Revealing novel and conservative T-cell epitopes with MHC B2 restriction on H9N2 avian influenza virus (AIV).

B2 haplotype major histocompatibility complex (MHC) has been extensively reported to confer resistance to various avian diseases. But its peptide-binding motif is unknown, and the presenting peptide is rarely identified. Here, we identified its peptide-binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) in vitro using Random Peptide Library-based MHC I LC-MS/MS analysis. To further clarify the structure basis of motif, we determined the crystal structure of the BF2∗02:01-PB2552-560 complex at 1.9 Å resolution. We found that BF2∗02:01 had a relatively wide antigen-binding groove, and the structural characterization of pockets was consistent with the characterization of peptide-binding motif. The wider features of the peptide-binding motif and increased number of peptides bound by BF2∗02:01 than BF2∗04:01 might resolve the puzzles for the presence of potential H9N2 resistance in B2 chickens. Afterward, we explored the H9N2 avian influenza virus (AIV)-induced cellular immune response in B2 haplotype chickens in vivo. We found that ratio of CD8+ T cell and kinetic expression of cytotoxicity genes including Granzyme K, interferon-γ, NK lysin, and poly-(ADP-ribose) polymerase in peripheral blood mononuclear cells were significantly increased in defending against H9N2 AIV infection. Especially, we selected 425 epitopes as candidate epitopes based on the peptide-binding motif and further identified four CD8+ T-cell epitopes on H9N2 AIV including NS198-106, PB2552-560, NP182-190, and NP455-463 via ELI-spot interferon-γ detections after stimulating memory lymphocytes with peptides. More importantly, these epitopes were found to be conserved in H7N9 AIV and H9N2 AIV. These findings provide direction for developing effective T cell epitope vaccines using well-conserved internal viral antigens in chickens.

The first report and biological characterization of Avian Orthoavulavirus 16 in wild migratory waterfowl and domestic poultry in China reveal a potential threat to birds.

RESEARCH HIGHLIGHTS: First confirmation of AOAV-16 in domestic and wild birds in China.AOAV-16 are low virulent viruses for chickens.Co-circulation/co-infection of AOAV-16 and H9N2 subtype AIV enhanced pathogenicity.Different intergenic sequences and recombination events exist within AOAV-16.

The role of transforming growth factor beta-1 protein in Escherichia coli secondary infection induced by H9N2 avian influenza virus in chickens.

The H9N2 subtype of avian influenza virus (AIV) is common in poultry production. It causes mild clinical signs but rarely leads to poultry mortalities. However, higher mortality can occur in chickens with co-infections, especially avian pathogenic Escherichia coli (APEC), which results in huge economic losses for the poultry industry. Unfortunately, the mechanism of co-infection remains unknown. Our previous studies screened several proteins associated with bacterial adhesion, including transforming growth factor beta-1 (TGF-ß1), integrins, cortactin, E-cadherin, vinculin, and fibromodulin. Herein, we investigated the contribution of TGF-ß1 to APEC adhesion after H9N2 infection. We first infected H9N2 and APEC in chicken, chicken embryo and DF-1 cells, and demonstrated that H9N2 infection promotes APEC adhesion to hosts in vitro and in vivo by plate count method. Through real-time fluorescence quantification and enzyme-linked immunosorbent assay, it was demonstrated that H9N2 infection not only increases TGF-ß1 expression but also its activity in a time-dependent manner. Then, through exogenous addition of TGF-ß1 and overexpression, we further demonstrated that TGF-ß1 can increase the adhesion of endothelial cells to DF-1 cells. Furthermore, the capacity of APEC adhesion to DF-1 cells was significantly decreased either by adding a TGF-ß1 receptor inhibitor or using small interfering RNAs to interfere with the expression of TGF-ß1. To sum up, H9N2 infection can promote the upregulation of TGF-ß1 and then increase the adhesion ability of APEC. Targeting TGF-ß1 and its associated pathway will provide valuable insights into the clinical treatment of E. coli secondary infection induced by H9N2 infection.

Improved pathogenicity of H9N2 subtype of avian influenza virus induced by mutations occurred after serial adaptations in mice.

H9N2 subtype, a low pathogenic avian influenza virus, is emerging as a major causative agent circulating poultry workplaces across China and other Asian countries. Increasing case number of interspecies transmissions to mammals reported recently provoked a great concern about its risks inducing global pandemics. In an attempt to understand the underlying mechanism of how the H9N2 virus disrupts the interspecies segregation to transmit to mammals. A mutant H9N2 strain was obtained by passaging the wildtype H9N2 A/chicken/Hong Kong/G9/1997 eight times from lung to lung in BALB/c mice. Our finding revealed that mice manifested severe clinical symptoms including losses of body weight, pathological damages in pulmonary sites and all died within two weeks after infected with the mutated H9N2, whereas all mice survived upon infected with wildtype strain in comparison, which suggested increased pathogenicity of the mutant strain. In addition, mice showed enhanced levels of proinflammatory cytokines in sera, including IL-6, TNF-α and IL-1ß compared to those subjected to wildtype viral infections. Sequence analysis showed that five amino acid substitutions occurred at PB2627, HA87, HA234, NP387 and M156, and a deletion mutation happened in the M gene (M157). Of these mutations, PB2 E627K played key roles in modulating lethality in mice. Taken together, the mutant H9N2 strain obtained by serial passaging of its wildtype in mice significantly increased its virulence leading to death of mice, which might be associated the accumulated mutations occurred on its genome.

Antiviral activity of diallyl trisulfide against H9N2 avian influenza virus infection in vitro and in vivo.

BACKGROUND: Diallyl trisulfide (DATS) is a garlic-derived organosulfur compound. As it has been shown to have anti-viral activity, we hypothesized that it may alleviate infections caused by H9N2 avian influenza virus (AIV), which is prevalent in poultry with pandemic potential. METHODS: Human lung A549 epithelial cells were treated with three different concentrations of DATS 24 h before (pre-treatment) or one hour after (post-treatment) H9N2 AIV infection. Culture supernatants were collected 24 h and 48 h post-infection and analyzed for viral titers and levels of inflammatory and anti-viral immune responses. For in vivo experiments, BABL/c mice were administered daily by intraperitoneal injection with DATS (30 mg/kg) for 2 weeks starting 1 day after H9N2 AIV infection. Clinical signs, lung pathology, and inflammatory and anti-viral immune responses were assessed 2, 4, and 6 days after infection. RESULTS: Both pre-treatment and post-treatment of A549 cells with DATS resulted in reduced viral loads, increased expression of anti-viral genes (RIG-I, IRF-3, and interferon-ß), and decreased expression of inflammatory cytokines (TNF-α and IL-6). These effects were also observed in H9N2 AIV-infected mice treated with DATS. Such treatment also reduced lung edema and inflammation in mice. CONCLUSIONS: Results suggest that DATS has anti-viral activity against H9N2 AIV and may be used as an alternative treatment for influenza virus infection.

The molecular evolutionary characteristics of new isolated H9N2 AIV from East China and the function of vimentin on virus replication in MDCK cells.

BACKGROUND: The low pathogenic H9N2 AIV caused the serious impact on the poultry industry and public safety. Our purpose was to investigate the molecular evolutionary characteristics of the new isolated H9N2 virus and investigate the intracellular target protein of H9N2 AIV replication in sensitive cells. METHODS: AIV A/chicken/Shandong/LY1/2017 (H9N2) was isolated from the cloaca of the healthy chicken in Shandong, and the full-length eight gene segments of this isolated H9N2 AIV were amplified by RT-PCR and analyzed. MDCK cells were used as the target cell model, and VOPBA assay and LC-MS/MS were carried out to identify the virus-binding protein of H9N2 AIV. MDCK cells were pre-treated with the special antibody and siRNA, and treated with H9N2 AIV to detect the virus replication. Additionally, Vimentin-pcDNA3.0 was successfully constructed, and transinfected into MDCK cells, and then H9N2 AIV mRNA was detected with RT-PCR. RESULTS: Phylogenetic analysis revealed that HA, NA, PB2, PB1, PA, NP and M seven genes of the isolated H9N2 AIV were derived from A/Chicken/Shanghai/F/98, while NS gene was derived from A/Duck/Hong Kong/Y439/97. The cleavage site sequence of HA gene of the isolated H9N2 AIV was a PARSSR G pattern, and the left side sequence (224 ~ 229) of receptor binding site was NGQQGR pattern, which were similar to that of A/Chicken/Shanghai/F/98. Following VOPBA assay, we found one protein of about 50KDa binding to H9N2 AIV, and the results of LC-MS/MS analysis proved that vimentin was the vital protein binding to H9N2 AIV. The pre-incubation of the specific antibody and siRNA decreased the viral RNA level in MDCK cells treated with H9N2 AIV. Furthermore, we found that over-expressed vimentin increased H9N2 AIV replication in MDCK cells. CONCLUSIONS: These findings suggested that the isolated H9N2 AIV might be a recent clinical common H9N2 strain, and vimentin protein might be one vital factor for H9N2 AIV replication in MDCK cells, which might be a novel target for design and development of antiviral drug.

Efficacy of an inactivated influenza vaccine adjuvanted with Toll-like receptor ligands against transmission of H9N2 avian influenza virus in chickens.

Avian influenza viruses (AIV), including the H9N2 subtype, pose a major threat to the poultry industry as well as to human health. Although vaccination provides a protective control measure, its effect on transmission remains uncertain in chickens. The objective of the present study was to investigate the efficacy of beta-propiolactone (BPL) whole inactivated H9N2 virus (WIV) vaccine either alone or in combination with CpG ODN 2007 (CpG), poly(I:C) or AddaVax™ (ADD) to prevent H9N2 AIV transmission in chickens. The seeder chickens (trial 1) and recipient chickens (trial 2) were vaccinated twice with different vaccine formulations. Ten days after secondary vaccination, seeder chickens were infected with H9N2 AIV (trial 1) and co-housed with healthy recipient chickens. In trial 2, the recipient chickens were vaccinated and then exposed to H9N2 AIV-infected seeder chickens. Our results demonstrated that BPL+ CpG and BPL+ poly(I:C) treated chickens exhibited reduced oral and cloacal shedding in both trials post-exposure (PE). The number of H9N2 AIV+ recipient chickens in the BPL+ CpG group (trial 1) was lower than in other vaccinated groups, and the reduction was higher in BPL+ CpG recipient chickens in trial 2. BPL+ CpG vaccinated chickens demonstrated enhanced systemic antibody responses with high IgM and IgY titers with higher rates of seroprotection by day 21 post-primary vaccination (ppv). Additionally, the induction of IFN-γ expression and production was higher in the BPL+ CpG treated chickens. Interleukin (IL)- 2 expression was upregulated in both BPL+ CpG and BPL+ poly(I:C) groups at 12 and 24 hr post-stimulation.

Hemagglutinin affects replication, stability and airborne transmission of the H9N2 subtype avian influenza virus.

H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.

Diversity of genotypes and pathogenicity of H9N2 avian influenza virus derived from wild bird and domestic poultry.

Introduction: The H9N2 subtype is a predominant avian influenza virus (AIV) circulating in Chinese poultry, forming various genotypes (A-W) based on gene segment origins. This study aims to investigate the genotypic distribution and pathogenic characteristics of H9N2 isolates from wild birds and domestic poultry in Yunnan Province, China. Methods: Eleven H9N2 strains were isolated from fecal samples of overwintering wild birds and proximate domestic poultry in Yunnan, including four from common cranes (Grus grus), two from bar-headed geese (Anser indicus), and five from domestic poultry (Gallus gallus). Phylogenetic analysis was conducted to determine the genotypes, and representative strains were inoculated into Yunnan mallard ducks to assess pathogenicity. Results: Phylogenetic analysis revealed that five isolates from domestic birds and one from a bar-headed goose belong to genotype S, while the remaining five isolates from wild birds belong to genotype A. These bird-derived strains possess deletions in the stalk domain of NA protein and the N166D mutation of HA protein, typical of poultry strains. Genotype S H9N2 demonstrated oropharyngeal shedding, while genotype A H9N2 exhibited cloacal shedding and high viral loads in the duodenum. Both strains caused significant pathological injuries, with genotype S inducing more severe damage to the thymus and spleen, while genotype A caused duodenal muscle layer rupture. Discussion: These findings suggest that at least two genotypes of H9N2 are currently circulating in Yunnan, and Yunnan mallard ducks potentially act as intermediaries in interspecies transmission. These insights highlight the importance of analyzing the current epidemiological transmission characteristics of H9N2 among wild and domestic birds in China.

MDA5 with Complete CARD2 Region Inhibits the Early Replication of H9N2 AIV and Enhances the Immune Response during Vaccination.

Chicken melanoma differentiation-associated gene 5 (MDA5) is a member of the RLRs family that recognizes the viral RNAs invading cells and activates downstream interferon regulatory pathways, thereby inhibiting viral replication. The caspase activation and recruitment domain (CARD) is the most important region in MDA5 protein. However, the antiviral and immune enhancement of MDA5 with the CARD region remains unclear. In this study, two truncated MDA5 genes with different CARD regions, namely MDA5-1 with CARD1 plus partial CARD2 domain and MDA5-2 with CARD1 plus complete CARD2 domain, were cloned via reverse transcription PCR and ligated into plasmid Flag-N vector to be Flag-MDA5-1 and Flag-MDA5-2 plasmids. DF-1 cells were transfected with two plasmids for 24 h and then inoculated with H9N2 virus (0.1 MOI) for 6 h to detect the levels of IFN-ß, PKR, MAVS, and viral HA, NA, and NS proteins expression. The results showed that MDA5-1 and MDA5-2 increased the expression of IFN-ß and PKR, activated the downstream molecule MAVS production, and inhibited the expression of HA, NA, and NS proteins. The knockdown of MDA5 genes confirmed that MDA5-2 had a stronger antiviral effect than that of MDA5-1. Furthermore, the recombinant proteins MDA5-1 and MDA5-2 were combined with H9N2 inactivated vaccine to immunize SPF chickens subcutaneously injected in the neck three times. The immune response of the immunized chicken was investigated. It was observed that the antibody titers and expressions of immune-related molecules from the chicken immunized with MDA5-1 and MDA5-2 group were increased, in which the inducing function of MDA5-2 groups was the highest among all immunization groups. These results suggested that the truncated MDA5 recombinant proteins with complete CARD2 region could play vital roles in antiviral and immune enhancement. This study provides important material for the further study of the immunoregulatory function and clinical applications of MDA5 protein.

Field efficacy of HVT-H9 vaccination after natural infection of H9N2 avian influenza virus in broilers.

H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.

Determining the Protective Efficacy of Toll-Like Receptor Ligands to Minimize H9N2 Avian Influenza Virus Transmission in Chickens.

Low-pathogenicity avian influenza viruses (AIV) of the H9N2 subtype can infect and cause disease in chickens. Little is known about the efficacy of immune-based strategies for reducing the transmission of these viruses. The present study investigated the efficacy of Toll-like receptor (TLR) ligands (CpG ODN 2007 and poly(I:C)) to reduce H9N2 AIV transmission from TLR-treated seeder (trial 1) or inoculated chickens (trial 2) to naive chickens. The results from trial 1 revealed that a low dose of CpG ODN 2007 led to the highest reduction in oral shedding, and a high dose of poly(I:C) was effective at reducing oral and cloacal shedding. Regarding transmission, the recipient chickens exposed to CpG ODN 2007 low-dose-treated seeder chickens showed a maximum reduction in shedding with the lowest number of AIV+ chickens. The results from trial 2 revealed a maximum reduction in oral and cloacal shedding in the poly(I:C) high-dose-treated chickens (recipients), followed by the low-dose CpG ODN 2007 group. In these two groups, the expression of type I interferons (IFNs), protein kinase R (PKR), interferon-induced transmembrane protein 3 (IFITM3), viperin, and (interleukin) IL-1ß, IL-8, and 1L-18 was upregulated in the spleen, cecal tonsils and lungs. Hence, TLR ligands can reduce AIV transmission in chickens.

Status and Challenges for Vaccination against Avian H9N2 Influenza Virus in China.

In China, H9N2 avian influenza virus (AIV) has become widely prevalent in poultry, causing huge economic losses after secondary infection with other pathogens. Importantly, H9N2 AIV continuously infects humans, and its six internal genes frequently reassort with other influenza viruses to generate novel influenza viruses that infect humans, threatening public health. Inactivated whole-virus vaccines have been used to control H9N2 AIV in China for more than 20 years, and they can alleviate clinical symptoms after immunization, greatly reducing economic losses. However, H9N2 AIVs can still be isolated from immunized chickens and have recently become the main epidemic subtype. A more effective vaccine prevention strategy might be able to address the current situation. Herein, we analyze the current status and vaccination strategy against H9N2 AIV and summarize the progress in vaccine development to provide insight for better H9N2 prevention and control.

Surveillance of avian influenza viruses in live bird markets of Shandong province from 2013 to 2019.

Avian influenza viruses (AIVs) seriously affect the poultry industry and pose a great threat to humans. Timely surveillance of AIVs is the basis for preparedness of the virus. This study reported the long-term surveillance of AIVs in the live bird market (LBM) of 16 cities in Shandong province from 2013 to 2019. A total of 29,895 samples were obtained and the overall positive rate of AIVs was 9.7%. The H9 was found to be the most predominant subtype in most of the time and contributed most to the monthly positve rate of AIVs as supported by the univariate and multivariate analysis, while H5 and H7 only circulated in some short periods. Then, the whole-genome sequences of 62 representative H9N2 viruses including one human isolate from a 7-year-old boy in were determined and they were genetically similar to each other with the median pairwise sequence identities ranging from 0.96 to 0.98 for all segments. The newly sequenced viruses were most similar to viruses isolated in chickens in mainland China, especially the provinces in Eastern China. Phylogenetic analysis showed that these newly sequenced H9N2 viruses belonged to the same clade for all segments except PB1. Nearly all of these viruses belonged to the G57 genotype which has dominated in China since 2010. Finally, several molecular markers associated with human adaptation, mammalian virulence, and drug resistance were identified in the newly sequenced H9N2 viruses. Overall, the study deepens our understanding of the epidemic and evolution of AIVs and provides a basis for effective control of AIVs in China.

Effect of Co-infection of Low Pathogenic Avian Influenza H9N2 Virus and Avian Pathogenic E. coli on H9N2-Vaccinated Commercial Broiler Chickens.

In the last 40 years, low pathogenic avian influenza virus (LPAIV) subtype H9N2 has been endemic in most Middle Eastern countries and of course Egypt which is one of the biggest poultry producers in the middle east region. The major losses with the H9N2 virus infections come from complicated infections in commercial broiler chickens, especially E. coli infection. In this work, 2,36,345 Arbor acres broiler chickens from the same breeder flock were placed equally in four pens, where two pens were vaccinated against LPAIV of subtype H9N2 virus, and the other two pens served as non-vaccinated controls. All were placed on the same farm under the same management conditions. A total of twenty birds from each pen were moved to biosafety level-3 chicken isolators (BSL-3) on days 21 and 28 of life and challenged with LPAIV-H9N2 or E. coli. Seroconversion for H9N2 was evaluated before and after the challenge. The recorded results revealed a significant decrease in clinical manifestations and virus shedding in terms of titers of shedding virus and number of shedders in vaccinated compared to non-vaccinated chickens. In groups early infected with LPAIV-H9N2 virus either vaccinated or not vaccinated, there was no significant difference in clinical sickness or mortalities in both groups, but in late infection groups with H9N2 alone, non-vaccinated infected group showed significantly higher clinical sickness in comparison with infected vaccinated group but also without mortality. In groups co-infected with E. coli (I/M) and H9N2, it showed 100% mortalities either in vaccinated or non-vaccinated H9N2 groups and thus reflect the high pathogenicity of used E. coli isolates, whereas in groups co-infected with E. coli (per os to mimic the natural route of infection) and LPAIV-H9N2, mortality rates were significantly higher in non-vaccinated groups than those vaccinated with H9N2 vaccine (15 vs. 5%). In conclusion, the use of the LPAIV H9N2 vaccine has significantly impacted the health status, amount of virus shed, and mortality of challenged commercial broilers, as it can minimize the losses and risks after co-infection with E. coli (orally) and LPAIV-H9N2 virus under similar natural route of infection in commercial broilers.

A general controllable release amplification strategy of liposomes for single-particle collision electrochemical biosensing.

As an important component of the COVID-19 mRNA vaccines, liposomes play a key role in the efficient protection and delivery of mRNA to cells. Herein, due to the controllable release amplification strategy of liposomes, a reliable and robust single-particle collision electrochemical (SPCE) biosensor was constructed for H9N2 avian influenza virus (H9N2 AIV) detection by combining liposome encapsulation-release strategy with immunomagnetic separation. The liposomes modified with biotin and loaded with platinum nanoparticles (Pt NPs) were used as signal probes for the first time. Biotin facilitated the coupling of biomolecules (DNA or antibodies) through the specific reaction of biotin-streptavidin. Each liposome can encapsulate multiple Pt NPs, which were ruptured under the presence of 1 × PBST (phosphate buffer saline with 0.05% Tween-20) within 2 min, and the encapsulated Pt NPs were released for SPCE experiment. The combination of immunomagnetic separation not only improved the anti-interference capabilities but also avoided the agglomeration of Pt NPs, enabling the SPCE biosensor to realize ultrasensitive detection of 18.1 fg/mL H9N2 AIV. Furthermore, the reliable SPCE biosensor was successfully applied in specific detection of H9N2 AIV in complex samples (chicken serum, chicken liver and chicken lung), which promoted the universality of SPCE biosensor and its application prospect in early diagnosis of diseases.

Real-Time Visualization of the Infection and Replication of a Mouse-Lethal Recombinant H9N2 Avian Influenza Virus.

H9N2 avian influenza viruses (AIVs) continuously cross the species barrier to infect mammalians and are repeatedly transmitted to humans, posing a significant threat to public health. Importantly, some H9N2 AIVs were found to cause lethal infection in mice, but little is known about the viral infection dynamics in vivo. To analyze the real-time infection dynamics, we described the generation of a mouse-lethal recombinant H9N2 AIV, an influenza reporter virus (VK627-NanoLuc virus) carrying a NanoLuc gene in the non-structural (NS) segment, which was available for in vivo imaging. Although attenuated for replication in MDCK cells, VK627-NanoLuc virus showed similar pathogenicity and replicative capacity in mice to its parental virus. Bioluminescent imaging of the VK627-NanoLuc virus permitted successive observations of viral infection and replication in infected mice, even following the viral clearance of a sublethal infection. Moreover, VK627-NanoLuc virus was severely restricted by the K627E mutation in PB2, as infected mice showed little weight loss and a low level of bioluminescence. In summary, we have preliminarily established a visualized tool that enables real-time observation of the infection and replication dynamics of H9N2 AIV in mice, which contributes to further understanding the mechanisms underlying the pathogenic enhancement of H9N2 AIV to mice.

Comparative analysis of key immune protection factors in H9N2 avian influenza viruses infected and immunized specific pathogen-free chicken.

H9N2 avian influenza viruses (AIV) continue to circulate in vaccinated chicken flocks in China, which prompted us to investigate the differential immune protection factors induced by H9N2 AIV infection and immunization for analyzing the reason of protection deficiency of H9N2 AIV inactivated vaccine. In this study, we firstly explored virus-induced optimal immune responses in chicken after H9N2 AIV infection. And, we found that H9N2 hemagglutination inhibition (HI) antibody level, antiviral interferon-stimulated genes including 2',5'-oligoadenylate synthetase-like and myxovirus resistance 1, CD8+ T cell response in peripheral blood lymphocytes (PBL) accompanied by the cytotoxicity-associated genes, including poly (ADP-ribose) polymerase and IFN-r play important roles in defending against H9N2 infection. Besides, we observed that vaccine immunization triggered the similar H9N2 HI antibody level as viral infection, the increase of CD4+ T cell percentage instead of CD8+ T cell percentage in PBL. Moreover, we further made a comparative analysis of immune-related gene expression profile in PBL and lung after H9N2 AIV infection and immunization, respectively. The results showed that vaccine immunization contributed to the up-regulation of Th2 cytokine. But the deficiency of cytotoxicity-associated genes induced by H9N2 AIV inactivated vaccine may be the potential key reason of protection deficiency. These findings provide evidence and direction for developing effective H9N2 AIV vaccines.

Variation and Molecular Basis for Enhancement of Receptor Binding of H9N2 Avian Influenza Viruses in China Isolates.

Currently, H9N2 avian influenza viruses (H9N2 AIVs) globally circulate in poultry and have acquired some adaptation to mammals. However, it is not clear what the molecular basis is for the variation in receptor-binding features of the H9N2 AIVs. The receptor-binding features of 92 H9N2 AIVs prevalent in China during 1994-2017 were characterized through solid-phase ELISA assay and reverse genetics. H9N2 AIVs that circulated in this period mostly belonged to clade h9.4.2. Two increasing incidents occurred in the ability of H9N2 AIVs to bind to avian-like receptors in 2002-2005 and 2011-2014. Two increasing incidents occurred in the strength of H9N2 AIVs to bind to human-like receptors in 2002-2005 and 2011-2017. We found that Q227M, D145G/N, S119R, and R246K mutations can significantly increase H9N2 AIVs to bind to both avian- and human-like receptors. A160D/N, Q156R, T205A, Q226L, V245I, V216L, D208E, T212I, R172Q, and S175N mutations can significantly enhance the strength of H9N2 AIVs to bind to human-like receptors. Our study also identified mutations T205A, D208E, V216L, Q226L, and V245I as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2002-2005 and mutations S119R, D145G, Q156R, A160D, T212I, Q227M, and R246K as the key sites leading to enhanced receptor binding of H9N2 AIVs during 2011-2017. These findings further illustrate the receptor-binding characteristics of avian influenza viruses, which can be a potential threat to public health.

Nitrate Is Crucial for the Proliferation of Gut Escherichia coli Caused by H9N2 AIV Infection and Effective Regulation by Chinese Herbal Medicine Ageratum-Liquid.

H9N2 avian influenza virus (AIV) infection in chickens is often accompanied by secondary bacterial infection, but the mechanism is unclear. The aim of the present study was to reveal that mechanism and explore non-antibiotic treatment. 16s rRNA sequencing and metabonomics were performed in the intestinal contents of chickens infected with H9N2 AIV or H9N2 AIV and fed with ageratum-liquid (AL) to reveal the metabolite that promote intestinal Escherichia coli (E. coli) proliferation caused by H9N2 AIV, as well as to determine the regulatory effect of AL. It was found that H9N2 AIV infection led E. coli to become the dominant gut microbe and promoted E. coli translocation from the intestinal tract to the visceral tissue through the damaged intestinal barrier. H9N2 AIV infection induces inflammation in the intestinal mucosa and promotes the secretion and release of nitrate from the host intestinal epithelium. In addition, nitrate promoted E. coli proliferation in the inflamed intestinal tract following H9N2 AIV infection. Furthermore, Chinese herbal medicine AL can restore intestinal homeostasis, inhibit the production of nitrate in the intestinal epithelium and effectively prevent the proliferation and translocation of E. coli in the intestines. This is the first report on the mechanism of E. coli secondary infection induced by H9N2 AIV, where herbal medicine AL was shown to have a good preventive effect on the secondary infection.