Evolution of the A/Chicken/Pennsylvania/83 (H5N2) influenza virus

The epidemiological features of the H5N2 outbreak of influenza in poultry were studied by sequencing the HA genes of several viruses isolated during the epidemic. Comparison of the nucleotide sequences of the HA genes indicated there was a single introduction of virulent virus. The variation rate (silent mutations) in the HA gene of the virulent Ck/Penn virus was 9.0 or 14.4% per 10 years depending on the viruses compared and was similar to that in H3 HA gene of human influenza A virus. The virulent and avirulent viruses isolated after October 1983 were derived from a common ancestoral virus and the virulent virus did not supersede the avirulent virus, instead, the virulent and avirulent viruses coexisted and evolved separately during the course of the epidemic. The evolutionary changes in the HA of H5N2 viruses that occurred during the epidemic permitted us to establish that a virus (A/Chick/Washington/84) that was isolated 8 months after the last H5N2 virus had been isolated from poultry in Pennsylvania belonged to the family of potentially dangerous H5N2 viruses and was a direct descendent of the virus that spread to Maryland and Virginia. All of the virulent Ck/Penn viruses retained the amino acid changes at residues 13 and 69 in the HA.     

The neuraminidases of the virulent and avirulent A/Chicken/Pennsylvania/83 (H5N2) influenza A viruses: sequence and antigenic analyses

To define the sequence changes that occurred in an avian influenza virus neuraminidase (NA) during the evolution of virulence, we have studied the NA of the virulent and avirulent A/Chick/Penn/83 (H5N2) influenza viruses. A comparison of the deduced amino acid sequence from these viruses shows that the virulent strain, which evolved from the avirulent by the accumulation of point mutations (Bean et al., 1985), acquired four amino acid changes in the NA: one in the transmembrane segment, one in the stalk, and two in the head. A comparison of the deduced amino acid sequences with those of the human N2 NAs indicates a 20-amino acid deletion in the stalk of the Chick/Penn/83 NA. Antigenic analysis of the NAs from the avirulent and virulent Chick/Penn/83 virus shows they are antigenically very closely related, but can be distinguished with two monoclonal antibodies at a site which probably involves at least one of the amino acid changes in the NA head. Antigenic analysis also shows the Chick/Penn/83 NAs are closely related to the NAs of other N2 avian influenza viruses isolated between 1965 and 1984, supporting previous studies which indicate a relative antigenic stability of the NA among avian N2 influenza viruses. The Chick/Penn/83 NAs are the first N2 NA genes of an avian virus to be sequenced. These NAs are antigenically closely related to the 1957 human N2 NAs, and show a high degree of amino acid sequence homology with the prototype 1957 human N2 NA. These data give further support to the view that the 1957 human H2N2 viruses were at least partially derived from an avian source.     

Changes in the hemagglutinin molecule of influenza type A (H3N2) virus associated with increased virulence for mice

Summary. The H3N2 influenza virus A/Philippines/82 (Phil82) and its bovine serum-resistant mutant, Phil82/BS, were used to investigate factors that influence virulence of influenza virus for mice. Phil82/BS, which lacks the high-mannose oligosaccharide at residue 165 of the hemagglutinin (HA) molecule, was found to replicate to a much higher titer in mouse lung than the parent Phil82, and had acquired lethality for mice. Further adaptation of Phil82/BS by sequential lung passage in mice yielded a strain of greater virulence, Phil82/BS/ML10, in which a change at residue 246 of HA resulted in loss of a second potential glycosylation site. Phil82 is highly sensitive to neutralization in vitro by murine serum- and lung-associated mannose-binding lectins (collectins). Characterization of the two mutant viruses indicated that resistance to murine collectins can account for the enhanced virulence of Phil82/BS but not for the further increase in virulence of Phil82/BS/ML10. Evidence is presented that residue 246 is not in fact glycosylated in Phil82/BS HA, nor presumably in the parent Phil82 virus. The HA molecule of Phil82/BS/ML10 displayed functional differences from Phil82/BS, including a change in the optimum pH of fusion and a minor change in receptor-binding specificity, which may allow improved efficiency of replication in the mouse lung. Received June 27, 1996 Accepted August 8, 1996     

Morphological changes in BHK-21 cells infected with S/N (H2N1) influenza virus

Summary Morphological changes developing in BHK-21 cells infected with the S/N virus (a recombinant of A₂/Singapore and A/NWS viruses), at the approximate m.o.i. of 1 TCD₅₀ per cell, were studied by electron microscopy. Substantial alterations were observed in both the nucleus and cytoplasm. Changes in the nuclei were first detected eight hours after infection. The nucleoli substance was reduced and dense inclusions consisting of fibrillar structures 40–60 Å in diameter were formed. The greatest number of inclusions and their distribution over the whole nuclear area were found 24 hours after infection. Electron-dense inclusions were also detected in the cytoplasm. They were identical in shape and size with those observed in the nuclei but they consisted of fibrils 35–40 Å in diameter. These inclusions were rare eight hours after infection. Their number increased with the incubation period, reaching a maximum 24 hours after infection. Six hours after infection filamentous structures 80–90 Å in diameter were observed in the cytoplasm. It is assumed that they represent viral ribonucleoprotein. They were at first (hour 6) distributed diffusely through the whole cytoplasm and were later (hour 8–10) found mainly near the cytoplasmic membrane. Twenty four hours after the infection the filamentous structures were abundant both in the central part of the cytoplasm and in the proximity of the cellular membrane. Sometimes they formed dense agglomerations 2000–4200 Å in width. Maturation of the virion took place at the plasma membrane and in cytoplasmic vacuoles in whose proximity the filamentous structures were found. The particles either budded directly from the smooth cell membranes or from processes formed on the cell surface. Numerous rod-like particles were observed.     

Mutations at the cleavage site of the hemagglutinin after the pathogenicity of influenza virus A/chick/Penn/83 (H5N2)

Six variants that form plaques in chick embryo cells in the absence of trypsin have been isolated from the apathogenic avian influenza virus A/chick/Pennsylvania/1/83 (H5N2). Unlike the wild-type, the plaque variants contain a hemagglutinin that is cleaved in chick embryo cells and MDCK cells. The variants differ also from the wild-type in their pathogenicity for chickens. Nucleotide sequence and oligosaccharide analysis of the hemagglutinin have revealed that, unlike natural isolates with increased pathogenicity (Y. Kawaoka et al., 1984, Virology 139, 303-316; Y. Kawaoka and R. G. Webster, 1985, Virology 146, 130-137), the variants obtained in vitro have retained an oligosaccharide at asparagine 11 that is believed to interfere with the cleavage site of the wild-type. However, all variants showed mutations in the hemagglutinin resulting in an increased number of basic groups at the cleavage site. These observations demonstrate that masking of the cleavage site by an oligosaccharide is overcome by an enhancement of the basic charge at the cleavage site.     

Molecular changes associated with the transmission of avian influenza a H5N1 and H9N2 viruses to humans.

In order to identify molecular changes associated with the transmission of avian influenza A H5N1 and H9N2 viruses to humans, the internal genes from these viruses were compared to sequences from other avian and human influenza A isolates. Phylogenetically, each of the internal genes of all sixteen of the human H5N1 and both of the H9N2 isolates were closely related to one another and fell into a distinct clade separate from clades formed by the same genes of other avian and human viruses. All six internal genes were most closely related to those of avian isolates circulating in Asia, indicating that reassortment with human strains had not occurred for any of these 18 isolates. Amino acids previously identified as host-specific residues were predominantly avian in the human isolates although most of the proteins also contained residues observed previously only in sequences of human influenza viruses. For the majority of the nonglycoprotein genes, three distinct subgroups could be distinguished on bootstrap analyses of the nucleotide sequences, suggesting multiple introductions of avian virus strains capable of infecting humans. The shared nonglycoprotein gene constellations of the human H5N1 and H9N2 isolates and their detection in avian isolates only since 1997 when the first human infections were detected suggest that this particular gene combination may confer the ability to infect humans and cause disease. J. Med. Virol. 66:107-114, 2002. Published 2002 Wiley-Liss, Inc.     

Heterogeneity of A/Singapore/1/57 (H2N2) influenza virus.

The population of A/Singapore/1/57 (H2N2) influenza virus was found to contain two types of infectious particle. "Normal" virions with a diameter from 100-130 nm, a buoyant density in sucrose of 1.21 g/cm3 and a sedimentation coefficient 770 S represented about 90 percent of the population. In addition, a considerable amount of larger particles (up to 1000 nm) with a buoyant density in sucrose of 1.18 g/cm3 and a sedimentation coefficient of over 1000 S were present.     

Molecular characterization and pathogenicity of swine influenza H9N2 subtype virus A/swine/HeBei/012/2008/(H9N2)

The H9N2 subtype influenza virus (IV) is a remarkable member of the influenza A viruses because it can infect not only chickens, ducks and pigs, but also humans. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. To further understand the genetic characteristics and evolution, we investigated the source and molecular characteristics of the H9N2 subtype swine influenza virus (SIV), and observed its pathogenicity in BALB/c mice. The BALB/c mice were inoculated intranasally with 100 median mouse infectious dose of A/swine/HeBei/012/2008/(H9N2) viruses to observe the pathogenicity. The HA, NP, NA and M gene were cloned, sequenced and phylogenetically analyzed with related sequences available in GenBank. The infected mice presented with inactivity, weight loss and laboured respiration, while the pathological changes were characterized by diffuse alveolar damage in the lung. The nucleotide and deduced amino acid sequence of HA, NP, NA and M gene was similar with that of A/chicken/Hebei/4/2008(H9N2). The HA protein contained 6 glycosylation sites and the motif of HA cleavage site was PARSSR GLF, which is characteristic of low pathogenic IV. In the HA, NP, M and NA gene phylogenetic trees, the isolate clustered with A/chicken/Hebei/4/2008(H9N2). The isolate possibly came from A/chicken/Hebei/4/2008(H9N2) and was partially varied during its cross-species spread.     

Replication and pathogenesis associated with H5N1, H5N2, and H5N3 low-pathogenic avian influenza virus infection in chickens and ducks

A comparative study examining replication and disease pathogenesis associated with low-pathogenic H5N1, H5N2, or H5N3 avian influenza virus (AIV) infection of chickens and ducks was performed. The replication and pathogenesis of highly pathogenic AIV (HPAIV) has received substantial attention; however, the behavior of low-pathogenic AIVs, which serve as precursors to HPAIVs, has received less attention. Thus, chickens or ducks were inoculated with an isolate from a wild bird [A/Mute Swan/MI/451072/06 (H5N1)] or isolates from chickens [A/Ck/PA/13609/93 (H5N2), A/Ck/TX/167280-4/02 (H5N3)], and virus replication, induction of a serological response, and disease pathogenesis were investigated, and the hemagglutinin and neuraminidase (NA) gene sequences of the isolates were determined. Virus isolated from tracheal and cloacal swabs showed that H5N1 replicated better in ducks, whereas H5N2 and H5N3 replicated better in chickens. Comparison of the NA gene sequences showed that chicken-adapted H5N2 and H5N3 isolates both have a deletion of 20 amino acids in the NA stalk region, which was absent in the H5N1 isolate. Histopathological examination of numerous organs showed that H5N2 and H5N3 isolates caused lesions in chickens in a variety of organs, but to a greater extent in the respiratory and intestinal tracts, whereas H5N1 lesions in ducks were observed mainly in the respiratory tract. This study suggests that the H5N1, H5N2, and H5N3 infections occurred at distinct sites in chicken and ducks, and that comparative studies in different model species are needed to better understand the factors influencing the evolution of these viruses.     

Attenuated ts-recombinants of influenza A/USSR/77 (H1N1) virus obtained by crossing with the cold-adapted donor A/Leningrad/134/57 (H2N2) virus

Conditions of obtaining attenuated influenza virus recombinants by crossing of a cold-adapted donor with A (H1N1) influenza virus that reappeared in 1977 were studied. Temperature-sensitive recombinants suitable for intranasal immunization of adults with low titres of anti-haemagglutinin and anti-neuraminidase antibodies, and possessing sufficiently high immunogenicity were obtained by crossing of native parent strains and cross-reactivation techniques. It was confirmed that the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus variant is a prospective donor of attenuation for obtaining recombinants--candidates for live influenza vaccine strains.     

Molecular signatures of virulence in the PB1-F2 proteins of H5N1 influenza viruses

The PB1-F2 protein of influenza A viruses contributes to pathogenesis in animal models. Specific molecular signatures of virulence within PB1-F2 have been mapped for some functions. The 66S polymorphism may modulate interferon activity, and four C-terminal amino acids, 62L, 75R, 79R, and 82L, contribute to cytokine release and inflammatory responses in specific virus backgrounds. All available PB1-F2 sequences from H5N1 subtype influenza A viruses were analyzed. The majority (82.5%) of H5N1 sequences available in the Influenza Research Database code for PB1-F2 proteins with 4 or more of these virulence associated amino acids. Most of these are avian sequences from highly pathogenic strains isolated in Asia or Africa. The 66S polymorphism was uncommon (5.3% of sequences) but was found in association with the other 4 inflammatory amino acids in select highly pathogenic strains in Asia. These analyses suggest that if an H5N1 virus were to emerge as a pandemic strain, the PB1-F2 protein will be a contributor to pathogenesis. Research on the pathogenic effect of these signatures in an H5N1 background should be undertaken. Surveillance efforts should include sequencing of the PB1 gene segment and analysis for these molecular signatures to allow for the potential prioritization of resources during pandemic planning.     

Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?

The A/Chick/Penn/83 (H5N2) influenza virus that appeared in chickens in Pennsylvania in April 1983 and subsequently became virulent in October 1983, was examined for plaque-forming ability and cleavability of the hemagglutinin (HA) molecule. The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. In contrast, the virulent virus produced plaques and cleaved the HA precursor into HA1 and HA2 in the presence or absence of trypsin. The apparent molecular weight of the HA1 from the avirulent virus was higher than that from the virulent virus, but when the viruses were grown in the presence of tunicamycin, the molecular weights of HA were indistinguishable. Two of nine monoclonal antibodies to the HA of the avirulent virus indicate that there is at least one epitope on the HA that is different between the virulent and avirulent viruses. The amino acid sequences of the HAs from the two viruses were compared by sequencing their respective HA gene. The nucleotide sequence coding for the processed HA polypeptide contained 1641 nucleotides specifying a protein of 547 amino acids. The amino acid sequences of the virulent and avirulent viruses were indistinguishable through the connecting peptide region, indicating that the difference in cleavability of the H5 HA is not directly attributed to the amino acid sequence of the connecting peptide. Four of seven nucleotide changes resulted in amino acid changes at residues 13, 69, and 123 of HA1 and at residue 501 of the HA2 polypeptide. Since there were no deletions or insertions in the amino acid sequence of the virulent or avirulent viruses, the possibility exists that the difference in molecular weight is due to loss of a carbohydrate side chain in the virulent strain. The amino acid change in the virulent strain at residue 13 is the only mutation that could affect a glycosylation site and this is in the vicinity of the connecting peptide. It is postulated that the loss of this carbohydrate may permit access of an enzyme that recognizes the basic amino acid sequences and results in cleavage activation of the HA in the virulent virus.     

Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus.

Nucleotide sequences were determined for the RNA segments coding for proteins other than the hemagglutinin and neuraminidase of the A/Leningrad/134/57 (H2N2) wild-type (A/Len/wt) virus and its two cold-adapted (ca) and attenuated variants, A/Leningrad/134/17/57 (A/Len/17/ca) and A/Leningrad/134/47/57 (A/Len/47/ca) that are used in the U.S.S.R. in the preparation of reassortant live attenuated vaccines. Ten nucleotide differences were detected between the sequences of the A/Len/wt and A/Len/17/ca viruses; of these, eight were deduced to encode amino acid (aa) substitutions. One aa substitution each was predicted for the PB2, M1, M2, and NS2 proteins, whereas two aa substitutions each were predicted for the PB1, and PA proteins of the A/Len/17/ca virus. Four additional nucleotide changes were found in the genome of the A/Len/47/ca virus; three of these were detected to code for one additional aa substitution each for the PB2, PB1, and NP proteins.     

Genetic characterization of canine influenza A virus (H3N2) in Thailand

In January 2012, several clinical cases of dogs with flu-like symptoms, including coughing, sneezing, nasal discharge, and fever, were reported in a small-animal hospital located in Bangkok, Thailand. One influenza A virus was identified and characterized as an avian-like influenza virus H3N2. The virus was named A/canine/Thailand/CU-DC5299/12. A phylogenetic analysis indicated that the canine virus belonged to an avian Eurasian lineage and was genetically related to the canine influenza viruses H3N2 from China and Korea. This canine virus displays a unique genetic signature with two amino acid insertions in the NA protein, which is similar to the canine influenza viruses from eastern China (Zhejiang and Jiangsu). This study constitutes the first report of H3N2 canine influenza virus infection in a small-animal hospital in Thailand.