Characteristics of Avian Influenza H9N2 Virus Isolated from Humans and its Seroprevalence Among Occupationally Exposed Populations in China

Background: The first human-infected H9N2 influenza case can be traced back to 1998. Although the H9N2 influenza virus has low pathogenicity in animals, it donated partial or whole cassettes of internal genes to reassort novel viruses, such as H7N9, H10N8 and H5N6 viruses, that caused human infections with high fatality. Since 2013, sporadic but increasingly frequent human cases caused by H9N2 influenza virus have been confirmed globally, and most of them were from China. Methods: Information on human infections with H9N2 influenza virus was collected. Viral molecular determinants were determined by deep sequencing, and phylogenetic analysis was performed using MEGA 6.06. Antigenic analysis was performed by a hemagglutination inhibition (HI) assay. Receptor binding preference analysis was conducted based on a solid-phase binding assay with synthetic sialylglycopolymers. Antiviral susceptibility was determined by a fluorescence-based neuraminidase (NA) inhibition assay. Serological study of occupationally exposed populations was performed by HI assay screening and confirmed by microneutralization assay.Results: From 2013 to 2018, 33 human H9N2cases were reported in China, among them 75.7% were children under 10 years old .The 22 viruses were isolated and concentrated in the Y280/G9 lineage of the HA and NA genes. All human H9N2 viruses belonged to the Y280/G9 antigenic lineage, presented a human-like receptor binding preference and remained susceptible to NA inhibitors, but most demonstrated resistance to M2 inhibitors. The seroprevalence of occupationally exposed populations was 2.15%, 3.17%, 2.93% and 1.54% from 2015 to 2018, respectively. A significant difference in seroprevalence was shown between provinces with human cases (3.66%) and provinces without human cases (2.18%). Conclusions: The continuous antigenic drift and human-like receptor binding preference of the H9N2 virus enable it to have a high risk of causing human infections. The status of the seropositivity in occupationally exposed populations implies a substantial threat to public health. Research on human infection with H9N2 influenza virus should be strengthened to monitor the emergence of sustainable human-to-human transmission and the possibility of an endemic or a pandemic related to it.

Polymerase acidic subunit of H9N2 polymerase complex induces cell apoptosis by binding to PDCD 7 in A549 cells

Background H9N2 influenza virus, a subtype of influenza A virus, can spread across different species and induce the respiratory infectious disease in humans, leading to a severe public health risk and a huge economic loss to poultry production. Increasing studies have shown that polymerase acidic (PA) subunit of RNA polymerase in ribonucleoproteins complex of H9N2 virus involves in crossing the host species barriers, the replication and airborne transmission of H9N2 virus. Methods Here, to further investigate the role of PA subunit during the infection of H9N2 influenza virus, we employed mass spectrometry (MS) to search the potential binding proteins of PA subunit of H9N2 virus. Our MS results showed that programmed cell death protein 7 (PDCD7) is a binding target of PA subunit. Co-immunoprecipitation and pull-down assays further confirmed the interaction between PDCD7 and PA subunit. Overexpression of PA subunit in A549 lung cells greatly increased the levels of PDCD7 in the nuclear and induced cell death assayed by MTT assay. Results Flow cytometry analysis and Western blot results showed that PA subunit overexpression significantly increased the expression of pro-apoptotic protein, bax and caspase 3, and induced cell apoptosis. However, knockout of PDCD7 effectively attenuated the effects of PA overexpression in cell apoptosis. Conclusions In conclusion, the PA subunit of H9N2 virus bind with PDCD7 and regulated cell apoptosis, which provide new insights in the role of PA subunit during H9N2 influenza virus infection.

Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain 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 2016-2017 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 sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating 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 co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 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 prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

Autophagy is involved in the replication of H9N2 influenza virus via the regulation of oxidative stress in alveolar epithelial cells

Background Oxidative stress is an important pathogenic factor in influenza A virus infection. It has been found that reactive oxygen species induced by the H9N2 influenza virus is associated with viral replication. However, the mechanisms involved remain to be elucidated. Methods In this study, the role of autophagy was investigated in H9N2 influenza virus-induced oxidative stress and viral replication in A549 cells. Autophagy induced by H9N2 was inhibited by an autophagy inhibitor or RNA interference, the autophagy level, viral replication and the presence of oxidative stress were detected by western blot, TCID50 assay, and Real-time PCR. Then autophagy and oxidative stress were regulated, and viral replication was determined. At last, the Akt/TSC2/mTOR signaling pathways was detected by western blot. Results Autophagy was induced by the H9N2 influenza virus and the inhibition of autophagy reduced the viral titer and the expression of nucleoprotein and matrix protein. The blockage of autophagy suppressed the H9N2 virus-induced increase in the presence of oxidative stress, as evidenced by decreased reactive oxygen species production and malonaldehyde generation, and increased superoxide dismutase 1 levels. The changes in the viral titer and NP mRNA level caused by the antioxidant, N-acetyl-cysteine (NAC), and the oxidizing agent, H2O2, confirmed the involvement of oxidative stress in the control of viral replication. NAC plus transfection with Atg5 siRNA significantly reduced the viral titer and oxidative stress compared with NAC treatment alone, which confirmed that autophagy was involved in the replication of H9N2 influenza virus by regulating oxidative stress. Our data also revealed that autophagy was induced by the H9N2 influenza virus through the Akt/TSC2/mTOR pathway. The activation of Akt or the inhibition of TSC2 suppressed the H9N2 virus-induced increase in the level of LC3-II, restored the decrease in the expression of phospho-pAkt, phospho-mTOR and phospho-pS6 caused by H9N2 infection, suppressed the H9N2-induced increase in the presence of oxidative stress, and resulted in a decrease in the viral titer. Conclusion Autophagy is involved in H9N2 virus replication by regulating oxidative stress via the Akt/TSC2/mTOR signaling pathway. Thus, autophagy maybe a target which may be used to improve antiviral therapeutics.

Polymerase Acidic Subunit of H9N2 Polymerase Complex Induces Cell Apoptosis by Binding to PDCD 7 in A549 Cells

Background: H9N2 influenza virus, a subtype of influenza A virus, can spread across different species and induce the respiratory infectious disease in humans, leading to a severe public health risk and a huge economic loss to poultry production. Increasing studies have shown that polymerase acidic (PA) subunit of RNA polymerase in ribonucleoproteins complex of H9N2 involves in crossing the host species barriers, the replication and airborne transmission of H9N2.Methods: Here, to further investigate the role of PA subunit during the infection of H9N2 influenza virus, we employed mass spectrometry (MS) to search the potential binding proteins of PA subunit of H9N2. Our MS results showed that programmed cell death protein 7 (PDCD7) is a binding target of PA subunit. Co-immunoprecipitation and pull-down assays further confirmed the interaction between PDCD7 and PA subunit. Overexpression of PA subunit in A549 lung cells greatly increased the levels of PDCD7 in the nuclear and induced cell death assayed by MTT assay.Results: Flow cytometry analysis and Western blot results showed that PA subunit overexpression significantly increased the expression of pro-apoptotic protein, bax and caspase 3, and induced cell apoptosis. However, knockout of PDCD7 effectively attenuated the effects of PA overexpression in cell apoptosis.Conclusions: In conclusion, the PA subunit of H9N2 bind with PDCD7 and regulated cell apoptosis, which provide new insights in the role of PA subunit during H9N2 influenza virus infection.