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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 6.0 EID 50 per mouse. We suppose that the phenotypic difference may probably be attributed to the amino acid substitutions in the polymerase genes between the two isolates that constitute of a subject of further ongoing research., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2021. Published by Elsevier B.V.)

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, γδ 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 γδ 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., Competing Interests: The authors declare that they have no conflict of interest.

Many H5 and H7 subtype avian influenza vaccines are poorly immunogenic in terms of inducing hemagglutination-inhibition (HI) antibody titers. Residue 227 (H3 numbering) in the receptor binding site in the hemagglutinin (HA) is critical for the detectability of HI antibodies induced by H5 influenza vaccines. However, whether the effect of residue 227 on immunogenicity can be generalized in different subtypes is unclear. In this study, the impact of HA residue 227 on immunogenicity of H5N1, H5N6, and H7N9 avian influenza vaccines was evaluated in chickens. Polymorphism analysis revealed that S227 is overwhelmingly dominant in HA of the H5N1 and H7N9 subtypes, whereas this amino acid is present in a small proportion of H5N6 viruses. The H5N1, H5N6, and H7N9 vaccines harboring S227 in HA induced relatively low HI titers at week 2 postimmunization (pi), and antibody titers increased at week 3 pi. S227N substitution in these vaccines consistently enhanced HI titers significantly. Another H5N6 vaccine harboring Q227 in HA elicited a robust HI antibody response, and Q227S substitution led to a significant drop of HI titers. Cross-HI testing against the wild-type and mutant viruses revealed that the amino acid at position 227 was associated with the detectability of HI titers induced by H5 and H7 avian influenza vaccines. The results indicate an important role of residue 227 in HA in immunogenicity of H5 and H7 subtype avian influenza vaccines in chickens. Our findings also provided useful information for vaccine seed virus selection and genetic engineering for immunogenicity enhancement of avian influenza vaccines.

Pre-existing immunity against the conserved haemagglutinin (HA) stalk underlies the elicitation of cross-group antibody induced by natural H7N9 virus infection and immunization in humans. However, whether broadly reactive antibodies can be induced by H7N9 infection and immunization in the absence of pre-existing stalk-specific immunity is unclear. In this study, antibody response induced by H7N9 virus infection and immunization with inactivated and viral-vectored H7N9 vaccines in naïve chickens was analysed. The results showed that H7N9 infection and immunization with inactivated vaccine resulted in potent induction of haemagglutination-inhibition (HI), virus neutralization (VN) and HA-binding antibodies, whereas Newcastle disease virus (NDV)-vectored H7N9 vaccine induced marginal HI and VN titres but high levels of HA-binding antibody. In addition, H7N9 infection and immunization induced stalk-specific antibodies in naïve chickens and these antibodies recognized different epitopes in the stalk. Virus infection and immunization with inactivated vaccine elicited antibodies cross-reactive with both group 1 and group 2 HAs, while antibodies induced by NDV-H7N9 vaccination showed a narrower cross-reactivity within group 2. Moreover, only homologous neutralizing activity of the sera against H7N9 virus was observed, and cross-binding antibodies did not show heterosubtypic neutralizing activity. Our results indicated that cross-group binding but non-neutralizing antibodies primarily targeting the stalk can be induced by natural H7N9 infection and immunization with inactivated vaccine in naïve chickens. This suggests that at least in a naïve chicken model, pre-existing stalk-specific immunity is not required for induction of broadly reactive antibodies. Additionally, H7N9-based immunogens may be explored as vaccine candidates or as a prime component to induce broadly protective influenza immunity., (© 2020 Blackwell Verlag GmbH.)

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a highly contagious disease that has led to tremendous economic losses worldwide. Pigeon paramyxovirus type 1 (PPMV-1) is an antigenic and host variant of NDV. However, limited in-depth studies are available concerning side-by-side comparison of pathogenicity of PPMV-1 and its phylogenetically close NDV both in chickens and pigeons. To this end, two phylogenetically closely related NDV isolates, Kuwait 256 and JS/07/04/Pi from chicken and pigeon respectively were pathotypically and genotypically characterized in this study. The results indicated that Kuwait 256 was a velogenic strain, while JS/07/04/Pi was a mesogenic strain based on the mean death time of chick embryos (MDT) and intracerebral pathogenicity index in 1-day-old chicks (ICPI). Pathogenicity tests showed that Kuwait 256 caused severe clinical signs and 100 % mortality, while JS/07/04/Pi caused no apparent disease in chickens. Interestingly, both Kuwait 256 and JS/07/04/Pi caused morbidity and mortality in pigeons. Notably, pigeons infected with JS/07/04/Pi exhibited viral shedding for longer time compared to Kuwait 256-infected pigeons. Collectively, the findings of this study suggested that PPMV-1 decreased the pathogenicity in chickens but gained a survival advantage over NDV of chicken origin after its adaptive variation in pigeons based on the previous evidence that PPMV-1 originated from chicken-origin viruses. This study laid the foundation for the elucidation of the molecularmechanism underlying difference in pathogenicity of PPMV-1 and chicken-origin NDV in chickens., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2019 Blackwell Verlag GmbH.)

In the fifth wave of the H7N9 avian influenza epidemic, highly pathogenic avian influenza (HPAI) A (H7N9) viruses have emerged and pose a great challenge to public health and the poultry industry. In addition, there are apparent genetic and antigenic variations between the classical H7N9 avian influenza virus and the newly-emerged H7N9 virus. Therefore, an antigenic-match vaccine is required for the prevention and control of H7N9 avian influenza in poultry in China. In this study, a recombinant Newcastle disease virus (NDV)-vectored vaccine expressing the HA derived from a prevailing HPAI H7N9 virus (GD15) was generated using reverse genetics. The recombinant virus (rAI4HA) showed virus yield and growth capacity in chicken embryos comparable to the parental virus (rAI4). Expression of the HA protein was detected in chicken embryo fibroblasts inoculated with rAI4HA. A chicken immunization study demonstrated that both rAI4HA and rAI4 induced similar anti-NDV hemagglutination inhibition (HI) antibody titers at weeks 2, 3, and 4 after a single immunization. However, rAI4HA-immunized chickens had a low seroconversion rate (20%) and negative HI titers against H7N9. Additionally, rAI4HA elicited high levels of H7N9-specifc IgY antibody as measured by ELISA. More importantly, the recombinant vaccine provided a complete protection against a lethal challenge with HPAI H7N9 virus and significantly inhibited virus shedding after a single immunization. Our results suggest that the recombinant NDV-vectored H7N9 vaccine expressing the antigenic-match HA can confer a complete protection against HPAI H7N9 challenge after a single immunization.

Two reassortant H5N2 viruses in which hemagglutinin (HA) was clustered into clade 2.3.4.4, were isolated from apparently healthy waterfowl in live poultry markets in Eastern China in 2016. We used specific pathogen-free chickens, mallard ducks, and BALB/c mice to evaluate the isolates' biological characteristics in different animal models. The newly isolated reassortant H5N2 viruses were able to cause severe disease in chickens and effective contact transmission, only at high doses. Our pathogenicity studies in ducks yielded an interesting result: the intravenous pathogenicity index (IVPI) indicated that isolate A/goose/Eastern China/1106/2016(1106) was low pathogenic and the other isolate A/duck/Eastern China/YD1516/2016(YD1516) was of highly pathogenicity in ducks. However, our 50% duck lethal dose (DLD 50 ) experiment demonstrated that these viruses were all of low pathogenicity (DLD 50  > 10 7.0 EID 50 ) in ducks. Additionally, despite the fact that reassortant H5N2 were of low pathogenicity in mice, they could bind to both avian-type (SAα-2,3 Gal) and human-type (SAα-2,6 Gal) receptors, suggesting that these isolates still present a high risk for human infection. Therefore, it is of great importance to implement continual surveillance of avian influenza virus (AIV) to protect both veterinary and public health., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Pathogenesis of genotype VII Newcastle disease virus (NDV) is characterized with remarkable immunopathology in the spleen in chickens. However, the mechanism for this unique pathological phenotype is not fully understood. Previous transcriptomics data showed that genotype VII NDV JS5/05 caused a greater downregulation of extracellular matrix (ECM) genes than genotype IV virus Herts/33 in the spleen. In this study, the role of ECM in pathology of genotype VII NDV was investigated using quantitative proteomics. Pathology studies showed that JS5/05 caused severe immunopathology characterized with remarkable necrosis in the spleen, whereas Herts/33 only induced mild pathological changes. The ECM was firstly enriched from the spleens and ECM proteins of different categories were identified by LC-MS/MS. Quantitative proteomic analysis showed that JS5/05 caused a significant disruption of ECM integrity and molecular composition compared to Herts/33. Particularly, JS5/05 induced a more remarkable collagen breakdown in the spleen compared to Herts/33. Moreover, matrix metalloproteinase (MMP)-13 and -14 were significantly upregulated by JS5/05 infection. KEGG pathway analysis suggested that differential regulation of ECM proteins by JS5/05 and Herts/33 may impact pathology through different pathways. Therefore, our results suggested that MMP upregulation and consequent ECM degradation contribute to immunopathology of genotype VII NDV in the spleen., Significance: Pathogenesis of genotype VII NDV is characterized with severe immunopathology in the spleen in chickens. Elucidating the mechanism of this pathology phenotype is critical to understand pathogenesis of genotype VII NDV. Here, we present the proteomic data of an important non-cellular compartment, the extracellular matrix (ECM), in the spleen from chickens infected with genotype VII and IV NDVs. Our results suggest that significant upregulation of matrix metalloproteinases by genotype VII NDV and consequent disruption of ECM integrity and composition may be associated with immunopathology in the spleen. Moreover, ECM degradation, represented by collagen breakdown, is an important pathology event in the process of genotype VII NDV infection. Our study for the first time presents evidence of ECM regulation by NDV and adds ECM remodeling as a new manifestation for NDV pathology. Our findings also deepen the understanding of NDV pathogenesis., (Copyright © 2018. Published by Elsevier B.V.)

Background: Newcastle disease (ND) is one of the most contagious and devastating diseases to poultry in the world. The causative agents are virulent strains of Newcastle disease virus (NDV), which belong to the genus Avulavirus, sub-family Paramyxoviridae, family Paramyxovirinae. Knowing the genomic and antigenic characteristics of virulent NDVs might contribute to ND control in China., Results: The results showed that all of the virulent strains belonged sub-genotype VIId shared the same cleavage site (112)RRQKR/F(117) in the fusion protein. At least 69 % (38 of 55) of the NDV strains possessed E347K variation in the hemagglutinin-neuraminidase protein. The cross-neutralization tests confirmed that the strains harboring 347 K showed lower antigenic relatedness with LaSota. Furthermore, the immune-challenge experiment indicated that LaSota could not provide complete protection against infection with the E347K variant NDVs as the vaccinated birds were still able to be infected and shed virulent challenge viruses., Conclusions: Currently, sub-genotype VIId NDVs are the prevalent virulent strains circulating among vaccinated chicken flocks in Eastern China. Our findings indicated that the E347K variation in HN gene would expand the antigenic difference with LaSota, which may be responsible for the increasing isolation rate of these strains from vaccinated chickens.

Unlabelled: PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus., Importance: Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode as many as 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly identified protein in segment 3 that acts to decrease the virulence of the 1918 H1N1 virus in mice by modulating host gene expression. Our study extends these functions of PA-X to H5N1 HPAIV. We demonstrated that loss of PA-X expression increases the virulence and replication of an H5N1 virus in mice and avian species and alters the host innate immune and cell death responses. Our report is the first to delineate the role of the novel PA-X protein in the pathogenesis of H5N1 viruses in avian species and promotes our understanding of H5N1 HPAIV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Avian influenza viruses of subtype H9N2 are widely prevalent in poultry in many Asian countries, and the segmented nature of the viral genome results in multiple distinct genotypes via reassortment. In this study, genetic evolution of H9N2 viruses circulating in eastern China during 2007-2013 was analyzed. The results showed that the diversity of the gene constellations generated six distinct genotypes, in which a novel genotype (S) bearing the backbone of A/chicken/Shanghai/F/98-like viruses by acquiring A/quail/Hong Kong/G1/97-like polymerase basic subunit 2 and matrix genes has gradually established its ecological niche and been consistently prevalent in chicken flocks in eastern China since its first detection in 2007. Furthermore, genotype S possessed the peculiarity to donate most of its gene segments to other emerging influenza A viruses in China, including the novel reassortant highly pathogenic avian influenza H5N2, the 2013 novel H7N7, H7N9 and the latest reassortant H10N8 viruses, with potential threat to poultry industry and human health., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The role of live bird markets (LBMs) in the perpetuation of avian influenza virus (AIV) has been well studied worldwide; however, there is a paucity of information on the prevalence of different AIV subtypes in LBMs in Eastern China. In this study, long-term surveillance was conducted to investigate the prevalence of AIV in chickens, ducks, and geese in an LBM in Yangzhou city, Jiangsu province, Eastern China, between July 2002 and May 2011. The study used primary virus isolation and subtype-specific reverse-transcription-PCR. In total, 23 different HA-NA subtype combinations were detected, mainly in domestic ducks. This result suggests that domestic ducks play a more important role in LBMs in Eastern China.

There are few data available for the trends of antimicrobial resistance of Salmonella enterica subspecies enterica serovar Pullorum (S. Pullorum) in China and other parts of the world. Thus, the objective of this study was to evaluate the changes in antimicrobial resistance of S. Pullorum isolated from diseased chickens in China from 1962 to 2007. A total of 450 S. Pullorum isolates were tested for their susceptibility to 17 antimicrobials in a disk diffusion method. 39-95% of the isolates displayed a high level of resistance, particularly against ampicillin, carbenicillin, streptomycin, tetracycline, trimethoprim and sulfafurazole. Isolates exhibited increased resistance to carbenicillin, spectinomycin, trimethoprim, trimethoprim/sulfamethoxazole and nalidixic acid during the study period. Moreover, 56.2% of the isolates exhibited multiple drug resistance (MDR; resistance> or =4 antimicrobials) and showed an increasing trend between 1970-1979 and 2000-2007. Therefore, the results suggest that certain measures, including continued surveillance of antimicrobial resistance and the rational use of antimicrobials, are necessary and important in order to control the rapid increase in antimicrobial resistance in S. Pullorum.

Simple Summary: Avian influenza viruses have the ability to breach species barriers and infect mammals, posing a significant threat to public health. The H7N9 subtype of avian influenza virus that emerged in China in 2013 resulted in 1568 human infections, with an alarming mortality rate of nearly 40%. While human infections caused by avian influenza viruses typically occur sporadically, the reasons behind the widespread impact of H7N9 remain unclear. During surveillance for avian influenza in live poultry markets in eastern China in 2013, we isolated two H7N9 avian influenza viruses from seemingly healthy chickens. One of these chicken-derived viruses demonstrated the ability to bind to mammalian receptors and naturally harbored the mammalian molecular marker PB2-627K. Furthermore, this virus proved to be highly pathogenic to mice. In summary, our findings suggest that the presence of a mammal-adapted H7N9 virus in poultry in 2013 could be a significant factor explaining the unusually high number of human infections during that period. Avian influenza viruses can cross species barriers and adapt to mammals. The H7N9 subtype AIV that emerged in China in 2013 caused 1568 human infections, with a mortality rate of nearly 40%. We conducted a retrospective analysis of H7N9 viruses that were isolated in live poultry markets in 2013. We found that two avian-origin H7N9 isolates, A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013, have a similar genetic background but exhibit different pathogenicity in mice. Whole-genome alignment of the two H7N9 viruses was carried out, and only six amino acid differences mapped in five genes, including the well-known virulence molecular marker PB2-E627K. Our retrospective analysis highlighted the importance of monitoring the adaptive mutations in avian influenza viruses with zoonotic potential. [ABSTRACT FROM AUTHOR]

Commercial inactivated vaccines against H9N2 avian influenza (AI) have been developed in China since 1990s and show excellent immunogenicity with strong HI antibodies. However, currently approved vaccines cannot meet the clinical demand for a live-vectored vaccine. Newcastle disease virus (NDV) vectored vaccines have shown effective protection in chickens against H9N2 virus. However, preexisting NDV antibodies may affect protective efficacy of the vaccine in the field. Here, we explored avian paramyxovirus serotype 2 (APMV-2) as a vector for developing an H9N2 vaccine via intranasal delivery. APMV-2 belongs to the same genus as NDV, distantly related to NDV in the phylogenetic tree, based on the sequences of Fusion (F) and hemagglutinin-neuraminidase (HN) gene, and has low cross-reactivity with anti-NDV antisera. We incorporated hemagglutinin (HA) of H9N2 into the junction of P and M gene in the APMV-2 genome by being flanked with the gene start, gene end, and UTR of each gene of APMV-2-T4 to generate seven recombinant APMV-2 viruses rAPMV-2/HAs, rAPMV-2-NPUTR-HA, rAPMV-2-PUTR-HA, rAPMV-2-FUTR-HA, rAPMV-2-HNUTR-HA, rAPMV-2-LUTR-HA, and rAPMV-2-MUTR-HA, expressing HA. The rAPMV-2/HAs displayed similar pathogenicity compared with the parental APMV-2-T4 virus and expressed HA protein in infected CEF cells. The NP-UTR facilitated the expression and secretion of HA protein in cells infected with rAPMV-2-NPUTR-HA. Animal studies demonstrated that immunization with rAPMV-2-NPUTR-HA elicited effective H9N2-specific antibody (6.14 ± 1.2 log2) responses and conferred complete immune protection to prevent viral shedding in the oropharyngeal and cloacal swabs from chickens challenged with H9N2 virus. This study suggests that our recombinant APMV-2 virus is safe and immunogenic and can be a useful tool in the combat of H9N2 outbreaks in chicken. [ABSTRACT FROM AUTHOR]

As the causative agent of Newcastle disease (ND), Newcastle disease virus (NDV) has seriously restricted the development of the poultry industry. Previous research has shown that miRNAs, members of the small noncoding RNA family, are implicated in the regulation NDV replication through extensive interactions with host mRNAs, but whether miRNAs affect NDV replication by directly binding to the NDV antigenome remains unclear. In this study, potential Gallus gallus miRNAs targeting the antigenome of NDV were bioinformatically predicted using the online software RegRNA 2.0, and gga-miR-1603 and gga-miR-1794 were identified as targeting the viral L gene directly through dual-luciferase reporter assays. Sequence alignment analysis demonstrated that multiple genotypes of NDVs harbored highly conserved binding sites for gga-miR-1603 and gga-miR-1794 in the viral antigenome located at 8611–8634 nt and 14,490–14,514 nt, respectively. Meanwhile, we found that gga-miR-1603 and gga-miR-1794 negatively regulated the expression of viral L gene at both the RNA and protein levels, as well as viral replication in vitro. Furthermore, NDV infection had no effect on endogenous gga-miR-1603 and gga-miR-1794 expression in various avian cell lines. Overall, our present study demonstrated that gga-miR-1603 and gga-miR-1794 directly bind to the viral L gene to facilitate ts degradation and inhibit the replication of multiple genotypes of NDVs in vitro. These findings will provide us with important clues for antiviral therapy against NDV infection. [ABSTRACT FROM AUTHOR]

Abstract Background The goose is usually considered to be resistant even to strains of Newcastle disease virus (NDV) that are markedly virulent for chickens. However, ND outbreaks have been frequently reported in goose flocks in China since the late 1990s with the concurrent emergence of genotype VIId NDV in chickens. Although the NDVs isolated from both chickens and geese in the past 15 years have been predominantly VIId viruses, published data comparing goose- and chicken-originated ND viruses are scarce and controversial. Results In this paper, we compared genotype VIId NDVs originated from geese and chickens genetically and pathologically. Ten entire genomic sequences and 329 complete coding sequences of individual genes from genotype VIId NDVs of both goose- and chicken-origin were analyzed. We then randomly selected two goose-originated and two chicken-originated VIId NDVs and compared their pathobiology in both geese and chickens in vivo and in vitro with genotype IV virus Herts/33 as a reference. The results showed that all the VIId NDVs either from geese or from chickens shared high sequence homology and characteristic amino acid substitutions and clustered together in phylogenetic trees. In addition, geese and chickens infected by goose or chicken VIId viruses manifested very similar pathological features distinct from those of birds infected with Herts/33. Conclusions There is no genetic or phenotypic difference between genotype VIId NDVs originated from geese and chickens. Therefore, no species-preference exists for either goose or chicken viruses and more attention should be paid to the trans-species transmission of VIId NDVs between geese and chickens for the control and eradication of ND.

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. • Genotype S H9N2 AIV donate their internal gene cassette to facilitate the generation of H5N6, H7N9, etc. • Genotype S H9N2 is the substitution of G1-like PB2 and M genes for the earlier F/98-like M and PB2 of genotype H. • G1-like H9N2 PB2 and M genes enhance virulence of reassortant H5Nx and H7N9 viruses in chickens. • Genotype S H9N2 internal gene cassette is superior to that of genotype H in optimizing viral fitness in chickens. [ABSTRACT FROM AUTHOR]

Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples. [ABSTRACT FROM AUTHOR]

Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013–2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk. • Highlighting novel key elements in the HA and PA protein required for H7N9 transmissibility in chickens. • Discovery of a single amino acid mutation, HA-D167N, influencing the airborne transmission of H7N9. • Elucidating the pathogenic factors underlying the impact of HA and PA segments on the transmission of H7N9. • Providing novel insights into the sharp reduction of H7N9 in China. [ABSTRACT FROM AUTHOR]

Since the national vaccination program was implemented with the H5/H7 bivalent vaccine in poultry in September 2017, the prevalence of H7N9 avian influenza viruses (AIVs) has been controlled effectively in China. However, highly pathogenic H7N9 viruses still exist, causing sporadic outbreaks especially in some regions of northern China. During our routine surveillance in poultry in 2020, we isolated two strains of H7N9 subtype AIV from breeder layer farms in northern China. We found that these two chicken-origin H7N9 isolates were both highly pathogenic (HP) with a four-amino-acid (KRTA) insertion and an I326V mutation (H3 numbering) in the cleavage site of HA to make the motif PEVPKRKRTAR↓GLF. Molecular markers associated with antigenic drift and enhanced pathogenicity in mammals and interspecies transmission were detected in both isolates. Remarkably, both strains gained the F102V and N157D mutations in their HA genes, which have never been reported before. Solid-phase direct binding assay showed that these two isolates both had dual-receptor binding characteristics, while thermal and acid stability assays indicated that they were relatively stable in high-temperature or acidic conditions. In addition, the animal experiments demonstrated that both strains were highly pathogenic to chickens but low pathogenic to mice. These results suggested that the evolution of H7N9 subtype AIV is still continuing, and they pose a potential threat to poultry and public health. Thus, attention should be paid to the importance of continual surveillance of the H7N9 AIVs. • Two highly pathogenic antigenic variant H7N9 strains were isolated from chickens in 2020. • Both strains possessed dual-receptor binding characteristics and were thermal and acid stable. • F102V and N157D mutations in HA (H3 numbering) were novelly detected in H7N9 viruses. [ABSTRACT FROM AUTHOR]

• H7N9 VLP composed of HA, NA and M1 genes was generated in BEVS via coinfection strategy. • H7N9 VLP has strong immunogenicity and excellent efficacy both in chickens and mice. • H7N9 VLP significantly reduces virus shedding and viral replication in chickens and mice. • H7N9 VLP dramatically alleviates pulmonary lesions induced by H7N9 virus in chickens and mice. • H7N9 VLP significantly diminishes key inflammatory cytokine expression in mouse lung. 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. [ABSTRACT FROM AUTHOR]