CHAPTER 14

1. CHAPTER 14 Orthomyxovirus

2. Definitions of the virus: • The family Orthomyxoviridae includes viruses with genomes composed of several (six to eight) segments of single stranded RNA. The most important members of the family are the influenza viruses, which are included in three genera (Influenzavirus A, B, and C). Influenza viruses that are pathogenic to domestic animals are included in the

3. genus Influenzavirus A, whereas viruses in the two other genera (B and C) circulate continuously in humans. Influenza A viruses infrequently are transmitted from their animal hosts to humans. The emergence of the highly pathogenic Eurasian–African H5N1 virus in southeast Asia led to a worldwide surveillance program to track this virus because of its virulence to humans.

4. Cross-species infections occur sporadically between birds and mammals, including swine, horses, mink, marine mammals, and humans. Incursions of influenza A virus from wild birds into domestic poultry occur much more frequently. Domestic swine are considered an important intermediate (“bridge”) host in those areas of the world where there is frequent contact between birds and swine.

6. Whole-genome sequencing of influenza viruses is used for phylogenetic analyses. Comparison of the hemagglutinin (HA) and neuraminidase (NA) genes shows no host-specific lineages because of gene reassortment. Analyses of the matrix protein gene (M) show two major avian lineages, two equine lineages, two gull lineages, two swine lineages and a human lineage. Analysis of the PB1 gene segregates human viruses between the North American swine and Eurasian avian groups.

7. The Eurasian–African H5N1 viruses, as a result of circulation in a variety of wild and domestic birds including species such as passerine birds that are not usually infected with influenza virus. Although the separation of swine and domestic poultry production from aquatic birds can interrupt the evolutionary progression, such approaches are often difficult to enforce, and the potential threat of epidemics of human influenza that emerge from avian or mammalian reservoirs will persist.

8. Eurasian–African H5N1 influenza virus, this newly emergent virus had its origin in triple reassortant swine viruses (H1N1, H3N2, H1N2) that had been circulating in pigs in North America since the late 1990s. This novel virus was an additional reassortment that replaced two gene segments (NA and M) of the North American swine virus with the respective segments of the Eurasian swine virus.

10. The family Orthomyxoviridae comprises the genera Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus, and Isavirus. The name of the family is derived from the Greek myxa, meaning mucus, and orthos, meaning correct or right. Influenza A viruses are pathogens of horses, swine, humans, and domestic poultry, but they are the cause of sporadic or geographically limited infections and disease in mink, seals, whales, and dogs. Influenza B viruses are pathogens of humans.

11. The emergence of variant viruses depends not only on genetic drift—that is, point mutations (nucleotide substitutions, insertions, deletions), but also on genetic shift—that is, genomic segment reassortment. In the current classification system, influenza A viruses are categorized into 16 hemagglutinin (H) and 9 neuraminidase (N) types. In naming virus strains, influenza virus species or type (A, B, or C), host, geographic origin, strain number, year of isolation and hemagglutinin and neuraminidase subtypes are included.

12. Examples of virus strain names included A/equine/Miami/1/1963 (H3N8), the prototypic equine influenza virus 2. By 2008, 10 distinct genetic clades were recognized (0–9), and genetic diversity within individual clades that have several subclades have been recognized (2.1.3, 2.1.1, etc.). This clade nomenclature system readily identifies the genetic linkage of the virus. The strain nomenclature system will continue to be maintained in repositories and be used to identify sequences deposited into databases such as GenBank.

15. Orthomyxovirus virions are pleomorphic, often spherical but sometimes filamentous, and 80–120 nm in their smallest dimension. They consist of a lipid envelope with large spikes surrounding eight (genera Influenzavirus A, Influenzavirus B, and Isavirus), seven (genus Influenzavirus C), or six (genus Thogotovirus) helically symmetrical nucleocapsid segments of different sizes. For influenza A and B viruses, there are two kinds of glycoprotein spikes: homotrimers of the hemagglutinin protein and homotetramers of the neuraminidase protein.

16. complementarity. This feature is essential for RNA synthesis. Influenza C viruses lack neuraminidase, and have only one type of glycoprotein spike that consists of multifunctional hemagglutinin- esterase molecules. Virion envelopes are lined by the matrix protein, M1. on the inner surface of the lipid bilayer and are spanned by a small number of ion channels composed of tetramers of a second matrix protein, M2. Genomic segments consist of a molecule of viral RNA enclosed within a capsid composed

17. of helically arranged nucleoprotein. Three proteins that make up the viral RNA polymerase complex (PB1, PB2, and PA) are associated with the genomic RNA and nucleoprotein. The genome consists of six to eight segments of linear negative-sense, single-stranded RNA, and is 10–14.6 kb in overall size. The genome segments have non-translated regulatory sequences at both the 5’ and 3’ ends. The 13 nucleotides at the terminal 5’

18. end and 12 at the 3’ end are identical for each of the genomic segments and show partial inverted. Because of their lipid envelope, influenza viruses are sensitive to heat (56°C, 30 minutes), acid (pH 3), and lipid solvents, and are thus very labile under ordinary environmental conditions. Infectious influenza A virus has been recovered after 30 days in cold lake water. Influenza virions attach to cells via the binding of their activated hemagglutinin to sialic-acid-containing receptors on the plasma

19. membrane. Different cells have different linkages of N-acetyl neuraminic acid (sialic acid) to a galactose residue, and the hemagglutinin recognizes these different linkages, which in turn determine the host range of the virus. The gut epithelium of ducks has a receptor with an 2,3 linkage (SA2,3Gal), whereas the predominant influenza virus receptor in the upper respiratory tract of humans is an  2,6 linkage (SA2,6Gal). The binding affinity for

20. the SA2,6Gal glycan varies with the length of the oligosaccharide. Human-adapted H1 and H3 viruses show binding preference for long oligosaccharides present on epithelial cells in the upper respiratory tract, and mutations that affect this binding alter transmissibility. A single amino acid change in the hemagglutinin protein of the 1918 H1 Spanish flu virus at position 190 (E190D) changes binding preference from SA2,3Gal to

22. SA2,6Gal. The low pH of the endosome triggers a conformational change in the hemagglutinin protein such that the hydrophobic domain of the HA2 trimer mediates fusion of the viral envelope with the endosomal membrane, releasing the RNA + nucleoprotein + polymerase proteins (RNP) into the cytoplasm. While in the endosome, the ion channel M2 tetramer transports protons into the virus particle to enable dissociation of the binding of

23. M1 to the RNP complex, thus freeing it from the viral envelope. Amantadine and rimantadine inhibit virus replication by blocking the ion channel activity of M2. The synthesis of all RNA of influenza virus synthesis takes places in the nucleus of the cell. This requires that the RNP, because of its size, be actively transported into the nucleus. Nuclear localization signals on the nucleoprotein interact with the nuclear transport machinery of the cells to transport the RNP into the nucleus.

24. For all viruses with negative-sense RNA genomes, the genome of orthomyxoviruses serves two functions: as a template for the synthesis of messenger RNAs (mRNAs), and as a template for the synthesis of positive-sense replicative intermediate RNA, which is the template for progeny genomic RNA synthesis. Primary transcription involves an unusual phenomenon known as cap snatching: the viral endonuclease activity of PB2 cleaves the 5’- methylguanosine cap plus about 10–13

25. nucleotides from heterogeneous cellular mRNAs that are captured by PB2. These caps are then used by the virus as primers for transcription by the viral RNA polymerase (transcriptase; PB1). The viral mRNAs thus are capped and also become polyadenylated through transcription of five to seven “U” residues on the virion RNA. All orthomyxoviruses extend the coding capacity of their genomes by producing two proteins from one gene by using an alternative splicing

26. mechanism. Influenza A virus uses splicing for gene segments 7 (M1+M2) and 8 (NS1+NEP/NS2). Influenza B virus also uses overlapping stop and start codons to generate two protein products. In certain influenza A virus strains, a PB1-F2 protein of 87–90 amino acids is generated by a +1 reading frameshift. Viral protein synthesis occurs in the cytoplasm using cellular translation machinery. Early in infection, there is enhanced synthesis of nucleoprotein and NS1, whereas synthesis of hemagglutinin, neuraminidase, and matrix protein 1 is delayed.

27. Replication of genomic RNA segments requires the synthesis of full-length, positive-sense RNA intermediates which must lack 5’ caps and 3’-poly(A) tracts. Newly synthesized nucleoprotein binds to these RNAs, facilitating their use as templates. Late in infection, the matrix protein, M1, enters the nucleus and binds to nascent genomic RNA-nucleoprotein, thereby downregulating transcription and permitting export from the nucleus. NEP/NS2 bind the M1–RNP complexes, providing nuclear export signals and interaction

28. for replication of the virion RNA, and NS1 has been shown to inhibit the antiviral response triggered by the infection. Nucleoprotein are transported to the nucleus to interact with the RNA to initiate replication. With the nuclear export machinery that moves the RNP into the cytoplasm. Virions are formed by budding, incorporating M1 protein and nucleocapsids that have aligned below patches on the plasma membrane in which hemagglutinin, neuraminidase, and

29. matrix protein M2 have been inserted. In polarized cells, influenza virus buds from the apical surface of the cell. The hemagglutinin and neuraminidase proteins each contain transmembrane domains that associate with areas of the membrane enriched for sphingolipids and cholesterol that are designated lipid rafts. That segment-specific packaging signals are contained in the initial protein coding region of each RNA segment. This mechanism provides

30. specificity to the packaging process. As virions bud, the neuraminidase spikes (peplomers) facilitate the “pinching off” and release of virions. In poultry, replication is predominantly within the respiratory tract, but it also can occur in the intestinal tract, suggesting transmission may be by either ingestion or inhalation. The hemagglutinin protein of influenza A viruses is synthesized as a single polypeptide designated HA0. The hemagglutinin protein had to be cleaved post-translationally for the virus

31. to be infectious, which established a clear link between cleavability of hemagglutinin and virulence. The avian viruses that were highly pathogenic or high-pathogenicity avian influenza (HPAI) had several basic amino acids at the hemagglutinin cleavage site or long insertions of amino acids, whereas those low- pathogenicity avian influenza (LPAI) viruses contain a single arginine at a short cleavage site.

32. The proteases that are capable of cleaving at the single arginine are tissue restricted. In birds and mammals, epithelial cells within the respiratory and gastrointestinal tracts contain trypsin-like enzymes which can cleave. Virus is produced in a non-infectious form and activation occurs extracellularly. In addition, certain respiratory bacteria, including normal flora, can secrete proteases that cleave the hemagglutinin of influenza A viruses. For HPAI, there are several basic amino acids

33. at the cleavage site, which expands the range of cells capable of producing infectious virus, because cleavage can be mediated by the ubiquitous family of endopeptidase furins that are located in the trans-Golgi network. The hemagglutinin is cleaved intracellularly and fully infectious virions are released from infected cells without any requirement for the extracellular activation that is necessary for LPAI virus strains. The HPAI viruses are not maintained in wild bird reservoirs, but arise following mutation in

34. the hemagglutinin cleavage site of LPAI viruses. Dimeric NS1 binds to double-stranded RNA, which is a potent interferon inducer. The NS1 mutants are lethal to mice lacking interferon response genes, but infection is restricted in normal mice. The PB2 protein is a key component of the RNA transcription and replication process of influenza viruses, and may exert an important role in determining virulence and host range. The specific amino acid at residue 627 defines whether influenza A viruses are highly virulent

35. to mice. Introduction of a PB1 gene from a pathogenic virus into swine influenza virus increased the virulence of the reassortant. A novel protein that was generated by a +1 frameshift was mapped to the PB1 gene. Virulence of influenza viruses clearly can be multifactorial. Host range is determined by receptor specificity and cleavability of the hemagglutinin protein, as well as the activity of the PB2 protein. The PB1-F2 protein apparently contributes to the virulence phenotype of

36. individual viruses, and NS1—and other proteins—interfere with innate host defenses. The influenza in chickens known as “fowl plague”. A very large epizootic centered in the Pennsylvania in 1983–1984, which at the time cost US $60 million to control. Avian influenza viruses are categorized, for international trade issues, as of either high or low pathogenicity. The definitions are: 1. For the purposes of international trade, avian influenza(AI) in its notifiable form [notifiable

37. avian influenza(NAI)] is an infection of poultry caused byany influenza A virus of the H5 or H7 subtypes or byany AI virus with an intravenous pathogenicity index(IVPI) greater than 1.2 (or at least75% mortality) as described below. a. HPNAI viruses have an IVPI in 6-week-old chickens greater than 1.2 or, as an alternative, cause at least 75% mortality in 4- to 8-week-old chickens infected intravenously. H5 and H7 viruses which do not have an IVPI of greater than 1.2 or cause less than 75% mortality in an

38. intravenous lethality test should be sequenced to determine whether multiple basic amino acids are present at the cleavage site of the hemagglutinin molecule (HA0); if the amino acid motif is similar to that observed for other HPNAI isolates, the isolate being tested should be considered as HPNAI; b. LPNAI are all influenza A viruses of H5 and H7 subtype that are not HPNAI viruses. Because all outbreaks of HPAI have been caused by H5 or H7 viruses, and H5 or H7

39. LPAI virus in a commercial rearing facility can possibly to mutate to HPAI virus. Because all outbreaks of HPAI have been caused by H5 or H7 viruses, and H5 or H7 LPAI virus in a commercial rearing facility can possibly to mutate to HPAI virus. Highly virulent strains of avian influenza A virus cause sudden death without prodromal symptoms. If birds survive for more than 48 hours, there is a cessation of egg laying, respiratory distress, lacrimation, sinusitis, diarrhea, edema of the head, face and neck,

40. and cyanosis of unfeathered skin, particularly the comb and wattles. Birds may show nervous signs such as tremors of the head and neck, inability to stand, and torticollis. The LPAI viruses may also cause considerable losses. Clinical signs in chickens may be exacerbated by concurrent infections, the use of live-attenuated virus vaccines, or environmental stress. Avian influenza virus is shed in high concentrations in the feces of wild birds, and

41. can survive for long periods in cold water. The virus is introduced into susceptible flocks periodically by interspecies transmission—that is, between chickens and from wild birds, especially wild ducks. Avian influenza viruses have been isolated from imported caged birds, such passerine and psittacine birds are not natural reservoirs and they become infected after exposure to infected domestic and captive ducks. Live markets may be critical to the epidemiology of influenza virus infections.

42. Pathogenic marker is the amino acid sequence at the cleavage site of the hemagglutinin protein. Changes in the amino acid sequence alter the rate of cleavage of the protein and the virulence. Most LPAI viruses have a single basic amino acid (arginine) at the cleavage site, with a glycosylation site that shields the cleavage site. Elimination of the glycosylation site, changes in the amino acids to basic ones, insertions that open the cleavage site, and deletions that shift basic amino acids to the cleavage site all change cleavability and alter pathogenicity.

43. There also is variation in the susceptibility or resistance of different bird species to individual HPAI virus strains. The virus replication occurs in the intestinal tract and the respiratory tract. In virulent HPAI virus strains, there is viremia and multifocal lymphoid and visceral organ necrosis that result in pancreatitis, myocarditis, myositis, and encephalitis. Laboratory testing involves RT-PCR assay to detect the matrix protein (M) gene, as this is conserved. Samples positive are tested for

44. specific H5 and H7 genes. If samples are H5 or H7 positive, sequence analysis is to determine the properties of the cleavage site. If several basic amino acids are detected, then regulatory action is taken. Virus isolation is used for in- vivo pathogenicity tests. Virus is best isolated from cloacal swabs (wild birds and poultry) and tracheal swabs (poultry). Specimens are inoculated into the allantoic cavity of 10–11-day-old embryonating eggs, or on to MDCK cells, and the presence of virus is indicated by hemagglutinating activity using

45. chorioallantoic or cell culture fluids and chicken red blood cells. Flocks infected with HPAI viruses are depopulated, to prevent spread to other facilities and to wild birds in the environment. Surveillance is used to monitor for the presence of H5 and H7 LPAI viruses in domestic poultry. These LPAI virus strains cause some production losses, but the concern is that continued passage of the virus in domestic poultry can select for more virulent viruses.

46. Vaccination has not been used to control outbreaks of HPAI in most developed countries, because of the potentially negative impact on their ability to conduct international trade, which requires that expensive surveillance programs be instituted to identify any infected birds within the vaccinated population. The H5N1 transmission to humans was linked to contact with infected poultry, and a few instances are person-to-person spread, but only amongst individuals within close proximity to one another.

47. Although the highly pathogenic H5N1 virus has the requisite basic amino acids at the cleavage site, this property has not enhanced human-to-human transmission and the virus also does not replicate well in pigs.