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Pandemic influenza, typically caused by the reassortment of human and avian influenza viruses, can result in severe or fatal infections in humans. Timely identification of potential pandemic viruses must be a priority in influenza virus surveillance. However, the range of host species responsible for the generation of novel pandemic influenza viruses remains unclear. In this study, we conducted serological surveys for avian and human influenza virus infections in farmed mink and determined the susceptibility of mink to prevailing avian and human virus subtypes. The results showed that farmed mink were commonly infected with human (H3N2 and H1N1/pdm) and avian (H7N9, H5N6, and H9N2) influenza A viruses. Correlational analysis indicated that transmission of human influenza viruses occurred from humans to mink, and that feed source was a probable route of avian influenza virus transmission to farmed mink. Animal experiments showed that mink were susceptible and permissive to circulating avian and human influenza viruses, and that human influenza viruses (H3N2 and H1N1/pdm), but not avian viruses, were capable of aerosol transmission among mink. These results indicate that farmed mink could be highly permissive “mixing vessels” for the reassortment of circulating human and avian influenza viruses. Therefore, to reduce the risk of emergence of novel pandemic viruses, feeding mink with raw poultry by-products should not be permitted, and epidemiological surveillance of influenza viruses in mink farms should be urgently implemented.

H5N6 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4 not only exhibits unprecedented intercontinental spread in poultry, but can also cause serious infection in humans, posing a public health threat. Phylogenetic analyses show that 40% (8/20) of H5N6 viruses that infected humans carried H9N2 virus-derived internal genes. However, the precise contribution of H9N2 virus-derived internal genes to H5N6 virus infection in humans is unclear. Here, we report on the functional contribution of the H9N2 virus-derived matrix protein 1 (M1) to enhanced H5N6 virus replication capacity in mammalian cells. Unlike H5N1 virus-derived M1 protein, H9N2 virus-derived M1 protein showed high binding affinity for H5N6 hemagglutinin (HA) protein and increased viral progeny particle release in different mammalian cell lines. Human host factor, G protein subunit beta 1 (GNB1), exhibited strong binding to H9N2 virus-derived M1 protein to facilitate M1 transport to budding sites at the cell membrane. GNB1 knockdown inhibited the interaction between H9N2 virus-derived M1 and HA protein, and reduced influenza virus-like particles (VLPs) release. Our findings indicate that H9N2 virus-derived M1 protein promotes avian H5N6 influenza virus release from mammalian, in particular human cells, which could be a major viral factor for H5N6 virus cross-species infection., Author summary H9N2 avian influenza viruses (AIVs), through reassortment of their internal genes with other circulating AIVs subtypes (H5N6, H7N9, H10N8 and H10N3 viruses), are known to enable the resulting novel reassortants to infect humans. However, the precise roles of H9N2 virus-derived internal genes in human infection remain unknown. Here, we found that H9N2 virus-derived (but not H5N1 virus-derived) M1 protein showed strong interaction with host GNB1 protein to enhance M1 protein transport to budding sites at the cell membrane to promote progeny virus release, hence facilitating H5N6 reassortants to infect human cells.

Adaptation of PB2 protein is important for the establishment of avian influenza viruses in mammalian hosts. Here, we identify I292V as the prevalent mutation in PB2 of circulating avian H9N2 and pandemic H1N1 viruses. The same dominant PB2 mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses. In human cells, PB2-292V in H9N2 virus has the combined ability of conferring higher viral polymerase activity and stronger attenuation of IFN-β induction than that of its predecessor PB2-292I. IFN-β attenuation is accompanied by higher binding affinity of PB2-292V for host mitochondrial antiviral signalling protein, an important intermediary protein in the induction of IFN-β. In the mouse in vivo model, PB2-292V mutation increases H9N2 virus replication with ensuing increase in disease severity. Collectively, PB2-292V is a new mammalian adaptive marker that promotes H9N2 virus replication in mammalian hosts with the potential to improve transmission from birds to humans.

Domestic ducks are considered as the interface between wild aquatic birds and terrestrial poultry and play an important role in the transmission and evolution of avian influenza viruses (AIVs). However, the infectivity of H9N2 AIVs in different domestic duck species has not been systematically evaluated. Here we investigated the infectivity of various genotypes of chicken H9N2 AIVs in Pekin duck (Anas Platyrhynchos), Mallard duck (Anas Platyrhynchos) and Muscovy duck (Cairina Moschata) through intranasal inoculation. We found that Pekin ducks and Mallard ducks were generally resistant to chicken H9N2 virus infection, while Muscovy ducks were relatively susceptible to H9N2 AIVs. All the tested viruses were isolated from oropharynx, trachea and lung tissues of Muscovy ducks. Additionally, genotype 57 (G57) H9N2 AIVs, which was predominant in chickens since 2010, showed increased virus replication in this duck species, indicating an improved interspecies transmission ability of recent H9N2 viruses from chickens to ducks. Our results demonstrated the role of Muscovy ducks in the ecology of H9N2 AIVs. More attentions should be paid to this host during viral surveillances. Additionally, inactivated H9N2 vaccine may be unnecessarily used in Pekin and Mallard ducks.

H9N2 avian influenza viruses (AIVs) were prevailing in chickens, causing great economic losses and public health threats. In this study, turkey herpesviruses (HVT) was cloned as an infectious bacterial artificial chromosomes (BAC). Recombinant HVT (rHVT-H9) containing hemagglutinin (HA) gene from H9N2 virus were constructed via galactokinase (galK) selection and clustered regularly interspaced short palindromic repeats/associated 9 (CRISPR/Cas9) gene editing system. The recombinant rHVT-H9 showed no difference with parent HVT in plague morphology and virus replication kinetics. H9 protein expression of rHVT-H9 could be detected by western blot and indirect immunofluorescence assay (IFA) in vitro and in vivo. Immunization with rHVT-H9 could induce robust humoral and cellular immunity in chickens. In the challenge study, no chicken shed H9N2 virus from oropharynx and cloaca, and no H9N2 virus was found in viscera in vaccination groups. The result suggests that rHVT-H9 provides effective protection against H9N2 AIV in chickens.

During 2012–2017, we collected throat swabs from dogs in China to characterize canine influenza virus (CIV) A(H3N2) isolates. A new antigenically and genetically distinct CIV H3N2 clade possessing mutations associated with mammalian adaptation emerged in 2016 and replaced previously circulating strains. This clade probably poses a risk for zoonotic infection.

H9N2 avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to cocirculating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain a sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016 to 2017 was by far the largest, but the mechanistic explanation behind the scale of infection is not clear. Here, we found that just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new subclades, changed antigenicity, and increased their prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus subclades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into cocirculating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C and increased viral replication in the upper and lower respiratory tracts of infected mice, which could account for the sharp increase in human cases of H7N9 virus infection in the 2016–2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential. IMPORTANCE Avian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to cocirculating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role as a donor of new subclade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (genotype 72 [G72]) with enhanced infectivity in humans during the 2016–2017 H7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of the prevalence and variants of H9N2 virus in chicken flocks is necessary for the detection of zoonotic mutations.

Avian influenza viruses (AIVs) are zoonotic viruses that exhibit a range of infectivity and severity in the human host. Severe human cases of AIV infection are often accompanied by neurological symptoms; however, the factors involved in the infection of the central nervous system (CNS) are not well known. In this study, we discovered that the avian-like sialic acid (SA)–α2,3-galactose (α2,3-Gal) receptor is highly presented in mammalian (human and mouse) brains. In the generation of a mouse-adapted neurotropic H9N2 AIV (SD16-MA virus) in BALB/c mice, we identified key adaptive mutations in its hemagglutinin (HA) and polymerase basic protein 2 (PB2) genes that conferred viral replication ability in the mouse brain. The SD16-MA virus showed binding affinity for the avian-like SA–α2,3-Gal receptor, enhanced viral RNP polymerase activity, and increased viral protein production and transport that culminated in elevated progeny virus production and severe pathogenicity. We further established that host fragile X mental retardation protein (FMRP), a highly expressed protein in the brain that is physically associated with viral nucleocapsid protein (NP) to facilitate RNP assembly and export, was an essential host factor for the neuronal replication of neurotropic AIVs (H9N2, H5N1, and H10N7 viruses). Our study identified a mechanistic process for AIVs to acquire neurovirulence in mice. IMPORTANCE Infection of the CNS is a serious complication of human cases of AIV infections. The viral and host factors associated with the neurovirulence of AIV infections are not well understood. We identified and functionally characterized specific changes in the viral HA and PB2 genes of a mouse-adapted neurotropic avian H9N2 virus responsible for enhanced virus replication in neuronal cells and pathogenicity in mice. Importantly, we showed that host FMRP was a crucial host factor that was necessary for neurotropic AIVs (H9N2, H5N1, and H10N7 viruses) to replicate in neuronal cells. Our findings provide insights into the pathogenesis of the neurovirulence of AIV infections.

Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infections due to host restriction. Current research about host restriction focuses mainly on the barriers of cell membrane, nuclear envelope, and host proteins; whether microRNAs (miRNAs) play a role in host restriction is largely unknown. In this study, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two Sus scrofa (ssc; swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and that P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222. Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial (NPTr) cells and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus. IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication; however, the role of miRNAs in host species specificity is unclear. Here, we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells is modulated by NF-κB P65 phosphorylation in response to AIV infection but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.

Equine-origin H3N8 and avian-origin H3N2 canine influenza viruses (CIVs) prevalent in dogs are thought to pose a public health threat arising from intimate contact between dogs and humans. However, our understanding of CIV virulence is still limited. Influenza A virus PA-X is a fusion protein encoded in part by a +1 frameshifted open reading frame (X-ORF) in segment 3. The X-ORF can be translated in full-length (61-amino-acid) or truncated (41-amino-acid) form. Genetic analysis indicated that the X-ORFs of equine H3N8 and avian H3N2 influenza viruses encoded 61 amino acids but were truncated after introduction into dogs. To determine the effect of PA-X truncation on the biological characteristics of CIVs, we constructed four recombinant viruses on H3N8 and H3N2 CIV backgrounds bearing truncated or full-length PA-Xs. We observed that truncation of PA-X increased growth of both H3N8 and H3N2 CIVs in MDCK cells and suppressed expression from cotransfected plasmids in MDCK cells. Furthermore, truncation of PA-X enhanced viral pathogenicity in dogs, as shown by aggravated clinical symptoms and histopathological changes, increased viral replication in the respiratory system, and prolonged virus shedding. Additionally, CIVs with truncated PA-Xs were transmitted more efficiently in dogs. Global gene expression profiling of the lungs of infected dogs revealed that differentially expressed genes were mainly associated with inflammatory responses, which might contribute to the pathogenicity of PA-X-truncated CIVs. Our findings revealed that truncation of PA-X might be important for the adaptation of influenza viruses to dogs. IMPORTANCE Epidemics of equine-origin H3N8 and avian-origin H3N2 influenza viruses in canine populations are examples of successful cross-species transmission of influenza A viruses. Genetic analysis showed that the PA-X genes of equine H3N8 or avian H3N2 influenza viruses were full-length, with X-ORFs encoding 61 amino acids; however, those of equine-origin H3N8 or avian-origin H3N2 CIVs were truncated, suggesting that PA-X truncation occurred after transmission to dogs. In this study, we extended the PA-X genes of H3N8 and H3N2 CIVs and compared the biological characteristics of CIVs bearing different lengths of PA-X. We demonstrated that for both H3N8 and H3N2 viruses, truncation of PA-X increased virus yields in MDCK cells and enhanced viral replication, pathogenicity, and transmission in dogs. These results might reflect enhanced suppression of host gene expression and upregulation of genes related to inflammatory responses. Collectively, our data partially explain the conservation of truncated PA-X in CIVs.

The retinoic acid-inducible gene I (RIG-I) receptor senses cytoplasmic viral RNA and activates type I interferons (IFN-I) and downstream antiviral immune responses. How RIG-I binds to viral RNA and how its activation is regulated remains unclear. Here, using IFI16 knockout cells and p204-deficient mice, we demonstrate that the DNA sensor IFI16 enhances IFN-I production to inhibit influenza A virus (IAV) replication. IFI16 positively upregulates RIG-I transcription through direct binding to and recruitment of RNA polymerase II to the RIG-I promoter. IFI16 also binds to influenza viral RNA via its HINa domain and to RIG-I protein with its PYRIN domain, thus promoting IAV-induced K63-linked polyubiquitination and RIG-I activation. Our work demonstrates that IFI16 is a positive regulator of RIG-I signalling during influenza virus infection, highlighting its role in the RIG-I-like-receptor-mediated innate immune response to IAV and other RNA viruses, and suggesting its possible exploitation to modulate the antiviral response.

Background H9N2 influenza viruses continuously circulate in multiple avian species and are repeatedly transmitted to humans, posing a significant threat to public health. To investigate the adaptation ability of H9N2 avian influenza viruses (AIVs) to mammals and the mutations related to the host switch events, we serially passaged in mice two H9N2 viruses of different HA lineages — A/Quail/Hong Kong/G1/97 (G1) of the G1-like lineage and A/chicken/Shandong/ZB/2007 (ZB) of the BJ/94-like lineage —and generated two mouse-adapted H9N2 viruses (G1-MA and ZB-MA) that possessed significantly higher virulence than the wide-type viruses. Finding ZB-MA replicated systemically in mice. Genomic sequence alignment revealed 10 amino acid mutations coded by 4 different gene segments (PB2, PA, HA, and M) in G1-MA compared with the G1 virus and 23 amino acid mutations in 5 gene segments (PB1, PA, HA, M, and NS) in ZB-MA compared to ZB virus, indicating that the mutations in the polymerase, HA, M, and NS genes play critical roles in the adaptation of H9N2 AIVs to mammals, especially, the mutations of M1-Q198H and M1-A239T were shared in G1-MA and ZB-MA viruses. Additionally, several substitutions showed a higher frequency in human influenza viruses compared with avian viruses. Conclusions Different lineages of H9N2 could adapt well in mice and some viruses could gain the ability to replicate systemically and become neurovirulent. Thus, it is essential to pay attention to the mammalian adaptive evolution of the H9N2 virus.

The most commonly utilized inactivated influenza vaccines (IIVs) are usually deficient in cross immunity against divergent viruses. On the other hand, live attenuated influenza vaccines (LAIVs) are proved to be more effective in cross-protective immunity. We previously developed a H9N2 LAIV and verified its effective protection against a broad spectrum of H9N2 strains. In the present study, we evaluated its cross-immunity against H5N2 virus, a representative subtype of currently predominant H5 highly pathogenic avian influenza viruses. All chickens vaccinated with this LAIV survived from challenge of H5N2 virus in a lethal dose, and viral proliferation was effectively inhibited, as well as pathological lesions. Vaccination of this LAIV significantly activated H5N2-reactive CD4+ and CD8+ T cells in lungs. These LAIV-activated cross-reactive T cells expanded robustly following H5N2 exposure, and the increasing tendency was temporally correlated with viral clearance. Besides cellular immunity, factors of humoral immunity also play a contributing role in cross-immunity. Passively transferring H9N2 LAIV anti-serum resulted in 100% survival rate to chickens against H5N2 virus. Within components of the anti-serum, cross-binding IgGs against nucleoprotein (NP) of H5N2 virus were found of a contributing role in the cross immunity. These results indicate that this H9N2 LAIV represents a promising strategy for controlling highly pathogenic H5N2 virus in chickens. The cross immunity was partly attributed to LAIV activated H5N2-cross-reactive T cells and partly attributed to cross-binding IgGs against NP.

In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses. IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

Influenza H9N2 subtype viruses and their reassortants (such as H7N9) are posing increasing threats to birds and humans in China. During 2009–2013, multiple novel subtype viruses with H9N2 original genes emerged in China. Yet, the genetic evolution of H9N2 viruses in various host organisms in China has not been systematically investigated since 2009. In the present study, we performed large-scale sequence analysis of H9N2 viral genomes from public databases, representing the spectrum of viruses isolated from birds, mammals and humans in China from 1994 to 2013, and updated the clade classification for each segment. We identified 117 distinct genotypes in 730 H9N2 viruses. We analyzed the sequences of all eight segments in each virus and found three important time points: the years 2000, 2006 and 2010. In the periods divided by these years, genotypic diversity, geographic distribution and host range changed considerably. Genotypic diversity fluctuated greatly in 2000 and 2006. Since 2010, a single genotype became predominant in poultry throughout China, and the eastern coastal region became the newly identified epidemic center. Throughout their 20-year prevalence in China, H9N2 influenza viruses have emerged and adapted from aquatic birds to chickens. The minor avian species and wild birds exacerbated H9N2 genotypes by providing diversified genes, and chickens were the most prevalent vector in which the viruses evolved and expanded their prevalence. It is the necessity for surveillance and disease control on live-bird markets, poultry farms and wild-bird habitats in China.

H9 subtype avian influenza virus (AIV) and infectious bronchitis virus (IBV) are major pathogens circulating in poultry and have resulted in great economic losses due to respiratory disease and reduced egg production. As similar symptoms are elicited by the two pathogens, it is difficult for their differential diagnosis. So far, no reverse transcription-polymerase chain reaction (RT-PCR) assay has been found to differentiate between H9 AIV and IBV in one reaction. Therefore, developing a sensitive and specific method is of importance to simultaneously detect and differentiate H9 AIV and IBV. In this study, a duplex RT-PCR (dRT-PCR) was established. Two primer sets target the hemagglutinin (HA) gene of H9 AIV and the nucleocapsid (N) gene of IBV, respectively. Specific PCR products were obtained from all tested H9 AIVs and IBVs belonging to the major clades circulating in China, but not from AIVs of other subtypes or other infectious avian viruses. The sensitivity of the dRT-PCR assay corresponding to H9 AIV, IBV and mixture of H9 AIV and IBV were at a concentration of 1×101, 1.5×101 and 1.5×101 50% egg infective doses (EID50) mL−1, respectively. The concordance rates between the dRT-PCR and virus isolation were 99.1 and 98.2%, respectively, for detection of samples from H9N2 AIV or IBV infected chickens, while the concordance rate was 99.1% for detection of samples from H9N2 AIV and IBV co-infected chickens. Thus, the dRT-PCR assay reported herein is specific and sensitive, and suitable for the differential diagnosis of clinical infections and surveillance of H9 AIVs and IBVs.

Swine influenza A virus (swine IAV) circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like (EA) H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription (RT)-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets (outer and inner) were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit (PFU) mL −1 which was over 10 4 PFU mL −1 for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Influenza virus hemagglutinin (HA) plays an important role in viral antigenicity, replication and host range. However, few amino acid positions in HA were reported to play multiple functions in both viral antigenicity and replication. In the present study, through analyzing the amino acid sequences of H9N2 avian influenza viruses (AIVs) isolated from China, we identified a multi-functional substitution of D200N in HA1 protein. Firstly, the substitution of D200N changed the antigenicity of H9N2 AIVs. Secondly, the D200N increased the HA cleavage efficiency and reduced acid and thermal stability of HA protein, which triggered viral-endosomal membrane fusion whereby promoted the release of viral genome into the host cytoplasm. Finally, residue 200-N increased the replication of H9N2 viruses in both chicken embryo fibroblast (CEF) cells and chicken embryonated eggs. In summary, the D200N substitution is a newly identified antigenicity and replication determinant of H9N2 AIVs, which should be paid more attention during surveillance.

Hemagglutinin (HA) cleavage is critical for virulence of influenza viruses. The amino acid residue at the P6 position of the HA cleavage site (HACS) has been shown to be most variable and to have a direct correlation with the cleavage efficiency and pathogenicity of H5N1 avian influenza viruses (AIVs) in mammals. Among these amino acid variants, serine has been associated with the highest virulence in mammals, and its detection may serve as an indicator for H5N1 AIVs with high pathogenicity and potential public risk. We developed a rapid detection method based on reverse-transcription (RT)-PCR and pyrosequencing to detect a mutation at the HACS that is associated with increased pathogenicity of H5N1 AIVs in mammals. Herein, we provide a specific, sensitive, and reliable method for rapid detection of one of the virulence determinants associated with increased pathogenicity of H5N1 AIVs in mammals.

H9N2 influenza viruses have been circulating worldwide in multiple avian species, and regularly infect pigs and humans. Recently, a novel protein, PA-X, produced from the PA gene by ribosomal frameshifting, was demonstrated to be an antivirulence factor in pandemic 2009 H1N1, highly pathogenic avian H5N1 and 1918 H1N1 viruses. However, a similar role of PA-X in the prevalent H9N2 avian influenza viruses has not been established. In this study, we compared the virulence and cytopathogenicity of H9N2 WT virus and H9N2 PA-X-deficient virus. Loss of PA-X in H9N2 virus reduced apoptosis and had a marginal effect on progeny virus output in human pulmonary adenocarcinoma (A549) cells. Without PA-X, PA was less able to suppress co-expressed GFP in human embryonic kidney 293T cells. Furthermore, absence of PA-X in H9N2 virus attenuated viral pathogenicity in mice, which showed no mortality, reduced progeny virus production, mild-to-normal lung histopathology, and dampened proinflammatory cytokine and chemokine response. Therefore, unlike previously reported H1N1 and H5N1 viruses, we show that PA-X protein in H9N2 virus is a pro-virulence factor in facilitating viral pathogenicity and that the pro- or antivirulence role of PA-X in influenza viruses is virus strain-dependent.

The H10 subtype of avian influenza viruses (AIVs) circulates globally in wild birds and poultry, and this subtype has been shown to be increasingly prevalent in China. Among the various H10 viruses, H10N7 AIVs have caused repeated mammal and human infections. To investigate their genetic adaptation in mammals, we generated a mouse-adapted avian H10N7 variant (A/mallard/Beijing/27/2011-MA; BJ27-MA) which exhibited increased virulence in mice compared to wild-type virus and acquired neurotropism. Sequencing showed the absence of the widely recognized mammalian adaptation markers of E627K and D701N in PB2 in the mouse-adapted strain; instead, five amino acid mutations were identified: E158G and M631L in PB2; G218E in haemagglutinin (H3 numbering); and K110E and S453I in neuraminidase (NA). The neurovirulence of the BJ27-MA virus necessitated the combined presence of the PB2 and NA mutations. Mutations M631L and E158G of PB2 and K110E of NA were required to mediate increased virus replication and severity of infection in mice and mammalian cells. PB2-M631L was functionally the most dominant mutation in that it strongly upregulated viral polymerase activity and played a critical role in the enhancement of virus replication and disease severity in mice. K110E mutation in NA, on the other hand, significantly promoted NA enzymatic activity. These results indicate that the novel mutations in PB2 and NA genes are critical for the adaptation of H10N7 AIV in mice, and they could serve as molecular signatures of virus transmission to mammalian hosts, including humans.

Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China. IMPORTANCE We recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens.

Virological and serological surveys have documented that H1N1/2009, avian-origin canine H3N2 (cH3N2), seasonal human-origin H3N2 (hH3N2), and equine-origin H3N8 influenza viruses are consistently circulating in dogs. In the present study, a multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was developed for simultaneous detection and differentiation of these influenza viruses. Four primer sets were designed to target the hemagglutinin genes of H1N1/2009, cH3N2, hH3N2, and H3N8 canine influenza viruses (CIVs). This mRT-PCR assay demonstrated high specificity and sensitivity for the four CIV subtypes. Additionally, mRT-PCR results obtained from 420 clinical samples were consistent with those obtained by the conventional virus isolation method. Our mRT-PCR assay is reliable for clinical diagnosis and rapid identification of CIVs.

The novel H7N9 influenza virus, which has caused severe disease in humans in China, is a reassortant with surface genes derived from influenza viruses in wild birds. This highlights the importance of monitoring influenza viruses in these hosts. However, surveillance of influenza virus in wild birds remains very limited in China. In this study, we isolated four H4N6 avian influenza viruses (AIVs) from mallard ducks in Beijing Wetland Park, which is located on the East Asia-Australasia migratory flyway. The gene segments of these Chinese H4N6 viruses were closest to AIVs in wild birds from Mongolia or the Republic of Georgia, indicating the interregional AIV gene flow among these countries. All of our isolates belonged to a novel genotype that was different from other H4N6 viruses isolated in China. We further evaluated the virulence and transmission of two representative H4N6 strains in mammalian models. We found that both of these H4N6 viruses replicated efficiently in mice without adaptation. Additionally, these two strains had a 100% transmission rate in guinea pigs via direct contact, but they had not acquired respiratory droplet transmissibility. These results reveal the potential threat to human health of H4N6 viruses in migratory birds and the need for enhanced surveillance of AIVs in wild birds.

We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus's 50% mouse lethal dose (LD 50 ), by almost 1,000-fold.

Two Viruses to Bind Structural studies of two different H7N9 influenza viruses isolated from humans—A/Shanghai/1/2013 and A/Anhui/1/2013—which have different amino acid sequences in the receptor binding site, provide data indicating that the virus is in transition with respect to host adaptation. The Shanghai virus was one of the first isolated in humans that binds avian receptor glycans with high affinity, but binds poorly to human receptors. However, the later Anhui isolates can bind both avian and human receptors at high affinity. Shi et al. (p. 243 , published online 5 September) show that four hydrophobic mutations contribute to acquisition of affinity for the human receptor by the virus hemagglutinin (HA) and confirm this effect in binding studies with virus particles. Further comparison of a mutant H7N9 A/Anhui/1/2013 HA with the bird flu H5N1 virus revealed the significance of some of the naturally occurring changes observed in circulating H7N9 viruses, which helps to explain how these viruses have been able to cause many severe human infections in a short time.

To prevent H9N2 avian influenza virus infection in chickens, a long-term vaccination program using inactivated vaccines has been implemented in China. However, the protective efficacy of inactivated vaccines against antigenic drift variants is limited, and H9N2 influenza virus continues to circulate in vaccinated chicken flocks in China. Therefore, developing a cross-reactive vaccine to control the impact of H9N2 influenza in the poultry industry remains a high priority. In the present study, we developed a live cold-adapted H9N2 influenza vaccine candidate (SD/01/10-ca) by serial passages in embryonated eggs at successively lower temperatures. A total of 13 amino acid mutations occurred during the cold-adaptation of this H9N2 virus. The candidate was safe in chickens and induced robust hemagglutination-inhibition antibody responses and influenza virus–specific CD4+ and CD8+ T cell immune responses in chickens immunized intranasally. Importantly, the candidate could confer protection of chickens from homologous and heterogenous H9N2 viruses. These results demonstrated that the cold-adapted attenuated H9N2 virus would be selected as a vaccine to control the infection of prevalent H9N2 influenza viruses in chickens.

Given the present extensive co-circulation in pigs of Eurasian avian-like (EA) swine H1N1 and 2009 pandemic (pdm/09) H1N1 viruses, reassortment between them is highly plausible but largely uncharacterized. Here, experimentally co-infected pigs with a representative EA virus and a pdm/09 virus yielded 55 novel reassortant viruses that could be categorized into 17 genotypes from Gt1 to Gt17 based on segment segregation. Majority of novel reassortants were isolated from the lower respiratory tract. Most of reassortant viruses were more pathogenic and contagious than the parental EA viruses in mice and guinea pigs. The most transmissible reassortant genotypes demonstrated in guinea pigs (Gt2, Gt3, Gt7, Gt10 and Gt13) were also the most lethal in mice. Notably, nearly all these highly virulent reassortants (all except Gt13) were characterized with possession of EA H1 and full complement of pdm/09 ribonucleoprotein genes. Compositionally, we demonstrated that EA H1-222G contributed to virulence by its ability to bind avian-type sialic acid receptors, and that pdm/09 RNP conferred the most robust polymerase activity to reassortants. The present study revealed high reassortment compatibility between EA and pdm/09 viruses in pigs, which could give rise to progeny reassortant viruses with enhanced virulence and transmissibility in mice and guinea pig models.

The PA-X protein is a fusion protein incorporating the N-terminal 191 amino acids of the PA protein with a short C-terminal sequence encoded by an overlapping ORF (X-ORF) in segment 3 that is accessed by + 1 ribosomal frameshifting and this X-ORF exists in either full length or a truncated form (either 61-or 41-condons). Genetic evolution analysis indicates that all swine influenza viruses (SIVs) possessed full-length PA-X prior to 1985, but since then SIVs with truncated PA-X have gradually increased and become dominant, implying that truncation of this protein may contribute to the adaptation of influenza virus in pigs. To verify this hypothesis, we constructed PA-X extended viruses in the background of a “triple-reassortment” H1N2 SIV with truncated PA-X and evaluated their biological characteristics in vitro and in vivo. Compared with full-length PA-X, SIV with truncated PA-X had increased viral replication in porcine cells and swine respiratory tissues, along with enhanced pathogenicity, replication and transmissibility in pigs. Furthermore, we found that truncation of PA-X improved the inhibition of IFN-I mRNA expression. Hereby, our results imply that truncation of PA-X may contribute to the adaptation of SIV in pigs.

Avian influenza viruses (AIVs) and Newcastle disease viruses (NDVs) co-circulate in the poultry population in China. These viruses cause repeated disease outbreaks that exhibit similar clinical symptoms and epidemiological patterns. H5 and H9 influenza viruses are the major pathogens infecting poultry stocks. Recently, H3 AIV (one of the main subtypes in waterfowl) has become endemic in chickens. A multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was designed for simultaneous detection and differentiation of avian H3, H5, H9 subtype AIVs and NDVs. Four primer sets were evaluated, three of which specifically targeted the hemagglutinin genes of H3, H5 and H9 AIVs, while the other targeted the NDV fusion gene. The sensitivity and specificity of the mRT-PCR assay was determined. The assay detected the major clades or genotypes of all of the reference AIVs and NDVs currently circulating in China. In addition, the mRT-PCR results obtained from screening 380 clinical swabs and 12 experimental tracheal samples were consistent with those obtained using conventional virus isolation methods. The mRT-PCR assay was established successfully for the detection and differentiation of avian H3, H5, and H9 subtype AIVs and NDVs. The method should, therefore, provide a valuable diagnostic tool for these infections.

Despite the long-term vaccination programs implemented in China, H9N2 avian influenza viruses (AIVs) continue to persist in chicken populations, even in vaccinated flocks. We previously demonstrated that H9N2 AIV isolated from chickens in China also underwent antigenic drift and evolved into distinct antigenic groups (C, D and E). To understand whether antigenic drift of viruses away from the vaccine strain partially contributed to the circulation of H9N2 AIV in China, we evaluated the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 AIV. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group C viruses, but not those of the more recent groups D and E. Vaccinated chickens, even those with vaccine-induced HI titers of 1:1024, shed virus after being infected with A/chicken/Shandong/ZB/2007, a representative virus of antigenic group D. Genetic analysis showed that the representative viruses of antigenic groups D and E possessed greater numbers of amino acid substitutions in the hemagglutinin protein compared to the vaccine strain and the antigenic group C virus, and many of which were located in antigenic sites. Our results indicated that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection, and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection. Furthermore, the avian influenza vaccination policy also needs to be re-assessed, and increased veterinary biosecurity on farms, rather than vaccine application alone, should be implemented to prevent and control avian influenza.

Evidence of H1N1/2009 influenza virus infection was identified in two domestic dogs in China in November 2009. Virus isolation and sequence analysis of all eight genes of the two isolates showed that they were related closely to the H1N1/2009 influenza virus circulating in humans, indicating that they were probably acquired from humans. To determine the pathogenicity and transmissibility of H1N1/2009 influenza virus in dogs, experimental infection and transmission were performed. Inoculated dogs were able to shed virus in nasal secretions, but symptoms were very mild. Uninoculated dogs were co-mingled to determine the transmissibility of the isolate, and one of three exposed dogs was shown to develop infection. The present findings indicate that human H1N1/2009 can infect dogs, but is transmitted inefficiently between dogs.

We previously demonstrated that H9N2 subtype avian influenza viruses (AIVs) isolated from 1994 to 2008 evolved into distinct antigenic groups (C, D, and E) and then underwent antigenic drift from commercial vaccines, causing a country-wide outbreak during 2010-2013. In this study, H9N2 AIVs isolated from chickens during 2009-2013 were antigenically analyzed by performing hemagglutination inhibition and neutralization assays using a panel of polyclonal antibodies. Our findings confirmed the antigenic drift of recent H9N2 viruses from the commercial vaccine and showed that most of these antigenic variants form a novel HI antigenic group, F, with a few belonging to groups D and E. Slight antigenic variation was observed in group F viruses. Genetic analysis of amino acid sequences deduced from hemagglutinin (HA) gene sequences indicated that 9 of 15 mutations predominant in the 2009-2013 viruses can be mapped to known antigenic sites, which might be responsible for the novel antigenicity of group F. These antigenic changes make it necessary to modify the influenza vaccine to ensure efficient protection. A vaccine candidate, Ck/HeB/YT/10, was selected and provided significant protection against viruses from different antigenic groups in terms of reduction in virus shedding, suggesting broad cross-reactivity. Taken together, our results indicate that the H9N2 chicken influenza viruses in China have evolved from distinct antigenic groups into a novel group F that became dominant during the country-wide outbreak and now seems to be undergoing new antigenic divergence. Systematic surveillance and timely updating of vaccine strains are important for viral prevention and control in the future.

The PA-X protein, arising from ribosomal frameshift during PA translation, was recently discovered in influenza A virus (IAV). The C-terminal domain ‘X’ of PA-X proteins in IAVs can be classified as full-length (61 aa) or truncated (41 aa). In the main, avian influenza viruses express full-length PA-X proteins, whilst 2009 pandemic H1N1 (pH1N1) influenza viruses harbour truncated PA proteins. The truncated form lacks aa 232–252 of the full-length PA-X protein. The significance of PA-X length in virus function remains unclear. To address this issue, we constructed a set of contemporary influenza viruses (pH1N1, avian H5N1 and H9N2) with full and truncated PA-X by reverse genetics to compare their replication and host pathogenicity. All full-length PA-X viruses in human A549 cells conferred 10- to 100-fold increase in viral replication and 5–8 % increase in apoptosis relative to corresponding truncated PA-X viruses. Full-length PA-X viruses were more virulent and caused more severe inflammatory responses in mice. Furthermore, aa 233–252 at the C terminus of PA-X strongly suppressed co-transfected gene expression by ∼50 %, suggesting that these terminal 20 aa could play a role in enhancing viral replication and contribute to virulence.

Worldwide dissemination of reassortant variants of H5 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses has posed a great threat to the poultry industry. Here, we systematically characterized the H5N2, H5N6 and H5N8 influenza viruses in poultry and compared them with those of previous clade 2.3.4 H5N1 virus. All the three H5 subtype reassortants caused systematic infection in ducks, and exhibited efficient direct transmission in ducks. All of them were highly pathogenic in chickens; however, the H5 reassortants have reduced virulence compared to the parental H5N1 virus. Antigenicity analysis revealed that the current vaccines that are widely used in China may fail to confer protection against the H5 reassortants.

Background The close contact between cats and humans poses a threat to public health because of the potential zoonotic transmission of influenza viruses to humans. Therefore, we examined the seroprevalence of pandemic H1N1/09, canine H3N2, and human H3N2 viruses in pet cats in northern China from 2010 to 2014. Finding Of 1794 serum samples, the seropositivity rates for H1N1/09, canine H3N2, and human H3N2 were 5.7%, 0.7%, and 0.4%, respectively. The seropositivity rate for H1N1/09 in cats was highest in 2010 (8.3%), and then declined continuously thereafter. Cats older than 10 years were most commonly seropositive for the H1N1/09 virus. Conclusions Our findings emphasize the need for continuous surveillance of influenza viruses in cats in China.

The emergence of human infection with a novel H7N9 influenza virus in China raises a pandemic concern. Chicken H9N2 viruses provided all six of the novel reassortant’s internal genes. However, it is not fully understood how the prevalence and evolution of these H9N2 chicken viruses facilitated the genesis of the novel H7N9 viruses. Here we show that over more than 10 y of cocirculation of multiple H9N2 genotypes, a genotype (G57) emerged that had changed antigenicity and improved adaptability in chickens. It became predominant in vaccinated farm chickens in China, caused widespread outbreaks in 2010–2013 before the H7N9 viruses emerged in humans, and finally provided all of their internal genes to the novel H7N9 viruses. The prevalence and variation of H9N2 influenza virus in farmed poultry could provide an important early warning of the emergence of novel reassortants with pandemic potential.

Outbreaks of pandemic H1N1 2009 (pH1N1) in turkeys have been reported in several countries. Co-infection of pH1N1 and avian H9N2 influenza viruses in turkeys provide the opportunity for their reassortment and novel reassortant viruses might further be transmitted to other avian species. However, virulence and transmission of those reassortant viruses in poultry remain unclear. In the present study, we generated 16 single-gene reassortant influenza viruses including eight reassortants on the pH1N1 background by individual replacement with a corresponding gene segment from H9N2 and eight reassortants on the H9N2 background replaced individually with corresponding gene from pH1N1 and characterized reassortants viruses in turkeys and chickens. We found that the pH1N1 virus dramatically increased its infectivity and transmissibility in turkeys and chickens after introducing any gene (except for PB2) from H9N2 virus and H9N2 virus acquired single gene (except for HA) of pH1N1 almost did not influence its replication and transmission in turkeys and chickens. Additionally, 13 reassortant viruses transmitted from turkeys to chickens. Our results indicate that turkeys and chickens are susceptible to pH1N1-H9N2 reassortant viruses and mixing breeding of different avian species would facilitate the transmission of these reassortant viruses.

H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2NS1230 and H9N2NS1237). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madin–Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2NS1230 and H9N2NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.

Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo . Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. IMPORTANCE We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility.

Since December 2013, at least three cases of human infections with H10N8 avian influenza virus have been reported in China, two of them being fatal. To investigate the epidemic potential of H10N8 viruses, we examined the receptor binding property of the first human isolate, A/Jiangxi-Donghu/346/2013 (JD-H10N8), and determined the structures of its haemagglutinin (HA) in complex with both avian and human receptor analogues. Our results suggest that JD-H10N8 preferentially binds the avian receptor and that residue R137-localized within the receptor-binding site of HA-plays a key role in this preferential binding. Compared with the H7N9 avian influenza viruses, JD-H10N8 did not exhibit the enhanced binding to human receptors observed with the prevalent H7N9 virus isolate Anhui-1, but resembled the receptor binding activity of the early-outbreak H7N9 isolate (Shanghai-1). We conclude that the H10N8 virus is a typical avian influenza virus.

Highly pathogenic avian influenza H5N1 virus clades 2.3.4, 2.3.2, and 7 are the dominant cocirculating H5N1 viruses in poultry in China. However, humans appear to be clinically susceptible mostly to the 2.3.4 virus clade. Here, we demonstrated that A549 cells and human macrophages infected with clade 2.3.4 viruses produced significantly more viruses than those infected with the other two clades. Likewise, clade 2.3.4-infected macrophages caused the most severe cellular damage and strongest proinflammatory response.

The D222N hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pdmH1N1) infections. The impact of this mutation was investigated in vitro and in vivo using recombinant viruses. The recombinant D222N virus grew to significantly lower viral titers than the WT in A549 but not in MDCK cells. A dose-dependent glycan array analysis with the D222N virus showed a modest increase in the binding avidity to human-like (α-2,6 sialylated glycan) receptors and avian-like (α-2,3 sialylated glycan) receptors than the WT virus. The D222N HA mutation resulted in slight weight loss, lower lung titers, inflammatory cytokines and alveolar inflammation in mice than the WT virus. This may or may not be associated with severe clinical outcomes reported in humans.

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.

Background Acute respiratory distress syndrome (ARDS) induced by pandemic 2009 H1N1 influenza virus has been widely reported and was considered the main cause of death in critically ill patients with 2009 H1N1 infection. However, no animal model has been developed for ARDS caused by infection with 2009 H1N1 virus. Here, we present a mouse model of ARDS induced by 2009 H1N1 virus. Methodology Principal Findings Mice were inoculated with A/swine/Shandong/731/2009 (SD/09), which was a 2009 H1N1 influenza variant with a G222D mutation in the hemagglutinin. Clinical symptoms were recorded every day. Lung injury was assessed by lung water content and histopathological observation. Arterial blood gas, leukocyte count in the bronchial alveolar lavage fluid and blood, virus titers, and cytokine levels in the lung were measured at various times post-inoculation. Mice infected with SD/09 virus showed typical ARDS symptoms characterized by 60% lethality on days 8–10 post-inoculation, highly edematous lungs, inflammatory cellular infiltration, alveolar and interstitial edema, lung hemorrhage, progressive and severe hypoxemia, and elevated levels of proinflammatory cytokines and chemokines. Conclusions/Significance These results suggested that we successfully established an ARDS mouse model induced by a virulent 2009 H1N1 variant without previous adaptation, which may be of benefit for evaluating the pathogenesis or therapy of human ARDS caused by 2009 H1N1 virus.

Vaccination for highly pathogenic avian influ- enza (HPAI) has been implemented in China for a decade, however, the virus is still present in poultry. A series of recombinant vaccines, Re-1 to Re-7, have been developed and used, and Re-8 will also be used in clinical settings to prevent the prevailing flu strains. The question remains, when can China eradicate the disease? Here, we review the epidemiology of H5 HPAI along with the development, usage and problems of vaccines. Further suggestions for controlling the disease in China are provided.

Background Influenza virus virulence can be exacerbated by bacterial co-infections. Swine influenza virus (SIV) infection together with some bacteria is found to enhance pathogenicity. Methods SIV-positive samples suspected of containing bacteria were used for bacterial isolation and identification. Antimicrobial susceptibility testing was performed by disc diffusion methods. To investigate the interaction of SIV and the bacteria in vitro, guinea pigs were used as mammalian hosts to determine the effect on viral susceptibility and transmissibility. Differences in viral titers between groups were compared using Student’s t-test. Results During surveillance for SIV in China from 2006 to 2009, seven isolates (24.14%) of 29 influenza A viruses were co-isolated with Stenotrophomonas maltophilia from nasal and tracheal swab samples of pigs. Antimicrobial susceptibility testing showed that the bacteria possessed a high level of resistance towards clinically used antibiotics. To investigate the interaction between these two microorganisms in influencing viral susceptibility and transmission in humans, guinea pigs were used as an infection model. Animals were inoculated with SIV or S. maltophilia alone or co-infected with SIV and S. maltophilia. The results showed that although no transmission among guinea pigs was observed, virus–bacteria co-infections resulted in higher virus titers in nasal washes and trachea and a longer virus shedding period. Conclusions This is the first report of influenza virus co-infection with S. maltophilia in the Chinese swine population. Increased replication of virus by co-infection with multidrug resistant bacteria might increase the infection rate of SIV in humans. The control of S. maltophilia in clinics will contribute to reducing the spread of SIV in pigs and humans.