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H9N2 influenza virus has been reported worldwide for several decades, and it has evolved into multiple genotypes among domestic poultry. However, the study involving ecology and evolution of low pathogenic avian influenza virus H9N2 in wild birds in China is limited. Here, we carried out surveillance of avian influenza virus H9N2 in wild birds along with the East Asian–Australian migratory flyway in China in 2017. To estimate the prevalence of H9N2 avian virus in wild birds, information on exposure of wild bird populations to H9N2 viruses using serology, in addition to virology, would greatly improve monitoring capabilities. In this study, we also present serological data of H9N2 among wild birds in China during 2013–2016. We report the identification of poultry‐derived H9N2 isolates from asymptomatic infected multispecies wild birds such as Common kestrel (Falco tinnunculus), Northern goshawk (Accipiter gentilis), Little owl (Athene noctua) and Ring‐necked Pheasant (Phasianus colchicus) in North China in June 2017. Phylogenetic analysis demonstrated that Tianjin H9N2 isolates belong to the G81 and carry internal genes highly homologous to human H10N8 and H7N9. The isolates could directly infect mice without adaptation but were restricted to replicate in the respiratory system. Glycan‐binding preference analyses suggested that the H9N2 isolates have acquired a binding affinity for the human‐like receptor. Notably, results from transmission experiment in guinea pigs and ferrets demonstrated the wild birds‐derived H9N2 influenza virus exhibits efficient transmission phenotypes in mammalian models via respiratory droplets. Our results indicate that the H9N2 AIVs continued to circulate extensively in wild bird populations and migratory birds play an important role in the spread and genetic diversification of H9N2 AIVs. The pandemic potential of H9N2 viruses demonstrated by aerosol transmission in mammalian models via respiratory droplets highlights the importance of monitoring influenza viruses in these hosts. [ABSTRACT FROM AUTHOR]

We previously obtained mouse-adapted variants of H1N2 avian influenza virus that contained PB2-L134H, PB2-I647L, PB2-D701N, HA-G228S, and M1-D231N mutations. Here, we analyzed the effects of these mutations on viral pathogenicity in a mammalian model. By evaluating the virulence of mouse-adapted H1N2 variants at different generations, we found that the PB2-D701N and HA-G228S mutations both contribute to the virulence of this virus in mammals. Furthermore, we found that the PB2-D701N and HA-G228S mutations both enhance the ability of the virus to replicate in vivo and in vitro and that the PB2-D701N substitution results in an expansion of viral tissue tropism. These results suggest that the PB2-D701N mutation and the HA-G228S mutation are the major mammalian determinants of H1N2 virus. These results help us to understand more about the mechanisms by which influenza viruses adapt to mammals, and monitoring of these mutations can be used in continuous influenza surveillance to assess the pandemic potential of avian influenza virus variants. [ABSTRACT FROM AUTHOR]

The worldwide circulation of H9N2 avian influenza virus in poultry, the greater than 2.3 % positive rate for anti-H9 antibodies in poultry-exposed workers, and several reports of human infection indicate that H9N2 virus is a potential threat to human health. Here, we found three mutations that conferred high virulence to H9N2 virus in mice after four passages. The PB2-E627K substitution rapidly appeared at the second passage and played a decisive role in virulence. Polymerase complexes possessing PB2-E627K displayed 16.1-fold higher viral polymerase activity when compared to the wild-type virus, which may account for enhanced virulence of this virus. The other two substitutions (HA-N313D and HA-N496S) enhanced binding to both α2,3-linked and α2,6-linked sialic acid receptors; however, the HA-N313D and N496S substitutions alone decreased the virulence of mouse-adapted virus. Furthermore, this mouse-adapted virus was still not transmissible among guinea pigs by direct contact (0/3 pairs). Our findings show that adaption in mice enhanced the viral polymerase activity and receptor-binding ability, which resulted in a virulent phenotype in mice but not a transmissible phenotype in guinea pigs, indicating that host factors play an important role in adaptive evolution of influenza in new hosts. [ABSTRACT FROM AUTHOR]

Several cases of humans infected with the H9N2 avian influenza virus (AIV) have been described since 1999; however, the infectivity and pathogenicity of H9N2 in humans is not well defined. A non-human primate model in rhesus macaques was developed to study H9N2 virus infections as a means of better understanding the pathogenesis and virulence of this virus, in addition to testing antiviral drugs. Rhesus macaques inoculated with H9N2 AIV presented with biphasic fever and viral pneumonia. H9N2 was recovered from nasal washes and pharyngeal samples up to days 7-9 postinfection, followed by an increase in HI (hemagglutination inhibition) antibody titers. Tissue tropism and immunohistochemistry indicated that H9N2 AIV replicated in the upper respiratory tract (turbinate, trachea, and bronchus) and in all lobes of the lung. Our data suggest that rhesus macaques are a suitable animal model to study H9N2 influenza virus infections, particularly in the context of viral evolution and pathogenicity. [ABSTRACT FROM AUTHOR]

Avian influenza virus (AIV) has caused serious epidemics all over the world. Notably, the low-pathogenic AIV H9N2 has been spreading widely, leading to enormous economic losses to the poultry industry. To rapidly monitor airborne H9 AIVs in chicken houses, a real-time RT-PCR method was established and used to detect virus in air samples, and it was also compared with the traditional RT-PCR. The results showed that the real-time RT-PCR possessed high specificity and sensitivity for H9 AIVs, and the sensitivity reached 100 copies/reaction, much higher than the traditional RT-PCR; airborne H9 AIVs were found in the six chicken houses by real-time RT-PCR, and their mean concentrations ranged from 1.25×10 to 6.92×10 copies/m air. Overall, the real-time PCR is a valuable tool for detecting airborne H9 AIVs. [ABSTRACT FROM AUTHOR]

H5N1 influenza virus is a threat to public health worldwide. The virus can cause severe morbidity and mortality in humans. We constructed an H5N1 influenza candidate virus vaccine from the A/chicken/Guizhou/1153/2016 strain that was recommended by the World Health Organization. In this study, we designed an H5N1 chimeric influenza A/B vaccine based on a cold-adapted (ca) influenza B virus B/Vienna/1/99 backbone. We modified the ectodomain of H5N1 hemagglutinin (HA) protein, while retaining the packaging signals of influenza B virus, and then rescued a chimeric cold-adapted H5N1 candidate influenza vaccine through a reverse genetic system. The chimeric H5N1 vaccine replicated well in eggs and the Madin-Darby Canine Kidney cells. It maintained a temperature-sensitive and cold-adapted phenotype. The H5N1 vaccine was attenuated in mice. Hemagglutination inhibition (HAI) antibodies, micro-neutralizing (MN) antibodies, and IgG antibodies were induced in immunized mice, and the mucosal IgA antibody responses were detected in their lung lavage fluids. The IFN-γ-secretion and IL-4-secretion by the mouse splenocytes were induced after stimulation with the specific H5N1 HA protein. The chimeric H5N1 candidate vaccine protected mice against lethal challenge with a wild-type highly pathogenic avian H5N1 influenza virus. The chimeric H5 candidate vaccine is thus a potentially safe, attenuated, and reassortment-incompetent vaccine with circulating A viruses. [ABSTRACT FROM AUTHOR]

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) caused poultry outbreaks in the Republic of Korea in 2014. The novel H5N8 HPAIV has spread to Asia, Europe, and North America and caused great public concern from then on. Here, we generated mouse-adapted variants of a wild waterfowl-origin H5N8 HPAIV to identify adaptive mutants that confer enhanced pathogenicity in mammals. The mouse lethal doses (MLD 50 ) of the mouse-adapted variants were reduced 31623-fold compared to the wild-type (WT) virus. Mouse-adapted variants displayed enhanced replication in vitro and in vivo , and expanded tissue tropism in mice. Sequence analysis revealed four amino acid substitutions in the PB2 (E627K), PA (F35S), HA (R227H), and NA (I462V) proteins. These data suggest that multiple amino acid substitutions collaboratively increase the virulence of a wild bird-origin reassortant H5N8 HPAIV and cause severe disease in mice. [ABSTRACT FROM AUTHOR]

Frequent emergence of low pathogenic avian influenza H3N2 viruses in the wild birds has caused concern for human health. Here, we generated mouse-adapted strains of a wild waterfowl-origin low pathogenic avian influenza H3N2 virus to identify adaptive mutations that confer enhanced virulence in mammals. The mouse lethal doses (MLD 50 ) of the adapted strains were reduced >562-fold compared to the parental virus. Mouse-adapted strains displayed enhanced replication in vitro and in vivo , and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H3N2 AIVs in mice. Genomic analysis revealed mutations in the PB2 (E192 K and D701N), PB1 (F269S, I475 V, and L598P), HA (V242E), NA (G170R), and M1 (M192 V) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H3N2 avian influenza A virus to replicate and cause severe disease in mammals. [ABSTRACT FROM AUTHOR]

Although H1N2 avian influenza virus (AIV) only infect birds, documented cases of swine infection with H1N2 influenza viruses suggest this subtype AIV may pose a potential threat to mammals. Here, we generated mouse-adapted variants of a H1N2 AIV to identify adaptive changes that increased virulence in mammals. MLD 50 of the variants were reduced >1000-fold compared to the parental virus. Variants displayed enhanced replication in vitro and in vivo , and replicate in extrapulmonary organs. These data show that enhanced replication capacity and expanded tissue tropism may increase the virulence of H1N2 AIV in mice. Sequence analysis revealed multiple amino acid substitutions in the PB2 (L134H, I647L, and D701N), HA (G228S), and M1 (D231N) proteins. These results indicate that H1N2 AIV can rapidly acquire adaptive amino acid substitutions in mammalian hosts, and these amino acid substitutions collaboratively enhance the ability of H1N2 AIV to replicate and cause severe disease in mammals. [ABSTRACT FROM AUTHOR]

Although H7N7 AIVs primarily circulate in wild waterfowl, documented cases of human infection with H7N7 viruses suggest they may pose a pandemic threat. Here, we generated mouse-adapted variants of a wild waterfowl-origin H7N7 virus to identify adaptive changes that confer enhanced virulence in mammals. The mouse lethal doses (MLD 50 ) of the adapted variants were reduced >5000-fold compared to the parental virus. Mouse-adapted variants viruses displayed enhanced replication in vitro and in vivo , and acquired the ability to replicate in extrapulmonary tissues. These observations suggest that enhanced growth characteristics and modified cell tropism may increase the virulence of H7N7 AIVs in mice. Genomic analysis of the adapted variant viruses revealed amino acid changes in the PB2 (E627K), PB1 (R118I), PA (L550M), HA (G214R), and NA (S372N) proteins. Our results suggest that these amino acid substitutions collaboratively enhance the ability of H7N7 virus to replicate and cause severe disease in mammals. [ABSTRACT FROM AUTHOR]

H6N1 avian influenza viruses (AIVs) may pose a potential human risk as suggested by the first documented naturally-acquired human H6N1 virus infection in 2013. Here, we set out to elucidate viral determinants critical to the pathogenesis of this virus using a mouse model. We found that the recombinant H6N1 viruses possessing both the PA-T97I and PB2-E627K substitutions displayed the greatest enhancement of replication in vitro and in vivo . Polymerase complexes possessing either PB2-E627K, PA-T97I, and PB2-E627K/PA-T97I displayed higher virus polymerase activity when compared to the wild-type virus, which may account for the increased replication kinetics and enhanced virulence of variant viruses. Our results demonstrate that PB2-E627K and PA-T97I enhance the ability of H6N1 virus to replicate and cause disease in mammals. Influenza surveillance efforts should include scrutiny of these regions of PB2 and PA because of their impact on the increased virulence of H6N1 AIVs in mice. [ABSTRACT FROM AUTHOR]

Highlights: [•] The susceptibility of domestic cats and dogs to H9N2 avian influenza virus was evaluated by intranasally or orally inoculating animals with an H9N2 influenza virus. [•] H9N2 AIV can efficiently infect cats and dogs via the upper respiratory tract. [•] H9N2 AIV transmission between cats and dogs is inefficient. [•] We suggested that the domestic dogs and cats may serve as an intermediate host in the transfer of H9N2 viruses to humans. [Copyright &y& Elsevier]