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PA-X protein arises from a ribosomal frameshift in the PA of influenza A virus (IAV). However, the immune regulatory effect of the PA-X protein of H1N1 viruses on the nasal mucosal system remains unclear. Here, a PA-X deficient H1N1 rPR8 viral strain (rPR8-△PAX) was generated and its pathogenicity was determined. The results showed that PA-X was a pro-virulence factor in mice. Furthermore, it reduced the ability of H1N1 viruses to infect dendritic cells (DCs), the regulator of the mucosal immune system, but not non-immune cells (DF-1 and Calu-3). Following intranasal infection of mice, CCL20, a chemokine that monitors the recruitment of submucosal DCs, was downregulated by PA-X, resulting in an inhibition of the recruitment of CD11b+ DCs to submucosa. It also attenuated the migration of CCR7+ DCs to cervical lymph nodes and inhibited DC maturation with low MHC II and CD40 expression. Moreover, PA-X suppressed the maturation of phenotypic markers (CD80, CD86, CD40, and MHC II) and the levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while enhancing endocytosis and levels of anti-inflammatory IL-10 in vitro, suggesting an impaired maturation of DCs that the key step for the activation of downstream immune responses. These findings suggested that the PA-X protein played a critical role in escaping the immune response of nasal mucosal DCs for increasing the virulence of H1N1 viruses.

We investigated genetic and biologic characteristics of 2 Eurasian avian-like H1N1 swine influenza viruses from pigs in China that belong to the predominant G4 genotype. One swine isolate exhibited strikingly great homology to contemporaneous human Eurasian avian-like H1N1 isolates, preferential binding to the human-type receptor, and vigorous replication in mice without adaptation.

Infectious bronchitis virus (IBV) has restricted cell tropism. Apart from the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered the development of IBV vaccines and research on mechanisms of IBV infection. In a previous study, a new Vero-cell-adapted strain HV80 was obtained via serial chicken embryo and cell passaging of strain H120 and 17 mutations leading to amino acid substitutions occurred in replication gene 1a, S gene and E gene. This study, we constructed recombinants that expressed chimeric S glycoprotein, S1 or S2 subunit of strain H120, and demonstrated that mutations in S2 subunit were related to the Vero cell adaption of strain HV80. With a genome backbone of strain HV80 or H120, and expression of chimeric S2′ cleavage site of H120 or HV80, two recombinants demonstrated that the RRRR690/S motif at the S2′ cleavage site played a key role in Vero cell adaption of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that F692V enhanced the capability of invasion of HV80 strain, and Q855H induced the formation of syncytia. A transient transfection assay demonstrated different mechanisms for virus-to-cell fusion and cell-to-cell fusion induced by S glycoprotein. The PRRR690/S motif at the S2′ cleavage site could be activated by proteases in the process of cell-to-cell fusion, while H855Q substitution did not affect the cell invasion of HV80, but hindered the cell-to-cell fusion by blocking activation of the S2′ cleavage site.IMPORTANCEInfectious bronchitis is an acute respiratory disease that has caused large economic losses to the poultry industry. As a member of the gamma-coronaviruses, the restricted cell tropism of infectious bronchitis virus (IBV) limits the development of cellular vaccines and research on infection mechanisms. As a strain that can replicate effectively in mammalian cell lines, studies of HV80’s adaptive mechanisms point a way for engineering other IBVs for adaptation in mammalian cell lines. In our study, different recombinants were constructed by reverse genetic techniques, and demonstrated the different mechanism between virus-to-cell and cell-to-cell fusion induced by HV80 S glycoprotein. The acquisition of a highly efficient S2′ cleavage site enabled the virus to invade Vero cells. The Q855H substitution played a key role in cell-to-cell fusion, and provided a more efficient model of infection in Vero cells. Our study provides new theoretical insights into mechanisms of IBV adaptation in mammalian cell lines.

H5 subtype avian influenza viruses can cause serious diseases in poultry and wild birds. On rare occasions, viruses can cause cross-species transmission to mammalian species, including humans, pigs, horses, canines, seals, and minks.

H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.

H9N2 subtype low pathogenicity avian influenza virus (AIV) poses a potential zoonotic risk. PA-X, a novel protein generated by PA gene ribosomal frameshift, is considered to be the virulence factor of H9N2 subtype AIVs. Our study found that rTX possessing PA-X protein enhanced the mammalian pathogenicity of H9N2 subtype AIVs compared with PA-X-deficient virus (rTX-FS). Furthermore, PA-X protein inhibited H9N2 subtype AIVs to infect dendritic cells (DCs), but not nonimmune cells (MDCK cells). Meanwhile, PA-X protein suppressed the phenotypic expression (CD80, CD86, CD40 and MHCII), early activation marker (CD69) and pro-inflammatory cytokines (IL-6 and TNF-α), whereas increased anti-inflammatory cytokine (IL-10) in DCs. After intranasally viral infection in mice, we found that PA-X protein of H9N2 subtype AIVs reduced CD11b

The emergence and prevalence of mcr-1 and its variants in humans, animals, and the environment pose a global public health threat. There is a pressing urgency to develop rapid and accurate methods to identify MCR-positive colistin-resistant bacteria in the clinical samples, providing a basis for subsequent effective antibiotic treatment.

Background Tigecycline and carbapenems are regarded as vital antimicrobials to treat serious bacterial infections. Co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomonas spp. was not investigated. Objectives To characterize a megaplasmid co-harbouring tmexCD1-toprJ1 and blaVIM-2 in Pseudomonas putida of migratory bird origin. Methods One tigecycline- and carbapenem-resistant strain was isolated and characterized by antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. Results The strain P. putida ZXPA-20 resistant to meropenem and tigecycline was positive for blaVIM-2 and tmexCD1-toprJ1 genes. The gene blaVIM-2 was inserted into the backbone of the megaplasmid pZXPA-20 within a Tn5090-like structure. The genetic context of tmexCD1-toprJ1 in the megaplasmid was identical to many chromosomal tmexCD1-toprJ1 of Pseudomonas species. Plasmid-mediated tmexCD1-toprJ1 gene cluster in Pseudomonas spp. was more common than that in Klebsiella pneumoniae. To the best of our knowledge, this is the first report of co-occurrence of blaVIM-2 and tmexCD1-toprJ1 in one plasmid. Conclusions Emergence of plasmid-mediated carbapenem and tigecycline resistance genes in P. putida from migratory birds highlighted the importance of surveillance of novel mobile resistance genes in migratory birds, which may play a vital role in global transmission of novel resistance genes.

For an investigation into the effects of glycosylation site modification on hemagglutinin (HA) on the biological characteristics of the H5N6 subtype avian influenza virus (AIV), the HA sequences of H5N6 AIVs from Global Initiative on Sharing All Influenza Data (GISAID) and the isolates in China were analyzed for genetic evolution and glycosylation site patterns. Eight recombinant H5N6 AIVs with different glycosylation site patterns were constructed, and their biological characteristics were determined. The results showed that H5N6 AIVs containing a 129-glycosylation site on HA are becoming prevalent strains in China. Acquisition of the 129-glycosylation site on the HA of H5N6 AIVs increased thermostability, decreased pH stability, and attenuated pathogenicity and contact transmission in chickens. Most importantly, H5N6 AIVs escaped the neutralization activity of the Re-8-like serum antibody. Our findings reveal that H5N6 AIVs containing the 129-glycosylation site affect antigenicity and have become prevalent strains in China.

The emergence of multidrug-resistant (MDR) bacteria harboring tet(X4), bla

Influenza A virus (IAV) polymerase protein PB2 has been shown to partially inhibit the host immune response by blocking the induction of interferons (IFNs). However, the IAV PB2 protein that regulates the downstream signaling pathway of IFNs is not well characterized. Here, we report that IAV PB2 protein reduces cellular sensitivity to IFNs, suppressing the activation of STAT1/STAT2 and ISGs. Furthermore, IAV PB2 protein targets mammalian JAK1 at lysine 859 and 860 for ubiquitination and degradation. Notably, the H5 subtype of highly pathogenic avian influenza virus with I283M/K526R mutations on PB2 increases the ability to degrade mammalian JAK1 and exhibits higher replicate efficiency in mammalian (but not avian) cells and mouse lung tissues, and causes greater mortality in infected mice. Altogether, these data describe a negative regulatory mechanism involving PB2-JAK1 and provide insights into an evasion strategy from host antiviral immunity employed by IAV.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4

In recent years in China, clade 2.3.4.4 H5N6 plus clade 2.3.2.1 H5N1 subtype highly pathogenic avian influenza (HPAI) viruses have gradually become endemic in poultry, and their co-circulation could inevitably facilitate the gene reassortment between each other. During our routine surveillance in live poultry markets (LPMs) in eastern China in 2017-2018, a novel reassortant H5N6 strain with the HA gene derived from clade 2.3.2.1 was isolated from the cloacal swabs of apparently healthy ducks. Phylogenetic tracing analysis indicated that another two clade 2.3.2.1 H5N1 strains with divergent lineages of PB1 gene and one clade 2.3.4.4 H5N6 isolate of the dominant genotype sharing spatio-temporal proximity were intimately involved in the generation of this rarely reported clade 2.3.2.1 H5N6 reassortant. Distinct with the other three HPAI H5 viruses showing moderate virulence in mice, the H5N1 strain of the homologous internal gene constellation against the clade 2.3.2.1 H5N6 reassortant was highly pathogenic, which might probably attribute to the H3 subtype-derived PB1 gene. However, as compared to the clade 2.3.4.4 H5N6 ancestor, the clade 2.3.2.1 H5N6 reassortant displayed a broader tissue distribution and higher viral titres in mice, which could likely facilitate the viral maintenance and spread in nature. Therefore, our results highlight that continuous epidemiological survey of H5 subtype HPAI viruses in LPMs needs to be strengthened to prevent the potential poultry or even public health threat of the novel reassortants from endemic viruses.

Antibody-antigen (Ab-Ag) interactions are canonically described by a model that exclusively accommodates noninteraction (0) or reproducible interaction (RI) states, yet this model is inadequate to explain often-encountered nonreproducible signals. Here, by monitoring diverse experimental systems using a peptide-protein hybrid microarray, we observed that Ab-probe interactions comprise a substantial proportion of nonreproducible antibody-based results. This enabled our discovery and capacity to reliably identify nonreproducible Ab-probe interactions (NRIs), as well as our development of a powerful explanatory model ("0-NRI-RI-Hook four-state model") that is mAb concentration-dependent, regardless of specificity, which ultimately shows that both nonspecific interactions and NRIs are not predictable yet certain to happen. Our discoveries challenge the centrality of Ab-Ag interaction specificity data in serology and immunology.

Preventing pathogenic viral and bacterial transmission in the human environment is critical, especially in potential outbreaks that may be caused by the release of ancient bacteria currently trapped in the permafrost. Existing commercial disinfectants present issues such as a high carbon footprint. This study proposes a sustainable alternative, a bioliquid derived from biomass prepared by hydrothermal liquefaction. Results indicate a high inactivation rate of pathogenic virus and bacteria by the as-prepared bioliquid, such as up to 99.99% for H1N1, H5N1, H7N9 influenza A virus, and Bacillus subtilis var . niger spores and 99.49% for Bacillus anthracis . Inactivation of Escherichia coli and Staphylococcus epidermidis confirmed that low-molecular-weight and low-polarity compounds in bioliquid are potential antibacterial components. High temperatures promoted the production of antibacterial substances via depolymerization and dehydration reactions. Moreover, bioliquid was innoxious as confirmed by the rabbit skin test, and the cost per kilogram of the bioliquid was $0.04427, which is notably lower than that of commercial disinfectants. This study demonstrates the potential of biomass to support our biosafety with greater environmental sustainability.

In recent years, clade 2.3.4.4 H5N6 subtype avian influenza virus (AIV) has been predominantly prevalent in poultry flocks in China. During our AIV surveillance in 2018-2019, 6 circulating strains of H5N6 that possess the natural loss of glycosylation site 154 due to N154D mutation in HA2 protein were isolated. In particular, 5 strains simultaneously carried the V202I mutation in NA protein. Based on the paired backbone H5N6 viruses Y6 and RY6, which just diverged in the glycosylation status at site 158 in HA1 protein, 8 reassortants of rY6-154 N/202 V, rY6-154D/202 V, rY6-154 N/202I and rY6-154D/202I plus rRY6-154 N/202 V, rRY6-154D/202 V, rRY6-154 N/202I and rRY6-154D/202I were constructed with different variation patterns at site 154 in HA2 and site 202 in NA. By determining those reassortants in growth performance on cells, plaque-forming ability, heat and low pH stability, and pathogenicity in mammals, the results showed that HA2 N154D and NA V202I could singly or jointly change the viral biological properties both in vitro and in vivo. Additionally, the effect of HA mutation was significantly more robust than that of NA, and the resulting increasing or reducing impact was closely related to the glycosylation at HA1 site 158. The present study provided a reference for further parsing the relevant mechanism of the functional match between HA and NA proteins of the influenza virus.

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Antibody-antigen (Ab-Ag) interactions are canonically described by a model which exclusively accommodates non-interaction (0) or reproducible-interaction (RI) states, yet this model is inadequate to explain often-encountered non-reproducible signals. Here, by monitoring diverse experimental systems and confirmed COVID-19 clinical sera using a peptide microarray, we observed that non-specific interactions (NSI) comprise a substantial proportion of non-reproducible antibody-based results. This enabled our discovery and capacity to reliably identify non-reproducible Ab-Ag interactions (NRI), as well as our development of a powerful explanatory model (“0-RI-NRI-Hook four-state model”) that is [mAb]-dependent, regardless of specificity, which ultimately shows that both NSI and NRI are not predictable yet certain-to-happen. In experiments using seven FDA-approved mAb drugs, we demonstrated the use of NSI counts in predicting epitope type. Beyond challenging the centrality of Ab-Ag interaction specificity data in serology and immunology, our discoveries also facilitated the rapid development of a serological test with uniquely informative COVID-19 diagnosis performance.

H9N2 subtype avian influenza virus has dramatically evolved and undergone extensive reassortment since its emergence in early 1990s in China. The genotype S (G57), emerging in 2007 with the substitution of F98-like PB2 and M gene by G1-like ones, has become the overwhelming predominant genotype for the past 11 years since 2010. Here, we found that virus with G1-like PB2 were more efficient in protein expression and in infectious virus production than that with F98-like PB2 gene. By coinfected MDCK cells with the reassortant virus, more survival opportunity for viruses with G1-like PB2 than that of F/98-like was observed. Besides, in animal experiments, we found that the G1-like PB2 increases virus infectivity, replication, and virus shedding of H9N2 in chickens. Our results suggested that the substitution of G1-like PB2 play important role in promoting the fitness of genotype S H9N2 virus in China.

As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade 2.3.4.4 variants in or around the recognized antigenic epitopes A-E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single-site or multi-site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade 2.3.4.4) as the HA backbone to study their antigenic variations, respectively. The hemagglutination-inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.

The H5N1 subtype of the avian influenza virus causes sporadic but fatal infections in humans. H5N1 virus infection leads to the disruption of the alveolar epithelial barrier, a pathologic change that often progresses into acute respiratory distress syndrome (ARDS) and pneumonia. The mechanisms underlying this remain poorly understood. Here we report that H5N1 viruses downregulate the expression of intercellular junction proteins (E-cadherin, occludin, claudin-1, and ZO-1) in several cell lines and the lungs of H5N1 virus-infected mice. H5N1 virus infection activates TGF-β-activated kinase 1 (TAK1), which then activates p38 and ERK to induce E3 ubiquitin ligase Itch expression and to promote occludin ubiquitination and degradation. Inhibition of the TAK1-Itch pathway restores the intercellular junction structure and function in vitro and in the lungs of H5N1 virus-infected mice. Our study suggests that H5N1 virus infection impairs the alveolar epithelial barrier by downregulating the expression of intercellular junction proteins at the posttranslational level.

The contamination of Salmonella Enteritidis in eggs and chicken meat via vertical transmission has become a worldwide public health concern. Biofilm formation by S. Enteritidis further enhances its antibacterial resistance. However, whether genes related to biofilm formation affect the level of vertical transmission is still unclear. Here, S. Enteritidis mutants ΔcsgD, ΔcsgA, ΔbcsA, and ΔadrA were constructed from wild type strain C50041 (WT), and their biofilm-forming ability was determined by Crystal violet staining assay. Then the median lethal dose (LD50) assay was performed to determine the effects of the selected genes on virulence. The bacterial load in eggs produced by infected laying hens via the intraperitoneal pathway or crop gavage was determined for evaluation of the vertical transmission. Crystal violet staining assay revealed that S. Enteritidis mutants ΔcsgD, ΔcsgA, and ΔbcsA, but not ΔadrA, impaired biofilm formation compared with WT strain. Furthermore, the LD50 in SPF chickens showed that both the ΔcsgD and ΔbcsA mutants were less virulent compared with WT strain. Among the intraperitoneally infected laying hens, the WT strain-infected group had the highest percentage of bacteria-positive eggs (24.7%), followed by the ΔadrA group (16%), ΔcsgA group (9.9%), ΔbcsA group (4.5%), and ΔcsgD group (2.1%). Similarly, among the crop gavage chickens, the WT strain group also had the highest infection percentage in eggs (10.4%), followed by the ΔcsgA group (8.5%), ΔadrA group (7.5%), ΔbcsA group (1.9%), and ΔcsgD group (1.0%). Our results indicate that the genes csgD and bcsA help vertical transmission of S. Enteritidis in chickens.

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Background Hemagglutinin is a major surface protein in influenza A virus (IAV), and HA2 is relative conserved among different IAVs. It will be meaningful to identify broad-spectrum epitopes based on the HA2 protein. Results Overlapping peptides of the HA2 protein of the H5N1 IAV A/Mallard/Huadong/S/2005 were synthesized and loaded on modified silica gel film to form a microarray, and antisera against different subtypes of IAVs were used to screen universal epitopes. The selected epitope was further confirmed by western blotting using anti-peptide immune serum and viruses rescued with amino acid substitution. The results showed that 485-FYHKCDNECME-495 of the H5 14th peptide in HA2 had broad-spectrum binding activity with antisera against H1, H3, H4, H5, H6, H7, H8, H9, and H10 subtype IAV. Substitution of amino acids (K or D) in rescued viruses resulted in decreased serum binding, indicating that they were critical residues for serum binding activity. In Immune Epitope Database, some epitopes containing 14–4 peptide were confirmed as MHC-II-restricted CD4 T cell epitope and had effects on releasing IL-2 or IFN. Conclusion The identified epitope should be a novel universal target for detection and vaccine design and its ability to generate immune protection needs further exploration.

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

The contamination of Salmonella Enteritidis in eggs and chicken meat via vertical transmission has become a worldwide public health concern. Biofilm formation by S. Enteritidis further enhances its antibacterial resistance. However, whether genes related to biofilm formation affect the level of vertical transmission is still unclear. Here, S. Enteritidis mutants ΔcsgD, ΔcsgA, ΔbcsA, and ΔadrA were constructed from wild type strain C50041 (WT), and their biofilm-forming ability was determined by Crystal violet staining assay. Then the median lethal dose (LD50) assay was performed to determine the effects of the selected genes on virulence. The bacterial load in eggs produced by infected laying hens via the intraperitoneal pathway or crop gavage was determined for evaluation of the vertical transmission. Crystal violet staining assay revealed that S. Enteritidis mutants ΔcsgD, ΔcsgA, and ΔbcsA, but not ΔadrA, impaired biofilm formation compared with WT strain. Furthermore, the LD50 in SPF chickens showed that both the ΔcsgD and ΔbcsA mutants were less virulent compared with WT strain. Among the intraperitoneally infected laying hens, the WT strain-infected group had the highest percentage of bacteria-positive eggs (24.7%), followed by the ΔadrA group (16%), ΔcsgA group (9.9%), ΔbcsA group (4.5%), and ΔcsgD group (2.1%). Similarly, among the crop gavage chickens, the WT strain group also had the highest infection percentage in eggs (10.4%), followed by the ΔcsgA group (8.5%), ΔadrA group (7.5%), ΔbcsA group (1.9%), and ΔcsgD group (1.0%). Our results indicate that the genes csgD and bcsA help vertical transmission of S. Enteritidis in chickens.

The extended co-circulation of H5 subtype highly pathogenic avian influenza (HPAI) viruses and H9N2 low pathogenic avian influenza (LPAI) viruses has inevitably facilitated gene reassortment between the two subtypes in fields. And, novel reassortant H5NX viruses harboring partial or even whole sets of H9N2 internal genes have continuously been detected, such as clade 2.3.4.4 H5N2 or H5N6 reassortants. Here, we report two novel H5N2 subtype HPAI isolates of HF9 and QY5 from chickens in live poultry markets during routine surveillance in 2018. Phylogenetic analysis showed that those two H5N2 strains both possessed the HA genes from clade 2.3.2.1e of H5N1 viruses but all the other seven gene segments consistently from the endemic S genotype of H9N2 subtype viruses. Further analysis revealed that HF9 and QY5 differed only in six sites including K353R, A588T and T661I in PB2, I682V and L704S in PB1 plus G631S in PA at the amino acid level. A chicken regression experiment confirmed that both HF9 and QY5 were lethal infection to all tested chickens via contact transmission. Moreover, those two isolates could immediately replicate in mice lungs without adaptation. However, mortality rate of those two variants were distinct in mice model, HF9 with 100% but QY5 with just 20% at the infection dosage of 10

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines' development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, &gamma, &delta, T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of &gamma, T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.

With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

The H9N2 avian influenza virus is not only an important zoonotic pathogen, it can also easily recombine with other subtypes to generate novel reassortments, such as the H7N9 virus. Although H9N2 live attenuated vaccines can provide good multiple immunities, including humoral, cellular, and mucosal immunity, the risk of reassortment between the vaccine strain and wild-type virus is still a concern. Here, we successfully rescued an H9N2 live attenuated strain [rTX-NS1-128 (mut)] that can interdict reassortment, which was developed by exchanging the mutual packaging signals of HA and truncated NS1 genes and confirmed by RT-PCR and sequencing. The dynamic growth results showed that rTX-NS1-128 (mut) replication ability in chick embryos was not significantly affected by our construction strategy compared to the parent virus rTX strain. Moreover, rTX-NS1-128 (mut) had good genetic stability after 15 generations and possessed low pathogenicity and no contact transmission characteristics in chickens. Furthermore, chickens were intranasally immunized by rTX-NS1-128 (mut) with a single dose, and the results showed that the hemagglutination inhibition (HI) titers peaked at 3 weeks after vaccination and lasted at least until 11 weeks. The cellular immunity (IL-6 and IL-12) and mucosal immunity (IgA and IgG) in the nasal and trachea samples were significantly increased compared to inactivated rTX. Recombinant virus provided a good cross-protection against homologous TX strain (100%) and heterologous F98 strain (80%) challenge. Collectively, these data indicated that rTX-NS1-128(mut) lost the ability for independent reassortment of HA and NS1-128 and will be expected to be used as a potential live attenuated vaccine against H9N2 subtype avian influenza.

Background: Establishment of scientific disinfection measures in domestic poultry farms and live-poultry markets (LPMs) are critical to prevent the spread of H7N9 subtype avian influenza that outbreaks in poultry and infection in humans. The current study was aimed to evaluate the efficacy of four common disinfectants to inactivate H7N9 subtype avian influenza virus (AIV). Methods: According to the disinfection technical specification, the average inactivation logarithmic values of four disinfectants against the epidemic H7N9 subtype AIV, including deciquam solution, glutaral and benzalkonium bromide solution, sodium dichloroisocyanurate powder, and peroxyacetic acid solution, were systematically evaluated. Result: Our data showed that incubation of 0.005% (w/v) deciquam solution for 30 min contact time, or 0.05% (v/v) glutaral and benzalkonium bromide solution for 10 min, or 0.24 g/L sodium dichloroisocyanurate powder for 30 min, or 0.05% (v/v) peroxyacetic acid solution for 10 min, can completely inactivate H7N9 subtype AIV. These results suggested that glutaral and benzalkonium bromide solution as well as peroxyacetic acid solution at recommended concentrations can be effectively used to cut off the spread of H7N9 subtype AIV in poultry farms and LPMs, however, deciquam solution and sodium dichloroisocyanurate powder need to increase their working concentration.

NS gene is generally considered to be related to the virulence of highly pathogenic avian influenza virus (AIV). In recent years, the strains with five amino acids added to the 80-84 positions of the NS1 protein have become prevalent in H5N1 subtype AIVs isolated from mammals. However, the pathogenicity and mechanism of this pattern in mammals remain unclear. In this study, H5N1 subtype AIVs without 80-84 amino acids of the NS1 protein (rNSΔ5aa ) and a mutant virus (rNS5aa-R ) with no deletion of 80-84 amino acids of the NS1 protein were used to determine the pathogenicity in mice. Our results showed that rNS5aa-R possessed an enhanced pathogenicity compared with rNSΔ5aa in vivo and in vitro, which was accompanied by high expression of IL-6, MX1 and CXCL10 in murine lungs. Furthermore, we found that rNS5aa-R increased the infection ability to dendritic cells (DCs). Besides, rNS5aa-R enhanced the expression of phenotypic markers (CD80, CD86, CD40 and MHCII), activation marker CD69, inflammatory cytokines (IL-6, TNF-α and IL-10) and antagonized interferon (IFN-α) of DCs, in comparison to rNSΔ5aa . Moreover, rNS5aa-R induced DCs to quickly migrate into nearby cervical lymph nodes by highly upregulating CCR7, and CD86 showed a high expression on the migrated DCs. We also found that rNS5aa-R -infected DCs significantly promoted the allogeneic CD4+ T-cell proliferation. These findings suggested that rNS5aa-R strongly induced the innate immune response compared with the rNSΔ5aa , which is conducive to activate a wide immune response, resulting in a strong cytokine storm and causing an enhanced pathogenicity of H5N1 subtype AIVs in mammals.

The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.

H5N8 subtype highly pathogenic avian influenza viruses (HPAIVs) pose a huge threat to poultry industry and general public health. Our previous study demonstrated that synergistic effect of 283M and 526R in PB2 gene was a critical factor for viral high pathogenicity in mammals. However, the potential pathogenic mechanism of the mutant virus is still unclear. Here, RNA-seq method was used to analyze the global host response of murine lungs after infecting with parental r-JY virus and JY-PB2-I283M-K526R mutant virus. We found that both amounts and the expression levels of host differentially expressed genes (DEGs) were higher in mutant virus-infected mice compared with the group of parental virus. Furthermore, the DEGs mainly related with innate immune response by GO and KEGG analysis. Especially, PB2-I283M-K526R mutation strongly induced a sharp expression of cytokine storm-related genes, including MX1, CXCL10, and IFN-γ, performed by qRT-PCR. We also found that PB2-I283M-K526R mutation accelerated the level of cell apoptosis by heat map analysis of apoptosis-related DEGs in lungs and apoptosis assay in vitro. Taken together, our data demonstrated that PB2-I283M-K526R of H5N8 subtype HPAIV exacerbated the innate immune response and the level of cell apoptosis, which might be a key pathogenic mechanism for the enhanced pathogenicity of mutants in mammals.

Pigeons were previously thought to be resistant to H5 viruses and to play a minimal role in spreading these viruses. In this study, we evaluated the pathogenicity of two clade 2.3.4.4 H5N6 viruses in pigeons and the potential viral transmissibility to specific-pathogen-free chickens in direct close contact with experimentally infected pigeons. No pigeons from the A/goose/Eastern China/Xin/2015 (GS/Xin) group exhibited clinical signs or mortality, and the virus was only detected in a few organs. However, 3 of 12 pigeons inoculated with the A/goose/Eastern China/0326/2015 (GS/0326) virus died, and 7 of 12 showed neurological symptoms and efficient viral replication in multiple organs. In both groups, viral shedding occurred in only some of the pigeons, the shedding period was relatively short, and the infection was not transmitted to the chickens. We also used chicken, duck, and BALB/c mouse models to evaluate the pathogenicity of the two H5N6 isolates. Both H5N6 isolates showed highly pathogenic to chickens but different degrees of pathogenicity in mice. Interestingly, in ducks, the intravenous pathogenicity index indicated that the GS/Xin isolate was low pathogenic, and the GS/0326 isolate was highly pathogenic, corresponding to the pathogenicity in pigeons. Our results indicated that the pathogenicity of the clade 2.3.4.4 H5N6 virus is diverse in pigeons, and pigeons contribute little to its transmission among poultry. However, pigeons may still be potential healthy reservoirs of the H5N6 highly pathogenic avian influenza virus.

Background Although influenza A virus (IAV) employs diverse strategies to evade IFN responses by inhibiting the synthesis of IFN, how IAV regulates signaling downstream of IFN is incompletely understood. Methods In this study, we used Western blot-based protein analysis coupled with RT-qPCR, overexpression and RNA interference to investigate the regulation of JAK1 by IAV infection. Results The results indicated that JAK1 was ubiquitinated and degraded, resulting in inhibition of type I and type II IFN responses, demonstrating that IAV antagonizes the IFN-activated JAK/STAT signaling pathway by inducing the degradation of JAK1. Furthermore. IAV infection upregulated the suppressor of cytokine signaling (SOCS) protein SOCS1, and SOCS1 mediated the ubiquitination and degradation of JAK1. Conclusion Collectively, our findings suggest that IAV infection induces SOCS1 expression to promote JAK1 degradation, which in turn inhibits host innate immune responses.

Hemagglutinin (HA) glycosylation of avian influenza virus (AIV) effects differently depending on the variation of glycosylation position and numbers. The natural mutation on the glycosylation sites of the AIV HA head occurs frequently. Our previous study shows that deletion of 158 or 169 glycosylation site on the HA head of the H5 subtype AIV strain rS-144-/158+/169+ increases the viral virulence in mammals; however, the mechanism remains unknown. In this study, several AIVs with different deletions at HA head glycosylation sites 144, 158 or 169 were tested for their biological characteristics to clarify the possible mechanism. We found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of inflammatory cytokines than rS-144-/158+/169+ did in the infected cells, but the TCID50 , EID50 and MDT of the viruses showed no difference. Moreover, we found that rS-144-/158-/169+ and rS-144-/158+/169- viruses induced higher levels of endoplasmic reticulum (ER) stress in the cells. Inhibition of inositol-requiring enzyme 1α (IRE1α) phosphorylation reduced the inflammation induced by AIV infection. Furthermore, we found that rS-144-/158-/169+ virus activated the c-Jun N-terminal kinase (JNK), X-box binding protein 1 (XBP1), and nuclear factor-κB pathways by activating IRE1α phosphorylation under ER stress, whereas the rS-144-/158+/169- virus activated only the JNK pathway by altering IRE1α phosphorylation. In vivo analysis of Kira6 intervention further confirmed that ER stress played a key role in higher virulence for HA head 158 or 169 site de-glycosylation AIV. Our findings reveal that deletion of additional HA head glycosylation sites 158 or 169 enhanced the AIV virulence via activating of strong ER stress and inflammation.

Before 2013, zoonotic influenza infections were dominated by H5N1 viruses in China. However, the emergence of the H7N9 viruses in early 2013 changed this dominance greatly, and more than 1,600 laboratory-confirmed human cases of H7N9 infections have been reported since then. To understand the underlying mechanism of the emergence of the fifth epidemic wave that shows an unexpected sharp increase, we systematically investigated the biological characteristics of the highly pathogenic (HP) and low-pathogenic (LP) H7N9 AIVs during this period. We first systematically analysed the haemagglutination assay gene of all the isolates available from the website and found that the HP and LP viruses differed a little in the well-established receptor binding sites and in other potentially important sites. Phylogenetic analysis showed that both the HP and LP viruses belong to the branch of the Yangtze River Delta, whereas they diverged to different small branches. To further compare the biological variations in the HP and LP viruses, we selected six HP and six LP strains for in-depth analysis, including receptor binding characteristics, thermal stability, viral replication and virulence in mice. The three major findings of this study were as follows: (a) Other potential site/sites may affect the receptor binding property of the H7N9 viruses; (b) the HP viruses displayed a higher thermostability than did the LP viruses, quite consistent with the epidemiological data during the summer period; and (c) one-third of the HP viruses were moderately pathogenic in mice, whereas all the LP viruses were nonpathogenic in this animal model. However, the LP viruses replicated more efficiently in the mouse lung and can spread to the extrarespiratory organs (spleen, kidney and brain). Taken together, our results suggest that both the HP and LP H7N9 viruses can pose a potential threat to public health, highlighting the importance of the continual surveillance of the H7N9 AIVs.

Genetic reassortments between highly pathogenic avian influenza (HPAI) H5 subtype viruses with different neuraminidase (NA) subtypes have increased in prevalence since 2010 in wild birds and poultry from China. The HA gene slightly evolved from clade 2.3.4 to clade 2.3.4.4, raising the question of whether novel clade 2.3.4.4 HA broke the balance with N1 but is matched well with NAx to drive viral epidemics. To clarify the role of compatibility between HA and NA on the prevalence of H5Nx subtypes, we constructed 10 recombinant viruses in which the clade 2.3.4 or clade 2.3.4.4 HA genes were matched with different NA (N1, N2 and N8) genes and evaluated viral characteristics and pathogenicity. Combinations between clade 2.3.4 HA and N1 or between clade 2.3.4.4 HA and NAx, but not between clade 2.3.4.4 HA and N1, or between clade 2.3.4 HA and NAx, promoted viral growth, NA activity, thermostability, low-pH stability and pathogenicity in chicken and mice. These findings suggest that both clade 2.3.4 HA/N1 and clade 2.3.4.4 HA/NAx displayed a better match, which could promote the increased prevalence of clade 2.3.4 H5N1 AIV (prior to 2010) and clade 2.3.4.4 H5Nx AIV (since 2010) in China, respectively.

H7N9 avian influenza virus poses a dual threat to both poultry industry and public health. Therefore, it is highly urgent to develop an effective vaccine to reduce its pandemic potential. Virus-like particles (VLP) represent an effective approach for pandemic vaccine development. In this study, a recombinant baculovirus co-expressing the HA, NA and M1 genes of the H7N9 virus was constructed for generation of H7N9 VLP. Single immunization of chickens with 15 μg of the VLP or the commercial whole virus inactivated vaccine stimulates high hemagglutination inhibition, virus neutralizing and HA-specific IgY antibodies. Moreover, the antiserum had a good cross-reactivity with H7N9 field strains isolated in different years. Within 14 days after a lethal challenge with highly pathogenic (HP) H7N9 virus, no clinical symptoms and death were observed in the vaccinated chickens, and no virus was recovered from the organs. Compared to the non-vaccinated chickens, H7N9 VLP significantly reduced the proportion of animals shedding virus. Only 30 % of the VLP-vaccinated birds shed virus, whereas virus shedding was detected in 50 % of the chickens immunized with the commercial vaccine. Moreover, both vaccines dramatically alleviated pulmonary lesions caused by HP H7N9 virus, with a greater degree observed for the VLP. Altogether, our results indicated that the H7N9 VLP vaccine candidate confers a complete clinical protection against a lethal challenge with HP H7N9 virus, significantly inhibits virus shedding and abolishes viral replication in chickens. The VLP generated in this study represents a promising alternative strategy for the development of novel H7N9 avian influenza vaccines for chickens.

Activation of c-Jun terminal kinase (JNK) by the nonstructural protein 1 (NS1) of the H5N1 subtype of influenza A virus (IAV) plays an important role in inducing autophagy and virus replication. However, the mechanisms of NS1-induced JNK activation remain elusive. Here we first confirmed the ability of H5N1 (A/mallard/Huadong/S/2005) to activate JNK and to induce autophagy in 293T cells, a human embryonic kidney cell line. We further showed that TAK1, MAP kinase kinase 4 (MKK4), and JNK were activated in 293T cells transfected with the NS1 gene of the H5N1 virus. JNK activation by the NS1 protein or by H5N1 virus was blocked by 5Z-7-Oxozeaenol (5Z), a TAK1-specific inhibitor, and by TAK1 siRNA. Further study showed that 5Z and TAK1 siRNA suppressed H5N1 virus-induced autophagy and inhibited virus replication. Our study unveiled a previously unrecognized role of TAK1 in IAV replication, IAV-induced JNK activation, and autophagy.

Summary: Influenza A virus (IAV) primarily infects the airway and alveolar epithelial cells and disrupts the intercellular junctions, leading to increased paracellular permeability. Although this pathological change plays a critical role in lung tissue injury and secondary infection, the molecular mechanism of IAV-induced damage to the alveolar barrier remains obscure. Here, we report that Gli1, a transcription factor in the sonic hedgehog (Shh) signaling pathway, is cross-activated by the MAP and PI3 kinase pathways in H1N1 virus (PR8)-infected A549 cells and in the lungs of H1N1 virus-infected mice. Gli1 activation induces Snail expression, which downregulates the expression of intercellular junction proteins, including E-cadherin, ZO-1, and Occludin, and increases paracellular permeability. Inhibition of the Shh pathway restores the levels of Snail and intercellular junction proteins in H1N1-infected cells. Our study suggests that Gli1 activation plays an important role in disrupting the intercellular junctions and in promoting the pathogenesis of H1N1 virus infections.

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1-like M and PB2 genes for the earlier F/98-like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1-like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98-like counterpart, while the G1-like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1-like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.

Genotype S H9N2 viruses frequently donate their internal genes to facilitate the generation of novel influenza viruses, e.g., H5N6, H7N9, and H10N8, which have caused human infection. Genotype S was originated from the replacement of F/98-like M and PB2 genes of the genotype H with those from G1-like lineage. However, whether this gene substitution will influence the viral fitness of emerging influenza viruses remains unclear. We found that H5Nx and H7N9 viruses with G1-like PB2 or M gene exhibited higher virulence and replication than those with F/98-like PB2 or M in chickens. We also determined the functional significance of G1-like PB2 in conferring increased polymerase activity and improved nucleus transportation efficiency, and facilitated RNP nuclear export by G1-like M. Our results suggest that G1-like PB2 and M genes optimize viral fitness, and thus play a crucial role in the genesis of emerging influenza viruses that cause rising prevalence in chickens.

Background Antigenic drift of H9N2 low pathogenic avian influenza viruses (AIV) may result in vaccination failure in the poultry industry and thus a cross-protective vaccine against H9N2 AIV is highly desirable. Methods A series of H9N2 recombinant viruses with the internal genes of A/Puerto Rico/8/34 (H1N1, PR8) were generated, based on the compatibility between HA and NA, the effect of HA deglycosylation, and protective antigenic epitopes in HA. After evaluation of their biological and immunological characteristics, three recombinant AIVs with the internal genes of the Y280-like strain SN were selected for protective efficacy studies. Results The recombinant viruses rHASNNA3, rHASN-△200, rHASN-△287, and rHASN-R92G-E93K displayed good cross reactivity and induced higher neutralization antibody titers against both SN and the F98-like strain YZ4. Furthermore, those recombinant viruses had a higher EID50 in chicken embryos after the replacement of internal-gene backbone from PR8 to SN. The rSNHA-△200 induced better protection in immunized chickens against challenge of homologous and heterologous H9N2 avian influenza viruses when compared with the wild type strain. Conclusion The recombinant virus rSNHA-△200 can be used as a potential broad-spectrum vaccine against H9N2 avian influenza.