Chapter 21. Orthomyxoviridae

Chapter 21

Orthomyxoviridae

Chapter Outline Properties of ORTHOMYXOVIRUSES 392 AVIAN INFLUENZA VIRUSES 403 Classification 392 Human Disease 407 Virion Properties 394 CANINE INFLUENZA VIRUSES 407 Virus Replication 395 BAT INFLUENZA VIRUSES 408 Molecular Determinants of Pathogenesis 397 BOVINE INFLUENZA D VIRUSES 408 MEMBERS OF THE GENUS INFLUENZAVIRUS A 398 HUMAN INFLUENZA VIRUSES 408 EQUINE INFLUENZA VIRUSES 398 MEMBERS OF THE GENUS ISAVIRUS 408 SWINE INFLUENZA VIRUSES 400 INFECTIOUS SALMON ANEMIA VIRUS 408 Human Disease 403 Other ORTHOMYXOVIRUSES 409

The family Orthomyxoviridae includes viruses with pandemic influenza viruses in wild and domestic animal genomes composed of several (six to eight) segments of species. Lastly, widespread use of sensitive and specific single-stranded RNA. The most important members of the RT-PCR assays that detect all influenza viruses has facili- family are the influenza viruses, which are included in four tated detailed surveillance without the need for virus iso- genera (Influenzavirus A, B, C, and D). Influenza viruses lation. Once a sample is identified as influenza A-positive that are pathogenic to wild and domestic animals and birds with this assay, the infecting virus rapidly can be further are included in the genus Influenzavirus A, whereas viruses characterized by gene-specific RT-PCR assays to deter- in the two other genera (B and C) circulate continuously in mine its hemagglutinin/neuraminidase subtype and cul- humans. Influenza A viruses infrequently are transmitted tured for subsequent antigenic analyses. from their animal hosts to humans, but human epidemics Although the original isolation of an influenza virus did and pandemics caused by influenza A viruses typically not occur until 1930 (from swine), associated diseases pre- have no animal involvement beyond the initial incursion. viously had been recognized in both animals and humans. Human influenza viruses sporadically are transmitted to Indeed, human influenza was described by Hippocrates swine, leading to establishment of virus lineages adapted to some 2400 years ago. Human pandemics have occurred this host species. Continuing surveillance, therefore, is throughout history, and the “Spanish flu” pandemic of 1918 essential to identify and detect influenza A virus variants was especially dramatic. The causative agent of highly that are capable of infecting humans while they are still pathogenic avian influenza (HPAI), “fowl plague,” was rec- confined either to their animal host or to a limited number ognized in the late 19th century as a filterable agent (ie, of human contacts. virus), but was not identified as an influenza virus until Recent developments have advanced significantly the 1955. High-pathogenicity avian influenza (HPAI) virus was understanding of the biology of influenza viruses. First, first isolated from wild birds in 1961—specifically, com- increased virologic surveillance and the rapid characteri- mon terns (Sterna hirundo) in South Africa—but until zation of viruses by sequencing have confirmed extensive recently this highly lethal virus has since been rarely genomic rearrangements between different influenza detected in wild birds. Low-pathogenicity avian influenza viruses. Secondly, the emergence of the highly pathogenic (LPAI) virus was first isolated from wild birds in 1972, and Eurasian H5N1 virus in Southeast Asia in 1997 and the such low-virulence viruses are common in aquatic species H1N1 virus pandemic of 2009 led to establishment of of wild birds. Aquatic birds (orders Anseriformes and worldwide surveillance programs to identify potentially Charadriiformes), especially ducks, shorebirds, and gulls,

are the essential reservoir hosts of low-pathogenicity influ- influenza A virus from wild aquatic birds into domestic enza A viruses (Table 21.1). Influenza viruses replicate in poultry occur much more frequently, but until recently the intestinal and upper respiratory epithelium of these birds most of these events went undetected as the immediate without producing overt disease, and are excreted in high consequences were limited. There have been only a very concentrations in feces and oral secretions. The viruses effi- limited number of outbreaks of highly pathogenic avian ciently are transmitted by the fecaloral route, and migrat- influenza in domestic poultry worldwide that resulted in ing aquatic birds carry viruses between their summer and high death losses or regulatory action to eradicate or man- winter habitats, which may span continents. Feeding stops age the infection, such as the recent epizootic of Eurasian along the flyways during the migrations provide further H5N1 HPAI virus infection. In addition, many human opportunity for spread of the viruses to resident contact infections with low-pathogenicity H7N9 avian influenza wild and domestic bird populations, and facilitate the con- virus reported by China in 2013 complicate surveillance tinuing process of evolution of these viruses. due to the lack of poultry mortality as a driver of diagnos- Cross-species infections occur sporadically between tic activities. Domestic swine are considered an important birds and mammals, including swine, horses, dogs, mink, intermediate (“bridge”) host in those areas of the world marine mammals, and humans (Fig. 21.1). Incursions of where there is frequent contact between poultry and

TABLE 21.1 Hemagglutinin Subtype Distributiona Between Different Birds (Class: Aves) and Mammals (Class: Mammalia)

HA Host of Origin Subtypec Mammalia Aves

Humans Swine Equine Anseriformes Charadriiformes and Galliformes (eg, dabbling ducks) Procellariiformes (eg, shorebirds, (Domestic gulls, seabirds) Poultry) H1 11 11 1 1 11 e H2 (11 )b 61 1 1 H3 11 11 11 11 1111e H4 6111 1 H5 66 1 1 11b H6 61111 H7 66(11 )b 11 11b H8 66 H9 66 1 11 11 H10 6111 H11 1111 H12 11 6 H13 1111 H14d 6 H15d 66 H16 1

a 6, sporadic; 1, multiple reports;11, most common. b(), Previously common but now not reported. cBoth LP and HP viruses. dRare subtypes. ePrimarily swine influenza virus infections of domestic turkeys. From Swayne, D.E. (Ed.). Animal Influenza. Copyright r John Wiley and Sons (2009), with permission. Orthomyxoviridae Chapter | 21 391

FIGURE 21.1 Interspecies transmission of influenza A viruses. Diagrammatic representation of the source and movement of influenza A viruses or their genes within avian and mammalian ecological and epidemiological situations. H, hemagglutinin subtype; those in ( ) were previously common but no longer are in circulation. From Swayne, D.E. (Ed.), Avian Influenza, p. 62. Copyright r John Wiley & Sons (2009), with permission.

swine, although the premise that swine are an essential and reassortment can complicate the interpretation of phy- intermediate host for the development of pandemic influ- logenetic trees. In contrast, analyses of the matrix protein enza virus strains has not been substantiated. Whole- gene (M) of field strains of influenza A virus show two genome sequencing of influenza viruses is now widely major avian lineages (North American and Eurasian), two used to reconstruct the phylogeny of each of the eight equine lineages, two gull lineages (North American and viral gene segments. For instance, comparison of the hem- European), two swine lineages (North American and agglutinin (HA) and neuraminidase (NA) genes shows Eurasian) and a human lineage. Analysis of the PB1 gene divergence into multiple subtypes (H1 through H16 and segregates human viruses between the North American N1 through N9, respectively) and host-specific lineages swine and Eurasian avian groups. Predictably, there are within some subtypes. However, interspecies transmission exceptions such as an outbreak of equine influenza in 392 PART | II Veterinary and Zoonotic Viruses

China in 1989 that was caused by an H3N8 virus from a PROPERTIES OF ORTHOMYXOVIRUSES contemporary Eurasian avian source, whereas the classi- cal H3N8 equine lineage has its origin from a North Classification American avian lineage virus. Influenza A viruses from The family Orthomyxoviridae comprises the genera wild birds are very diverse, comprising the majority of Influenzavirus A, Influenzavirus B, Influenzavirus C, the viral gene pool, and all gene segments evolve con- Thogotovirus, Quaranjavirus, and Isavirus. The name of stantly. However, the virion surface genes accumulate the family is derived from the Greek myxa, meaning amino acid changes most frequently. Evolution is detected mucus, and orthos, meaning correct or right. The name not only in mammalian (eg, equine) and domestic poultry was intended to distinguish the orthomyxoviruses from viruses, but also in viruses from wild birds. the paramyxoviruses. Influenza is the Italian form of With renewed appreciation of the natural history of Latin, from influentia, “influence,” so used because epi- influenza viruses as “species jumpers,” prevention efforts demics were believed to be caused by astrological or logically will continue to focus on those situations in other occult influences. Influenza A viruses are common which high densities of birds and mammals are main- pathogens of horses, swine, humans, and domestic poultry tained in close proximity and in which there are rapid throughout much of the world, but they also are the cause turnover in populations, such as live-animal markets. The of sporadic or geographically limited infections and dis- latter often bring together a wide variety of poultry and ease in mink, seals, whales, and dogs. Influenza B viruses other birds such as chickens, ducks, turkeys, pheasants, are pathogens of humans, but there are reports of influ- guinea fowl, and chukars as well as mammals, such as enza B virus infection in seals. Influenza C viruses infect pigs, rabbits, and civets, without biosecurity practices. humans and swine, and reassortants have been detected, Live bird markets amplify, diversify (reassort), and per- but influenza C viruses rarely cause serious disease in petuate viruses within them, and serve as a source of either species. The thogotoviruses are tick-borne viruses infection of poultry farms via the movement of empty that sporadically infect livestock and humans in Africa, cages and personnel. Surveillance also is required for Europe, Asia, and most recently North America, but their early identification of emerging reassortant viruses. pathogenic significance remains conjectural. The recently Although improved agricultural biosecurity practices that established genus Quaranjavirus includes viruses isolated separate swine and domestic poultry production from wild from ticks and birds predominantly, but sometimes also aquatic birds can reduce the frequency of newly emergent from humans with febrile disease. The sole member of influenza virus variants, such approaches are often diffi- the genus Isavirus is infectious salmon anemia virus, a cult to enforce, and the potential threat of epidemics of highly fatal disease of marine-farmed Atlantic salmon. human influenza that emerge from avian or mammalian Newly discovered orthomyxoviruses from cows (proposed reservoirs will persist. new genus Influenzavirus D) and bats are awaiting defini- The need for intensive surveillance for the emergence tive taxonomic classification. of novel influenza viruses was demonstrated dramatically A classification system was developed for influenza in April 2009, with the appearance of a new pandemic viruses because of the practical need to assess the risk strain of influenza A virus in Mexico. Instead of the represented by the emergence of new variant viruses, and widely anticipated emergence of a new pandemic virus the need to determine herd or population immunity based on the highly pathogenic Eurasian H5N1 influenza against previously circulating strains so that vaccine virus, this newly emergent virus had its origin in triple requirements can be assessed. The emergence of variant reassortant swine viruses that had been circulating in viruses depends not only on genetic drift—ie, point muta- pigs in North America since the late 1990s. The novel tions (nucleotide substitutions, insertions, deletions), but feature of this new virus was an additional reassortment also on genetic shift—ie, genomic segment reassortment. that replaced two gene segments (NA and M) of the Previously, drift and shift in only the viral hemagglutinin North American swine virus with the respective segments and the neuraminidase were intensively monitored, but, of the Eurasian swine virus. The origin and the date of with the advent of enhanced sequencing technology, other development of this novel H1N1 virus have not been viral genes may assume more importance in assessing definitively established, as the first indication of its exis- risk. In the current classification system, influenza A tence was from human infections. Initial investigations viruses are categorized into 16 hemagglutinin (H) and 9 were unable to detect the novel H1N1 infection in swine neuraminidase (N) subtypes, although recently described in the absence of contact with infected workers. This bat influenza viruses may increase the number of hemag- recent event further emphasizes the unpredictable evolu- glutinin sub types. In naming virus strains, influenza virus tion and emergence of influenza virus as a mammalian type (A, B, or C), host if other than humans (swine, pathogen. equine, chicken, turkey, mallard, etc.), geographic origin Orthomyxoviridae Chapter | 21 393

(at province or state level), strain number, year of sample (H5N1). The initial outbreak viruses from Hong Kong collection, and hemagglutinin and neuraminidase subtypes from 1997 were included in a single clade with the proto- are included. Thus the full identification of an influenza type virus, based on the hemagglutinin sequence. virus looks like a secret code but is precise and informa- However, since 2003 the viruses have spread to progres- tive. Examples of virus strain names include: A/equine/ sively more regions beyond China and have evolved into Miami/1/1963 (H3N8), the prototypic equine influenza several independent but related clades. By 2004, 10 dis- virus 2; A/swine/Iowa/15/1930 (H1N1), the prototypic tinct first order genetic clades were recognized (09), strain of swine influenza virus; A/Hong Kong/1/1968 and continued evolution in subsequent years has resulted (H3N2), the virus that caused the human pandemic of in a total of 30 additional second, third, and fourth order 1968; A/chicken/Scotland/1959 (H5N1), the first HPAI clades (eg, 2.1, 2.2, 2.1.3, 2.2.1, 2.1.3.2 and 2.2.1.1, etc.) virus of the H5 subtype. (Fig. 21.2). This clade nomenclature system readily iden- Changes recently have been implemented in the tifies the genetic linkage of the virus regardless of the nomenclature for Eurasian H5N1 hemagglutinin, and for geographic location, source of the isolate, or year of the other influenza A viruses as appropriate, because the link- isolate. The strain nomenclature system will continue to ing of the isolates to a specific geographic location be maintained in repositories and be used to identify becomes confusing and uninformative as the virus spreads sequences deposited into databases such as GenBank. globally. A numerical clade system has been adopted to Although any gene constellation and any combination better relate the evolutionary changes in these related of HA and NA genes can arise by genetic reassortment, H5N1 isolates over time; a clade is a taxonomic group only a limited range of combinations are recognized as comprising a single common ancestor and all descendants important and naturally occurring subtypes responsible of that ancestor. For the Eurasian H5N1 hemagglutinin for animal infections: (1) enzootic H7N7 and H3N8 gene, the reference isolate is A/Goose/Guangdong/1/1996 viruses (previously designated equine influenza viruses 1

1996–2004 2005 2008 2011 2014

2.3.4.3 2.3.4.4

2.3.4 2.3.4.2 2.3.4.2 2.3.3 2.3.4.5 2.3 2.3.2 2.3.4.1 2.3.3 2.3.1 2.3.3 2.3.2.1a 2.3.2.1 2.3.2 2 2.2 2.3.2 2.3.2.1b 2.2 2.3.2.1c 2.5 2.5 2.3.1 2.3.1 2.2.1.1 2.2.1.1a 1 2.1 2.2.1 2.1.3 2.2.1 9 2.4 2.1.2 1 2.1.1 8 2.2.2.1 2.4 2.2.2 8 2.5 6 1 2.5 2.1.3.3 9 2.1.3.2 2.1.3.2a 8 2.1.3.1 2.1.3 5 6 2.1.3 2.1.2 2.1.2 2.1.1 9 2.1.1 5 6 2.4 2.4 7 1.1.2 7 5 1.1 1.1.1 9 1 1 4 7 8 9 4 6 8 5 6 3 3 4 7.2 5 7.1 7.2 3 7.1 0 0 0 4 4 3 3 0 0 0.005 Not detected since at least 2008 (n=13)

FIGURE 21.2 Genetic evolution of the HA gene of H5N1 high-pathogenicity avian influenza (HPAI) viruses, A/goose/Guangdong/1/1996 lineage, since its emergence in 1996. The constant genetic divergence of HA leading to emergence of new genetic groups (shown in pink shading) is illustrated by phylogenetic trees supporting updated clade classifications proposed by the World Health Organization, World Organization for Animal Health [Office International des Epizooties: International Office of Epizootics, Paris (OIE)], and the United Nations Food and Agriculture Organization, H5N1 Evolution Working Group. Viruses not detected since 2008 are marked with a red star. From Updated unified nomenclature system for the highly pathogenic H5N1 avian influenza viruses, WHO (adapted and updated). http://www.who.int/influenza/gisrs_laboratory/h5n1_nomenclature/en/. 394 PART | II Veterinary and Zoonotic Viruses

and 2, respectively) that cause respiratory disease in functions are linked to the surface proteins: receptor bind- horses; (2) enzootic H1N1, H1N2, and H3N2 viruses that ing, receptor cleavage, and membrane fusion. Virion cause influenza in swine; (3) sporadic H7N7 and H4N5 envelopes are lined by the matrix protein, M1 on the inner viruses that cause respiratory and systemic disease in surface of the lipid bilayer with a small number of inter- seals; (4) sporadic H10N4 viruses that cause respiratory spersed ion channels composed of tetramers of the second disease in mink; (5) historically endemic H1N1, H2N2, matrix protein, M2. Genomic segments consist of a mole- H3N2, and more recently sporadic or limited H5N1, cule of viral RNA enclosed within a capsid composed of H7N3, H7N7, H7N9, and H9N2 viruses that cause helically arranged nucleoprotein. Three proteins that respiratory disease in humans; (6) geographically make up the viral RNA polymerase complex (PB1, PB2, restricted H3N8 and H3N2 viruses that cause respiratory and PA) are associated with the genomic RNA and nucle- disease in dogs; (7) nearly all genetic combinations occur oprotein (NP). The genome consists of six to eight seg- in wild aquatic birds, but particular emphasis is placed on ments of linear negative-sense, single-stranded RNA, and detection of H5 and H7 viruses in domestic poultry because they can be associated with the high-pathogenicity phenotype (HPAI viruses). TABLE 21.2 Properties of Influenza Viruses Virion Properties Six genera: Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus, Isavirus, and Quaranjavirus Orthomyxovirus virions are pleomorphic, filamentous but Virions are pleomorphic, spherical, or filamentous, 80120 nm become spherical upon laboratory cultivation, and in diameter, and consist of an envelope with large spikes 80120 nm in their smallest dimension (Fig. 21.3). They surrounding six to eight helically symmetrical nucleocapsid consist of a lipid envelope with large glycoprotein spikes segments of different sizes surrounding eight (genera Influenzavirus A, Influenzavirus The genome consists of linear negative-sense, single-stranded RNA, B, and Isavirus), seven (genus Influenzavirus C), or six divided into six to eight segments, 1014.6 kb in overall size (genera Thogotovirus and Quaranjavirus) helically sym- There are two kinds of spikes (influenza A & B virus); rod metrical nucleocapsid segments of different sizes shaped, consisting of homotrimers of the hemagglutinin (Table 21.2). For influenza A and B viruses, there are two glycoprotein, and mushroom shaped, consisting of kinds of spikes: homotrimers of the hemagglutinin glyco- homotetramers of the neuraminidase protein protein and homotetramers of the neuraminidase glyco- Transcription and RNA replication occur in the nucleus; capped protein. Influenza C viruses lack a distinct neuraminidase 50 termini of cellular precursor mRNAs are cleaved and used as spike, and have a single kind of glycoprotein spike that primers for mRNA transcription; budding takes place on the consists of multifunctional hemagglutinin-esterase mole- plasma membrane cules. Infectious salmon anemia virus (genus Isavirus) Defective interfering particles and genetic reassortment occur also has a hemagglutinin-esterase and a fusion or F pro- frequently tein. Regardless of the configurations, at least three

HA M1

NA M2

NP PA PB1 PB2

vRNA FIGURE 21.3 (Left) Diagram of an influenza A virus virion in section. The indicated glycoproteins embedded in the lipid membrane are the trimeric hemagglutinin (HA), which predominates, and the tetrameric neuraminidase (NA). The envelope also contains a small number of M2 membrane ion channel proteins. The internal components are the M1 membrane (matrix) protein and the viral ribonucleoprotein (RNP) consisting of RNA segments, associated nucleocapsid protein (NP), and the PA, PB1, and PB2 polymerase proteins. (Right) negative contrast electron micrograph of particles of influenza A virus Courtesy of N. Takeshi. The bar represents 100 nm. From Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, p. 681. Copyright r Elsevier (2005), with permission. Orthomyxoviridae Chapter | 21 395

is 1014.6 kb in overall size. The genome segments have preference for long oligosaccharides present on epithelial nontranslated regulatory sequences at both the 50 and 30 cells in the upper respiratory tract, and mutations that affect ends. The 13 nucleotides at the terminal 50 end and 12 at this binding alter transmissibility. A single amino acid the 30 end are identical for each of the genomic segments change in the hemagglutinin protein of the 1918 H1 and show partial inverted complementarity. This feature Spanish flu virus at position 190 (E190D) changes binding is essential for RNA synthesis. preference from SAα2,3Gal to SAα2,6Gal. Because of their structural and biochemical properties, Influenza viruses enter cells via receptor-mediated influenza viruses are sensitive to heat (56C, 30 minutes), endocytosis. While in the endosome, the ion channel M2 acid (pH 3), and lipid solvents, and are thus very labile (matrix protein 2) tetramer allows the flow of protons under ordinary environmental conditions. However, infec- into the virus particle to enable dissociation of the tious influenza A virus has been recovered after 30 days other matrix protein (M1) from the RNP complex (which in cold lake water. includes viral genomic RNA, nucleoprotein, and polymerase proteins), thus freeing it from the viral envelope. The low pH (acidic) of the endosome triggers a conforma- Virus Replication tional change in the hemagglutinin (HA) protein such Influenza virions attach to cells via the binding of their acti- that the hydrophobic domain of the HA2 trimer mediates vated hemagglutinin to sialic-acid-containing receptors on fusion of the viral envelope with the endosomal mem- the plasma membrane, as depicted and described for influ- brane, releasing the RNA, nucleoprotein, and polymerase enza A virus (Fig. 21.4). Different host cells have surface proteins (RNP) into the cytoplasm (Fig. 21.4). Amantadine glycans with different linkages of N-acetyl neuraminic acid and rimantadine inhibit virus infection by blocking the ion (sialic acid) to a galactose residue, and the hemagglutinin channel activity of M2. A unique feature of influenza virus recognizes these different linkages, which in turn determine is that all RNA synthesis takes places in the nucleus of the the host range of the virus. The gut epithelium of ducks has cell. This requires that the RNP, because of its size, be a receptor with an α2,3 linkage (SAα2,3Gal), whereas the actively transported into the nucleus. Nuclear localization predominant influenza virus receptor in the upper respira- signals on the nucleoprotein interact with the nuclear tory tract of humans is an α2,6 linkage (SAα2,6Gal). There transport machinery of the cells to transport the RNP into is also evidence that binding affinity for the SAα2,6Gal the nucleus. glycan varies with the length of the oligosaccharide. As with all viruses with negative-sense RNA genomes, Human-adapted H1 and H3 viruses show binding the genome of orthomyxoviruses serves two functions: as

FIGURE 21.4 Schematic diagram of the influenza viral life cycle. ER, endoplasmic reticu- lum; M1, M2, matrix proteins; mRNA, messenger RNA; NP, nucleoprotein; NS1, NS2, nonstructural proteins 1, 2; PA, PB1, PB2, proteins of the viral RNA polymerase complex; PB1-F2, nonessential lineage- dependent protein linked to virulence. From Neumann, G., Noda, T., Kawaoka, Y., 2009. Emergence and pandemic potential of swine-origin H1N1 influenza virus. Nature 459, 931939, with permission. 396 PART | II Veterinary and Zoonotic Viruses

(A) Influenza A virus PB1 segment 2 a template for the synthesis of messenger RNAs (mRNAs), and as a template for the synthesis of positive- 0 PB1 A(n) 757 aa sense replicative intermediate RNA, which is the template 2341 +1 PB1-F2 A(n) 11, 57, or 79-90 aa for progeny genomic RNA synthesis. Primary transcription involves an unusual phenomenon known as cap 0 PB1 N40 A(n) 718 aa snatching: the viral endonuclease activity of the polymerase (PA) cleaves the 50-methylguanosine cap plus about (B) Influenza A virus segment 7 1013 nucleotides from cellular precursor mRNAs that M1 A(n) 252 aa are captured by PB2, another component of the polymer- 1027 ase complex. These caps are then used by the virus as pri- A(n) 97 aa M2 mers for transcription by the viral RNA polymerase (transcriptase; PB1). The viral mRNAs thus are capped (C) Influenza A virus segment 8 and also become polyadenylated through template slip- NS1 A(n) 237 aa page and repeated transcription of five to seven “U” resi- 890 dues on the virion RNA. All orthomyxoviruses extend the NS2 (NEP) A(n) 121 aa coding capacity of their genomes by producing two proteins from certain genes by using a splicing mechanism. (D) Influenza B virus segment 6 NB NA Influenza A virus uses splicing for gene segments 7 (M1 0 NB A(n) 100 aa AAAAAUGAACAAUGCUA 1557 and M2) and 8 (NS1 and NEP/NS2) (Fig. 21.5); influenza +1 NA A(n) 466 aa NB B virus uses gene segment 8 (NS1 and NEP/NS2); influ- NA enza C virus uses gene segments 6 (CM1 and CM2) and 7 (E) Influenza B virus segment 7

M1 A(n) 248 aa M1 BM2 uuaUAAUGc 1191 M1 second AUG, present in many but not all viruses, in frame in the PB1 A(n) 109 aa L BM2 ORF as the initiation codon encodes the polypeptide PB1 N40, the C terminal 718 amino acids of PB1. (B) Influenza A virus segment 7 showing (F) Influenza C virus segment 6 M1 and M2 mRNAs and their coding regions. M1 and M2 share 9 P42 A(n) 374 aa amino-terminal residues, including the initiating methionine; however, the ORF of M2 mRNA (nt 7401004) differs from that of M1. Signal peptidase 1181 (C) Influenza A virus segment 8 showing the initiating methionine. M1’ (p31) 259aa CM2 115aa The ORF2 of NS2 (NEP) mRNA (nt 529861) differs from that of NS1. TG A M1 A(n) 242 aa (D) ORFs in influenza B virus RNA segment 6, illustrating the overlapping ORFs of NB and NA. Nucleotide sequence surrounding the 2 AUG (G) Thogotovirus segment 6 initiation codons, in the mRNA sense, is shown. (E) influenza B virus RNA segment 7 ORFs and the organization of the ORFs used to translate ML A(n) 304 aa the M1 and M2 proteins. A stop-start pentanucleotide, thought to couple 995 translation between the 2 ORFs, is illustrated. (F) Influenza C virus M A(n) 266 aa mRNAs derived from segment 6. The unspliced and spliced mRNAs TG A encode P42 and M1, respectively. The cleavage of P42 by a signal pepti- (H) Isavirus segment 7 dase produces M10 (p31) and CM2. (G) Thogot virus segment 6 showing M and ML. M is translated from a spliced mRNA with a stop codon that NS A(n) 301 aa 966 is generated by the splicing process itself, asin influenza C virus M1 s7ORF2 A(n) 160 aa mRNA. ML is translated from the unspliced transcript and represents an elongated form of M with a C-terminal extension of 38 aa. (H) Isavirus (I) Isavirus segment 8 mRNAs derived from segment 7. The unsliced mRNA encodes the NS protein and the unspliced mRNA encodes a protein of unknown function. 0 M1 A(n) 197 aa (I) Isavirus mRNAs derived from segment 8. ORF1 starts at nt 22 and 736 encodes the M1 protein, ORF2 starts at nt 36, in the 12 reading frame A(n) 242 aa +2 s8ORF2 relative to ORF1 and encodes a protein of unknown function. For all FIGURE 21.5 Orthomyxovirus genome organization. The genomic panels, the boxes represent different coding regions. Introns in the 0 organization and open reading frames (ORFs) are shown for genes that mRNAs are shown by the V-shaped lines; filled rectangles at the 5 ends encode multiple proteins. Segments encoding the polymerase, hemagglu- of mRNAs represent heterogeneous nucleotides derived from cellular 0 tinin, and nucleoprotein genes are not depicted as each encodes a single RNAs that are covalently linked to viral sequences. Lines at the 5 and 0 protein. (A) Influenza A virus PB1 segment 2 ORFs. Initiation of PB1 3 termini of the mRNAs represent untranslated regions. Modified from translation is thought to be relatively inefficient based on Kozak’s rule, Lamb and Horvath, 1991, and Garcia-Rosado et al., 2008. From King, likely allowing initiation of PB1-F2 translation by ribosomal scanning A.M., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus and results in PB1-F2 proteins of different size. In addition, the use of a Taxonomy: Ninth Report of the International Committee on Taxonomy of (Continued) Viruses, p. 752753 Copyright r Elsevier (2012), with permission. Orthomyxoviridae Chapter | 21 397

(NS1 and NEP/NS2); thogotoviruses use gene segment 6 For wild aquatic birds, the virus is shed in the feces (M and ML); isavirus uses gene segments 7 and 8. The and transmission is fecaloral, but respiratory replication NS1 and NS2 (HEP) proteins are defined as nonstructural recently has been documented, indicating the potential for proteins whereas the various M, CM, and ML proteins are inhalational transmission. In poultry, replication is pre- all membrane associated. Influenza B virus also uses a dominantly within the respiratory tract, but it also can different strategy for gene segment 7, involving overlap- occur in the intestinal tract, suggesting transmission may ping stop and start codons to generate two protein pro- be by either ingestion or inhalation. In mammals, trans- ducts. In certain influenza A virus strains, a PB1-F2 mission is by aerosol, droplets, and fomites. The thogoto- protein of 8790 amino acids is generated by a ribosomal viruses are transmitted by ticks and replicate in both ticks 11 reading frameshift. and mammals. The isaviruses may be transmitted in Viral protein synthesis occurs in the cytoplasm using water, with the gills of susceptible fish being the principal the host cellular translation machinery. There is clear evi- site of virus uptake and infection. dence for temporal regulation of gene expression, but the mechanism is unresolved. Early in infection, there is enhanced synthesis of nucleoprotein and NS1, whereas Molecular Determinants of Pathogenesis synthesis of hemagglutinin, neuraminidase, and matrix The hemagglutinin protein of influenza A viruses is protein 1 is delayed. Nucleoprotein is required for replica- synthesized as a single polypeptide designated HA0. tion of the virion RNA, and NS1 has been shown to A key event in the history of influenza virus biology inhibit the antiviral response triggered by the infection. was the discovery that the hemagglutinin protein had to Nucleoprotein and the polymerase proteins must be trans- be cleaved posttranslationally for the virus to be infec- ported to the nucleus to interact with the RNA to initiate tious, which established a clear link between cleavability genome replication. of hemagglutinin and virulence. The HA0 from avian Replication of genomic RNA segments requires the viruses that were designated as highly pathogenic or high- synthesis of full-length, positive-sense RNA intermedi- pathogenicity avian influenza (HPAI) viruses due to their ates, which, unlike the corresponding mRNA transcripts, lethality for chickens had several basic amino acids at the must lack 50 caps and 30-poly(A) tracts. Newly synthe- hemagglutinin cleavage site or long insertions of amino sized nucleoprotein binds to these RNAs, facilitating their acids. In contrast viruses designated as low pathogeni- use as templates for the synthesis of genomic RNAs. Late city avian influenza (LPAI) and currently circulating in infection, the matrix protein, M1, enters the nucleus mammalian-adapted influenza viruses contain a single and binds to nascent genomic RNA-nucleoprotein, arginine at a short cleavage site. The proteases that are thereby downregulating transcription and permitting capable of cleaving at the single arginine are tissue export from the nucleus. NEP/NS2 bind the M1RNP restricted, and access to the appropriate protease deter- complexes, thus providing nuclear export signals and mines tissue tropism. In birds and mammals, epithelial interaction with the nuclear export machinery that moves cells within the respiratory and gastrointestinal tracts pro- the RNP into the cytoplasm. duce trypsin-like enzymes that can cleave the hemaggluti- Virions are formed by budding, incorporating M1 pro- nin of their respective LPAI and mammalian viruses. tein and nucleocapsids that have aligned below patches on Virus is produced in a noninfectious form and activation the plasma membrane in which hemagglutinin, neuramini- of infectivity occurs extracellularly. In addition, certain dase, and matrix protein M2 have been inserted. In polar- respiratory bacteria, including normal flora, can secrete ized epithelial cells, influenza virus buds from the apical proteases that cleave and activate the hemagglutinin of surface of the cell. The hemagglutinin and neuraminidase influenza A viruses. In contrast, the hemagglutinin of proteins each contain transmembrane domains that associ- HPAI viruses, features several basic amino acids at the ate with areas of the membrane enriched for sphingolipids cleavage site, which expands the range of organs capable and cholesterol that are designated lipid rafts. These lipid of producing infectious virus, because cleavage can be rafts have altered fluidity that appears to be critical for the mediated by the ubiquitous family of endopeptidase furins budding process and infectivity of the mature virus particle. that are located in the trans-Golgi network. In this man- Recent studies showed that segment-specific packaging ner, the hemagglutinin is cleaved intracellularly and fully signals contained in each RNA segment mediate selective infectious virions are released from infected cells without incorporation of only one copy of each RNA segment into any requirement for the extracellular activation step that each budding virion. As virions complete budding (“pinch- is necessary for LPAI virus strains. Thus continuous mon- ing off”), the neuraminidase spikes (peplomers) facilitate itoring of the sequence at the cleavage site of circulating the release of virions by destroying receptors on the plasma strains of LPAI viruses is used to identify hemagglutinin membrane that would otherwise recapture virions and hold cleavage site mutations that might predict the emergence them at the cell surface. of highly virulent viruses. In general, the HPAI viruses 398 PART | II Veterinary and Zoonotic Viruses

are not maintained in wild bird reservoirs, but arise fol- determined by receptor specificity and cleavability of the lowing mutation in the hemagglutinin cleavage site of hemagglutinin protein, as well as the activity of the PB2 LPAI viruses as a result of sustained circulation in terres- polymerase complex protein. The novel PB1-F2 protein trial birds including chicken, turkey, ostrich, and quail, apparently also contributes to the virulence phenotype among others. of individual viruses, and NS1—and perhaps other As with most viruses, influenza viruses have devel- proteins—interfere with innate host defenses. Studies in oped mechanisms to counteract innate antiviral host laboratory animals also show protracted alteration of defenses. NS1 is the key influenza virus protein responsi- innate immune responses mediated by pattern recognition ble for blocking the response. Dimeric NS1 protein binds receptors after influenza virus infection, and these altera- to double-stranded RNA, which is a potent interferon tions presumably can predispose affected animals to inducer (see Chapter 4: Antiviral Immunity and Virus secondary respiratory infections. Vaccines). The exact mechanism by which NS1 blocks interferon responses is not known, but virus strains with mutations of NS1 are attenuated, and cells infected with MEMBERS OF THE GENUS these NS1 mutants contain increased levels of interferon INFLUENZAVIRUS A response gene transcripts as compared with those infected with wild-type virus. The NS1 mutants are lethal to mice lacking interferon response genes, whereas infection is EQUINE INFLUENZA VIRUSES restricted in normal mice. Mutation of a single amino Although outbreaks of a respiratory disease in horses that acid at position 42 (P42S) of NS1 greatly enhances viru- probably was influenza have been described throughout lence of influenza virus by attenuating the anti-interferon history, including the so-called Great Epizootic of 1872 capability of NS1. In isaviruses, it is the 7i protein that amongst horses in North America, the differentiation of assumes the role of interferon antagonist, whereas in tho- equine influenza from other equine respiratory diseases gotoviruses it is the ML protein; the sites of action of was not definitively established until 1956, when influ- these proteins in the antiviral pathway may be different. enza virus A/equine/Prague/1/56 (H7N7) (equine influ- The PB2 protein of the polymerase complex is a key enza virus 1) was isolated during an epizootic in central component of the RNA transcription and replication pro- Europe. Subsequently in the United States, a second virus, cesses of influenza viruses, and may exert an important A/equine/Miami/1/63 (H3N8) (equine influenza virus 2), role in determining virulence and host range. The specific was isolated in 1963. Since then, the disease has been amino acids at residues 591, 627 and 701 determine reported in horses, and also in donkeys and mules, in vir- whether avian influenza A viruses will replicate well in tually all parts of the world, although certain island coun- mammalian cells. These residues also determine virulence tries, including Iceland and New Zealand, have of the virus to experimentally infected laboratory mice. In maintained their freedom from infection. Australia and avian influenza viruses (H5N1, H7N7, and H10N8) South Africa are both currently free of equine influenza viruses, a lysine at residue 627, or asparagine at position after experiencing recent outbreaks. 701, or basic amino acid at position 59, enhances growth Influenza is considered to be the most important cause of the virus in mammalian cells, and this change from of viral respiratory disease in horses. H3N8 virus has glutamic acid is selected when avian viruses successfully been identified in all recent outbreaks; the last outbreak cross the mammalian species barrier. caused by subtype H7N7 virus was in 1979 and this virus In addition to changes in the glycoproteins in the no longer circulates in equine populations. The H3N8 viruses responsible for the human influenza pandemics of virus has undergone modest genetic drift since it was first 1957 and 1968, there was also an incorporation of the isolated, and there are now discernible branches in the PB1 gene from an avian virus. Furthermore, introduction evolutionary tree: a Eurasian branch and an American of a PB1 gene from a pathogenic virus into swine influ- branch. The American branch is further subdivided into enza virus increased the virulence of the reassortant. A Argentinean, Florida, and Kentucky sublineages. Both novel protein that was generated by translation from a 11 American sublineages also circulate in horses in Europe open reading frame of the PB1 gene, designated PB1-F2, and Asia, but the Eurasian lineage has been detected in localized to mitochondria in infected cells and induced North America only once. Although only modest anti- apoptosis in monocyte/macrophages. Although PB1-F2 is genic changes have occurred in the H3N8 virus over time, dispensable for replication in cell culture and embryo- failure to upgrade vaccines to include currently circulat- nated eggs, it can affect virulence of the virus in mice ing strains has resulted in significant outbreaks of respira- and, potentially, other mammals. tory disease in these vaccinated animals. Continuing From the preceding, virulence determinants of influ- surveillance to isolate new variants is necessary to opti- enza viruses clearly can be multifactorial. Host range is mize vaccine efficacy. Orthomyxoviridae Chapter | 21 399

Clinical Features and Epidemiology Apart from one outbreak in China in 1989 that was derived from an avian source, equids are the only known Influenza virus characteristically spreads very rapidly source of equine influenza viruses. In 1989, a severe out- amongst susceptible horses, and causes disease of high break of H3N8 influenza A virus infection occurred in morbidity 24 48 hours after infection. The clinical signs are horses in northeastern China, with morbidity of 80% and the result of infection of the respiratory tract: there is redden- mortality of 20%. In a second epizootic in the following ing of the nasal mucosa, conjunctivitis, and serous; later, year, morbidity was about 50%, but there was little or no mucopurulent nasal discharge. The serous nasal discharge mortality, probably because of the immune status of develops at the same time as a characteristic harsh, dry, par- horses in the region. Of particular interest was the discov- oxysmal cough that may persist for up to 3 weeks. Infected ery that, although the causative virus had the same anti- horses develop fever (39.5 41 C) lasting for 4 5daysand genic composition as viruses circulating among horses in become anorexic and depressed. Mortality is rare, but pro- other parts of the world, its genes were of recent avian longed fever in pregnant mares may result in abortion. origin. Serological studies indicated that the virus was not Clinical diagnosis of acute cases is straightforward, but diag- present in horses in China before 1989; thus it represents nosis in partially immune horses is more difficult, as the dis- the transfer of an avian influenza virus to mammals with- ease must be differentiated from other respiratory infections, out reassortment. This serves to emphasize that, however including those caused by equine herpesviruses, equine rhini- uncommon the emergence of new strains of influenza tis viruses, and a variety of bacteria. Subclinical infections virus, ongoing surveillance is required to detect such with virus shedding are frequently seen in vaccinated horses events and develop interventions. in which the immune response is weak or a poor match for the circulating virus. Secondary bacterial infections may occur, characterized by purulent nasal exudates and broncho- Pathogenesis and Pathology pneumonia. In the absence of such complications, the disease Equine influenza viruses replicate in epithelial cells of the is self-limiting, with complete recovery occurring within upper and lower respiratory tract. Infection causes 23 weeks after infection. destruction of the ciliated epithelial lining, which induces Equine influenza viruses are highly contagious and are inflammation and subsequent formation of exudate and spread rapidly in stables or studs by infectious exudate that nasal discharge. The most important changes occur in the is aerosolized by frequent coughing. Virus is excreted dur- lower respiratory tract and include laryngitis, tracheitis, ing the incubation period and horses remain infectious for bronchitis, and bronchointerstitial pneumonia that are at least 5 days after clinical disease begins. Close contact accompanied by pulmonary congestion and alveolar between horses facilitates rapid transmission; however, con- edema. Secondary infections may result in conjunctivitis, taminated clothing of stable personnel, equipment, and pharyngitis, bronchopneumonia, and chronic respiratory transport vehicles may also contribute to virus dissemina- disease. Fatal bronchointerstitial pneumonia was tion. Equine populations that are moved frequently, such as described amongst foals less than 2 weeks of age during racehorses, breeding stock, show jumpers, and horses sent the 2007 equine influenza epizootic in Australia; as this to sales, are at special risk. The rapid international spread of country previously was free of the virus, the epizootic equine influenza is caused by the year-round transport of occurred in an immunologically naı¨ve population of horses for racing and breeding purposes between Europe, horses. The severe disease encountered in these foals was North America, Japan, Hong Kong, South Africa, attributed to lack of protection from colostral antibodies Australasia, and elsewhere. Although clinical manifesta- specific for equine influenza virus, rather than an infections normally begin in the cold season, epizootics gener- tion with an unusually pathogenic virus. ally occur during the main racing season—that is, between Factors that contribute to innate resistance of horses April and October in the northern hemisphere. include: (1) the mucus blanket that protects the respiratory The highly contagious nature of equine influenza virus epithelium, and the continuous beating of cilia that clears was graphically illustrated in 2007, when an H3N8 virus virus from the respiratory tract; (2) soluble lectins, lung (American lineage) spread amongst horses in Australia, a surfactants, and sialoglycoproteins present in mucus and country that previously was free of the virus. From an ini- transudates that bind virions; and (3) alveolar macro- tial incursion at a quarantine station in New South Wales, phages. If the horse has been infected previously, antihe- the virus spread within 3 months to some 10,000 premises magglutinin antibodies may intercept and neutralize the in New South Wales and Queensland. The epizootic was virus if the challenge virus is antigenically closely controlled by movement restrictions and vaccination, but matched to the immunizing virus. Secretory immunoglob- this extraordinary event emphatically confirmed the ulin A is proposed to be the most relevant antibody in the impact of equine influenza virus in an immunologically upper respiratory tract, but serum-derived antibodies also naı¨ve population of horses. provide protection. Levels of vaccine-induced serum 400 PART | II Veterinary and Zoonotic Viruses

antibodies as measured by single radial hemolysis corre- Vaccination is extensively practiced in influenza virus late well with protection in challenged animals. In labora- enzootic countries. Vaccination was previously carried out tory animal models, activated macrophages, natural killer exclusively with inactivated vaccines that now contain two cells, and virus-specific T cells are crucial to clearance of different lineages of A/equine (H3N8) viruses, preferably virus from the lower respiratory tract, as are interferon-γ including strains that match the prevalent field virus. and interleukin-2. Efforts to undertake similar studies in Several vaccinations are necessary to achieve full protec- horses are complicated by the lack of necessary reagents. tion of individual horses, although the optimal timing of booster immunizations is conjectural. The H7N7 equine influenza virus has been eliminated from most reformu- Diagnosis lated vaccines since 2000, when an OIE expert panel con- The clinical presentation of influenza in horses is highly cluded that there was no epidemiological evidence to characteristic. In most laboratory settings, detection of support inclusion of H7N7 in equine vaccines. A continu- equine influenza virus is achieved using RT-PCR assays. ing concern with inactivated vaccines is their inability to Nasal or oropharyngeal swabs taken early in the infection induce a cellular immune response equivalent to that which (within 35 days of onset of clinical signs) are the samples occurs after natural infections. Various formulations have of choice, and virus need not be viable for detection by RT- attempted to correct this problem, including the use of PCR assay. Similar samples can be used for virus isolation, immunostimulatory adjuvants (oil- and polymer-adjuvanted but the swabs should be placed in a virus transport medium preparations), Quil-A-based immune-stimulating com- to preserve infectivity. H3N8 viruses replicate in 10-day- plexes, DNA vaccines for use in prime-boost strategies, old embryonated eggs, using either the amniotic or the poxvirus-vectored vaccines, and a live-attenuated, cold- allantoic route of inoculation and incubating at 3537C adapted virus vaccine. These approaches can induce a for 34 days, but a blind passage may be necessary to pro- more durable immune response as compared with previous duce a detectable level of virus by standard hemagglutina- whole-virus and subunit vaccines. Commercially available tion tests. Isolation of the virus can be performed in cell canarypox virus vectored recombinant vaccines containing culture systems. MadinDarby canine kidney (MDCK) the hemagglutinin protein and an immune-stimulating com- cells are preferred; trypsin must be included in the culture plex have been demonstrated to produce a cellular immune medium, because most cells lines cannot cleave the hemag- response in horses. Recombinant canarypox virus vectored glutinin protein, which is necessary for virus replication. vaccines were used in combination with animal movement Virus replication can be detected by the demonstration of restrictions to quickly control incursions of equine influ- hemagglutination activity in the harvested amniotic or enza into both South Africa and Australia. allantoic fluid or cell culture fluid by antigen-capture A major potential problem with inactivated vaccines is ELISA or by RT-PCR. Isolates are now identified by hem- their inability to provide a protective immune response for agglutinin- and neuraminidase-specific RT-PCR assays or, foals with maternally derived antibodies. Current recom- less frequently, by hemagglutination-inhibition using a mendations are to withhold vaccination until at least panel of subtype-specific reference antisera. Retrospective 6 months of age. The canarypox virus vectored equine serologic diagnosis of equine influenza virus infection can influenza vaccines can prime foals even in the presence of be made using paired serum samples. maternal antibody, which may afford enhanced protection Outbreaks of respiratory disease in horses caused by of young animals. influenza virus infection rapidly can be confirmed in the field Regardless of the vaccine type, regulatory agencies (nonlaboratory settings) using lateral-flow antigen-capture must develop flexible regulations that can allow rapid tests. Although these tests can be relatively insensitive incorporation of new strains of virus into the vaccines in in detecting infection of individual horses, they are very a manner similar to that adopted for human vaccines. specific and useful for rapid confirmation of epizootics. SWINE INFLUENZA VIRUSES Immunity, Prevention, and Control Swine influenza was first recognized and described in the Control of incursions of equine influenza virus into previ- north central United States at the time of the catastrophic ously virus-free countries involves isolation and vaccina- 1918 pandemic of human influenza, and for a long time tion, as practiced during outbreaks earlier this century in was reported only from this area, where annual outbreaks both Australia and South Africa. Similarly, stables and occurred each winter. The first isolation of swine influenza racing facilities where equine influenza outbreaks occur virus was by Richard Shope in 1930 [A/swine/Iowa/15/ should be quarantined. After all horses have recovered, 1930 (H1N1)]. Although the 1918 pandemic affected cleaning and disinfection of boxes and stables, equipment, humans throughout Europe, swine influenza was not and transport vehicles is necessary. observed in Europe until the 1940s and 1950s in Orthomyxoviridae Chapter | 21 401

Czechoslovakia, the United Kingdom, and West Germany. and PB1 genes from a human virus strain; PB2 and PA from The virus then apparently disappeared until 1976, when it an avian strain; and the NP, M,andNS genes from the clas- reappeared in northern Italy and spread to Belgium and sical swine virus (Fig. 21.6). In China, H3N2 human-like southern France in 1979; since then it has occurred in virus, H3N2 double reassortants, and H3N2 triple reas- Europe rather regularly. However, the virus causing these sortants have circulated in the pig population. Other combi- more recent epizootics in swine was of avian origin, and nations also exist, and novel reassortants may develop closely related to a duck virus. frequently, as occurred with the emergence of the novel pan- Two distinct variants of the H1N1 swine influenza virus demic H1N1 virus in 2009. In addition, there is continuing now circulate in the world—specifically, the avian variant accumulation of amino acid changes within the hemaggluti- found in Europe since 1979, and the variant found in the nin proteins of circulating strains of swine influenza virus. United States that is similar to the original virus strain. Pigs have been considered the “mixing vessel” for influ- Swine have also become infected with other types of influenza virus because of their susceptibility to infection with enza A virus, including human H3N2 strains in China, both avian and human influenza viruses. Pigs possess both Europe, and North America, and reassortant H1N2 viruses avian-type (SAα2,3Gal) and human-type (SAα2,6Gal) have emerged that contain genes from both swine and receptors. The reassortant influenza viruses that currently human virus strains; there are even triple reassortants that circulate in swine throughout the world support this interme- contain genes from swine, human, and avian viruses. For diary role, although only the 2009 pandemic H1N1 human example, a virus that caused a significant epizootic of respi- influenza viruses are related to swine viruses. Curiously, ratory disease in North American swine has: the HA, NA, although pigs can become infected with various avian

FIGURE 21.6 Genomic composition of the predominant influenza A viruses endemic in swine populations around the world. Although all IAV established in swine populations are of the H1N1, H1N2, or H3N2 subtypes, multiple lineages and whole-genome constellations distinguish viruses from different countries and regions. The major lineages include swine-adapted viruses of North America, Europe, Asia, and human seasonal viruses that have were transmitted to swine and become established by achieving sustained transmission, including the H1N1 2009 pandemic virus designated H1N1pdm09. The triple reassortant internal gene (TRIG) genome constellation emerged in 1997 and has become dominant in many countries. From Vincent, A., et al. (2014). Review of Influenza A virus in swine worldwide: a call for increased surveillance and research. Zoonoses and Public Health 61, 417. doi:10.1111/zph.12049, with permission. 402 PART | II Veterinary and Zoonotic Viruses

viruses, enzootic swine viruses possess only the H1 or H3 and there is a rapid progression of the infection in the epi- hemagglutinins. The current Eurasian H5N1 avian viruses thelium of the nasal cavity and large airways. Infection can sporadically can infect pigs, but replicate poorly. Swine progress to involve all airways in just a few hours. Animals influenza, subtypes H3N2 virus has been transmitted to tur- develop bronchointerstitial pneumonia that is characterized keys. Turkeys were also infected with the 2009 pandemic by sharply demarcated lung lesions in the apical and cardiac H1N1 virus by exposure through artificial insemination. lobes, with hyperemia, consolidation, and the presence of inflammatory exudates in airways. Histologically, epithelial surfaces are denuded, with accumulation of intraluminal Clinical Features and Epidemiology debris within affected airways. There is collapse of adjacent After an incubation period of 2472 hours, the onset of dis- airspaces, interstitial pneumonia, and emphysema. ease is abrupt, often appearing in many animals in a herd at the same time. There is fever (42C), with apathy, inap- petence, huddling, and a reluctance to move, and signs of Diagnosis respiratory distress: paroxysmal coughing, sneezing, rhinitis Swine influenza is characterized by sudden onset of with nasal discharge, labored breathing, and bronchial rales highly contagious respiratory disease that may be con- at auscultation. After 36 days, swine usually recover fused with infectious diseases such as those caused by quickly, eating normally by 7 days after appearance of the Actinobacillus pleuropneumoniae and Mycoplasma hyop- first clinical signs. If sick swine are kept warm and free of neumoniae. Indeed, the gross lung lesions in swine with stress, the course of disease is benign, with few complica- influenza can closely resemble those of swine with tions and a case-fatality rate of less than 1%; however, M. hyopneumoniae infection. For routine diagnosis, the some animals develop severe bronchopneumonia, which RT-PCR test has replaced virus isolation, thanks to its may result in death. Reproductive losses have occurred speed and capability for automation. Isolation is still used amongst pregnant sows infected with some H3N2 triple to provide viruses for genetic analyses; isolations can be reassortant viruses. Although most pigs recover unevent- achieved using embryonated eggs or by cell culture fully, the economic consequences of swine influenza are using MDCK cells with a trypsin-containing overlay. considerable, in that sick swine either lose weight or their Identification of the virus subtype is by hemagglutinin- and weight gains are reduced. neuraminidase-specific RT-PCR tests with confirmation by Outbreaks of swine influenza are observed mostly in sequence analysis of the amplified products, or with spe- late fall and winter, or after the introduction of new swine cific monoclonal antibodies. Virus can be detected in tissue into susceptible herds. Infections can occur year-round in samples by immunofluorescence or by immunohistochem- confinement swine facilities. Frequently, the disease appears istry. Serological tests (hemagglutination-inhibition and simultaneously on several farms within an area; outbreaks ELISA) can be used to detect infection of unvaccinated are explosive, with all swine in a herd becoming sick at vir- swine. Full genome sequencing and phylogenetic analyses tually the same time. The mechanism of the interepizootic can provide additional information regarding the virus survival of swine influenza virus has been a matter of inten- genotype; such analyses are performed as part of molecular sive investigation for many years, but remains unsolved. epidemiology and viral ecology studies. Swine influenza virus clearly can become enzootic in large herds in which there is continuous infusion of newly susceptible animals, but there is no credible evidence of a true car- Immunity, Prevention, and Control rier state in any species infected with an influenza virus. Swine influenza is controlled by vaccination and strict The novel 2009 pandemic strain of H1N1 influenza biosecurity measures that prevent introduction of the virus. virus can infect pigs, but the associated clinical disease is Many commercial producers of swine now use an mild. However, substantial economic losses have been “all-inall-out” system of production. In this type of facil- incurred through the quarantine of infected herds because ity, biosecurity may be sufficient to exclude influenza virus of public health concerns. As frequently occurs when zoo- infection, provided there is reliable access to virus-free notic diseases appear, consumption of pork dropped sub- replacement stock. Vaccines can be used to control disease stantially after the emergence of the “swine flu” virus in in facilities where exclusion is not practical. Because of the 2009, although studies indicated absence of infection risk number of reassortant influenza viruses that currently circu- to pork consumers. late in the commercial swine population, vaccines are now being formulated with a minimum of two different viral antigens, and some with three. As with all influenza virus Pathogenesis and Pathology vaccines, the vaccines do not prevent infection, or Swine influenza virus infection follows the typical pattern completely suppress the shedding of virus following natural for respiratory viral infections: virus entry is via aerosol, infection. For production facilities, the simple goal is to Orthomyxoviridae Chapter | 21 403

reduce viral spread, prevent significant clinical disease and HPAI viruses that became extinct within a few months or the associated economic losses. Vaccination does confuse years since emergence, the HPAI virus lineage derived the interpretation of serological testing, either for diagnos- from A/goose/Guangdong/1/1996 (H5N1) has persisted tic purposes or in seroprevalence studies. for two decades, gained nearly global distribution, and has affected more countries and birds than the other 34 Human Disease outbreaks or epizootics combined. This Eurasian H5N1 virus lineage has reassorted with different neuraminidase Infection of humans with swine influenza virus can occur subtypes giving rise to H5N2, H5N3, H5N5, H5N6 and among abattoir workers exposed to virus-infected pigs, H5N8 HPAI viruses, and therefore it is herein designated and may cause respiratory disease. Human infections with as H5Nx. The recent introduction of Eurasian lineage H3N2 viruses from swine have been detected at agricul- HPAI H5N8 into Europe (United Kingdom, Netherlands, tural fairs in the United States, often when these viruses Italy, and Germany) as well North America (Canada and were circulating the show swine population. Human infec- United States) was attributed to infected wild bird migration is otherwise rare, and person-to-person spread is lim- tions. The resulting in H5Nx outbreaks in commercial and ited. However, because of fears of another pandemic like backyard poultry in Europe, Canada and the United States that of 1918, swine influenza virus infections of humans raised considerable public health concerns. are the subject of considerable public health concern. For Avian influenza viruses are categorized, for interna- example, the isolation of H1N1 swine influenza virus tional trade issues, as of either high or low pathogenicity. from military recruits at Fort Dix in the United States in The definitions (as found in the World Organisation for 1976 led to a massive human immunization campaign in Animal Health (OIE) Terrestrial Animal Health Code the United States. Many believe the response in 1976 was (2015), Chapter 10.4) are: an overreaction, but the emergence of the novel pandemic H1N1 in 2009 clearly validates concerns regarding zoo- 1. For the purposes of the Terrestrial Code, avian influ- notic H1N1 influenza virus infections. Human infections enza is defined as an infection of poultry caused by any with swine H3N2 triple reassortant viruses have resulted influenza A virus of the H5 or H7 subtypes or by any in limited person-to-person transmission. influenza A virus with an intravenous pathogenicity index (IVPI) greater than 1.2 (or as an alternative at AVIAN INFLUENZA VIRUSES least 75% mortality) as described below. These viruses are divided into HPAI viruses and LPAI viruses: The devastating form of influenza in chickens known as a. HPAI viruses have an IVPI in 6-week-old chickens “fowl plague” was recognized as a distinct disease entity greater than 1.2 or, as an alternative, cause at least as early as 1878, in Northern Italy. The disease spread 75% mortality in 4- to 8-week-old chickens rapidly in Europe and Asia, and was reported in both infected intravenously. H5 and H7 viruses which North and South America by the mid-1920s. The causa- do not have an IVPI of greater than 1.2 or cause tive agent was isolated in 1901, but it was not identified less than 75% mortality in an intravenous lethality as an influenza virus until 1955. In 1961, an outbreak of test should be sequenced to determine whether high mortality occurred in common terns (Sterna hirundo) multiple basic amino acids are present at the cleav- in South Africa that provided the first evidence for direct age site of the hemagglutinin molecule (HA0); if involvement of wild birds in virus ecology. From the the amino acid motif is similar to that observed for 1970s onward, avian influenza came into ecological focus other HPAI isolates, the isolate being tested should when surveillance indicated the ubiquitous presence of be considered as HPAI virus asymptomatic infections in wild waterfowl with LPAI b. LPAI viruses are all influenza A viruses of H5 and viruses, and sporadically with HPAI viruses, posing a H7 subtypes that are not HPAI viruses. constant threat to commercial chicken industries. A very 2. This standard was established because all outbreaks of large epizootic centered in the commercial industries of HPAI have been caused by H5 or H7 viruses, and the Pennsylvania in 19831984, which at the time cost presence of any H5 or H7 LPAI virus in a commercial approximately US $60 million to control (loss of an esti- rearing facility is cause for concern because of the mated 17 million chickens and turkeys), brought sub- inherent potential of these viruses to mutate to the stance to this risk. Since 1959, there have been at least 35 highly pathogenic variant. outbreaks or epizootics of highly pathogenic avian influenza virus infection caused by distinct viruses (or virus lineages) in poultry and wild birds, with losses from disease or culling in excess of 500 million birds. All out- Clinical Features and Epidemiology breaks were caused by mutants derived from LPAI The disease caused in chickens and turkeys by HPAI viruses found in wild bird populations. Unlike all previous viruses has historically been called “fowl plague.” Today, 404 PART | II Veterinary and Zoonotic Viruses

the term should be avoided, except where it is part of the markets. The first indication of a potentially new epizo- name of well-characterized strains [eg, A/fowl plague otic of avian influenza virus was the isolation of an HPAI virus/Rostock/1934 (H7N1)]. The HPAI viruses cause virus from a goose in Guangdong, China, in 1996, with sudden death without prodromal symptoms. If birds sur- subsequent spread and outbreaks among poultry in Hong vive for more than 48 hours (which is more likely in older Kong in 1997, 2001, and 2002. In addition, this unique birds), there is a cessation of egg laying, respiratory dis- virus caused 18 human infections, with 6 deaths; the first tress, lacrimation, sinusitis, diarrhea, edema of the head, document human fatal infections by an HPAI virus. face and neck, and cyanosis of unfeathered skin, particu- Efforts to control the outbreak by depopulation and some larly the comb and wattles. Birds may show nervous signs vaccination with an H5N2 vaccine eliminated the disease such as tremors of the head and neck, inability to stand, and infection in Hong Kong, but by 2003 this H5N1 torticollis, and other unusual postures if surviving more HPAI virus had spread to Korea, Japan, Indonesia, 35 days after exposure. Thailand, and Vietnam. Wild water fowl experimentally The LPAI viruses may also cause considerable losses, infected with HPAI viruses isolated before 1997 did not particularly in turkeys, because of anorexia, lethargy, show clinical signs. However, in 2002, waterfowl in two decreased egg production, respiratory disease, and sinusi- parks in Hong Kong developed neurological disease after tis. Clinical signs in chickens and turkeys may be exacer- infection with this Goose/Guangdong-(Gs/GD)-lineage bated markedly by concurrent infections (eg, various H5N1 virus. Furthermore, captive tigers and lions in viral, bacterial, and mycoplasma infections), the use of Thailand died after being fed infected poultry, which con- live-attenuated virus vaccines, or environmental stress firmed its unusual properties. The H5N1 HPAI virus that (eg, poor ventilation and overcrowding). Low-pathogenicity circulated in 2002 showed multiple gene reassortments (LPAI) H9N2 viruses are ubiquitous in terrestrial poultry and mutations as compared with the 1997 virus. In early throughout Asia, and parts of North Africa and the 2005, H5N1 HPAI virus was isolated from dead wild Middle East. H9N2 have been identified as a source birds in Qinghai Lake of central China and the virus then of poultry-adapted genes for reassortment with new was detected in Mongolia, Siberia, Kazakhstan, and hemagglutinin subtypes from aquatic birds, eg, H10Nx Eastern Europe later that year. This Eurasian H5N1 HPAI and H7N9, enabling circulation of new viruses in poultry virus was detected in most countries of Asia, Europe, and flocks. parts of Africa in 2006, although the “virus” has under- Avian influenza virus is shed in high concentrations in gone many changes since the initial isolate from the goose the feces of wild birds, and can survive for long periods in 1996 (Fig. 21.7). The Eurasian lineage H5N1 virus has in cold water. The virus is often introduced into suscepti- become enzootic in poultry populations from China, ble flocks periodically by interspecies transmission—that Vietnam, Cambodia, Bangladesh, India, Indonesia, Egypt, is, from wild aquatic birds, especially wild ducks, to pre- and other countries in these regions. In addition, sporadic mises with mixed poultry species; thus facilities where outbreaks were detected in the Korean Peninsula, Japan, wild birds have access facilitate this type of transmission. Laos, and Nepal. Eurasian-lineage H5N8 viruses were It is unclear how the many subtypes of avian influenza A also detected in poultry in Germany, the Netherlands, viruses are maintained in wild birds from year to year; it Italy, and the United Kingdom. The first outbreak was is hypothesized that the viruses are maintained by circula- detected on November 5, 2014 at a turkey farm in tion at low levels in large wild bird populations, even dur- Mecklenburg-Vorpommern, Germany. ing migration and overwintering. Studies of wild ducks in Intercontinental wild bird migrations introduced a Canada have shown that up to 60% of juvenile birds are related H5N8 virus from Asia into North America during already infected silently as they congregate before their late 2014. A wild bird surveillance program identified in southern migration. Avian influenza viruses have also fre- early December 2014 a wholly Eurasian 2.3.4.4 H5N8 quently been isolated in many countries from imported from a gyrafalcon (Falco rusticolus) in the state of caged birds, although such passerine and psittacine birds Washington. Reassortment between the Eurasian H5N8 are not natural reservoirs of LPAI viruses and they proba- and North American aquatic bird low pathogenic viruses bly only become infected after exposure to infected vil- resulted in the emergence of new subtypes; eg, H5N1 and lage poultry, especially domestic and captive ducks. H5N2 and new genotypes with three to four additional Live markets also may be critical to the epidemiology genes of North American origin; PB1, PA, NA, and NS of influenza virus infections. The Eurasian H5N1 epizo- (H5N1) and PB1, NP, NA (H5N2). These viruses are des- otic clearly confirms the risk associated with the continu- ignated EurasianAmerican (EAAM) H5Nx. In March ous influx of susceptible animals and mixing of multiple 2015, a highly pathogenic H5N2 virus (HPAI) was avian species (including terrestrial and aquatic birds), detected in commercial poultry facilities in Minnesota, leading to viral amplification, reassortment and rapid evo- Missouri, Arkansas, and Kansas. The outbreak pattern lution through serial transmission in birds at these supported the introduction of the virus into the Midwest Orthomyxoviridae Chapter | 21 405

FIGURE 21.7 Global detection of H5N1 high-pathogenicity avian influenza viruses, A/goose/Guangdong/1/1996 lineage, in animals and humans, including reassortants with N2, N3, N5, and N8 neuraminidase gene subtypes, from January 2003 to Jan 2015. Courtesy of G. Belot, Food and Agriculture Organization of the United Nations. Reproduced with permission, http://empres-i.fao.org/eipws3g/. by migrating waterfowl in the Mississippi flyway. By the the amino acid sequence at the proteolytic cleavage site end of the outbreak, over 200 commercial facilities in 16 of the hemagglutinin protein. Changes in the amino acid states were depopulated with a loss of over 48 million tur- sequence can alter the rate of cleavage of the protein and keys and chickens with a direct loss of 1.6 billion dollars, drastically alter the virulence of these viruses. Most LPAI clearly the most expensive “foreign” animal disease out- viruses have a single basic amino acid (arginine) at the break in US history. The HPAI H5N2 appears to now be cleavage site. In addition, some hemagglutinins have a endemic in the waterfowl using the North American fly- glycosylation site that shields the cleavage site. ways. While rapid depopulation of infected premises is Elimination of the glycosylation site, plus changes of non- still considered the preferred control strategy, limited use basic amino acids to basic ones, insertions that open the of vaccines may be used as a temporary measure to con- cleavage site, and additions of basic amino acids to the tain an outbreak. cleavage site all change cleavability and often increase The role of wild birds in the transmission of the pathogenicity. Eurasian H5Nx virus is intimately linked to its differential The phenomenon of hemagglutinin cleavage is a major pathogenicity for at least some species of wild aquatic determinant of the virulence of avian influenza viruses; birds. Legal and illegal trade in poultry and wild birds however, other portions of the hemagglutinin protein, in must also be carefully monitored, as H5Nx infection has addition to other gene products, also can contribute been detected in imported birds at international borders. through their respective roles on virus binding and repli- Intense surveillance for the Eurasian H5Nx (x 5 1, 2, 3, 6 cation efficiency. There also is variation in the suscepti- or 8) viruses has been reinitiated in Europe, North bility or resistance of different bird species to individual America, and elsewhere since late 2014. In North HPAI virus strains. For example, A/chicken/Scotland/59 America, initial efforts targeted Alaska, western Canada, (H5N1) virus is more virulent for chickens, whereas and the west coast of the United States, because of the A/turkey/Ontario/7732/66 (H5N9) virus is more virulent overlapping migration routes of Asian and North for turkeys. In nature, ducks historically have been refrac- American wild birds, but now the program has expanded tory to clinically severe outcomes with the most virulent into the Mississippi and Atlantic flyways. HPAI viruses, but severe disease has been described following infection with some Gs/GD-lineage H5N1 viruses since 2002. The GS/GD-lineage H5N1 HPAI viruses also Pathogenesis and Pathology cause disease in humans. However, despite millions of The extraordinary virulence of some avian influenza virus potential human exposures to infected birds carrying this strains reflects the properties of several viral gene pro- virus, only B694 people were hospitalized and 402 deaths ducts. A key pathogenic marker that is monitored with have been documented in 15 countries (as of January 23, avian influenza viruses, particularly H5 and H7 viruses, is 2015). This suggests that, although highly fatal, human 406 PART | II Veterinary and Zoonotic Viruses

infections with Gs/GD-lineage H5N1 are rare, and that by gene-specific RT-qPCR assays or with monospecific this HPAI virus is principally a pathogen of birds and not antisera using hemagglutination-inhibition (HI) tests. a pandemic human virus. Infection of flocks can also be assessed using serologic The pathogenesis of avian influenza is quite different tests such as agar gel immunodiffusion, ELISA tests, and from that in mammals, in that virus replication occurs in hemagglutination-inhibition tests to detect antibodies to the intestinal tract as well as the respiratory tract. In infec- influenza antigens. The initial screening is with a broad tions with most HPAI virus strains, there is viremia and serological test for influenza viruses (such as agar gel systemic spread, with hypercytokinemia preceding multi- immunodiffusion or ELISA), followed by 16 different focal lymphoid necrosis and vasculitis and thrombosis hemagglutinin- and nine neuraminidase-specific tests for that results in necrosis and inflammation in many organs subtyping. including pancreas, heart, brain, skeletal muscle, and skin. Chickens, quail, and turkeys, which succumb after several days of illness, exhibit petechial hemorrhages and serous Immunity, Prevention, and Control exudates in respiratory, digestive, and cardiac tissues. Control of avian influenza virus infections of domestic Turkeys may also have air sacculitis and pulmonary con- poultry is reliant on biosecurity, surveillance, and depopu- gestion. In all avian species that survive HPAI virus infec- lation whenever HPAI viruses are detected. Biosecurity is tion, or following LPAI virus infection, neutralizing critical to prevent potentially catastrophic economic loss antibodies are detectable within 510 days, peak during as a result of epizootics of HPAI virus infections, and to the second to third week, and persist for up to 18 months. prevent the evolution of H5 and H7 LPAI viruses to Experimentally, few gallinaceous poultry survive infec- HPAI viruses by segregating domestic poultry from wild tion by HPAI viruses, as infection nearly always results in birds. Commercial facilities in southeast Asia that utilized death. appropriate biosecurity measures suffered no losses during the recent epizootic of Gs/GD-lineage H5N1 virus infection, whereas losses were substantial in those facili- Diagnosis ties that permitted the mixing of poultry with wild birds Clinical diagnosis is at best presumptive and only used or those that had links to live poultry markets. Co- during epizootics, because of the extreme variability in mingling of domestic and wild aquatic birds clearly also the clinical signs accompanying influenza virus infections promotes the introduction of new LPAI viruses into poul- in birds. At the flock level, mortality, egg production, and try with subsequent evolution into more virulent forms. body weight charts are often early indicators of nonspe- Flocks that are found to be infected with HPAI viruses cific infectious and noninfectious illness, including avian are depopulated, to prevent spread to other commercial influenza virus infection. Laboratory-based testing typi- facilities and to wild birds in the environment. This cally involves real-time (quantitative) RT-PCR (RT- approach initially failed to contain the 2015 HPAI out- qPCR) assay to detect the matrix protein (M) gene, as this break in the US because the biosecurity measures in place is highly conserved in all avian and mammalian influenza were inadequate to account for the movement of equip- viruses. Samples positive by this assay then are tested for ment and personnel between commercial facilities. specific H5 and H7 genes by RT-qPCR. If samples are Surveillance is used to monitor for the presence of H5 H5 or H7 positive by RT-qPCR, sequence analysis is and H7 LPAI viruses in domestic poultry. These LPAI undertaken to determine the properties of the cleavage virus strains may cause some production losses, but the site. H5/H7 negative samples can be sequenced to deter- concern is that continued passage of the virus in domestic mine the HA subtype. If several basic amino acids are poultry can lead to mutations in virulence determinants detected at the cleavage site, then regulatory action is that result in HPAI viruses. Depopulation of facilities taken to eliminate the focus of infection. Virus isolation is infected with H5 and H7 LPAI virus is now routinely used to obtain viruses for antigenic analyses and for done to eliminate potential trade implications and con- in vivo pathogenicity tests; isolations are also performed cerns regarding the presence of avian influenza A virus. for non-H5 or -H7 viruses, especially if there is any mor- Most countries with developed industries endeavor to tality associated with the sampled premise. Virus is best prevent infection and limit outbreaks of avian influenza isolated from cloacal swabs (wild birds and aquatic poul- through a combination of biosecurity practices, including try) and tracheal swabs (terrestrial poultry). Specimens education of workers, quarantine, surveillance with appro- are inoculated into the allantoic cavity of 1011-day-old priate diagnostic procedures, and rapid depopulation when embryonating eggs, or on to MDCK cells, and the pres- indicated. Vaccination has not been used to control out- ence of virus is indicated by hemagglutinating activity breaks of HPAI in most developed countries, because of the using chorioallantoic or cell culture fluids and chicken or potentially negative impact on their ability to conduct inter- turkey red blood cells. Isolates are routinely characterized national trade, which requires that expensive surveillance Orthomyxoviridae Chapter | 21 407

programs be instituted to identify any infected birds within readily transmitted between intensively housed dogs. the vaccinated population. In certain situations, principally Canine influenza virus is continuing to evolve, as recent those associated with H5 and H7 LPAI virus infections, isolates exhibit an accumulation of amino acid changes in vaccination in combination with strict quarantines can be the hemagglutinin protein. Initial isolates had multiple used to prevent serious economic losses by limiting depopu- amino acid changes in the hemagglutinin, as compared lation and allow the opportunity to market meat or eggs with the current circulating equine influenza virus, H3N8. from H5 and H7 free or recovered flocks. Uncontrolled H5 Infection of hounds by equine influenza virus H3N8 has or H7 vaccination will probably never be permitted in coun- been detected in several instances in England and tries involved in international trade. occurred during the recent equine influenza outbreak in Australia. However, the infections were limited, and there is no evidence that these were a result of canine influenza Human Disease virus. These cases appear to be simply equine influenza Avian influenza viruses are thought to be the original virus infection in dogs. source of all mammalian influenza A virus genes; how- In 2008, an avian-origin H3N2 virus able to cause sig- ever, direct infection of individual humans by avian influ- nificant clinical disease in dogs was identified in Korea. enza viruses is sporadic. Sustained transmission of avian Subsequently, the presence of this canine influenza virus influenza viruses in humans has never been documented. was documented in China and Thailand. In addition to Only the hemagglutinin, neuraminidase, and basic poly- dogs, this virus is also capable of infecting domestic cats. merase 1 genes from the avian gene pool have entered the Disconcertingly, the detection in dogs of reassortants human population by reassortment, leading to the 1957 between avian-origin H3N2 and H1N1 viruses suggests H2N2 and 1968 H3N2 pandemics. Among the avian that dogs might play a role in the emergence of new influenza viruses, two Eurasian viruses, H5N1 and H7N9, strains of influenza virus. In March of 2015, an epizootic are unique in producing human infections and fatalities. of H3N2 canine influenza virus infection occurred in The vast majority of these cases of H5N1 and H7N9 Chicago, IL, and, unexpectedly, complete genomic analy- transmission to humans can be directly linked to contact sis showed the causative virus to be directly related to the with infected poultry, and in very rare instances are con- Korean virus. This H3N2 canine influenza virus was later sistent with person-to-person spread, but only amongst detected in Atlanta, GA, and with the movement of individuals in very intimate contact to one another. infected dogs, cases have since been reported from the Although the highly pathogenic H5N1 virus has the requi- Eastern seaboard of the United States. There is now con- site basic amino acids at the cleavage site, this property cern that reassortment will occur between the new H3N2 alone has not enhanced human-to-human transmission virus and the preexisting H3N8 canine influenza virus. and the virus also does not replicate well in pigs. Thus The signs of influenza virus infection in dogs regard- additional changes clearly will have to occur for these less of strain are similar to or indistinguishable from those viruses to acquire person-to-person transmissibility and of canine respiratory disease complex (“kennel cough”). start an influenza pandemic. The major difference is that up to 5070% of dogs in a kennel or shelter may be affected with influenza, whereas kennel cough typically would affect fewer than 10% of the CANINE INFLUENZA VIRUSES population. All ages and breeds of dog are susceptible. In 2004, an outbreak of respiratory disease in a group of Recovery is usually uneventful, unless there is a secondary greyhounds in Florida resulted in the death of several infection; the influenza virus infection destroys the ciliated dogs, some of which had remarkably severe hemorrhagic epithelial cells of the respiratory tract, which greatly pneumonia. Comprehensive sequence analyses of an enhances the chances of a secondary bacterial infection. influenza virus, now known as canine influenza virus, There are some data to indicate that the H3N2 virus repli- which was isolated from one dog indicated the virus was cates to higher titers in infected dogs, and is shed for lon- from the H3N8 equine lineage. Serological surveys of ger periods as compared to H3N8 influenza virus. greyhound populations throughout the United States The canine respiratory tract does express the sialic showed very high seroprevalence rates for this H3N8 acid molecules (both α-2,3- and α-2,6-sialic acid-linked virus, consistent with a recent history of respiratory dis- receptors) that serve as receptors for influenza viruses in ease outbreaks in racing greyhounds. The virus was iso- birds and humans, and canines are susceptible infection lated from nonracetrack resident dogs in New York State with Eurasian H5N1 virus. Furthermore, dogs in the epi- in 2005. Incursions into several other states subsequently zootic areas for this virus were seropositive for H5 antibo- were described, although the virus has spread very slowly dies, and dogs exposed to H5N1 virus experimentally and virtually all cases to date have involved dogs in ken- became infected, shed virus, but showed no obvious clini- nels, animal shelters, or day-care centers. The virus is cal signs. A swine origin H5N2 was isolated from a dog 408 PART | II Veterinary and Zoonotic Viruses in China, and sporadic isolations of human pandemic HUMAN INFLUENZA VIRUSES H1N1 have been identified in dogs. All of these occur- rences highlight the need for continuing surveillance of The relationship of animal influenza viruses to human canines for the potential emergence of any highly patho- infections has been described in the preceding sections, genic influenza virus. including the emergence of influenza A viruses from animal reservoirs. In general, epidemic human influenza is BAT INFLUENZA VIRUSES seldom maintained among animals, although there are exceptions. The pandemic H1N1 virus that emerged in An influenza virus was first discovered in little yellow- 2009 was transmitted to pigs, and has been circulating in shouldered bats (Sturnira lilium) in Guatemala during a swine in many parts of the world. The 2009 H1N1 virus virologic surveillance study conducted in 2009 and 2010. was also found to be transmitted from infected humans to The viruses detected in two bats were found to share domestic cats, dogs, and pet ferrets, with the occurrence genetic and functional properties that resemble type A of significant clinical disease in all species. Ferrets can be influenza viruses. The hemagglutinin and neuraminidase infected and transmit both human influenza A and B proteins of the bat virus were phylogenetically related to viruses; they are used as laboratory models for pathogene- those of avian influenza viruses and the bat virus was pro- sis studies, because the associated disease closely mimics visionally designated as subtype H17N10. However, the that in humans. Nonhuman primates, including gibbons, bat virus hemagglutinin does not mediate agglutination of baboons, and chimpanzees, can also be naturally infected red blood cells and the neuraminidase does not cleave with human influenza A virus, and many species of New sialic acid, suggesting utilization of a different receptor to World and Old World monkeys are susceptible to experi- enter cells. Furthermore, the virus cannot be grown in cell mental infection. culture. Related bat influenza viruses were subsequently detected in other bats species in Peru and these viruses were provisionally designated as H18N11 subtype. MEMBERS OF THE GENUS ISAVIRUS Serologic surveys conducted in Central and South America indicated that these viruses are common in bats, INFECTIOUS SALMON ANEMIA VIRUS with up to 30% of seropositive samples from some spe- Infectious salmon anemia is a serious disease of cultured cies. It is not known if bat influenza infection causes dis- Atlantic salmon (Salmo salar) that has caused significant ease in its natural hosts or whether the virus can be mortality among salmon farms in Northern Europe, eastern transmitted to other mammalian species. Canada, Maine, and Chile. Globally, economic losses due to infectious salmon anemia have been in the billions of BOVINE INFLUENZA D VIRUSES dollars. Because of the severity of the disease, the A new virus recovered from the respiratory tract of cattle European Union includes infectious salmon anemia in its in the United States was found to share many characteris- list of the most dangerous diseases of fish, and it is one of tics in common with human influenza type C viruses. just 10 virus infections of finfish that is reportable to the However, detailed genetic and antigenic analyses sup- World Organization for Animal Health (OIE). The causa- ported the creation by the International Committee on tive agent, infectious salmon anemia virus, is the only Taxonomy of Viruses (ICTV) of a new genus member of the genus Isavirus. The virus has a segmented Influenzavirus D with the bovine influenza D virus as its genome, with eight distinct segments that encode at least type species. These viruses were commonly detected in 10 proteins. Like influenza virus, these segments undergo samples collected from cattle with respiratory disease mutation, reassortment, or recombination events that gener- (bovine respiratory disease complex). Seroprevalence ate a large variety of strains. One of the surface proteins, often exceeded 80% in the study populations of cattle. the hemagglutinin-esterase (HE) encoded by segment 6 is Closely related viruses were isolated subsequently from responsible for receptor binding and receptor destroying cattle in China and virus was also detected in Europe. activity, whereas the fusion protein (F) encoded by seg- The virus infects swine, albeit with much lower frequency ment 5 is responsible for membrane fusion. Variants of than cattle. Genetic analysis identified two distinct infectious salmon anemia virus that cause disease in lineages of the surface glycoprotein hemagglutinin- farmed Atlantic salmon can be isolated in salmonid cell esterase-fusion gene. These virus lineages showed mini- lines and have deletions in a highly polymorphic region mal antigenic cross-reactivity but can reassort their genes (HPR) of the HE and an insertion or amino acid change with one another. Although the bovine influenza D near the cleavage site of the F protein. Wild-type strains of viruses can infect and be transmitted in other mammalian infectious salmon anemia virus lack these deletions in the species, eg, sheep and goats, its host range remains to be stalk region of the HE protein and are commonly referred determined as does its significance as a primary pathogen. to as HPR0 types. These wild-type strains typically infect Orthomyxoviridae Chapter | 21 409

the gill tissues, are generally of low virulence for salmon, Interestingly, however, the esterase activity of the HE and have proven resistant to isolation in fish cell lines. protein can dissolve the hemagglutination reaction with Such HPR0 (wild-type) strains can only be identified using fish erythrocytes, with the exception of those from a molecular assay targeting sequences in segment 6 or Atlantic salmon, the fish most severely affected by infec- other portions of the virus genome. While other wild sal- tious salmon anemia virus infection. This enhanced bind- monids have been shown to carry the virus, clinical disease ing is speculated to have a role in the severe anemia that outbreaks of infectious salmon anemia have only been develops in these fish. As with other orthomyxoviruses, observed in farmed Atlantic salmon. infectious salmon anemia virus has at least one protein— The hallmark of the disease is a profound anemia, 7i—that is able to block the innate antiviral defense sys- with hematocrit values less than 10% (normal value, tem. Infectious salmon anemia virus is a strong inducer of approximately 40%). The severe form of the disease in interferon response genes, but is insensitive to the actions infected fish is characterized by exophthalmia, pale gills, of the interferon response. hemorrhagic ascites and hemorrhagic liver necrosis, and The diagnosis of infectious salmon anemia virus infec- renal interstitial hemorrhage and tubular necrosis. tion is made on the basis of the characteristic gross and Histological lesions include filamental arteriole conges- histopathologic lesions, immunofluorescence staining of tion and lamellar telangiectasia (aneurisms) in the gills, tissue samples, isolation of virus using cell lines, and one diffuse sinusoidal congestion and erythrophagia in the of several RT-PCR assays. Because the presence of infec- spleen, and multifocal regions of congestion and hemor- tious salmon anemia virus has regulatory consequences, rhage in the pyloric ceca. In the highly sensitive farmed the tests and testing protocols that are acceptable for an salmon, viremia develops, with the virus targeting blood official diagnosis may vary, but RT-PCR tests should cells, endothelial cells, and macrophage-like cells. become the standard because of their sensitivity and rapid Initially endemic among wild salmonids in the North turnaround time. Antibody tests to detect exposure to Atlantic Ocean, wild-type (HPR0) strains of infectious infectious salmon anemia virus have been developed, but salmon anemia virus are maintained through both hori- have not been commonly applied. Control of infectious zontal and vertical transmission. However, like orthomyx- salmon anemia virus infections are complicated by the oviruses, wild-type infectious salmon anemia virus issue of dealing with fish in an open environment in infections in captive populations of Atlantic salmon which the virus may be circulating in native wild fish reared at high densities can give rise to mutants with without evidence of infection. Outbreaks have been man- much higher virulence that may spread rapidly to nearby aged through a combination of regulatory measures and farms. The outbreaks of disease that devastated the husbandry practices, including restricted movements of Chilean Atlantic salmon farming industry are thought to fish between farms, enforced slaughtering, use of all- be a result of the introduction of infectious salmon anemia inall-out programs at farms, and disinfection of slaugh- virus from Norway via contaminated eggs. terhouses and processing plants. Inactivated whole-virus There is great variation in the mortality produced by vaccine preparations provide partial protection, but their infectious salmon anemia virus infections, which reflects use is limited by difficulties associated with vaccine host resistance and strain variation in the virus. Using the delivery to large numbers of fish and development of HE gene, isolates of infectious salmon anemia virus can asymptomatic carrier infections of some vaccinated fish. be grouped into a North American lineage and a European lineage. These lineages can be further subdivided into genotypes based on HPR deletion patterns that OTHER ORTHOMYXOVIRUSES are associated with viral virulence. In experimental infections of Atlantic salmon with the most virulent virus Thogotovirus and Dhori viruses respectively contain six or strains, death commences within 1013 days after infec- seven gene segments, but both viruses are included in the tion and continues for a further 915 days, ultimately genus Thogotovirus. These viruses are transmitted between yielding mortality rates greater than 90%. Moderately vir- vertebrates by ticks, and likely have a global distribution. ulent viruses show mortality rates between 50% and 89% Thogotovirus infections of ticks, humans, and a variety and protracted killing, whereas low virulent strains have of animals have been described in Africa and southern mortality rates less than 50% in Atlantic salmon. Europe. The range and species tropism of Dhori virus is However, experimentally infected coho salmon (a dis- apparently similar, but also includes India and eastern tantly related Pacific species) were resistant to the devel- Europe. Dhori virus infection can cause a febrile illness opment of disease with isolates of infectious salmon and encephalitis in humans, but its significance as an ani- anemia virus from Europe and North America. As with mal pathogen is uncertain. Two additional orthomyxo- influenza A viruses, there does not appear to be a single viruses isolated over 40 years ago from ticks were recently viral protein that is always predictive of virulence. characterized, Upolu virus from Australia and Aransas Bay 410 PART | II Veterinary and Zoonotic Viruses

virus from the United States. These two viruses have mor- years ago. Although Quaranfil virus has been isolated phological, serologic, and genetic properties that support from febrile children, the impact of these viruses on ani- their classification within the genus Thogotovirus. A third mal and human health remains uncertain. However, two potential thogotovirus, named Bourbon virus, was recently more recently identified viruses, Cygnet River virus from isolated from a human in Kansas who subsequently died Australia and Wellfleet Bay virus from the United States, from the complications of the infection. were respectively linked to significant die-offs of captive Quaranjavirus is a newly recognized genus in the Muscovy ducks (Cairina moschata) and common eiders family Orthomyxoviridae. Quaranfil virus, Johnston Atoll (Somateria mollissima). Genetic analysis of these two virus, Jos virus, Araguari virus, and Lake Chad virus were viruses suggests they may be geographical variants of the isolated from ticks, birds, and mammals in Africa, Central same species. The involvement of ticks in the infection Asia, South America, and the Pacific islands over 45 cycle is as yet undefined. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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