Martin DA, Mitchell CJ, Bowen R, Bunning ML: West Nile virus recombinant DNA vaccine protects mouse Submit your next manuscript to BioMed Central and horse from virus challenge and expresses in vitro a noninfectious and take full advantage of: recombinant antigen that can be used in enzyme-linked immunosorbent assays. J Virol 2001, 75:4040-4047. 127. Zhao Z, Wakita T, Yasui K: Inoculation of plasmids encoding Japanese • Convenient online submission encephalitis virus PrM-E proteins with colloidal gold elicits a protective • Thorough peer review immune response in BALB/c mice. J Virol 2003, 77:4248-4260. • No space constraints or color ﬁgure charges 128. Prow TW, Chen X, Prow NA, Fernando GJ, Tan CS, Raphael AP, Chang D, Ruutu MP, Jenkins DW, Pyke A, Crichton ML, Raphaelli K, Goh LY, Frazer IH, • Immediate publication on acceptance Roberts MS, Gardner J, Khromykh AA, Suhrbier A, Hall RA, Kendall MA: • Inclusion in PubMed, CAS, Scopus and Google Scholar Nanopatch-targeted skin vaccination against West Nile Virus and • Research which is freely available for redistribution Chikungunya virus in mice. Small 2010, 6:1776-1784.

Submit your manuscript at www.biomedcentral.com/submit