Human Antibody Responses to Hantavirus Recombinant Proteins and Development of Diagnostic Methods
A kadem isk A v h a n d l in g som för avläggande av doktorsexamen i medicinsk vetenskap vid Umeå Universitet, offentligen kommer att försvaras i föreläsningssalen Major Groove, Institutionen för Mikrobiologi, Umeå Universitet, fredagen den 15 november 1996, klockan 9 f.m.
av
Fredrik Elgh
Avdelningen för Virologi Umeå Universitet
Umeå 1996 Hu m a n A n tibo d y R esponses t o Hantavirus Re c o m b in a n t P ro tein s a n d D ev elo pm en t o f D ia g n o stic M eth o d s
Fredrik Elgh 1996
U m e å U n iv e r s it y M e d ic a l D issertations New series No 482 - ISSN 0346-6612
Rodent-borne hantaviruses (family Bunyaviridae) cause two distinct human infections; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is a common viral zoonosis, characterized by fever, renal dysfunction and hemostatic imbalance. Four HFRS-associated hantaviruses have been described: Hantaan virus and Seoul virus mainly found in Asia, Dobrava virus, encountered in the Balkan region and Puumala virus (PUU), causing mild HFRS (nephropathia epidemica; NE) in Europe. HPS, recently discovered in the Americas, involves adult respiratory distress syndrome with a high mortality rate and is caused by Sin Nombre virus. Hantaviruses are enveloped and carry a RNA genome which encodes a polymerase, two glycoproteins and a nucleocapsid protein. The latter elicits a strong humoral immune response in infected patients. The clinical diagnosis of hantavirus infections has until recently relied on serological confirmation by immunofluorescense assay (IFA) and enzyme-linked immunosorbent assay (ELISA) using cell culture derived viral antigens. Due to the hazardous nature of hantaviruses and variable virus yield in cell culture we aimed at using recombinant hantavirus proteins for serological purposes. We expressed PUU N in E. coli (PUU rN) and found that high levels of IgM to this protein could be detected at onset of NE. This indicated that it was useful as the sole antigen for serodiagnosis. Our finding was confirmed by comparing IFA and PUU rN ELISA using 618 sera collected at the regional diagnostic laboratory. Full-length PUU rN is difficult to purify due to aggregation to E. coli remnants. We therefore located the important domain for the humoral immune response by utilizing truncated PUU rN proteins to its amino-terminal region (amino acid 7-94). Amino acid 1-117 of N of the five major human hantavirus pathogens were produced in £. coli. Serological assays based on them could detect IgM and IgG serum responses in 380 HFRS and HPS patients from Sweden, Finland, Slovenia, China, Korea and the USA with high sensitivity. In an epidemiological investigation of hantavirus serum responses in European Russia we unexpectedly found antibody responses to the hantaviruses found in east Asia and the Balkan region in 1.5 %, speaking in favour for the presence of such virus in this region. The degree of cross reactivity within the hantavirus genus was adressed by following the serum responses in NE patients. We found an increase of cross reactivity during the maturation of the immune response from onset of disease up to three years by comparing the IgG reactivity towards the hantavirus aminoterminal rN proteins. The first human isolate of the causative agent of NE in Scandinavia was recovered in cell culture from phytohemagglutinin stimulated leukocytes. Serological analysis revealed that this virus belongs to the PUU hantavirus serotype, distinct from the rodent prototype PUU Sotkamo. The human PUU Umeå is unique but genetically similar to rodent isolates from northern Sweden.
Key words: Puumala Virus, Hantavirus Infections, Recombinant Proteins, Capsid, Humoral Immunity, Immunoassay
ISBN 91-7191-229-0 Human antibody responses to hantavirus recombinant proteins and development of diagnostic methods
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No 482 - ISSN 0346-6612
From the Department of Virology Umeå University, Umeå, Sweden
Human Antibody Responses to Hantavirus Recombinant Proteins
& Development of Diagnostic Methods
Fredrik Elgh
Umeå 1996 Copyright © 1996 by Fredrik Elgh ISBN 91-7191-229-0
Printed in Sweden by Solfjädern Offset AB, Umeå, 1996 To the memory of Lisa Elgh
TABLE OF CONTENTS
PREFACE ...... vii
SUMMARY ix
SAMMANFATTNING AV AVHANDLINGEN (summary in Swedish) ..... x
PAPERS ...... xiii
ABBREVIATIONS ...... xiv
INTRODUCTION ...... 1
The link between hemorrhagic fevers with renal syndrome on the Eurasian continent 2
HFRS in the Balkan region ...... 3
Potential for the presence of other hantaviruses than the Puumala serotype in Europe 4
New world hantaviruses ...... 4
Epidemiology and ecology of hantaviruses ...... 5
Hantavirus virology ...... 9
Evolution of hantaviruses ...... 11
Transmission of hantaviruses to man ...... 15
Clinical manifestations ...... 16
Korean hemorrhagic fever ...... 16
Nephropathia epidemica ...... 17
Hantavirus pulmonary syndrome ...... 18
Diagnosis of hantavirus infections ...... 19
Virus isolation ...... 19
Serological diagnosis ...... 19
Detection of viral antigen ...... 22
Genetic assays ...... 22
AIMS OF THIS THESIS ...... 23
RESULTS AND DISCUSSION ...... 25
Cloning and expression of the Puumala virus, strain Sotkamo, S gene segment .... 25 Study design to compare rN based ELISA and IFA ...... 27
Kinetics of Puumala virus IgM and IgG responses by rN based ELISA and IFA .... 27
Clinical evaluation of Puumala virus rN based ELISA ...... 28
Identification of a major antigenic domain in Puumala virus rN ...... 31
Amino-terminal rN of five hantaviruses as antigens in serological assays ...... 33
Polyclonal antibody responses in animals to hantavirus aminoterminal rN proteins 34
Detection of specific antibodies in sera from different parts of the world by
multi-valent hantavirus ELISA ...... 34
Seroepidemiology in European Russia by multi-valent hantivirus rN ELISA .... 38
Kinetics of antibody responses to recombinant hantavirus nucleocapsid proteins
in patients with nephropathia epidemica ...... 39
Isolation and characterization of the causative virus in NE 43
CONCLUSIONS ...... 47
REFERENCES ...... 49
APPENDIX: Papers I-VII ...... 61
vi PREFACE
In science, there are, as you know, a wide variety of topics to choose among and I have tried a few of them through the years. Among them, fibrinolytic genes at least took me half-way. mRNA seemed to fragile and therefore I turned to robust clinical work. After a few years of practice in reality I came back to research and now hantaviruses have become the topic of my book. It has been both a pleasure and a pain to write the pamphlet you're holding. A pleasure because I found so many new (to me) aspects of virology while digging into the enormous resources of knowledge that are hidden on the shelves of our University and Hospital libraries here in town and elsewhere (thanks UB and Vårdbiblioteket for all help!). N ot to be forgotten is also the great feeling that it is finally, finalmento, finished!!! A slight pain because of all the long nights that turn into morning too quickly when the computer is humming and because I have had too few moments with my family lately. Plus and minus - when you read this book it is definitely out of m y hands! Puuh! A few days of rest will follow for me and my family after the final delivery of this manuscript to the printers! First of all I must tell you that the final months of word- carving had not been possible without the total and constant support from Eva (all my love to You, Jonathan, Gustaf and Petter). There are (very) many more friends and colleagues that deserve acknowledgements for having made it possible for me to get this work to where it is now. I would like to mention some of them here: It has been a joyful opportunity to spend the last five years at the Department of Virology and Laboratory of Clinical Virology in Umeå. First of all I want to thank Åke Gustafsson for pointing out to me that I was ment for clinical virology back in '91 when I was doing my clinical training down south. Per Juto, in charge of the constantly growing and energetic clinical lab and the pioneering NE team, has been a constant source of support throughout this work. Without his belief in and patience with me including help with all the sometimes rather fuzzy manuscripts, not much had been achieved. Göran Wadell, professor and as such sometimes hidden behind shiploads of different typographical matter, has shared all possible resources and ideas in benefit for the present work. Arne Tärnvik, professor of infectious diseases, is greatly acknowledged for creating resources for our group and for very constructive help and ideas while I was trying to get the last manuscript into order. I am very happy about the ongoing collaboration with Åke Lundkvist (SMI). Thanks for friendship, invaluable help and stimulating discussions! Back to Umeå - D o you think I gathered the data all by myself??? O h no! Kristina Lindman, Madeleine Hägglund, Anci Verlemyr and Karin Edlund have fought bravely with different aspects of muroid fevers (and me). I hope that you can take some more.... To all of you at Staten and Viruslab I wish to express my gratitude for your helpfulness and the open-minded atmosphere that are the signums of our lab. Since the first three years of my work was performed in parallell with clinical duties, I sometimes had-to-do-a-lot-of-things at the same time. That had not been possible without your help and understanding. Thanks, Torgny, Katrine and Rolf for helping out with many practical details! I am grateful to Jens Boman for many nice discussions on science and life at the lab and to Urban Kumlin for standing in for me and for many valuable ideas. Long-time hacker Åke Gustafsson, deserves yet another special section for putting me on/off the net (work) and for trying to teach me DOS and that. All the members of the NE team are greatly acknowledged for stimulating round- table-conferences on antibodies, Swedish/Russian party culture, trap-nights, phylogenies, and even more important matters. I would especially like to thank Mats Linderholm for being a great friend and for helping me to put things in order (both words, family and figures). It has been a pleasure to work together with Oleg Alexeyev and Clas Ahlm - 1 must admit that I am very grateful that you did not involve me so much in rodent pathology... Since I have had the opportunity to do labwork in the neighbourhood of my present location already in the early eighties I have come to know quite a number of great people of the Umeå scientific frontier. Among them I would especially like to thank Henrik Semb for being a true friend and back-up. Henrik and the members of his lab has helped me a lot with different practicalities which I am very grateful for. Another old sport from my fibrinolytic era is Gunnar Pohl who took his time and thaught me what is up-and-down on a chromatography column. Thanks!!! In this context I would also like to thank Torgny Stigbrand and Berith Nilsson for all help to produce valuable Mabs. It has ben very amusing to communicate ideas and reagents with colleagues, co- authours and collaborators outside of the Umeå territory. A special thanks to Brian Hjelle for putting up with all my email questions and to him and Donna Wiger for extensive and valuable discussions and criticism on Paper IV. I have really appreciated the kind gifts of hantavirus DNA and antisera from Connie Schmaljohn and sequence information on various viruses that I have received prior to publication from Janne Hörling, Tatjana Avsic-Zupanc, Olli Vapalahti and Alex Plyusnin. Without your help I had not achieved much of this. Almost every research project is in urgent need for money. I would like to take the oppportunity to thank my supporters by mentioning them here: the Medical Faculty, Umeå University, the Swedish Medical Research Council, the Kempe Foundation, the Joint Committee of the Northern Swedish Health Care Region, Förenade Liv Mutual Group Life Insurance Company, the Swedish Society for Medical Research and the Swedish Society for Medicine. Finally, I would like to thank my entire family for understanding, encouragement and love. I hope that pages x-xii will explain to you some of what we have been doing here.
Umeå, October 17, 1996 A SUMMARY
Rodent-borne hantaviruses (family Bunyaviridae) cause two distinct human infections; hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is a common viral zoonosis, characterized by fever, renal dysfunction and hemostatic imbalance. Four HFRS-associated hantaviruses have been described: Hantaan virus and Seoul virus mainly found in Asia, Dobrava virus, encountered in the Balkan region and Puumala virus (PUU), causing mild HFRS (nephropathia epidemica; NE) in Europe. HPS, recently discovered in the Americas, involves adult respiratory distress syndrome with a high mortality rate and is caused by Sin Nombre virus. Hantaviruses are enveloped and carry a RNA genome which encodes a polymerase, two glycoproteins and a nucleocapsid protein. The latter elicits a strong humoral immune response in infected patients. The clinical diagnosis of hantavirus infections has until recently relied on serological confirmation by immunofluorescense assay (IFA) and enzyme-linked immunosorbent assay (ELISA) using cell culture derived viral antigens. Due to the hazardous nature of hantaviruses and variable virus yield in cell culture we aimed at using recombinant hantavirus proteins for serological purposes. We expressed PUU N in Escherichia coli (PUU rN) and found that high levels of IgM to this protein could be detected at onset of NE. This indicated that it was useful as the sole antigen for serodiagnosis. Our finding was confirmed by comparing IFA and PUU rN ELISA using 618 sera collected at the regional diagnostic laboratory. Full-length PUU rN is difficult to purify due to aggregation to E. coli remnants. We therefore located the important domain for the humoral immune response by utilizing truncated PUU rN proteins to its amino-terminal region (amino acid 7-94). Amino acid 1-117 of N of the five major human hantavirus pathogens were produced in E. coli. Serological assays based on them could detect IgM and IgG serum responses in 380 HFRS and HPS patients from Sweden, Finland, Slovenia, China, Korea and the USA with high sensitivity. In an epidemiological investigation of hantavirus serum responses in European Russia we unexpectedly found antibody responses to the hantaviruses found in east Asia and the Balkan region in 1.5 %, speaking in favour for the presence of such virus in this region. The degree of cross reactivity within the hantavirus genus was adressed by following the serum responses in NE patients. We found an increase of cross reactivity during the maturation of the immune response from onset of disease up to three years by comparing the IgG reactivity towards the hantavirus aminoterminal rN proteins. The first human isolate of the causative agent of NE in Scandinavia was recovered in cell culture from phytohemagglutinin stimulated leukocytes. Serological analysis revealed that this virus belongs to the PUU hantavirus serotype, distinct from the rodent prototype PUU Sotkamo. The human PUU Umeå is unique but genetically similar to rodent isolates from northern Sweden. SAMMANFATTNING AV AVHANDLINGEN Brief summary of the thesis (in Swedish).
Hantavirus orsakar sannolikt flera hundratusen sjukdomsfall årligen runt om i världen. Sjukdomspanoramat sträcker sig från mycket svåra tillstånd till milda sådana. De svåra ger blödningar, shock och njurfunktionsnedsättning och de drabbar främst människor i Kina, Korea och andra länder i Fjärran Östern. I Europa finns liknande sjukdomstillstånd men dessa har oftast ett mildare förlopp. En nyligen upptäckt sjukdomsform i Nord- och Sydamerika ger upphov till svår lungfunktionsnedsättning i form av bland annat lungödem. Denna sjukdom tar livet av mer än hälften av de människor som drabbas. Två svenska läkare (Gustaf Myhrman och S. G. Zetterholm) beskrev 1934 en sjukdom som drabbade människor i Norrland. Myhrman och Zetterholm beskrev en sjukdom som startade med hög feber, muskelvärk och svåra smärtor i rygg och mage. Många gånger misstolkades sjukdomstecknen och man opererade patienterna i buken för vad man trodde var en kirurgisk åkomma. Senare i sjukdomsförloppet fick patienterna en njurfunktionsnedsättning vilket gav minskade urinmängder och förändringar i blodet som också tydde på att njurarna inte fungerade som de skulle. Efter några dagar började njurarna fungera igen och patienterna återhämtade sig så småningom i de allra flesta fall. Under våren och sommaren 1942, i samband med andra världskriget, fann tyska och finska militärläkare samma typ av sjukdom hos mer än 1000 soldater vid Salla-fronten i finska Lappland (i nuvarande nordvästra hörnet av Ryssland). Under 50-, 60- och 70-talen beskrevs den här sjukdomen flitigt i nordiska medicinska tidskrifter och sjukdomen namngavs av Myhrman. Den fick heta nephropathia epidemica; nephropatia på grund av att den drabbade njurarna och epidemica för att den uppträdde säsongsvis. Liknande sjukdomar beskrevs i Ryssland och av de japanska ockupationsstyrkorna i Kina under 30-talet. Man lyckades överföra smittämnet mellan människor och man förstod att sjukdomen inte orsakades av bakterier eller större mikroorganismer. I övrigt förblev smittämnet okänt. Under Koreakriget (1951-54) insjuknade tusentals soldater (ffa Förenta Nationernas förband) i en liknande, svår, sjukdom som kom att kallas Koreansk hemorrhagisk feber. Dödligheten bland de insjuknade soldaterna var hög; 5-10%. Man hade från början ingen som helst aning om vad detta sjukdomstillstånd kunde orsakas av. 1953 föreslog den blivande nobelpristagaren Carleton Gajdusek att det fanns ett samband mellan de skandinaviska, ryska, kinesiska och koreanska sjukdomar som nämnts. Koreanska och amerikanska forskare arbetade intensivt under flera decennier för att ta reda på orsaken men inte förrän 1978 stod det klart att sjukdomen orsakades av ett virus som härbärgerades i smågnagare (Professor Ho Wang Lee och medarbetare i Seoul, Korea). Det visade sig att ryska, finska och svenska varianter var orsakade av liknande virus. Man hade en längre tid misstänkt att skogssork var orsaken till den skandinaviska varianten av sjukdomen och Kurt Nyström, läkare i Umeå, kunde i sin avhandling 1977 visa att antalet fall av sjukdomen nephropathia epidemica (sorkfeber) ökade och minskade i takt med variationen av mängden sorkar i naturen. I en avhandling från Umeå (1989) kunde Bo Settergren bekräfta att sorkfeber i vår del av världen är mycket lik andra hantavirusorsakade sjukdomar men att vår inhemska variant är lindrigare till sin natur. De senaste 20 årens forskning har inneburit mycket för förståelsen av de här sjukdomstillstånden och de virus som orsakar dem. De hantavirus som orsakar sjukdomarna i Asien, Europa och Amerika har isolerats och deras arvsmassa har analyserats. Ett flertal andra gnagarburna virus som tillhör samma grupp men inte orsakar sjukdom har också identifierats och undersökts. Det har visat sig att den här virustypen är spridd över i stort sett hela världen och att virus och gnagare verkar ha utvecklats tillsammans under lång tid. Hantavirusorsakade sjukdomar är relativt vanliga och ibland allvarliga. Därför har det varit viktigt att ta fram diagnostiska metoder som är känsliga och inte ger upphov till felaktiga tolkningar. Virus som orsakar de här sjukdomarna kan odlas i cellkultur på laboratorium. Ur sådana infekterade cellkulturer har man renat fram virus. Dessa har sedan använts för att ta reda på om sjuka människor har antikroppar riktade mot virus. På så sätt kan man få en uppfattning om patienten har drabbats av ett hantavirus. På senare tid har det också utvecklats metoder för att identifiera virusets arvsmassa i kroppsvätskor och vävnader från människor med sjukdom orsakad av hantavirus. Det var denna metod som gjorde det möjligt att snabbt ta reda på orsaken till den sjukdom som drabbade många människor i ett indianreservat i sydvästra USA under våren 1993 och som kom att kallas hantavirus pulmonary syndrome. Denna sjukdom visade sig också vara orsakad av ett virus som härbergeras i gnagare. Man kunde visa att sjukdomsframkallande virus för den amerikanska sjukdomsvarianten är närbesläktat med det som drabbar skandinaviska sorkfeberpatienter. Att producera hantavirus i cellkultur för användning i antikropps-undersökningar av de ovan nämnda sjukdomstillstånden går ganska bra. Det finns dock en del svårigheter;
I. det är farligt att arbeta med hantavirus i laboratorium eftersom de kan orsaka sjukdom, ü. de preparationer av virus som framställs kan variera i kvalité och m. avläsning av de metoder som traditionellt har använts (immun- fluorescens-mikroskopi) är beroende av speciellt tränad personal. Detta gjorde att vi 1992 beslutade oss för att framställa hantavirusproteiner i genetiskt modifierade bakterier. I min avhandling (den 3:e sorkfeberrelaterade avhandlingen från Umeå) visas att virusets nukleokapsidprotein (det vill säga det protein som binder till och sannolikt skyddar virus arvsmassa) är mycket användbart för identifiering av antikroppar vid de här sjukdomstillstånden. Vi har också kunnat visa att det är en del av proteinet som reagerar starkt med patienternas antikroppar och denna kunskap har vi utnyttjat när vi tagit fram metoder för att undersöka förekomsten av antikroppar mot alla kända sjukdomsframkallande hantavirus. Vi har testat den här metoden på patientmaterial från Sverige, Norge, Finland, Slovenien, Korea, Kina och USA och kunnat visa den är snabb, känslig och inte ger upphov till falska diagnoser. Antikroppssvaret hos patienter med sorkfeber har studerats med hjälp av de framställda proteinerna. Vi har kommit fram till att man bör välja att testa om patienten har antikroppar av IgM-typ mot de i bakterier framställda hantavirusproteinerna, eftersom sådana finns hos de allra flesta alldeles i början av sjukdomsförloppet. Med de metoder som vi utvecklat är det också möjligt att visa hur antikroppssvaret mot dessa proteiner förändras med tiden. I en undersökning fann vi att svenska patienter som haft sorkfeber får alltmer antikroppsreaktivitet riktad mot hantavirus med ursprung i Asien ju längre tid som förlöper mellan sjukdoms- och provtagningstillfällena. Detta är viktig kunskap, framförallt för tolkning av svar som erhålls i undersökningar som görs en längre tid efter sjukdomsdebuten. Det har varit svårt att isolera det europeiska sorkfeber-orsakande viruset från människa. Ett flertal virusisolat från skogssorkar finns beskrivna varav några från Sverige. När det gäller isolat från patienter finns flera beskrivna från Ryssland och ett från Frankrike. I Skandinavien har det inte förrän nu gått att hitta levande virus i vävnader eller kroppsvätskor från sjuka patienter. I avhandlingen beskrivs hur vi från vita blodkroppar, med ursprung i en sorkfeber-patient som infekterats utanför Umeå, har fått virus att växa fram i cellkultur. En ny metod som innebär att vi stimulerat patientens vita blodkroppar att växa till och dela sig i snabb takt har använts. Troligen har det gjort att även virus har formerat sig och kunnat infektera våra cellkulturer. Vi har analyserat virus med hjälp av antikroppar från djur som vaccinerats med hantavirus och hantavirusproteiner samt med antikroppar från sjuka människor. Umeåvirusets arvsmassa har också analyserats och det står klart att det är av samma typ (Puumalavirus) som tidigare identifierats i sorkar här i landet. PAPERS
This thesis is based on the following papers, which will be referred to in the text by their Roman numerals.
I. Elgh F, Wadell G, and Juto P. Comparison of the kinetics of Puumala virus specific IgM and IgG antibody responses in nephropathia epidemica as measured by a recombinant antigen-based enzyme-linked immunosorbent assay and an immunofluorescence test. Journal of Medical Virology 1995;45:146-150.
II. Elgh F, Linderholm M, Wadell G, and Juto P. The clinical usefulness of a Puumalavirus recombinant nucleocapsid protein based enzyme-linked immunosorbent assay in the diagnosis of nephropathia epidemica as compared with an immunofluorescence assay. Clinical and Diagnostic Virology 1996;6:17-26.
III. Elgh F, Lundkvist Å, Alexeyev OA, Wadell G, and Juto P. A major antigenic domain for the human humoral response to the Puumala virus nucleocapsid protein is located at the amino-terminus. Journal of Virological Methods 1996;59:161-172.
IV. Elgh F, Lundkvist Å, Alexeyev OA, Avsic-Zupanc T, Hjelle B, Lee HW, Smith KJ, Vainionpää R, Wiger D, Wadell G, and Juto P. Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on the detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes. Submitted.
V. Alexeyev OA, Elgh F, Zhestkov AV, Wadell G, and Juto P. Hantaan and Puumala virus antibodies in blood donors in Samara, an HFRS-endemic region in European Russia. Lancet 1996;347:1483.
VI. Elgh F, Linderholm M, Hägglund M, Wadell G, Tärnvik A, and Juto P. Development of humoral cross reactivity to recombinant nucleocapsid proteins of five pathogenic hantaviruses in nephropathia epidemica. Submitted.
VII. Juto P, Elgh F, Ahlm C, Alexeyev OA, Edlund K, Lundkvist Å, and Wadell G. The first human isolate of Puumala virus in Scandinavia as cultured from phytohemagglutinin stimulated leucocytes. Submitted. ABBREVIATIONS* aa amino acid A U arbitrary units CDC Centers for Disease Control and Prevention cDNA complementary DNA CFT complement fixation test CNS central nervous system DIC disseminated intravascular coagulation DNA deoxy-ribonucleic acid ELISA enzyme-linked immunosorbent assay EM electron microscopy Gl glycoprotein 1 G2 glycoprotein 2 HDPA high density particle agglutination assay HFRS hemorrhagic fever with renal syndrome HI hemagglutination-inhibition assay HPS hantavirus pulmonary syndrome IAHA immune adherence hemagglutination asssay IFA indirect fluorescent antibody assay IgA immunoglobulin A IgG immunoglobulin G IgM immunoglobulin M kDa kilo Daltons KHF Korean hemorrhagic fever L large gene segment M middle size gene segment Mab monoclonal antibody mRNA messenger RNA N nucleocapsid protein NE nephropathia epidemica N TA neutralization assay OD optical density ORF open reading frame PCR polymerase chain reaction RDRP RNA-dependent RNA polymerase rN recombinant nucleocapsid protein rNA truncated recombinant nucleocapsid protein RNA ribonucleic acid S small gene segment SDS sodium dodecyl sulphate ssRNA single-stranded RNA WHO World Health Organization
* Abbreviations of hantaviruses are given in Table 1 and Figure 3.
xiv INTRODUCTION
In 1933 and 1934 two Swedish physicians, working in northern Sweden, independently of each other observed a previously not described disease syndrome (Myhrman, 1934; Zetterholm, 1934). It was characterized by rapid onset of high fever, malaise, chills, headache, abdominal, back and often generalized pain and a renal syndrome with proteinuria and oliguria followed by a diuretic phase. A spontaneous recovery followed all cases described. An epidemic with characteristics similar to this disease was observed during World War II among both Finnish and German troops at the Salla front in Finnish Lappland in the spring and summer of 1942 (Stuhlfauth, 1943; Hording, 1946). Among a Finnish contingent of 550 men, 60 fell ill while 1000 cases were reported among the German soldiers, located in a nearby area. In this region an abundance of rodents was reported in 1942. In 1943 the German troops remained in the same region and in this year only a few cases appeared in parallel with a low frequency of rodents. Both authors speculated in a disease causing agent spread by rodents. The symptomatology mimicked leptospirosis (Weil’s disease) and Leptospira was therefore proposed by Stuhlfauth as a putative pathogen. The pathogenic role of leptospira was not possible to prove serologically neither by Stuhlfauth nor Myhrman (1951) in later investigations. During the forthcoming decades a large number of cases of this disease were reported from Sweden, Norway, Finland and Denmark (Oldberg, 1941; Myhrman, 1945, 1948 and 1951; Knutrud, 1949; Muri, 1950 and 1953; Tungland, 1954; Hansen, 1958; Ornstein and Söderhjelm, 1963; Lähdevirta, 1971; Nyström, 1977). In 1945 Myhrman proposed nephropathia epidemica (NE) as the name for this disease, which is now internationally acknowledged. During the Korean conflict more than 3000 United Nation soldiers stationed in the demilitarized zone near the fighting front on the Korean peninsula acquired a severe disease characterized by fever, hemorrhagic manifestations and renal insufficiency (1951-1954) (Gajdusek, 1953, 1956 and 1962; Smadel, 1953; Sheedy et al., 1954). This disease, at that time largely unknown to western medicine, was recognized as the Korean hemorrhagic fever (KHF). Mortality rates were high, 5-10%, mainly due to severe bleedings, shock and renal failure. However, evidence of a similar disease was documented as early as 1913 in medical records from Vladivostok (Casals et al., 1970). Russian and Japanese littérature, as reviewed by Gajdusek (1953) and Smadel (1953), also revealed the
1 presence of a similar disease along the lower Amur river valley in the southeastern part of Russia and across the Amur in Manchuria. This was described by the Russians and the Japanese Army stationed in Manchuria in 1932. Chinese authorities reported about the existence of a similar disease in Inner Mongolia (Gajdusek, 1962). In the late 1950’s Soviet physicians reported about diseases with similar features in European Russia. Identical disease patterns had been observed already in the 1930's under the name of Tula fever. In the 1940's, Soviet and Japanese researchers provoked the disease in humans by parenteral injections of bacterial-free filtrates of body fluids from patients into previously healthy individuals, thus suggesting a viral etiology (Gajdusek, 1962). The discovery of KHF during the Korean war marked the starting point of intense studies in search of the causative agent of these diseases. Circumstantial evidence pointed towards the role of rodents in the spread of them (Hortling, 1946; Casals et al., 1970; Lähdevirta, 1971; Nyström, 1977). An agent that reacted strongly in indirect fluorescent antibody assay (IFA) to antibodies from patients that had acquired KHF was identified in lung sections from the rodent Apodemus agrarius coreae (field mouse) (Lee & Lee, 1976). These rodents had been captured in rural endemic areas. The responsible virus, named Hantaan (HTN) after the original site of rodent capture at the Hantaan river in Korea, was isolated and could later be propagated in cell culture (Lee et al., 1978; French et al., 1981). This greatly simplified serological diagnosis of the disease.
The link between hemorrhagic fevers with renal syndrome on the Eurasian continent
In 1953 D. C. Gajdusek postulated the relationship between KHF and the Chinese, Russian and European hemorrhagic fevers, including NE. Shortly after the discovery of IFA reactivity in sera of KHF patients to an agent in Apodemus agrarius coreae lung sections (Lee & Lee, 1976 and 1977; Lee et al., 1978), it was possible to show that sera of NE patients from Sweden and Finland reacted with an identical fluorescence pattern to the KHF antigen (Svedmyr et al., 1979; Lee et al., 1979). When similar NE antigen preparations derived from Finnish Chlethrionomys glareolus (bank vole) were available (Brummer-Korvenkontio et al., 1980) it was possible to compare reactivities of KHF and NE patient sera with both types of antigens. It was then found that a
2 high proportion of NE sera reacted with both agents as opposed to KHF sera of which reactivity towards the NE agent was regularly absent (Svedmyr et al., 1980 and 1982). Serological investigations performed in the former Soviet Union described evidence for the circulation of several distinct viruses responsible for similar diseases (Gavrilovskaya et al., 1981; Tkachenko et al., 1982). Furthermore, evidence of several serotypes in Europe were put forward the same year (Lee, P.W. et al., 1982b). The NE causing virus was isolated from lungs of C. glareolus captured in Finland, adapted to growth in cell culture and named Puumala virus (PUU) after the original site of rodent capture (Brummer-Korvenkontio et al., 1982; Schmaljohn et al., 1985). Similar viruses have been isolated from C. glareolus in Sweden (Niklasson & LeDuc, 1984; Yanagihara et al., 1984a and b). HFRS was also reported from cities in Korea where A. agrarius does not exist. A virus with immunological properties similar but distinct from HTN was isolated from Rattus norvégiens and R. rattus (rat) in urban areas in Korea and has become known as the Seoul virus (SEO) (Lee, H.W. et al., 1982). This virus has been shown to be pathogenic to man and represent a serotype of its own (Chu et al., 1994; Li, Y.L. et al., 1995). It thus became evident that the postulation by Gajdusek was correct, i.e. that these diseases, found throughout the Eurasian landmass, were caused by similar rodent-borne viruses. At a WPIO meeting in Tokyo 1982 the name hemorrhagic fever with renal syndrome (HFRS) was recommended for the viral diseases within this group (WHO, 1983). In 1985 hantavirus became the accepted term for HFRS-causing and related viruses (Schmaljohn et al., 1985).
HFRS in the Balkan region
HFRS was initially reported from the former Yugoslavia in the early 1950's and has since been accounted for frequently (Avsic-Zupanc et al., 1989 and 1990; Gligicet al., 1989b). Serological analysis has revealed the circulation of at least two serotypes (Avsic-Zupanc et al., 1989 and 1990; Gligic et al., 1989a; Hukic et al., 1996). A hantavirus, similar but serologically distinct from HTN, was isolated from the lungs of an Apodemus flavicollis (yellow-necked field mouse) captured in an area of Slovenia where a number of severe HFRS cases had occurred (Avsic-Zupanc, 1992). This virus, named Dobrava (DOB), has been
3 characterized genetically (Avsic-Zupanc et al., 1995). Recently, RNA of viruses much resembling DOB has been amplified from Greek and Albanian patients with HFRS (Antoniadis et al., 1996). Furthermore, high titers of neutralizing antibodies to DOB has been found in sera of FÎFRS patients from Bosnia- Hercegovina (Lundkvist et al., 1996a). These data are strong evidence for the pathogenetic significance of DOB throughout the Balkan region. HFRS- patients from Greece have been shown to react stronger to HTN- than to PUU-like viruses in IFA and plaque-reduction neutralization tests, suggesting a pathogenetic role for HTN-like viruses in this country (Antoniadis et al., 1987b). The genetic character of a DOB/HTN-like virus isolate from a severe HFRS case in Greece remains to be elucidated (Porogia virus) (Antoniadis et al., 1987a).
Potential for the presence of other hantaviruses than the Puumala serotype in Europe
The spread of A. agrarius, A. flavicollis and Rattus species throughout large parts of Europe and European Russia might be the explanation for the not uncommon finding of seroreactivity to HTN-, SEO- and DOB-like viruses in this part of the world (Gligic et al., 1989a; Gresikova et al., 1990 and 1994; Groen et al., 1991b; Alexeyev et al., 1996b [Paper V]). However, the significance of these observations awaits further investigation.
New world hantaviruses
The existence of hantaviruses in American rodents has been known since 1982 (Tsai et al., 1982; LeDuc et al., 1982; Gibbs et al., 1982; Lee, P.W. et al., 1982a; Yanagihara, 1990). The presence of SEO-like viruses was demonstrated by serology and virus isolation in Rattus species in the USA. By serology, SEO has been shown to infect humans. In addition, a hantavirus representing a novel serotype, designated Prospect Hill virus (PH), was isolated from Microtus pennsylvanicus (meadow vole) (Lee, P.W. et al., 1982a and 1985a). This virus has not yet been implicated in human disease. A role for HTN/SEO-like hantaviruses in the development of hypertensive disease in the USA has been
4 claimed and domestic cases of HFRS have been presented (Glass et al., 1993 and 1994). In the spring and summer of 1993 a series of deaths and cases of critical illness were reported by health-care workers in the southwestern USA (Four- corners area) (CDC, 1993a). The patients presented an influenza-like illness, succeeded by rapidly progressing pulmonary edema, respiratory insufficiency and shock. Laboratory tests for a wide arrray of viral and bacterial pathogens revealed that the common denominator in these patients was the presence of antibodies to a hantavirus not previously recognized in the USA (CDC, 1993b). Immunohistochemical and polymerase chain reaction (PCR) techniques confirmed these observations (CDC, 1993b and c; Nichol et al., 1993b; Ksiazek et al., 1995). Genetic information showed that the pathogen was a hantavirus, most closely related to the American PH and European PUU (the latter causing NE) (Nichol et al., 1993b; Spiropoulou et al., 1994; Hjelle et al., 1994b). Several names have been suggested; Four Corners virus and Muerto Canyon virus have been discarded for several reasons. The presently used name is Sin Nombre virus (SNV), i.e. the virus without a name. Recombinant proteins specific for SNV were promptly produced for diagnostic and epidemiological purposes in rodents and man in parallell with PCR (Feldmann et al., 1993; Hjelle et al., 1993).
Epidemiology and ecology of hantaviruses
The rodent role in NE was studied by Nyström in Västerbotten county, northern Sweden (1977). In the investigated region, during 1959 to 1975, he found that NE was of endemic nature with no apparent person-to-person spread. He found a congruent variation in seasonal incidence of disease and seasonal prevalence of small rodents. The possibe role of rodents in NE was also discussed by Finnish researchers (Lähdevirta, 1971, Korpela & Lähdevirta, 1978). They noticed the seasonal distribution of the rodent population in parallel with the incidence of NE, and also circumstances that linked contact with rodents to cases of disease. A striking finding in Sweden was that albeit a high incidence in the north, almost no cases of NE occur south of the 60th parallell (Nyström, 1977; Niklasson & LeDuc, 1987; Settergren et al., 1988). More recent epidemiological investigations show that PUU and/or related viruses are responsible for the majority of hantavirus caused disease in northern
5 and central Europe and European Russia, which is the habitat for its predominant host C. glareolus (Fig. 1; Table 1) (LeDuc, 1987; Niklasson & LeDuc, 1987; Settergren et al., 1988; Ahlm et al., 1994; Groen et al., 1995a; Zollerete/., 1995).
PU U ▲ HIN ■ SEO □ DOB 0 SNV • NYVO BAYV © BCCVO ANDEV ®
Fig. 1 . Schematic distribution of hantaviruses associated with disease.
Disease caused by PUU is especially common in northern Scandinavia, Finland and European Russia (Lähdevirta et al., 1984; Niklasson & LeDuc, 1987; Settergren et al., 1988; Niklasson et al., 1993). The C. glareolus population density in northern Sweden varies during 3-4 year cycles and can vary more than 300-fold (Hörnfeldt, 1994). In this region the incidence of serologically verified NE can reach up to 37 per 100,000 inhabitants during peak years (Niklasson et al., 1995). The mean annual incidence rate for the most endemic area in northern Sweden, Västerbotten county, is 21/100,000 with a seroprevalence of 5.4% (Ahlm et al., 1994). The seroprevalence is higher for farmers, forestry workers, and other professions that live and work in rural areas. Comparisons of total incidence and seroprevalence figures suggest that 8 infections occur for each verified case of NE.
6 Table 1 . Presentation of hantaviruses discovered to this date. JQ X * u Jk JQ < X o c o E E o c c o E 3 a> o C O > i i ^ a> -O "O "0 1 ! t £ 3 > < < to < < > U < ». LU «2 Q.LU .2 LU * ? ' ? £ ° -s5,® °* R ? < o J? i û ï î= ï= û in X 5 I . ] 3 ] .! I § J> o o 2 < .2 o 0 J o'c Z2 ÜE E o £ = 1 ~ © M J| J ® Ò X M > O ° o © E > . c -o a. o > p o c d O ■ f ?■ d> OO n v lu v> ) 5 O t f " °
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' O ' O ' J - O o = > a> a> = s ' O' O' * *© o* in 2 o U C* .2 g M S < g S < < m O S o 3 S D w <3 ^ w I I E £ o § O ^ ,2 ^ o o o o z .2 ® > o > t; e -® c -p 5 03 3 g - -o -r ojg = O 3 3 0 O O O O O _o -O _o ■o -o 3 U u O' ^ c o o r ö “P X tö 'r ^ “O O' .1 5 o * E 5 o o © u _r u © _© s J I 1 a -o ^ < > o' => Ü o in U £ I -2 © ^ © ^ o © o c o o “O c o -o ' O 3 .2 - 1 -i o I ? > Û > w Î? < CÛ U ^ 5 5 « = 03 i= «« —5 . .2 .2 — O b O O ^ LU > s; > > lu u o E O c o X 03 o i ^ in ® § 03 o 2 5 c2 -O "O O' ' O ' O ' O ' O ' O ' O = :=“ :=* in r ! -O O © Q. © ® © © © 3 0 n o o E °~ “O O X t © O C o X o I I 2 0 . £ f < Ofi £ © =£ o 2 5 .25 3 O O C * N E O z '0 Ì _ ~o “O "O U Od z 3 3 0 O C © O j o 2 Disease linked to HTN is mainly found in the Far East (Fig. 1; Table 1) (eastern Russia, China, Korea) (Lee et al., 1990; Lundkvist & Niklasson, 1994). The geographic distribution of HTN is not known in detail but is likely to follow the distribution of its host A. agrarius which is found throughout Eurasia (LeDuc, 1987). SEO-like viruses have been isolated from urban areas in Korea, Japan and the USA (Fig. 1; Table 1) (Lee, H.W. et al., 1982; Kitamura et al., 1983; Sugiyama et al., 1984c; Tsai et al., 1985). Antibodies directed towards this hantavirus have been detected in many parts of the world possibly due to the wide distribution of its Rattus host (LeDuc et al., 1986; Childs et al., 1988). Both HTN and SEO co-circulate and cause disease in China and Korea (Liang et al., 1994; Li, Y.L. et al., 1995). In east Asia HFRS is an important disease entity with 70-130,000 cases reported from China annually in 1980-1982 and 500-900 incidents of disease occuring per year in Korea (Lee, 1989; Lee et al., 1990; Lundkvist & Niklasson, 1994). SNV and genetically related viruses have been found to infect rodents and man throughout North and South America (Fig. 1; Table 1) (Hjelle et al., 1996b; Khan et al., 1996b; Lopez et al., 1996; Rawlings et al., 1996; Werker et al., 1995). A large number of species of small rodents have been shown to carry different hantaviruses throughout the Americas. There are several indications that SNV and related viruses have existed in rodents and caused human disease long before the 1993 outbreak (Frampton et al., 1995; Nerurkar et al., 1993 and 1994; Wilson et al., 1994). The rodent Peromyscus maniculatus (deer mouse) was identified as the major reservoir for SNV in North America (Childs et al., 1994). This rodent is spread throughout the region with some exceptions (Khan et al., 1996b). Viruses that are genetically related to SNV have been detected and isolated from the southeastern and northeastern USA where other rodents than P. maniculatus have been shown to be the reservoir (Fig. 1; Table 1) (New York virus, Bayo virus, Black Creek Canal virus [BCCV]) (Hjelle et al., 1995c; Morzunov et al., 1995; Rollin et al., 1995; Torrez-Martinez et al., 1995; Huang, et al., 1996). Two teams of researchers managed to isolate SNV in cell-culture in 1994 (Elliot et al., 1994; Schmaljohn et al., 1995). Cross-neutralization experiments of hantaviruses with immune sera from experimentally infected animals and HFRS and HPS patient sera showed that SNV and BCCV represent unique serotypes (Chu et al., 1995). 8 Hantavirus virology Rodent-borne hantaviruses compose, together with the arthropod-borne bunyavirus, nairovirus, phlebovirus and tospovirus, the Bunyaviridae family of viruses, consisting of more than 350 serologically distinct viruses, among which several other human pathogens can be identified, such as Crimean-Congo hemorrhagic fever, Rift Valley fever, Sandfly fever and La Crosse viruses (Murphy et al., 1995). By electron microscopy (EM) and by biochemical analysis of their genetic material hantaviruses have been shown to be bunyavirus-like (McCormick et al., 1982; White et al., 1982; Hung et al., 1983a and b; Schmaljohn & Dalrymple, 1983; Schmaljohn et al., 1983; Goldsmith et al., 1995) (Fig. 2). Their virions are spherical or pleomorphic with a diameter of 80-110 nm and numerous hollow projections (7 nm in length, 11-13 nm in diameter), representing the viral glycoprotein complexes, can be seen at the surface. The virion lipid bilayer envelope is derived from the Golgi complex. Virus particles are formed here and bud into vesicles that are exported to the cell surface where viruses are released (Petterson, 1991; Ruusala et al., 1992). Fig. 2. a Electron micrograph of Hantaan virus (from Hung et al., 1988). b Hantavirus particle (schematic). 9 The genome of all members of the Bunyaviridae is in the form of single stranded RNA (ssRNA) and for the hantaviruses it is solely negative-stranded as opposed to some other members that also have ambisense ssRNA (Elliot, 1996). The tri-partite genome consists of a large (L), a medium (M) and a small (S) segment of approximately 6.5, 3.7 and 1.8 kb, respectively, forming individual nucleocapsids that appear to be of helical symmetry, in analogy with other Bunyaviridae. The L segment encodes the viral replicative enzyme which is an RNA-dependent RNA polymerase (RDRP) (Schmaljohn & Dalrymple 1983; Elliott et al., 1984). According to sequence comparison, the hantavirus polymerase (—250 kDa) contains several motifs found in RDRPs of other RNA viruses (Müller et al., 1994; Poch et al., 1989). The terminal nucleotide sequences of the three ssRNA genome segments are conserved within each genus of the Bunyaviridae (Schmaljohn & Dalrymple, 1983). The gene segments are complementary in a short stretch at their ends and double-stranded RNA can thus be formed into a panhandle structure, enabling circularization (Schmaljohn et al., 1985; Schmaljohn, 1996). These panhandle structures are thought to be involved in a prime- and realign mechanism that initiates viral transcription (Garcin et al., 1995). Transcription is believed to start by a RDRP-associated endonucleolytic cleavage of host cell 5'-capped mRNA. Ten to eighteen nucleotide long heterogenous capped cellular mRNA fragments are found at the 5' termini of transcripts and are believed to prime transcription. L, M and S nucleocapsids are formed by complex formation of negative ssRNA and multiple copies of nucleocapsid proteins (N) (Schmaljohn & Dalrymple, 1983). The RNA-binding N ( — 55 kDa) is encoded by the S segment (Schmaljohn et al., 1986b; Gött et al,. 1993). In the S segment of most members of Bunyaviridae a small open reading frame encodes a non-structural protein. Similar ORFs can be identified in some hantaviruses but no related protein has yet been identified (Schmaljohn et al., 1986b; Plyusnin et al., 1994a; Spiropoulou et al., 1994; Bowen et al., 1995). The coding capacity for the G l and G2 glycoproteins ( — 70 and 55 kDa, respectively) which are inserted in the virion envelope, is contained in the M segment (Schmaljohn & Dalrymple, 1983; Schmaljohn et al., 1987; Arikawa et al., 1990). These proteins are most probably produced as a precursor that is co- translationally cleaved (Kamrud & Schmaljohn, 1994). Several conserved glycosylation sites have been found in the G1/G2 amino acid sequence of hantaviruses (Antic et al., 1992). For HTN Gl and G2, the oligosacharides were found to be mainly of the high-mannose type, reflecting the probable Golgi 10 complex located maturation and assembly of virus (Hung et al., 1988; Schmaljohn et al., 1986a). Localization of Bunyaviridae glycoproteins to the Golgi appears to require association between Gl and G2 and localization signals have been found in the glycoproteins of several members of the Bunyaviridae (Ruusala et al., 1992; Lappin et al., 1994; Melin et al., 1995). Virions are transported in vesicles to the cellular surface, where they are released (Hung et al., 1988). On the other hand, EM investigations of SNV have shown accumulation and budding of viruses to be located at the cellular membrane although accumulation of virus antigens were also identified in close relation to the Golgi (Goldsmith et al., 1995). Cellular receptors for hantaviruses have not been identified and, so far, no information concerning the mode of viral entry into cells is available. The physicochemical properties of all hantaviruses are similar. Infectivity of the virus is stable at pH 7-9 and 4-20°C. Virus is inactivated at pH 5 and rapidly at 37°C. Furthermore, the virus is sensitive to 0.1% deoxycholate, 70% ethanol, ether, chloroform and acetone most probably due to its lipid envelope. It can be stored at -60°C for at least 4 years with preserved infectivity in a balanced salt solution (Lee, 1982). Evolution of hantaviruses To date a large base of genetic information is available on hantavirus gene sequences and the complete genomic sequences of five hantaviruses (HTN, SEO, PUU, SNV and TUL) have been determined (Plyusnin et al., 1996b). Hantaviruses that belong to different sero- or genotypes differ by 30-40% by nucleotide sequence comparison. Their protein products vary by 10-30% for the L segment, 15-40% for N and 20-50% for G1/G2. It has been proposed that hantaviruses are closely linked to and have evolved in parallel with their hosts since a close correspondence can be seen for the phylogeny of hantaviruses and the relationship of the rodents that harbour them (Fig. 3; Table 2) (Antic et al., 1992; Hjelle et al., 1995b; Hörling et al., 1996a; Plyusnin et al., 1994a, b and 1996b; Spiropoulou et al., 1994; Wilson and Reeder, 1993). The HTN/SEO/DOB-like hantaviruses are hosted by rodents of the Murinae subfamily of the Rodentia family Muridae and appear to have evolved separately within this type of rodents. PUU strains are carried by Cletbrionomys species and form, together with the Microtus related hantaviruses 11 PU U/Umeå Sweden PUU/Vindeln Sweden PUU/Hällnäs Sweden PUU/Berkel Germany PUU/Sotkamo Finland PUU/Evo Finland PUU/Puumala Finland PUU/Bashkiria W. Russia PUU/Udmurtia W. Russia PUU/Paris France KBR E. Russia PH MD, USA BLLLV MN, USA - ISLAV CA, USA TUL/Malacky Slovakia TUL/Moravia W. Russia TUL/Tula W. Russia SNV/NM-H10 NM, USA SNV/CC107 CA, USA MONV WV, USA NYV NY, USA MULEV TX, USA BAYV LA, USA BCCV FL, USA ELMCV CA, USA RIOSV Costa Rica RIOMV Bolivia ANDEV Argentina DOB Slovenia HTN S. Korea SEO Japan 30 20 10 0 12 Fig. 3. Phylogenetic tree of hantaviruses, created by comparing nucleotide 68-369 of the S genome segment (position relative to PUU Sotkamo) (Clustal program, Lasergene package, DNASTAR). Virus carried by 1 Arvicolinae rodents, subgenus Clethrionomys; 2 Arvicolinae rodents, subgenus Microfus; 3 Sigmondontinae rodents; 4 Murinae rodents. ANDEV (Andes virus; Lopez et al., 1996; U51042); BAYV (Bayo virus, strain Lousiana; Morzunov et al., 1995; L36929); BCCV (Black Creek Canal virus; Ravkov et al., 1995; L39949); BLLLV (Bloodland Lake virus, strain MO- 46; Hjelie et al., unpublished); DOB (Dobrava virus, strain 3970; Avsic-Zupanc et al., 1995; L41916); ELMCV (El Moro Canyon virus, strain RM-97; Hjelie et al., 1994a; Ul 1427); HTN (Hantaan virus, strain 76/11 8; Schmaljohn et al., 1986b; M l4626); ISLAV (Isla Vista virus, strain MC-SB-1; Song et al., 1995; U31534); KBR (Khabarosk virus, strain Khabarovsk/43/Mf; Hörling et al., 1996a; U35255); MONV (Monongahela virus, strain Monongahela-1; Song et al., 1996; U32591); NYV (New York virus-1; Hjelie et al., 1995c; U09488); PH (Prospect Hill virus-1; Parrington & Kang, 1990; M 34011, X55128); PUU/Vindeln (strain Vindeln/L20/Cg/83; Hörling et al., 1995a; Z48586); PUU/Hällnäs (strain Hällnäs/Bl/Cg/83 [Vranica]; Reipeta/., 1995; U14137); PUU/Umeå (strain Umeå/305/human/95; Juto et al., 1996 [Paper VII]; PUU/90-13 (strain 90-13 [Paris]; Bowen et al., 1995; U22423); PUU/Berkel (strain Berkel/human/94; Pilaski et al., 1994; L36943); PUU/Evo (strain Evo/12/Cg/93; Plyusnin et al., 1995b; Z30702); PUU/Sotkamo (strain Sotkamo/V-2969/Cg/81; Vapalahti et al., 1992; X61035); PUU/Udmurtia (strain Udmurtia/894/Cg/91; Plyusnin et al., 1994b; Z21497); PUU/Bashkiria (strain Bashkiria/CG-1 820; Stohwasser et al., 1990; M32750); PUU/Puumala (strain Puumala/1324/Cg/79; Plyusnin et al., 1995b; Z46942); RIOMV (Rio Mamore virus, strain OM-556; Hjelie et al., unpublished); RIOSV (Rio Segundo virus, strain RMx-Costa-1; Hjelie et al., 1995a; Ul 8100); SEO Seoul virus, strain SR-11; Arikawa et al., 1990; M34881 ); SNV/CCl 07 (Sin Nombre virus, strain Convict Creek 107; Li, D. et al., 1995; L33683); SNV/NM-H10 (strain NM-H10; Spiropoulou et al., 1994; L25784); TUL/Malacky (Tula virus, strain Tula/M alacky/M a/32/94; Sibold et al., 1995; Z48235); TUL/Moravia (strain M oravia/5286/M a/94; Plyusnin et al., 1995a; Z48573); TUL/Tula (strain Tula/76M a/87; Plyusnin et al., 1994a; Z30941 ). of both the Old and the New world, a separate phylogenetic clade. Clethrionomys and Microtus species are both members of the Arvicolinae subfamily of rodents. The recently discovered Topografov virus, found in Lemmus sibiricus (Siberian lemming; Arvicolinae) is also related to this group of viruses (Plyusnin et al., 1996a). Finally, a large number of hantaviruses that are phylogenetically linked to SNV are found in a wide variety of rodents of the Sigmondontinae subfamily in both North and South America, further strengthening the theory that hantaviruses have evolved separately in their respective carrier from a common ancestor (see references in Table 1). This type of co-evolution is not specific for hantaviruses and has been proposed for the development of other viruses (Chan et al., 1992; Nichol et al., 1993a). Analysis of PUU nucleotide and amino acid sequences in C. glareolus captured in different regions of Sweden has revealed two distinct lineages of PUU; one dominating the northern third and another in the middle part of the country (Hörling et al., 1996b). By rodent mitochondrial DNA analysis it was shown that these two PUU variants harboured in two distinct C. glareolus lineages. These two populations colonized Sweden in the beginning of the 13 Table 2. Taxonomic relationship between rodents and associated hantaviruses. Adapted from Khan et a l 1 996 Order Rodentia Family Bunyaviridae Suborder Sciurognathi Genus Hantavirus FamilyMuridae Subfamily Murinae Genus Apodemus Species agrarius Hantaan virus HFRS flavicollis Dobrava virus HFRS Genus Rattus Species rattus Seoul virus HFRS norvégiens Seoul virus HFRS Subfamily Arvicolinae Genus Clethrionomys Species glareolus Puumala virus NE/HFRS rufocanus Tobetsu virus (and Puumala) Genus Lemmus Species Sibiriens Topografov virus Genus Microtus Species arvalis Tula virus rossimeridialis Tula virus fortis Khabarovsk virus pennsylvanicns Prospect Hill virus californiens Isla Vista virus ochrogaster Bloodland Lake virus Subfamily Sigmondontinae Genus Peromyscns Species maniculatns Sin Nombre virus HPS manicnlatus nnbiterrae Monongahela virus lencopns New York virus HPS Genus Oryzomys Species palustris Bayou virus HPS Genus Reithrodontomys Species megalotns El Moro Canyon virus mexicanns Rio Segundo Genus Sigmodon Species hispidns Black Creek Canal virus HPS hispidus Muleshoe virus Genus Oligoryzomys Species microtis Rio Mamore virus 14 present post-glacial period (8-13,000 years ago); one into the southern part of Sweden through a land-bridge from Denmark and the other from Finland and Russia in the north. The absence of PUU in C. glareolus in the southern-most part (south of the 60th parallell) of Sweden remains to be explained since rodents from this region are easily infectable with the virus (Niklasson & Lundkvist, unpublished). The expansive phase of the rodent population cycle in the north might admit a rapid dissemination of virus among a dense population of animals with a low herd immunity against PUU infection. The lack of spread of PUU in southern Sweden might therefore be a consequence of absence of cyclicity of the C. glareolus population (Hansson & Henttonen, 1985). The presence of a more fragmented landscape than in the north might also be a factor that prevents the spread of virus. The discovery of a PUU-like virus in C. rufocanus in Japan is intriguing (Tobetsu virus) (Kariwa et al., 1995). PUU is known to cause NE in Europe and the dominating host is C. glareolus, although antibodies to PUU have been detected in C. rufocanus in Sweden (Niklasson & LeDuc, 1987). Future investigations of the genetic relationships of PUU-like viruses and their Chlethrionomys hosts in the entire Eurasia will possibly shed light on the proposed co-evolution of hantaviruses and their hosts (Plyusnin et al., 1994a, b and 1996b). Transmission of hantaviruses to man Hantavirus disease in man is often linked to close contact with rodents. The infection in the rodents is believed to be chronic but asymptomatic and accompanied by persistent shedding of virus (Lee et al., 1981; Lee et al., 1986; Tanishita et al., 1986; Yamanouchi et al., 1984; Yanagihara et al., 1985). Epidemiological observations, including outbreaks of PIFRS among personnel and visitors at laboratories that conducted experiments with infected animals, suggest that humans can acquire disease by inhalation of aerosolized excreta from infected animals (Desmyter et al., 1983; Lee & Johnson, 1982; Tsai, 1987; Umenai et al., 1979). The local host inflammatory response found in the lower respiratory tract of NE patients strengthens the hypothesis that the primary entry of virus is through this route (Linderholm et al., 1993). The rate of exposure to infected rodents is most probably important since it has been shown that the incidence of hantavirus caused disease clearly follows 15 the cycling of the size of the rodent population (Nyström, 1977; Lee, 1982). This was especially notable in the 1993 outbreak of HPS in the southeastern USA when the outbreak coincided with a 10-fold increase above normal levels of its principal rodent host, Peromyscus maniculatus (Stone, 1993). Such increases in the Peromyscus population has only ocurred three times during the 20th century: 1918, 1933-34 and 1993 (Chapman & Khabbaz, 1994). It is interesting to note that Navajo tribe leaders in the area of the 1993 outbreak linked the outbreak of disease to an unusually high pinon nut harvest, which is the major food for Peromyscus. They could recall similar incidents of unexplained, sudden, respiratory illnesses and death among previously healthy young Navajo, that coincided with the earlier unusual richness in pinon nut harvest and dramatic Peromyscus population increases. Human to human or nosocomial transmission is not known to occur in hantavirus disease (Tsai, 1987). Clinical manifestations Korean hemorrhagic fever Five phases in the course of disease were recognized among patients suffering from KHF during the Korean conflict; a febrile, hypotensive, oliguric, diuretic, and a convalescent phase (Fig. 4) (Gajdusek, 1953; Smadel, 1953; Sheedy et al., 1954; Cosgriff, 1991). Illness presents by abrupt onset of chills, followed by high fever, combined with headache, myalgias, back and abdominal pain, nausea and vomiting. It was not uncommon that these patients were explored surgically due to their acute abdominal state. Fever is high for several days and during this period injections and petechial bleedings can be present in the conjunctivae, skin and mouth. At the end of this phase fever decreases and the patients condition may appear to improve. This marks the onset of the hypotensive phase in which the blood pressure can decrease dramatically, resulting in shock, syncope and eventually in cardiac arrest and death. Hemostatic disturbances are now common with petechia throughout the skin and hemorrhagic manifestations, including hematemesis, epistaxis and bleeding from sites of injury and mucous membranes. Central nervous system (CNS) symptoms can appear and intracerebral hemorrhages may follow. In parallel with, or shortly after the hypotensive / hemorrhagic phase, renal involvement 16 becomes marked with massive proteinuria, oliguria and elevations in serum urea and creatinine. Fig. 4. The clinical manifestations of Korean hemorrhagic fever (from Sheedy et al., 1954). During the second week of illness blood pressure and renal function steadily improve. This phase is initiated with a marked polyuria and the patient recovers gradually during several weeks of convalescence. Mortality due to KHF caused by HTN has been estimated to 5-10% (Sheedy et al., 1954; Gajdusek, 1982) and for the milder form, caused by SEO < 1% (Lee et al., 1990; Tang et al., 1991). Nephropathia epidemica NE follows a similar disease progression as KHF, although symptoms are milder and death is only seen in a minority of the patients (< 0.2%) (Settergren, 1991; Linderholm et al., 1991). As for KHF, the onset of NE is abrupt with chills and accompanying, high fever (Settergren, 1991). Hypotension is rare but milder signs of hemorrhages, such as epistaxis and conjunctival injection or bleeding are not uncommon. Disseminated intravascular coagulation (DIC) is present in as many as 5%. The renal involvement in NE, as in KHF, results in oliguria due to interstitial nephritis and acute tubular necrosis in severe cases. The majority of NE patients regain their renal function during the first month to months of convalescence, 17 although renal impairment can be prolonged (Settergren et al., 1990). NE without renal failure has been observed in both mild and severe cases (Alexeyev & Baranov, 1993). Interestingly, respiratory symptoms are common in NE. Thus, 50% of NE patients were reported to have pulmonary infiltrates and/or pleural effusions during the acute stage of disease as demonstrated by chest radiography (Settergren et al., 1989; Linderholm et al., 1992). CNS-related symptoms occur in NE (Alexeyev & Morozov, 1995). This was reported for two patients already by Zetterholm in his original description of the disease in 1934. Mild and subclinical infections due to PUU are most probably common as shown by epidemiological investigations (Niklasson et al., 1987; Ahlm et al., 1994). Hantavirus pulmonary syndrome The prodromal phase of HPS is similar to the onset of HFRS (Butler & Peters, 1994; Duchin et al., 1994; Levy & Simpson, 1994). After a few days, a cardiopulmonary phase follows with tachypnea, tachycardia, fever and hypotension. DIC, as seen in HFRS, is uncommon. The renal failure of HFRS is not present in typical cases although proteinuria is commonly seen and elevations in creatinine levels occur in severe cases, as found in the southeastern part of USA where other serotypes than SNV, such as BCCV and BAYV, seem to predominate (Khan et al., 1995 and 1996a; Rollin et al., 1995; Hjelle et al., 1996a). Large volumes of semi-transparent, proteinaceous fluids are secernated from the airways of many HPS patients. Severe gas exchange abnormalities are common and a majority of patients require mechanical ventilation due to progression of hypoxemia. Pulmonary edema is the most probable cause for the respiratory failure. A high ratio of edema fluid to serum protein suggest a non- cardiogenic etiology for the pulmonary edema. A common cause of death is intractable hypotension terminating in cardiac dysrythmia. The cause of this is most probably an atypical septic shock resulting in myocardial depression and hypovolemia. The diagnose of hantavirus pulmonary syndrome has been confirmed by serology and/or PCR in more than 130 patients up to now and the overall mortality is approximately 50% (CDC, 1996; Khanet al., 1996b). 18 Diagnosis of hantavirus infections Before the discovery of HTN and related viruses as cause of HFRS (Lee & Lee, 1976; Lee, H.W. et al., 1982; Brummer-Korvenkontio et al., 1980), the diagnosis of diseases such as KHF and NE relied solely on clinical findings as described above. In highly endemic areas of HFRS, such as northern Scandinavia, the disease is recognized by a majority of physicians and the diagnosis, in this region NE, can be acquired solely by means of interpretation of clinical information in severe cases (Lähdevirta, 1971; Lähdevirta et al., 1984; Nyström, 1977). On the other hand, in mild and inapparent cases, and in many regions throughout the world where the incidence of hantaviruses caused disease is low a definite diagnosis can only be obtained by means of specific laboratory tests. A wide variety of such tools has been developed. Virus isolation The ultimate proof of an infection is the isolation of the causative agent. However, in hantavirus infections this is not possible in clinical practice, since the virus is dificult to recover in cell culture. When successful, it requires weeks or even months of blind passages before virus can be detected in cell culture (Juto et al., 1996 [Paper VH.]). Furthermore, hantaviruses are not cyto- pathogenic and require specific methods to allow detection. Serological diagnosis Indirect fluorescent antibody assay (IFA). The classic and widely used technique to detect hantavirus specific antibodies is the IFA, in which the serum is incubated on hantavirus infected rodent lung tissue or cells in tissue culture (in most methods Vero E6, i.e. transformed green monkey kidney epithelial cells) (Lee & Lee, 1976; French et al., 1981). Bound antibodies are visualized by microscopy, using a fluorescence-conjugated anti-human antibody specific conjugate. The presence of fluorescent, particulate, cytoplasmatic inclusions in hantavirus infected cells is pathognomonic. A variant of this methodology is the IFA IgG-avidity test. This method can distinguish between specific, low avidity antibodies representing the acute phase and specific high 19 avidity antibodies developed during the late convalescent phase of NE (Hedman et al., 1991). Hemagglutination-inhibition assay (HI). IFA is simple, sensitive and reliable but requires a fluorescence microscope, species-specific conjugates, experienced observers, and furthermore imposes a risk of subjective interpretation of results. This led to the employment of HI (Clarke & Casals, 1958), which is utilizing the hemagglutinating ability of hantavirus antigens of goose erythrocytes (Tsaiet al., 1984; Brummer-Korvenkontio et al., 1986; Okuno et al., 1986; Takahashi et al., 1986; Dantas et al., 1987; Qunying et al., 1996). Virus antigen prepared from both infected suckling mice brain and cell culture have been utilized. This method proved suitable, and as sensitive as IFA in most investigations. The advantages of HI are that it is more specific than IFA and can be applied for serological investigations in less well equipped laboratories. The disadvantage is that it is somewhat laborious and consumes large amounts of antigen. Immune adherence hemagglutination assay (IAHA). The complement system component C3b is produced when antibodies bind to virions. C3b- covered virions have the ability to bind to C3b-receptors on human erythrocytes. This is used to bridge antigen and erythrocytes to form agglutinates. The inhibition of this agglutination by virus-specific antibodies in complement-depleted human sera forms the basis for application of IAHA in hantavirus serology (Inouye et al., 1981; Sugiyama et al., 1984a and b; Tang et al., 1991). HI and IAHA techniques are used both to differentiate among hantavirus strains and the antibody reactivity to them in humans and animals. Complement fixation test (CFT). Complement fixation to antibodies bound to antigen inhibits lysis of sensitized indicator erythrocytes. This serological technique has also been applied in hantavirus serology (Gresikova et al., 1988; Sugiyama et al., 1984b). High density particle agglutination assay (HDPA). High density composite particles coated with HTN antigen has been used to detect specific antibodies in HFRS by visually detectable agglutination; a method that has been shown to be more sensitive than IFA (Tomiyama & Lee, 1990). 20 Enzyme-linked immunosorbent assay (ELISA). ELISA utilizing non denatured virus proteins has become an increasingly important diagnostic method for the detection of hantavirus specific antibodies in acute disease and for epidemiological purposes (Gavrilovskaya et al., 1981; Tkachenko et al., 1981; Ivanov et al., 1988; Niklasson & Kjelsson, 1988; Groen et al., 1991a; Lundkvist et al., 1993). Different formats, such as indirect detection of antibodies bound to hantavirus antigens that are coated directly to ELISA wells or captured by poly- or monoclonal antibodies, have been applied. The latter method enables the detection of antibodies directed to specific viral proteins (Groen et al., 1992; Lundkvist et al., 1993). Enzyme-conjugated antibodies with different specificities allow differentiation between different antibody isotypes. Another ELISA variant is the IgM-capture method as described by Niklasson & Kjelsson (1988). Test serum IgM is captured to ELISA wells by an anti-IgM antiserum and bound antibodies are detected by applying hantavirus antigen and a detecting polyclonal anti-hantavirus conjugate. This method has been improved by the introduction of monoclonal antibodies for detection of bound antigen (Lundkvist, 1993). IgM detecting methods are important tools to establish diagnosis in highly endemic areas with a high prevalence of IgG due to previous infections. Furthermore, the majority of hantavirus infected patients present IgM antibodies at onset of disease (LeDuc et al., 1990; Groen et al., 1992; Lundkvist et al., 1993; Elghet al., 1995 and 1996a [Paper I and VI]). Neutralization assays (NTA). NTA have been widely used in hantavirus research to differentiate viral strains and to define the origin of human seroresponses in HFRS and HPS (Tanishita et al., 1984; Lee et al., 1985b; Takenaka et al., 1985; Niklasson et al., 1991; Hörling et al., 1992 and 1996a; Chu et al., 1992 and 1995; Vapalahti et al., 1996b). Hantaviruses rarely, if ever, produce CPE when propagated in cell culture. They have been shown to produce plaques when overlayed with agarose or methylcellulose after prolonged growth in cell culture (French et al., 1981). This phenomenon prompted the development of plaque-reduction neutralization tests based on specific neutralization of infectivity by anti-sera. Since the process of plaque formation is slow and sometimes hard to detect, tests were developed involving specific staining of infected foci by antisera and visualizing them under the microscope by immunostaining (Tanishita et al., 1984; Niklasson et al., 1991). These techniques have later been adapted to ELISA format which enables large 21 numbers of sera to be tested for screening purposes (Hörling et al., 1992 and 1996a). Detection of viral antigen Antigen detection in the acute phase of hantavirus infections in humans has proven difficult, especially in milder forms of the disease such as NE (Temonen et al., 1996). There are reports of succesful immunohistochemical identification of hantavirus antigens in biopsies and post mortal specimens of severe HFRS and HPS patients (Poljak et al., 1994; Zaki et al., 1995). Immunohistochemistry is an established method to identify viral antigen in infected animals (Lee & Lee, 1976; Brummer-Korvenkontio et al., 1980). To enable investigations of viral antigen presence in tissues of animals, ELISA methods have been developed (Gavrilovskaya et al., 1981; Lundkvist et al., 1995b; Alexeyevet al., 1996a). Genetic assays Polymerase chain reaction (PCR). Sensitive diagnostic tools have recently been established for the detection of hantavirus genetic material in body fluids and tissues of patients with hantavirus related diseases (Grankvist et al., 1992; Nichol et al., 1993b; Hjelle et al., 1994c; Hörling et al., 1995b). These are based on reverse transcription of hantavirus RNA and subsequent amplification by PCR (Saiki et al., 1985; Mullis & Faloona, 1987). PCR and molecular sequencing of obtained amplimers have revolutionized the possibilities for rapid genetic differentiation among viral strains and genetic material detected in infected patients (Giebel et al., 1990; Tang et al., 1990; Arthur et al., 1992; Puthavathana et al., 1992; Xiao et al., 1992 and 1994; Kim et al., 1994; Spiropoulou et al., 1994; Hjelle et al., 1995c; Antoniadis et al., 1996). Dot spot hybridization assay. To detect viral RNA in tissues a dot spot hybridization assay was developed, based on extraction of total RNA from PUU infected macaques, immobilization on nitrocellulose and hybridization with labeled oligonucleotides specific for the PUU M and S segments (Groen et al., 1995b). 22 AIMS OF THIS THESIS To produce recombinant nucleocapsid proteins of Puumala virus and other hantaviruses that are pathogenic in humans. To localize domains within recombinant Puumalavirus nucleocapsid proteins that are important for antibody binding. To develop and evaluate serological assays based on recombinant hantavirus proteins for use in clinical diagnosis of hemorrhagic fevers with renal syndrome and hantavirus pulmonary syndrome and for epidemiological purposes. To investigate the kinetics and development of the humoral immune response to recombinant nucleocapsid proteins of different hantaviruses in patients with nephropathia epidemica. To isolate and characterize the causative virus of nephropathia epidemica. Pooh began to feel a little more comfortable, because when you are a Bear of Very Little Brain, and you Think of Things, you find sometimes that a Thing which seemed very Thingish inside you is quite different when it gets out in the open and has other people looking at it. A. A. Milne The House at Pooh Corner (1928) 24 RESULTS AND DISCUSSION Until recently, the classic and widely used method for serological diagnosis of hantavirus diseases has been EFA (French et al., 1981). In addition, ELISA based on cell cultured hantaviruses as the source of antigen has been used (Ivanov et al., 1988; Niklasson & Kjelsson, 1988). However, these assays are laborious and require containment laboratory facilities for production of viral antigen. Furthermore, interpretation of IFA results may be difficult, especially when comparing reactivities of individual serum samples to different hantavirus serotypes, since growth in cell culture and yield of different viruses vary, leading to antigen preparations of unpredictable quality (Elgh, unpublished observation). Yet another prerequisite for IFA is skilled personnel for the microscopic evaluation and the inherent subjective element in interpretation. This led us to seek a different approach to acquire hantavirus antigens for serological purposes. We wanted to use ELISA for diagnostic and epidemiological purposes, since it allows processing of large numbers of samples. This is of special interest for our laboratory, since it is responsible for hantavirus serology in the northern part of Sweden, an area endemic for PUU infection (NE). We test approximately 800-1400 patients annually (1991-1995) for the presence of hantavirus antibodies of which 100-200 yield a positive result, depending on the cyclicity of the incidence of NE. From previously published work it was evident that the PUU N was a promising candidate to be produced in recombinant systems. This protein is highly immunogenic and gives rise to high titers of antibodies in NE patient sera (Sheshberadaran et al., 1988; Zöller et al., 1989; Gött et al., 1991; Lundkvist et al., 1993). Based on sequence information of the PUU Sotkamo S genome segment, kindly provided to us by Vapalahti and co-workers prior to its publication (1992), we decided to clone and express the PUU S ORF in E. coli and test its efficacy as an antigen in serological assays. Cloning and expression of the Puumala virus, strain Sotkamo, S gene segment in E. coli We produced cDNA of PUU Sotkamo, amplified and cloned the ORF of its S gene segment into the E. coli expression vector pET-3a (Novagen Inc., Madison, 25 WI, USA). A recombinant PUU nucleocapsid protein (PUU rN) was produced upon induction of exponentially growing E. coli transformed with the recombinant plasmid (pPSX). This protein was shown to be highly reactive with sera from patients with NE in immuno-blots. Conventional purification methods including ion-exchange chroma tography, heparin-affinity chromatography and gel filtration, yielded only partially purified PUU rN. Among the experiences from this work we found that PUU rN co-purified with various E. coli proteins, that it had a significant affinity for heparin and that a substantial amount of nucleic acids were bound non-covalently to PUU rN under non-denaturing conditions. The affinity for heparin was most probably due to nucleic acids associated with PUU rN. To acquire a highly purified PUU rN we cloned the pET-3a containing PUU Sotkamo S ORE into the poly-histidine-fusion protein expression vector pET-19b (pPSX-His) (Fig. 5) that produced amino-terminal fusions containing 10 histidines for efficient and rapid purification on media containing bivalent metal ions such as Cu2+ and Ni2+ (Elgh et al., 1995 [Paper I]). Purification under denaturing conditions (6 M urea) yielded a recombinant nucleocapsid protein (rN) of approximately 55 kDa as determined by SDS-polyacrylamide gel electrophoresis. IgG in sera from NE patients was found to react specifically with PUU rN in western blot (WB) (Fig. 6). pPSX-his 7033 bp T7 promotor ]--T7 terminator— »• • ' U A A h Fig. 5. Presentation of the pPSX-His expression vector. cDNA of the PUU Sotkamo nucleocapsid protein encoding S gene open reading frame was cloned under the T7 promoter of the pET-19b vector. The amino-terminal part of the resulting fusion protein contains six histidine residues for convenient affinity purification on matrices containing bivalent metal ions. 26 1 10 20 Fig. 6 . Immuno-blot staining of purified recombinant Puumala Sotkamo virus nucieocapsid protein separated by 12 % SDS-polyacrylamide gel electrophoresis. Lanes 1-10 represent acute and convalescent sera from five nephropathia epidemica patients. Lanes 11-20 represent acute and convalescent sera from five patients with other viral infections. Study design to compare rN based ELISA and I FA To evaluate the reliability of E. coli expressed poly-histidine/PUU Sotkamo rN fusion protein as antigen in ELISA for the detection of IgM and IgG antibody responses in sera from NE patients we compared it with IFA using methanol/acetone treated PUU Sotkamo infected Vero E6 cells as antigen. The kinetics of the humoral response by the two methods was studied in a limited number of consecutive sera of NE patients and the clinical usefulness in achieving an early serological diagnosis in a large number of acute phase sera (Elgh et al., 1995 and 1996b [Paper I and II]). For the latter purpose the study was performed in a clinically relevant setting by including all 618 sera admitted to the local Laboratory of Clinical Virology, from patients with suspected NE, during a four-month period. Kinetics of Puumala virus IgM and IgG responses by rN based ELISA and IFA IgM and IgG responses were followed up to 6 months in 17 NE patients by PUU rN based ELISA and PUU based IFA (Elgh et al, 1995 [Paper I]). Inclusion criteria were presence of specific IgM as measured by IFA in the first serum sample obtained after onset of fever and at least one consecutive serum sample. In total 41 samples were available. 27 In all 17 patients an IgM response was demonstrated by ELISA in the first serum sample. The median titer obtained by IgM ELISA was 20 times higher than by IFA. As expected, a significant decrease of both ELISA and IFA IgM was seen between the acute and late convalescent phases of the disease. The two methods differed as regards the IgG results, since nine of the patients lacked a specific response by ELISA in the first serum sample, whereas IFA yielded merely two negative results. In those patients who displayed an IgG response by ELISA the titers ranged from 1/400 to 1/25.600 (median titer 1/6.400). These titers were 40 times higher by ELISA than results obtained by IFA in the same serum samples. There were 10 significant titer rises with both assays in the paired serum samples obtained within 3 to 38 days after onset of fever. A significant increase in IgG titer was noted between the acute and early convalescent phase (i.e. day 15-60 after onset of fever) by IFA, whereas such an increase was not detected by ELISA (Paper I; Fig. 2). All patients were IgG positive by both methods in at least one serum sample. In conclusion, we obtained similar results concerning the kinetics of the IgM response by both methods. The kinetics of the IgG response differed by the two methods and IgG could not be detected by PUU rN based ELISA nor by IFA in the first serum sample obtained after onset of disease in some patients. This information and the prevalence of IgG of > 5% of the population in northern Sweden suggested that it is necessary to base the serological diagnosis of NE on specific IgM detection as pointed out by others (Niklasson & Kjelsson, 1988; LeDuc et al., 1990) and that PUU rN is a reliable antigen for this purpose. Furthermore, we believe that assays based solely on IgG detection such as the avidity assays as proposed by Hedman et al. (1991) and Kallio- Kokko et al. (1993) may produce false negative results in those patients who have not mounted an IgG response early in the course of the disease. Clinical evaluation of Puumala virus rN based ELISA IgM. Out of the 618 serum samples described above, 101 sera were positive and 508 were negative for IgM by ELISA (Elghet al., 1996b [Paper II]). The concordance of ELISA and IFA was 98.5%. Nine sera were positive by IFA and negative by rN ELISA. However, three of the patients showed no IgG seroconversion by either rN ELISA or IFA in convalescent sera and did not 28 fulfil the clinical definition of NE infection. We therefore interpreted them as false positive by IgM IFA. Four of the remaining sera could be shown to be collected from patients with NE by demonstration of seroconversion and two by clinical criteria (only one serum available). Specificity and sensitivity of IgM rN ELISA as compared to IFA was found to be 100 and 94.4%, respectively (Table 3). The predictive value of a positive result by IgM rN ELISA for presence of NE was 100% (Table 4a). The predictive value of a negative result was 98.6%. Since three sera turned out to be falsely positive by IgM IFA, the predictive value of a positive result for this test was 97.3% (Table 4b). Table 3. Relation between IgM and IgG responses of 618 serum samples as measured by Puumala virus immunofluorescence assay (IFA) and recombinant nucleocapsid protein based enzyme-linked immunosorbent assay (rN ELISA), respectively. IgM rN ELISA IgG rN ELISA + - n + - n IFA + 101 91 110 IFA + 131 8 139 - 0 508 508 - 0 479* 479 n 101 517 618 n 131 487 618 sensitivity 94.4% * sensitivity 94.2% * specificity 100% * specificity 100% * n - number of sera. T This figure includes three sera false-positive in IFA IgM according to clinical evaluation and abscence of seroconversion. * of rN ELISA as compared to IFA. * This figure includes six sera negative by rN ELISA after serum absorbtion to E. co li antigen. IgG. By both rN ELISA and IFA, 131 were determined as positive and 473 as negative for IgG by IFA and PUU rN based ELISA (Elgh et al., 1996b [Paper II]). In total, 604 of 618 (97.7%) sera showed concordant results. In the investigated material we found paired sera drawn with an interval of 3-60 days from 13 patients. Among these patients, six (46%) had a four-fold or higher increase in IgG IFA titer and ten (77%) patients had a more than 50% increase of IgG ELISA values. As compared to IFA there were eight negative samples by rN ELISA. All of them were drawn within eight days after onset of fever and most presented low IgG titers by IFA (median 1/40; range 1/40- >.1/640). All eight patients fulfilled serological and clinical criteria of NE infection. One of these sera, obtained on 29 day five after onset of fever, lacked IgM by both methods, but there was a subsequent seroconversion. The remaining seven patients were positive by IgM rN ELISA (median 36 arbitrary units [AU], range 22-70). Table 4. (a) Puumala virus recombinant nucleocapsid protein based enzyme-linked immunosorbent assay (rN ELISA) and (b) immunofluorescence assay (IFA) IgM and IgG responses as compared to the diagnosis of nephropathia epidemica (NE) infection*. a IgM NE infection IgG NE infection + n + n rN + 1 0 1 0 101 rN + 95 44 139 ELISA - 7 510 517 ELISA • 13 466 479 n 108 510 618 n 108510 618 Positive predictive value 100% Positive predictive value 68.3% Negative predictive value 98.6% Negative predictive value 97.2 % b IgM NE infection IgG NE infection + n + n IFA + 107 3 110 IFA + 100 40 140 1 507 508 - 8 470 478 n 108 510 618 n 108 510 618 Positive predictive value 97.3 % Positive predictive value 71.4% Negative predictive value 99.8% Negative predictive value 98.3% * NE infection is defined as the presence of concordant positive IgM and IgG results in IFA and ELISA or, in the case of discordant results, seroconversion in IgG in convalescent sera and/or presence of all of the following three criteria: 1. sudden onset of fever, 2. at least one of transient thrombocytopenia (normal value > 1 5 0 x 1 0’/L) or transient serum creatinine elevation (normal value < 100 pmol/L) with protein- and hematuria, 3. no evidence of other infectious agents as cause of disease. There were six positive samples by IgG rN ELISA that were negative by IFA. These six rN ELISA positive samples became negative after serum preabsorption with an E. coli antigen lysate. When taking this into account, the specificity and sensitivity of the IgG rN ELISA, as compared to IFA, was 100 and 94.2%, respectively (Table 3). Positive predictive values for NE infection by the IgG tests were only 68.3% for rN ELISA and 71.4% for IFA (Table 4). By IFA and rN ELISA, 8 and 13 of 108 sera from patients with NE, respectively, lacked IgG in the first serum. However, the negative predictive value of PUU specific IgG by IFA and ELISA was 97.2% and 98.3% respectively. 30 To conclude, PUU IgG/IgM rN ELISA correlated well with PUU IF A. Detection of specific IgM was found to be the serological method of choice to diagnose NE by rN based ELISA as well as by IF A. During the course of this work several studies were published that confirmed our findings. PUU and HTN N proteins produced in E. coli were found to be reliable antigens in indirect ELISA for the detection of both IgG and IgM and also in IgM capture ELISA (Zöller et al., 1993 a and b). Despite the fact that these studies were based on a limited number of sera the result was essentially the same. Another study demonstrated the efficacy of E. coli produced PUU rN in ELISA based on IgG avidity testing (Kallio-Kokko et al., 1993). The authors found that this approach worked well for the diagnosis of acute NE in most patients but must be supplemented with an IgM test as has been mentioned above. Furthermore baculovirus produced rN has proven to be a reliable antigen for serological purposes (Rossi et al., 1990; Schuldt et al., 1994; Yoshimatsu et al., 1995; Vapalahti et al., 1996a). Vapalahti and co-workers (1996a) showed that baculovirus produced PUU Sotkamo rN had indistinguishable properties as compared to cell culture acquired N. When these antigens were compared by Mab analysis with E. coli rN it was found that two epitopes present in baculo and cell culture produced N were missing in the bacterial antigen. This discrepancy between cell cultured N and E. coli produced rN has also been found by us (Elgh et al., 1996d [Paper III]). Finally, we studied the IgA response of the 618 sera. In the described serum material we found no clinical benefit by including this type of analysis in the diagnostic arsenal concerning NE. The IgA response has been studied by others and detectable levels were found to correlate well with NE infection and the IgM response (Groen et al., 1994). Identification of a major antigenic domain in Puumala virus rN To purify full-length PUU Sotkamo rN it was necessary to use denaturing agents such as 6 M urea since the recombinant protein readily formed inclusion bodies within bacteria. It was also difficult to acquire large amounts of the protein and precipitation of expressed protein was difficult to avoid during renaturation. Furthermore, E. coli impurities co-purified to some extent with PUU rN even under denaturing conditions, which led us to use E. coli antigen as absorbent in ELISA buffers (Elgh et al., 1996b [Paper II]). We suspected that 31 this phenomenon was linked to the problem of aggregation of bacterial proteins to PUU rN which was observed for the non-his-containing PUU rN, as expressed by the pET-3a vector. We therefore wanted to know whether the humoral response to PUU rN was localised in a portion that was less inclined to auto-aggregation than the entire rN. To determine whether this was the case, five truncated, partially overlapping recombinant PUU N proteins were expressed in E. coli using the pRSET vector system (Fig. 8b) (R&D Systems Europe Ltd., Oxford, UK) (Elgh et al., 1996d [Paper IH]). Using immuno-blots and ELISA we could show that an amino-terminal region within PUU rN (aa 7-94) was responsible for the major response to this protein in sera from NE patients (Fig. 7). Recombinant protein aa 16 18 1— ...... 1 rN 1-433 r .'i rNA5 7-137 □ rNAl 1 95-137 1 rNA4 1— ' 137-433 1 "] rNA6 137-308 1 1 rNA3 308-433 Fig. 7. Immuno-blots demonstrating IgG reactivities of nephropathia epidemica (NE) and control sera to the following PUU virus antigens: rN, rNA5, rNAl 1, rNA4, rNA6 and rNA3. Serum samples in lanes 1-8 represent acute and convalescent sera from four NE patients, 9-12 represent acute and convalescent sera from two patients with acute mycoplasma infection and lanes 13-16 acute and convalescent sera of two patients with mononucleosis. Lane 18 demonstrates the reactivity of T7 Tag™ Mab to recombinant fusion proteins. Furthermore we could show that a majority of monoclonal antibodies (Mab) produced against PUU N (Lundkvist & Niklasson, 1991; Lundkvist et al., 1992) and PUU Sotkamo rN (Alexeyev et al., 1996a; Elgh et al., 1996d [Paper III]) reacted to this region. A panel of 110 HFRS sera and 62 control sera were used to compare the reliability of a truncated version of rN (aa 7-137) and full-length PUU rN as antigen in ELISA for the detection of IgM and IgG responses. We could conclude that their reactivity and efficacy as antigens in this type of assays were equal. During the course of this work it was also shown that the human humoral reactivity to E. coli produced SNV rN protein was localised to the amino-terminus 0enison et al., 1994). Similar recombinant N proteins of PUU, SEO and HTN serotypes, as expressed in E. coli and 32 containing parts of the amino-termini of these proteins worked well as antigens in ELISA (Wang et al., 1993). Epitope screening using synthetic peptides also revealed IgG reactivity in NE sera towards the amino-terminal region of PUU N, although several epitopes were also localised in other parts of PUU N (Lundkvist et al., 1995a; Vapalahti et al., 1995). Two 15-mer peptides specific for the amino-terminal region of PUU N were found to react with IgG in acute phase sera from NE patients (Lundkvist et al., 1995a). It was not possible to acquire IgM specific assays using such antigens. Furthermore, the humoral response to one of these 15-mers decreased dramatically in the convalescence of NE and two years after onset of disease. Hantavirus N aa 1 117 429-433 HTN rNA SEO rNA DOB rNA SNV rNA PUU rNA 6 X His T7 tag Hantavirus rNA Fig. 8. a) Schematic representation of the 433 amino acid (aa) hantavirus nucleocapsid protein (N) compared to the 117 amino acid (aa) truncated recombinant hantavirus N (rNA) proteins, b) Schematic representation of the E. coli produced hantavirus rNA fusion protein with amino-terminal histidine-tag (óxHis) and T7 Tag Mab epitope (T7 tag). Amino-terminal rN of five hantaviruses as antigens in serological assays As has been mentioned earlier, E. coli produced PUU rN is well suited for the detection of virus specific antibody responses in NE (Kallio-Kokko et al., 1993; Wang et al., 1993; Zöller et al., 1993a, b and 1995; Elgh et al., 1995 and 1996b [Paper I and H]). In analogy with our finding that a major domain for the humoral response in NE against PUU rN is situated at the amino-terminus of the protein, we cloned the genes encoding the 117 first amino acids (aa) of the 33 five major pathogenic hantaviruses known to date, namely HTN, SEO, DOB, SNV and PUU, in poly-histidine fusion protein expression vectors (pRSET [R&D Systems]; Fig. 8) (Elghet al., 1996c [Paper IV]). Their gene products (rNA) were affinity purified and utilized in ELISA. To enable comparison of responses to the different serotypes involved, the individual hantavirus antigens were coated at near equimolar concentrations. To be able to monitor this, the amount of bound antigens to the solid phase was detected by use of a Mab with specificity directed to the 11 aa bacteriophage T7 gene 10 peptide present in all fusion proteins (Fig. 8b). Polyclonal antibody responses in animals to hantavirus amino-terminal rN proteins We found that both hantaviruses and hantavirus rNs induced high antibody titers in animals (rabbit and mouse) as detected by rNA based ELISA. The most prominent antibody responses were directed to the homologous virus (Table 5). The antibody activity could generally be grouped into a HTN/SEO/DOB or a SNV/PUU type response. The rabbit immunized with native PUU seemed to give a more specific response than the animals immunized with PUU rN. The differences in the resulting antigen specificity might be a reflection of differences in the tertiary structure of the N amino-terminal region. Epitopes that induced a cross-reactive antibody response may be hidden in the structure of the native viral N protein but exposed in the denatured E. coli expressed rN. Similar differences in the specificity of the antibody responses induced by native or recombinant PUU and Tula virus N proteins have been reported previously in both bank voles and mice (Lundkvist et al., 1996b and c). Detection of specific antibodies in sera from different parts of the world by multi-valent hantavirus ELISA The am ino-term inal hantavirus rNAs were assayed by using sera from hantavirus infected patients in WB for their usefulness as antigens to detect IgG antibodies. We found that the antibody responses to the antigens were specific, but substantial cross reactivity could be seen within the HTN/SEO/DOB and SNV/PUU groups, respectively (Fig. 9). 34 Table 5. Reactivity of pol/clonal rabbit and mouse sera to E. co li produced, recombinant, truncated hantavirus nucleocapsid proteins (rNA) (aa 1-117). a) Reciprocai endpoint titers are shown. Titers of homologous virus and recombinant antigen are underlined, b) Relative values of reciprocal endpoint titers are shown. Titers of homologous virus and recombinant antigen are underlined. a Antiserum Recombinant antigen Species HTN’ rNA SEO3 rNA DOB4 rNA SNV5 rNA PUU4 rNA HTN' virus rabbit 20.480 20.480 20.480 1.280 1.280 SEO3 virus rabbit 40.960 40.960 40.960 5.120 5.120 DOB4 virus rabbit 10.240 10.240 20.480 640 1.280 SNV5 rN mouse 2.560 5.120 2.560 163.840 40.960 PUU7 virus rabbit 1.280 1.280 1.280 5.120 20.480 PUU4 rN rabbit 20.480 20.480 20.480 81.920 81.920 PUU4 rN mouse 640 1.280 1.280 20.480 81.920 not immunized rabbit <40 <40 <40 <40 <40 not immunized mouse <40 <40 <40 <40 <40 b Antiserum Recombinant antigen Species HTN rNA SEO rNA DOB rNA SNV rNA PUU rNA HTN virus rabbit 100 100 100 6.25 6.25 SEO virus rabbit 100 100 100 12.5 12.5 DOB virus rabbit 50 50 100 3 6 SNV rN mouse 2 3 2 100 25 PUU virus rabbit 6 6 6 25 100 PUU rN rabbit 25 25 25 100 100 PUU rN mouse 1 2 2 25 100 ' Hantaan virus (strain 7 6 /1 1 8 ),3 Seoul virus (strain SR-11 ),3 Seoul virus (strain 80-39), 4 Dobrava virus (strain 3 9 7 0 ),5 Sin Nombre virus (strain CCI 0 7 ),6 Puumala virus (strain Sotkamo),7 Puumala virus (strain K-27) 35 123456789 12 HTN rNA SEOrNA DOBrNA m mm SNVrNA PUU rNA Fig. 9. Western blot detecting immunoglobulin G reactivity in sera from hantavirus infected patients from Sweden (1-4), USA (5 and 6) and Korea (7 and 8) plus sera from four non-immune blood donors (9-12) to Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SNV) and Puumala (PUU) virus truncated amino-terminal recombinant poly-histidine containing nucieocapsid (N) fusion proteins (amino acid 1-117 of N) (rNA). RuMia (Samara); n=49 Stovanla (PUlUltoaarwwponae); n=21 d USA (Naw Mod co, Arizona); n=13 Aala (China, Kona); n=Z7 HTN SEO DOB HTN DOB Fig. 10. Immunoglobulin G reactivity in ELISA in sera of patients with an acute or recent hantavirus infection from a) Scandinavia (Norway, Sweden, Finland), b) Russia, c) and f) Slovenia, d) USA and e) Asia, to truncated amino- terminal recombinant poly-histidine containing nucieocapsid (N) fusion proteins (amino acid 1-117 of N) specific for Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN) and Puumala (PUU) virus. ELISA values are shown as arbitrary units. Vertical bars represent the activity of all sera. The boxes include the eight middle percentiles and the median value (horizontal bar). 36 The hantavirus specific response of the sera in WB was compared to ELISA utilizing the same five hantavirus rNAs and to the results in IF A. These three methods showed a high degree of correlation. Using the five rNA antigens in ELISA we were able to detect hantavirus specific IgM and IgG antibody responses in sera originating from patients with acute or retrospectively diagnosed hantavirus infections from many regions of the world in a rapid, specific and sensitive manner. We found that Scandinavian, Russian and American sera reacted mainly to the SNV/PUU group and that Asian sera reacted to the HTN/SEO/DOB group (Fig. 10). It is especially rewarding that the discrimination can be done by the IgM isotype already in the acute phase of hantavirus disease. An interesting finding was that both reactivity patterns were encountered in sera from Slovenia (Balkan region). This is a reflection of the presence of DOB- and PUU-like viruses circulating in this region (Avsic-Zupanc et al., 1990; Antoniadis et al., 1996; Lundkvist et al., 1996a). Since seroreactivity to either of the two groups of viruses is clearly different it is most often possible to determine whether a reactivity in a single serum is due to a virus from the one group or the other. At present, a more accurate virus type specific diagnosis can only be achieved by a neutralization test (Niklasson et al., 1991; Chu et al., 1994 and 1995; Lundkvist et al., 1996a). The substantial cross reactivity between the N proteins from different hantavirus serotypes can be explained by the presence of at least three distinct, hantavirus cross-reactive epitopes as has been mapped previously with Mabs (Lundkvist et al., 1991, 1992 and 1996b; Elgh et al., 1996d [Paper III]). The degree of cross reactivity to the different strains of viruses in different sera is possible to monitor using the approach shown here, in which a diagnostic method was developed that monitors seroreactivity to near equimolar amounts of antigens derived from the five different hantaviruses serotypes. It was shown that the seroreactivities to the closely related HTN, SEO and DOB group of viruses are almost indistinguishable and that a similar pattern was also observed with the SNV and PUU group of viruses. Our results show that a major domain for the human humoral immune response in hantavirus infections is situated in the amino-terminal of rN of all investigated viruses. Our findings were compatible with what has been shown for SNV in which the major antigenic determinants for the humoral immune response to rN was found between aa 17-59 (Jenison et al., 1994; Yamada et al., 1995). Comparisons of the amino acid sequence from hantavirus N protein 37 amino-terminal regions (aa 17-59) also show a high degree of homology within the two groups of viruses; 83.7-90.7% within the HTN/SEO/DOB group and 74.4% for SNV and PUU N. In contrast, amino acid homologies between the two groups of viruses only showed 44.2-46.5% homology. We believe that this relationship within and between the two groups of viruses is the basis for the serological reactivity pattern that was found in our study. Seroepidemiology in European Russia by multi-valent hantivirus rN ELISA Samara is a highly endemic region for NE in European Russia with more than 1000 patients hospitalized during the period 1988-1992. The Samara region along with the nearby region of Bashkirtostan are the highest endemic areas for NE in European Russia. Despite sim ilar clinical characteristics of NE of northern Europe and European Russia, there seems to be a higher frequency of severe and complicated forms of the disease in the latter (Alexeyev et al., 1993; Niklasson et al., 1993). To test the hypothesis for the presence of other causative hantaviruses than PUU in this region we investigated hantavirus antibodies in 206 healthy blood donors (155 male, 51 female; median age 41, range 19-60) from the city of Samara with 1.3 million inhabitants, situated on the Volga river in the eastern part of European Russia (Alexeyev et al., 1996b [Paper V]). We applied the multi-valent hantavirus ELISA described above with the modification that the antigens tested were HTN, SEO, DOB and PUU rN (aa 1-117). Sera were diluted 1/50. The upper limit of a negative serum to the recombinant proteins was determined by use of serum from 150 children (age 0-4 years) (median value 0.01; range 0.00-0.18). Samples from 15 (7.2%) individuals contained anti hantavirus antibodies (net absorbance >0.2). Seven (3.4%) of these sera reacted with a PUU antibody profile; i.e. the median absorbance value to the PUU antigen was 0.38 (range 0.26-1.89) and to the HTN antigen 0.08 (range 0.02- 0.17). Three (1.5%) of the sera reacted with a HTN antibody profile; i.e. median absorbance value to the HTN antigen was 0.82 (range 0.37-1.85) and to the PUU antigen 0.01 (range 0.0-0.02). Five (2.1%) sera reacted with equal intensity to both PUU and HTN antigens (median 0.26, range 0.20-0.44; median 0.21, range 0.20-0.47, respectively). Samples reactive to the HTN antigen were equally reactive to the SEO and DOB antigens. 38 In contrast to previous reports presenting PUU as the cause of hantavirus disease in European Russia, our data indicate that both HTN- and PUU-like viruses circulate in this region. The presence of hantavirus antibodies in patients with NE/HFRS in the Samara region has until now been diagnosed by IFA with the PUU CG 18-20 strain as antigen. Therefore, it seems likely that some HFRS cases, caused by HTN, SEO, DOB or related hantaviruses, might have been overlooked in the past. Another explanation is that HTN antibody positive sera had been drawn from individuals that had been exposed to this or similar agents while visiting other regions of the country, i.e. far east Russia. On the other hand, a Rattus borne form of the disease caused by a SEO-like virus or an Apodemus flavicollis or agrarius carried, DOB- or HTN-like hantavirus may well account for the presence of anti-HTN/SEO/DOB antibodies in sera from patients residing in this region. The recent finding by Lundkvist and coworkers (1996a) of PUU and DOB, but lack of HTN and SEO seroreactivities, as determined by neutralization test, in Bosnia- Hercegovina speaks in favour for DOB as a probable cause of HTN-like seroreactivities also in European Russia. An expanded sero-survey using the methods outlined here paralleled with neuralization tests and investigations of the nature of hantavirus antibodies, antigen and nucleic acids present in rodents in this region are in progress and will possibly shed more light to our preliminary findings. Kinetics of antibody responses to recombinant hantavirus nucleocapsid proteins in patients with nephropathia epidemica We wanted to investigate the kinetics and cross reactivity of hantavirus nucleocapsid protein-specific IgM, IgA and IgG antibody isotypes in NE patient sera up to 2-3 years after disease onset by using the recently developed, recombinant N based, multi-valent, ELISA (Elgh et al., 1996a and c [Paper IV and VI]). For this purpose we collected sera from 17 patients with NE on admission, at discharge and at follow-ups 1-2 months, 2-5 months and 2-3 years after. On day 2-8 after onset of NE, IgG antibodies to the PUU recombinant protein were detected in 15/17 patients (Table 6). On day 5-15, all 17 patients had IgG antibodies and at this time, the median value was maximally increased. In samples obtained 1-2 months and 2-5 months after onset, levels were still 39 Table 6. Number of patients with detectable IgG (a), IgM (b), and IgA (c) antibodies to recombinant nucleocapsid proteins of different hantaviruses at various intervals after onset of nephropathia epidemica (NE). a) IgG Time after onset of NE HTN* SEO DOB SNV PUU 2-8 days 1 / 17T 1/17 2/17 12/17 15/ 17 5-15 days 3/17 8/17 6/17 15/ 17 17/17 1-2 months 4 / 17 6/17 5/17 16/ 17 17/ 17 2-5 months 11/16 11/16 11/16 15/ 16 16/ 16 2-3 years 7/16 6/16 5/16 12/16 14/16 2 days to 3 years 12/17 12/17 12/17 17/ 17 17/ 17 b) IgM Time after onset of NE HTN SEO DOB SNV PUU 2-8 days 5/17 5/17 4/ 17 11/17 14/17 5-15 days 5/17 4/17 4/17 13/17 16/17 1-2 months 2/17 2/17 0/17 5/17 10/17 2-5 months 0/16 0/16 0/16 1 / 16 2/16 2-3 years 0/16 0/16 0/16 0/ 16 1 / 16 2 days to 3 years 5/17 5/17 4/17 14/17 17/ 17 c) IgA Time after onset of NE HTN SEO DOB SNV PUU 2-8 days 4/17 3/17 3/17 5/17 13/17 5-15 days 4/17 5/17 5/17 11/17 17/ 17 1-2 months 0/17 0/17 0/17 3/17 9/17 2-5 months 1 / 16 1 / 16 1 / 16 1 / 16 4/16 2-3 years 0/16 0/16 0/16 2/16 3/16 2 days to 3 years 5/17 6/17 7/17 11/17 17/ 17 * Recombinant nucleocapsid protein (aa 1-117) based indirect ELISA specific for Hantaan 76/118 virus (HTN), Seoul SR-11 virus (SEO), Dobrava virus (DOB), Sin Nombre CCI 07 virus (SNV) and Puumala Sotkamo virus (PUU). T Number of patients with antibodies / number of patients tested near maximum (Fig. 11a). At 2-3 years after onset, levels had declined considerably but were still detectable at the 1/400 dilution in 14/16 patients. IgM antibodies to the PUU protein were found on day 2-8 after onset in 14/17 patients (Table 6). All NE patients had detectable IgM by day 10 after onset of disease. This further strengthens the importance of specific IgM detection as the method of choice for the early diagnosis of NE. Levels were maximal on day 5-15. After 2-5 months IgM antibodies were detectable only in 2 of 16 patients. The IgA response showed kinetics similar to that of the IgM 40 response. On day 2-8 after onset, 13/17 patients had IgA antibodies to the PUU protein. Two patients with an early IgA response lacked detectable IgM antibodies to the protein at the initial interval of testing. Furthermore, only one of these two contained specific IgG. In a previous study, specific IgA detection did not convey any additional value to IgG and IgM in diagnostic serology (Elghet al., 1996b [Paper II]). One difference between that study and the present was the use of conjugates of different manufacturers. According to the present data, the inclusion of an IgA assay might be valuable at least in the few patients that fail to react with IgG or IgM antibodies in the early phase of NE. Detectable IgG reactivity to the SNV derived protein occurred in sera from all 17 patients (Table 6). The ratio between optical density (OD) values in assay of SNV antibodies and PUU antibodies increased by time after onset of disease (Fig. lib). The cross reactivity of serum IgG of the NE patients to HTN, SEO, and DOB derived recombinant proteins were lower than those to the SNV protein (Fig. 11a). Again, cross reactivity increased with time after onset of disease. On day 2-8 after onset of disease, 1-2 patients showed IgG reactivity to HTN, SEO, and DOB proteins whereas at 2-5 months, 11 of 16 patients had a reactivity to each of the three antigens (Table 6). The ratio of reactivities of the heterologous viruses to PUU OD values showed a pronounced increase by time (Fig. lib). FIG. 11. Chart depicting (a) median IgG reactivities (net optical density [OD]) of consecutive sera from nephropathia epidemica patients in hantavirus recombinant nucleocapsid protein (amino acid 1-117) based ELISA and (b) median ELISA values relative to Puumala virus specific ELISA results (net optical density relative to PUU reactivity [OD %]). Hantaan 76/118 virus (HTN), Seoul SR-11 virus (SEO), Dobrava virus (DOB), Sin Nombre CCI 07 virus (SNV) and Puumala Sotkamo virus (PUU). Sera were drawn 2-8 days (A), 5-15 days (B), 1-2 months (C), 2-5 months (D) and 2-3 years (E) after onset of disease. 41 The mechanism behind the increase in cross reactivity with time is unknown. One possibility might be the occurrence of a differential exposure of viral antigens during the course of infection. Some epitopes of the viral nucleocapsid might be exposed effectively to cells of the immune defense only after the initial stage of infection. According to studies on Mabs raised to the PUU N, there are several epitopes, both continuous short synthetic peptides and discontinuous epitopes (Lundkvist et al., 1995a; Vapalahti et al., 1995). When serum from several patients obtained in the acute phase and 2 years later were examined for IgG antibodies to synthetic peptides comprising amino acids 16-30 and 409-423, a decline in level was consistently found with time, whereas the response to native PUU increased (Lundkvist et al., 1995a). The present results are in line with these observations, insofar as they may suggest the presence of a difference in kinetics of response to different epitopes in PUU N. Possibly, the present increase in cross reactivity may involve affinity maturation. An increase in the affinity of IgG antibodies is well established from determinations of the binding of IgG to haptens after immunization with hapten-carrier complexes. Recently, however, IgG antibodies of high affinity have been found to be present early after viral immunization in mice and the affinity did not increase further during a period of several months (Roost et al., 1995). The antibodies were directed to one single protective epitope of the virus and the authors speculated that an early presence of high-affinity neutralizing antibodies to viruses may be important for host survival. If this can be extrapolated to other viruses such as hantaviruses, there might be an early presence of IgG antibodies to some epitopes of the homologous virus, and a more gradually developing response directed also to heterologous hantaviruses. These, and other explanations, might be tested further by use of synthetic peptides and/or recombinantly expressed proteins. Also IgM and IgA antibodies showed cross reactivity, mainly to the SNV protein. However, in the early phase of the disease the number of patients with measurable, specific antibodies of the IgM and IgA isotypes directed to the HTN, SEO, and DOB proteins was higher than for IgG (Table 6). Similar to the responses to the PUU recombinant protein, the antibody responses to the four heterologous antigens were transitory and did not last for more than a few weeks. Some important practical implications may be drawn from the present results. The multi-valent hantavirus rNA ELISA distinguished to an expected extent between various hantavirus diseases, irrespectively of time of sampling. 42 Even though cross reactivity increased with time, the reactivity was still higher to the homologous virus than to related viruses as late as 2-3 years after onset of disease (Fig 11a). This study shows that seroepidemiological screening must be done with antigens of several hantavirus serotypes and should not rely on single-antigen based assays. Furthermore, it is preferred that these antigens can be equalized in terms of number of epitopes that are exposed to the antibodies in the tested serum sample. The multi-valent ELISA affords an objective and economically favourable alternative to IF A. It should be noted that 3/17 sera drawn day 2, 2 and 4 after onset of disease did not present IgM antibodies to the amino-terminal PUU rN. This finding implies that such an antigen might be slightly less sensitive than full-length PUU rN which was shown in an earlier study to be reliable in diagnosing nephropathia epidemica (Elgh et al., 1995 [Paper IJ. Furthermore, in this study we found that the IgG response to the amino-terminal PUU rN decreases with time which implies that some sera from patients who have encountered a PUU virus infection can be missed in seroepidemiological investigations when using to high serum dilution. Accordingly, we found that PUU rNA ELISA, when used in 1/50 dilution, is more sensitive than IF A, as tested on a large material of sera from the PUU endemic region of northern Sweden (Ahlm et al., unpublished). In conclusion, we confirmed that ELISA based on recombinant hantavirus N is useful for detection of an antibody response in humans to PUU. A novel finding was an increase by time after onset of NE of cross reactivity with related hantaviruses. This knowledge is important to take into consideration when designing serological assays, especially for epidemiological screening purposes. It is of importance to be able to base such assays on controlled amounts of antigen which can be managed by techniques as outlined herein. Our results furthermore indicated that inclusion of determination of IgA antibodies may be valuable for early diagnosis of NE. Isolation and characterization of the causative virus in NE No human hantavirus isolate has been reported from northern Europe, which is one of the most endemic areas for NE (Lundkvist & Niklasson, 1994). NE is a milder form of HFRS, which might imply that the viral load in the patient is lower in this disease than in the more severe Asian form of HFRS, consequently making virus isolation more demanding. Another reason as to 43 why PUU is difficult to isolate from patients may be dependent on the fact that almost all patients have a strong immune response in the form of neutralizing IgM and IgG antibodies present at onset of symptoms rendering released virions non-infectious (Hörling et al., 1992; Lundkvist et al., 1993). In an attempt to cirmcumvent inhibiting effects of antibodies we aimed at isolating cell-bound virus from leucocytes after lectin stimulation of T cells with phytohemagglutinin (PHA) 0uto et al., 1996 [Paper VU]). Dextran- purified leukocytes acquired from a NE patient from Umeå, Sweden, were stimulated with PHA and co-cultured with monolayers of Vero E6 cells for 72 hours. Antigen was detected in the Vero E6 cells after 6 months and 2 weeks of incubation (passage 4). Identification of virus antigen was performed by IF A using rabbit anti-serum with specificity for PUU Sotkamo full-length rN (Elgh et al., 1996d [Paper IH]) and a human convalescent NE serum. By Mab analysis, the PUU Umeå isolate (PUU Umeå/305/human/95) revealed a reactivity pattern compatible with a virus of the PUU serotype. It did not, however, react with one of the two Mabs, that were raised by immunization with PUU Sotkamo rN. This indicates that PUU Umeå lacks the N epitope that is recognized by this antibody, situated between aa 7 and 94 of PUU Sotkamo N. Human HFRS sera from Sweden and Russia reacted with high IgG titres to the isolate, whereas sera from China failed to do so. The PUU Umeå S and M genome sequences showed high similarity, 97.7% and 90.7%, respectively, to PUU strain 83-L20, which is a rodent isolate obtained from the same region as PUU Umeå (Fig. 12) (Hörling et al., 1995a, 1996b). It is interesting to note that PUU Umeå is clearly distinct from the Finnish PUU strains (Vapalahti et al., 1992, Plyusnin et al., 1994b and 1995b). The S and M genome sequences of the PUU Sotkamo prototype strain differed significantly; sequence homology was 88.7% and 79.3%, respectively (Fig. 12). Accordingly PUU Umeå seems to be a member of a northern Swedish lineage of PUU, which in turn clusters with the other western European partially sequenced strains. When PUU Umeå was compared to geograhically even more distant PUU strains than the Finnish one, namely the Russian C. glareolus CG- 1820 and human strains P360 and K27, the differences were, as expected, even more pronounced (Giebel et al., 1989; Stohwasser et al., 1990; Xiao et al, 1993). It is therefore remarkable to note that the geographically remote PUU Vranica strain from Bosnia-Hercegovina (Reip et al., 1995) has a higher degree of similarity to PUU Umeå than the geographically much more close Finnish strain. The corresponding S and M sequence comparison with the PUU strain 44 CG-1820 K27 P360 - Sotkamo U m e å ------83-L20 - Vranica / Hällnäs B1 ■90-13 ~ T ~ 14 12 10 K27 j TP360p L - CG-1820c Udmurtia894 Evo12 - Sotkamo — Puumala B erk e l 83-L20 — U m e å ■C - Vranica / Hällnäs B1 90-13 r 10 Fig. 12. Clustal phylogram showing (a) the 333 nt partial M (nt 79-411) and (b) 302 nt partial S (nt 68-369) segment nucleotide sequences of Puumalavirus isolates. Nucleotide positions correlate to Puumala virus, strain Sotkamo. Distances along the horizontal axis between two viral strains are relatively proportional to the differences between sequences, whereas distances along the vertical axis are for graphical presentation only. Vranica to the human isolate revealed 97.4 and 92.8% similarity, respectively (Fig. 12). At the M segment level the similarity between the Vranica strain and the recently sequenced local vole strains is even higher than with PUU Umeå (Hörling et al., 1995a). The Vranica strain has also been shown by partial sequencing of its M segment to be identical to another rodent isolate from Västerbotten county; i.e. Hällnäs Bl (Yanagihara et al., 1984a and b; Xiao et al., 1994; Reip et al., 1995). We therefore believe, as has been suggested by others, that the real origin of PUU Vranica is northern Sweden and that its real identity is PUU strain Hällnäs Bl (Yanagihara et al., 1984a and b; Reip et al 1995, Hörling et al., 1996b). No reliable PUU sequences from the Balkan 45 region has so far been reported and the matter therefore awaits further investigation. In conclusion, we have isolated the first human PUU strain of northern Europe from PHA-stimulated blood leucocytes from a patient with acute NE. The isolate belongs, as shown by immunological and genetic means, to the PUU serotype of hantaviruses. It resembles, but is not identical to, previous rodent isolates from the same region as the patient. Phylogenetic analyses indicate that it clusters together with other western European PUU isolates (Fig. 12). 46 CONCLUSIONS The main results of this thesis include; *^> the cloning of the entire S gene open reading frame and expression of the nucleocapsid protein of Puumala Sotkamo hantavirus as a poly-histidine fusion protein in E. coli, the efficient affinity purification of recombinant Puumala virus nucleocapsid protein on metal chelating media, the identification of a major domain for the human humoral response in nephropathia epidemica patients in the amino- terminal region of the Puumala Sotkamo virus nucleocapsid protein, the cloning of the 5’ portion of the S genes and expression of the amino-terminal portion of the nucleocapsid proteins of Hantaan, Seoul, Dobrava and Sin Nombre hantaviruses, *^> the monitoring of equimolar amounts of antigen in solid phase based immunoassays by use of a monoclonal antibody with specificity for the fusion part of recombinant proteins, ^ the efficacy of recombinant hantavirus amino-terminal nucleocapsid proteins in serological assays for the detection of antibody responses in hemorrhagic fevers with renal syndrome and hantavirus pulmonary syndrome patients in different regions of the world, the value of specific IgM determination with some additional benefit of specific IgA in diagnosing nephropathia epidemica by recombinant Puumala virus nucleocapsid protein based ELISA, 47 the development of increased cross reactivity to heterologous hantavirus nucleocapsid proteins by time after onset of disease in nephropathia epidemica patients, ^ the successful isolation of the first human Puumala virus in Fenno-Scandinavia from blood leukocytes, the immunological and partial genetic characterization of the human Puumala Umeå virus isolate, which revealed that it resembles, but is not identical to, earlier isolated rodent viruses from the same region as the patient and that it is distinct from the Finnish rodent prototype strain Sotkamo. “Interesting; Hmmm, interesting if true?! 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Journal of hemorrhagic fever with renal syndrome (HFRS) virus. Medical Virology 39,200-207. Microbiology and Immunology 28,1345-1353. Zöller, L. G., Yang, S., Gött, P., Bautz, E. K. & Darai, G. Yanagihara, R. 1990.Hantavirus infection in the United 1993b.A novel ji-capture enzyme-linked immunosorbent States: epizootiology and epidemiology. Reviews of assay based on recombinant proteins for sensitive and Infectious Diseases 12,449-457. specific diagnosis of hemorrhagic fever with renal Yanagihara, R., Amyx, H. L. & Gajdusek, D. C. 1985. syndrome. Journal of Clinical Microbiology 31,1194- Experimental infection with Puumala virus, the etiologic 1199. agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus). Journal of Virology 55,34-38. 59 To write it, it took three months; to conceive it - three minutes; to collect the data in it - all my life. F. Scott Fitzgerald 60 APPENDIX Papers I-VII 61 I like work; it fascinates me. I can sit and look at it for hours. I love to keep it by me; the idea of getting rid of it nearly breaks my heart. Jerome K. Jerome Three Men in a Boat (1889) 62