Genetics,Receptor Binding Property,and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.     

Molecular basis of efficient replication and pathogenicity of H9N2 avian influenza viruses in mice

H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.     

Phylogenetic diversity and genotypical complexity of H9N2 influenza A viruses revealed by genomic sequence analysis

H9N2 influenza A viruses have become established worldwide in terrestrial poultry and wild birds, and are occasionally transmitted to mammals including humans and pigs. To comprehensively elucidate the genetic and evolutionary characteristics of H9N2 influenza viruses, we performed a large-scale sequence analysis of 571 viral genomes from the NCBI Influenza Virus Resource Database, representing the spectrum of H9N2 influenza viruses isolated from 1966 to 2009. Our study provides a panoramic framework for better understanding the genesis and evolution of H9N2 influenza viruses, and for describing the history of H9N2 viruses circulating in diverse hosts. Panorama phylogenetic analysis of the eight viral gene segments revealed the complexity and diversity of H9N2 influenza viruses. The 571 H9N2 viral genomes were classified into 74 separate lineages, which had marked host and geographical differences in phylogeny. Panorama genotypical analysis also revealed that H9N2 viruses include at least 98 genotypes, which were further divided according to their HA lineages into seven series (A-G). Phylogenetic analysis of the internal genes showed that H9N2 viruses are closely related to H3, H4, H5, H7, H10, and H14 subtype influenza viruses. Our results indicate that H9N2 viruses have undergone extensive reassortments to generate multiple reassortants and genotypes, suggesting that the continued circulation of multiple genotypical H9N2 viruses throughout the world in diverse hosts has the potential to cause future influenza outbreaks in poultry and epidemics in humans. We propose a nomenclature system for identifying and unifying all lineages and genotypes of H9N2 influenza viruses in order to facilitate international communication on the evolution, ecology and epidemiology of H9N2 influenza viruses.     

Mouse-adapted H9N2 influenza A virus PB2 protein M147L and E627K mutations are critical for high virulence

H9N2 influenza viruses have been circulating worldwide in multiple avian species and have repeatedly infected humans to cause typical disease. The continued avian-to-human interspecies transmission of H9N2 viruses raises concerns about the possibility of viral adaption with increased virulence for humans. To investigate the genetic basis of H9N2 influenza virus host range and pathogenicity in mammals, we generated a mouse-adapted H9N2 virus (SD16-MA) that possessed significantly higher virulence than wide-type virus (SD16). Increased virulence was detectable after 8 sequential lung passages in mice. Five amino acid substitutions were found in the genome of SD16-MA compared with SD16 virus: PB2 (M147L, V250G and E627K), HA (L226Q) and M1 (R210K). Assessments of replication in mice showed that all of the SD16-MA PB2, HA and M1 genome segments increased virus replication; however, only the mouse-adapted PB2 significantly increased virulence. Although the PB2 E627K amino acid substitution enhanced viral polymerase activity and replication, none of the single mutations of mouse adapted PB2 could confer increased virulence on the SD16 backbone. The combination of M147L and E627K significantly enhanced viral replication ability and virulence in mice. Thus, our results show that the combination of PB2 amino acids at position 147 and 627 is critical for the increased pathogenicity of H9N2 influenza virus in mammalian host.     

The Potential of Avian H1N1 Influenza A Viruses to Replicate and Cause Disease in Mammalian Models

H1N1 viruses in which all gene segments are of avian origin are the most frequent cause of influenza pandemics in humans; therefore, we examined the disease-causing potential of 31 avian H1N1 isolates of American lineage in DBA/2J mice. Thirty of 31 isolates were very virulent, causing respiratory tract infection; 22 of 31 resulted in fecal shedding; and 10 of 31 were as pathogenic as the pandemic 2009 H1N1 viruses. Preliminary studies in BALB/cJ mice and ferrets showed that 1 of 4 isolates tested was more pathogenic than the pandemic 2009 H1N1 viruses in BALB/cJ mice, and 1 of 2 strains transmitted both by direct and respiratory-droplet contact in ferrets. Preliminary studies of other avian subtypes (H2, H3, H4, H6, H10, H12) in DBA/2J mice showed lower pathogenicity than the avian H1N1 viruses. These findings suggest that avian H1N1 influenza viruses are unique among influenza A viruses in their potential to infect mammals.     

Reassortant H9N2 influenza viruses containing H5N1-like PB1 genes isolated from black-billed magpies in Southern China

H9N2 influenza A viruses have become endemic in different types of terrestrial poultry and wild birds in Asia, and are occasionally transmitted to humans and pigs. To evaluate the role of black-billed magpies (Pica pica) in the evolution of influenza A virus, we conducted two epidemic surveys on avian influenza viruses in wild black-billed magpies in Guangxi, China in 2005 and characterized three isolated black-billed magpie H9N2 viruses (BbM viruses). Phylogenetic analysis indicated that three BbM viruses were almost identical with 99.7 to 100% nucleotide homology in their whole genomes, and were reassortants containing BJ94-like (Ck/BJ/1/94) HA, NA, M, and NS genes, SH/F/98-like (Ck/SH/F/98) PB2, PA, and NP genes, and H5N1-like (Ck/YN/1252/03, clade 1) PB1 genes. Genetic analysis showed that BbM viruses were most likely the result of multiple reassortments between co-circulating H9N2-like and H5N1-like viruses, and were genetically different from other H9N2 viruses because of the existence of H5N1-like PB1 genes. Genotypical analysis revealed that BbM viruses evolved from diverse sources and belonged to a novel genotype (B46) discovered in our recent study. Molecular analysis suggested that BbM viruses were likely low pathogenic reassortants. However, results of our pathogenicity study demonstrated that BbM viruses replicated efficiently in chickens and a mammalian mouse model but were not lethal for infected chickens and mice. Antigenic analysis showed that BbM viruses were antigenic heterologous with the H9N2 vaccine strain. Our study is probably the first report to document and characterize H9N2 influenza viruses isolated from black-billed magpies in southern China. Our results suggest that black-billed magpies were susceptible to H9N2 influenza viruses, which raise concerns over possible transmissions of reassortant H9N2 viruses among poultry and wild birds.     

Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K

H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P ?+ ?M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P ?+ ?M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.     

Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin

A novel H1N1 influenza virus emerged in 2009 (pH1N1) to become the first influenza pandemic of the 21st century. This virus is now cocirculating with highly pathogenic H5N1 avian influenza viruses in many parts of the world, raising concerns that a reassortment event may lead to highly pathogenic influenza strains with the capacity to infect humans more readily and cause severe disease. To investigate the virulence of pH1N1-H5N1 reassortant viruses, we created pH1N1 (A/California/04/2009) viruses expressing individual genes from an avian H5N1 influenza strain (A/Hong Kong/483/1997). Using several in vitro models of virus replication, we observed increased replication for a reassortant CA/09 virus expressing the hemagglutinin (HA) gene of HK/483 (CA/09-483HA) relative to that of either parental CA/09 virus or reassortant CA/09 expressing other HK/483 genes. This increased replication correlated with enhanced pathogenicity in infected mice similar to that of the parental HK/483 strain. The serial passage of the CA/09 parental virus and the CA/09-483HA virus through primary human lung epithelial cells resulted in increased pathogenicity, suggesting that these viruses easily adapt to humans and become more virulent. In contrast, serial passage attenuated the parental HK/483 virus in vitro and resulted in slightly reduced morbidity in vivo, suggesting that sustained replication in humans attenuates H5N1 avian influenza viruses. Taken together, these data suggest that reassortment between cocirculating human pH1N1 and avian H5N1 influenza strains will result in a virus with the potential for increased pathogenicity in mammals.     

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.     

Virulence and genetic compatibility of polymerase reassortant viruses derived from the pandemic (H1N1) 2009 influenza virus and circulating influenza A viruses

Gene mutations and reassortment are key mechanisms by which influenza A virus acquires virulence factors. To evaluate the role of the viral polymerase replication machinery in producing virulent pandemic (H1N1) 2009 influenza viruses, we generated various polymerase point mutants (PB2, 627K/701N; PB1, expression of PB1-F2 protein; and PA, 97I) and reassortant viruses with various sources of influenza viruses by reverse genetics. Although the point mutations produced no significant change in pathogenicity, reassortment between the pandemic A/California/04/09 (CA04, H1N1) and current human and animal influenza viruses produced variants possessing a broad spectrum of pathogenicity in the mouse model. Although most polymerase reassortants had attenuated pathogenicity (including those containing seasonal human H3N2 and high-pathogenicity H5N1 virus segments) compared to that of the parental CA04 (H1N1) virus, some recombinants had significantly enhanced virulence. Unexpectedly, one of the five highly virulent reassortants contained a A/Swine/Korea/JNS06/04(H3N2)-like PB2 gene with no known virulence factors; the other four had mammalian-passaged avian-like genes encoding PB2 featuring 627K, PA featuring 97I, or both. Overall, the reassorted polymerase complexes were only moderately compatible for virus rescue, probably because of disrupted molecular interactions involving viral or host proteins. Although we observed close cooperation between PB2 and PB1 from similar virus origins, we found that PA appears to be crucial in maintaining viral gene functions in the context of the CA04 (H1N1) virus. These observations provide helpful insights into the pathogenic potential of reassortant influenza viruses composed of the pandemic (H1N1) 2009 influenza virus and prevailing human or animal influenza viruses that could emerge in the future.     

A single amino acid at the hemagglutinin cleavage site contributes to the pathogenicity and neurovirulence of H5N1 influenza virus in mice

H5 influenza viruses containing a motif of multiple basic amino acids at the hemagglutinin (HA) cleavage site (HACS) are highly pathogenic in chicken but display different virulence phenotypes in mammals. Previous studies have shown that multiple basic amino acids of H5N1 influenza virus are a prerequisite for lethality in mice. However, it remains unclear which specific residue at the cleavage site affects the pathogenicity of H5N1 in mammals. A comprehensive genetic analysis of the H5N1 HACS showed that residues at P6 (position 325, by H3 numbering) were the most polymorphic, including serine (S), arginine (R), deletion (*), glycine (G), and isoleucine (I). To determine whether a single residue at P6 could affect virulence, we introduced different mutations at P6 of an avirulent clade 7 H5N1 strain, rg325G, by reverse genetics. Among the recombinant viruses, the rg325S virus showed the highest cleavage efficiency in vitro. All these viruses were highly pathogenic in chicken but exhibited different virulences in mice. The rg325S virus exhibited the highest pathogenicity in terms of unrestricted organ tropism and neurovirulence. Remarkably, the HA-325S substitution dramatically increased the pathogenicity of H5N1 viruses of other clades, including clades 2.2, 2.3.2, and 2.3.4, indicating that this residue impacts genetically divergent H5N1 viruses. An analysis of predicted structures containing these mutations showed that the cleavage site loop with 325S was the most exposed, which might be responsible for the efficient cleavage and high virulence. Our results demonstrate that an amino acid substitution at the P6 cleavage site alone could modulate the virulence of H5N1 in mice.     

Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005

H9N2 influenza viruses have become established and maintain long-term endemicity in terrestrial poultry in Asian countries. Occasionally these viruses transmit to other mammals, including humans. Increasing epidemiological and laboratory findings suggest that quail may be an important host, as they are susceptible to different subtypes of influenza viruses. To better understand the role of quail in influenza virus ecology and evolution, H9N2 viruses isolated from quail during 2000 to 2005 were antigenically and genetically characterized. Our results showed that H9N2 viruses are prevalent year-round in southern China and replicate mainly asymptomatically in the respiratory tract of quail. Genetic analysis revealed that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail since 2000. Phylogenetic analyses demonstrated that most of the isolates tested were double- or multiple-reassortant variants, with four G1-like and 16 Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became predominant in quail since 2003, while multiple Ck/Bei-like genotypes were introduced into quail, wherein they incorporated G1-like gene segments, but none of them became established in this host. Those Ck/Bei-like reassortants generated in quail have then been introduced into other poultry. These complex interactions form a two-way transmission system between quail and other types of poultry. The present study provides evidence that H9N2 and H5N1 subtype viruses have also exchanged gene segments to generate currently circulating reassortants of both subtypes that have pandemic potential. Continuing influenza virus surveillance in poultry is critical to understanding the genesis and emergence of potentially pandemic strains in this region.     

H9N2 influenza viruses possessing H5N1-like internal genomes continue to circulate in poultry in southeastern China

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.     

Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.     

Clue to the molecular mechanism of virulence of highly pathogenic H5N1 avian influenza viruses isolated in 2004

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia since 2003. These viruses are highly lethal to birds and humans. Of the 74 confirmed human cases, 49 were fatal (as of Mar 30, 2005), raising concerns of a possible pandemic by these viruses. Despite the well-established pathogenicity of these viruses, the molecular mechanism for expressing such high virulence remains elusive. Thus, we examined the pathogenicity of the H5N1 viruses isolated in Vietnam in 2003-2004 using animal models (mouse, duck, and ferret). Viruses from humans were generally more pathogenic in mice and ferrets than those from birds. Indeed, one human isolate was even lethal to ferrets. The human isolate possessing Lys at amino acid position 627 of PB2 was more virulent than that possessing Glu at this position, underscoring the importance of Lys at this position 627 of PB2 for efficient growth in mammals.     

Detection and Genetic Characteristics of H9N2 Avian Influenza Viruses from Live Poultry Markets in Hunan Province,China

H9N2 avian influenza viruses (AIVs) are highly prevalent and of low pathogenicity in domestic poultry. These viruses show a high genetic compatibility with other subtypes of AIVs and have been involved in the genesis of H5N1, H7N9 and H10N8 viruses causing severe infection in humans. The first case of human infection with H9N2 viruses in Hunan province of China have been confirmed in November 2013 and identified that H9N2 viruses from live poultry markets (LPMs) near the patient’s house could be the source of infection. However, the prevalence, distribution and genetic characteristics of H9N2 viruses in LPMs all over the province are not clear. We collected and tested 3943 environmental samples from 380 LPMs covering all 122 counties/districts of Hunan province from February to April, 2014. A total of 618 (15.7%) samples were H9 subtype positive and 200 (52.6%) markets in 98 (80.3%) counties/districts were contaminated with H9 subtype AIVs. We sequenced the entire coding sequences of the genomes of eleven H9N2 isolates from environmental samples. Phylogenetic analysis showed that the gene sequences of the H9N2 AIVs exhibited high homology (94.3%-100%). All eleven viruses were in a same branch in the phylogenetic trees and belonged to a same genotype. No gene reassortment had been found. Molecular analysis demonstrated that all the viruses had typical molecular characteristics of contemporary avian H9N2 influenza viruses. Continued surveillance of AIVs in LPMs is warranted for identification of further viral evolution and novel reassortants with pandemic potential.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Distinct pathogenesis of hong kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses

In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian influenza viruses were analyzed for their pathogenicity in 6- to 8-week-old BALB/c mice. Both the avian and human HK H5 influenza virus isolates caused severe disease in mice, characterized by induced hypothermia, clinical signs, rapid weight loss, and 75 to 100% mortality by 6 to 8 days postinfection. Three of the non-HK-origin isolates caused no detectable clinical signs. One isolate, A/tk/England/91 (H5N1), induced measurable disease, and all but one of the animals recovered. Infections resulted in mild to severe lesions in both the upper and lower respiratory tracts. Most consistently, the viruses caused necrosis in respiratory epithelium of the nasal cavity, trachea, bronchi, and bronchioles with accompanying inflammation. The most severe and widespread lesions were observed in the lungs of HK avian influenza virus-infected mice, while no lesions or only mild lesions were evident with A/ck/Scotland/59 (H5N1) and A/ck/Queretaro/95 (H5N2). The A/ck/Italy/97 (H5N2) and the A/tk/England/91 (H5N1) viruses exhibited intermediate pathogenicity, producing mild to moderate respiratory tract lesions. In addition, infection by the different isolates could be further distinguished by the mouse immune response. The non-HK-origin isolates all induced production of increased levels of active transforming growth factor beta following infection, while the HK-origin isolates did not.     

Origin and molecular characterization of the human-infecting H6N1 influenza virus in Taiwan

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain’s pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.