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H7N9 subtype avian influenza virus (AIV) poses a great challenge to poultry industry. Virus-like particle (VLP) is a prospective alternative for the traditional egg-based influenza vaccines. N-linked glycosylation (NLG) regulates the efficacy of influenza vaccines, whereas the impact of NLG modifications on the efficacy of influenza VLP vaccines remains unclear. Here, H7N9 VLPs were assembled in insect cells through co-infection with the baculoviruses expressing the NLG-modified hemagglutinin (HA), neuraminidase and matrix proteins, and the VLP vaccines were assessed in chickens and mice. NLG modifications significantly enhanced hemagglutination-inhibition and virus neutralization antibody responses in mice, rather than in chickens, because different immunization strategies were used in these animal models. The presence of dual NLG at residues 133 and 158 significantly elevated HA-binding IgG titers in chickens and mice. The VLP vaccines conferred complete protection and significantly suppressed virus replication and lung pathology post challenge with H7N9 viruses in chickens and mice. VLP immunization activated T cell immunity-related cytokine response and inhibited inflammatory cytokine response in mouse lung. Of note, the presence of dual NLG at residues 133 and 158 optimized the capacity of the VLP vaccine to stimulate interleukin-4 expression, inhibit virus shedding or alleviate lung pathology in chickens or mice. Intriguingly, the VLP vaccine with NLG addition at residue 133 provided partial cross-protection against the H5Nx subtype AIVs in chickens and mice. In conclusion, dual NLG at residues 133 and 158 in HA can be potentially used to enhance the efficacy of H7N9 VLP vaccines in chickens and mammals., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fc-fusion proteins (FCPs), a new generation biological medicine, have revolutionized the practice of medicines that treat diseases. However, complex manufacturing techniques are required for FCP production, casting the affordability and accessibility issues in low- and middle-income economies (LMIEs). Virus-vectored system may serve as a simple and cost-effective platform for FCP delivery. As a proof-of-concept study, Newcastle disease virus (NDV), a widely-used vector for vaccine generation, was used as a vector to express and deliver a model FCP composed of the hemagglutinin (HA) and IgG Fc. A recombinant NDV expressing the HA-Fc fusion protein was generated using reverse genetics, which had comparable replication and virulence to the parental virus. High levels of expression of soluble HA-Fc were detected in cell culture and embryonated chicken eggs inoculated with the recombinant NDV. In addition, the recombinant NDV replicated in the lung of mouse, delivering the HA-Fc protein to this organ. The HA-Fc expressed by NDV specifically bound to murine FcγRI, which was dependent on the presence of the Fc tag. The recombinant NDV induced high vector-specific antibody response, whereas it failed to elicit H7N9-specific antibody immunity in mice. The absence of HA-specific antibodies may be attributed to deficient incorporation of the HA-Fc protein into NDV virion particles. Our results indicated that NDV may be potentially used as a vector for FCP expression and delivery. This strategy may help to enhance the affordability and equal accessibility of FCP biological medicines, especially in LIMEs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

H7N9 subtype avian influenza virus (AIV) is endemic in poultry in China, and vaccination is used as the primary strategy for disease control. However, by current serological tests, monitoring H7N9 virus infection in vaccinated poultry is difficult because vaccine-induced antibodies are not readily distinguishable from field viruses. Therefore, a test differentiating infected and vaccinated animals (DIVA) is critical for monitoring H7N9 virus. However, no DIVA test is available for the H7N9 subtype AIV. This study investigated the potential of an epitope (peptide 11) spanning the haemagglutinin (HA) cleavage site as a DIVA antigen for the H7N9 virus. The results showed that the H7N9 virus infection sera and post-challenge sera obtained from H7N9-vaccinated chickens reacted with peptide 11, whereas the sera elicited by inactivated and viral-vectored H7N9 vaccines had no reactivity with this peptide. Peptide 11 was further split into two peptides at the HA cleavage site, and the truncated peptides failed to discriminate H7N9 infected and vaccinated chickens. Peptide 11 is located in a main surface loop in the HA protein, and contains highly conserved residues in the HA cleavage site among the H7N9 subtype and different subtypes of groups 1 and 2, suggesting the potential of this peptide as a broad DIVA antigen for influenza viruses. Our study highlighted that peptide 11 is a promising DIVA antigen, and serological tests based on this peptide may serve as useful tools for monitoring H7N9 virus infection in vaccinated poultry. RESEARCH HIGHLIGHTSThe epitope spanning the HA cleavage site is a potential DIVA antigen for H7N9 AIV.The epitope reacted with LP and HP H7N9 viruses.The epitope has potential as a broad DIVA antigen for influenza viruses.

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.

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence ( 431 W- 433 Y- 437 L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Avian influenza virus (AIV) H7N9 that emerged in 2013 in eastern China is a novel zoonotic agent mainly circulating in poultry without clinical signs but causing severe disease with high fatality in humans in more than 5 waves. Since the emergence of highly pathogenic (HP) H7N9 variants in 2016, it has induced heavy losses in the poultry industry leading to the implementation of an intensive nationwide vaccination program at the end of wave 5 (September 2017). To characterize the ongoing evolution of H7N9 AIV, we conducted analyses of H7N9 glycoprotein genes obtained from 2013 to 2019. Bayesian analyses revealed a decreasing population size of HP H7N9 variants post wave 5. Phylogenetic topologies revealed that two novel small subclades were formed and carried several fixed amino acid mutations that were along HA and NA phylogenetic trees since wave 5. Some of the mutations were located at antigenic sites or receptor binding sites. The antigenic analysis may reveal a significant antigenic drift evaluated by hemagglutinin inhibition (HI) assay and the antigenicity of H7N9 AIV might evolute in large leaps in wave 7. Molecular simulations found that the mutations (V135T, S145P, and L226Q) around the HA receptor pocket increased the affinity to α2,3-linked sialic acid (SIA) while decreased to α2,6-linked SIA. Altered affinity may suggest that HP H7N9 variations aggravate the pathogenicity to poultry but lessen the threat to public health. Selection analyses showed that the HP H7N9 AIV experienced an increasing selection pressure since wave 5, and the national implementation of vaccination might intensify the role of natural selection during the evolution waves 6 and 7. In summary, our data provide important insights about the genetic and antigenic diversity of circulating HP H7N9 viruses from 2017 to 2019. Enhanced surveillance is urgently warranted to understand the current situation of HP H7N9 AIV., (Copyright © 2021 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.)

H7N9 avian influenza vaccines induce high levels of non-neutralizing (nonNeu) antibodies against the haemagglutinin (HA). However, the antigenic epitopes underlying this particular antibody response are still undefined. In this study, a panel of 13 monoclonal antibodies (mAbs) against the HA protein of H7N9 virus was generated and 12 of them had no hemagglutination inhibition and virus neutralizing activities. One linear epitope in the stalk (373-TAA-375) recognized by three mAbs and one conformational epitope in the head (220Q-225S-227G) targeted by one mAb were identified using peptide-based enzyme-linked immunosorbent assay (ELISA) and biopanning of phage display random peptide library. In addition, competition ELISA revealed that the mAb targeting the head epitope strongly inhibited HA-binding of chicken nonNeu anti-H7N9 sera, whereas lower inhibition was observed for chicken neutralizing antisera, indicating the immunodominance of this epitope in the elicitation of nonNeu antibodies. Moreover, the stalk epitope is conserved among the H1-H17 subtypes and the mAb recognizing this epitope exhibited cross-reactivity with different subtypes. In conclusion, two novel nonNeu epitopes in H7N9 HA were identified, and an epitope in the head was identified as an immunodominant epitope underlying the induction of nonNeu H7N9 antibodies. Our results add new knowledge to the molecular basis for antibody immunity against H7N9 vaccines and provide useful implications for vaccine design and modification., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.)

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.

The emergent highly pathogenic avian influenza A (H7N9) (HPAI) virus is a major public concern in China. Therefore, it is crucially important to develop an effective vaccine against this virus. In this study, we constructed a baculovirus vaccine expressing the hemagglutinin (HA) of H7N9 strain A/Chicken/Jiaxing/148/2014 (JX148). The recombinant baculovirus (rBac-JX148HA) generated in this study showed good growth in insect cells and good safety, and it stably expressed the HA protein. We compared the immunogenicity and efficacy of the inactivated whole-virus vaccine JX148 and rBac-JX148HA. One chicken in the JX148-treated group died on day 4 post-challenge, and three chickens had typical clinical symptoms (survival rate, 90%; morbidity, 40%). However, no chickens immunized with rBac-JX148HA showed clinical signs during the 14-day observation period. An analysis of viral shedding and viral replication demonstrated that rBac-JX148HA more efficiently inhibited viral shedding and viral replication than the inactivated whole-virus vaccine. Taken together, these results indicate that the inactivated recombinant baculovirus vaccine induces a high hemagglutination inhibition antibody titer, provides complete protection against challenge with the highly pathogenic H7N9 virus, and effectively inhibits viral shedding. Therefore, the candidate vaccine has potential utility in the prevention and control of H7N9 avian influenza and is also appropriate for veterinary vaccines using cell suspension culture technology.

A chimeric Newcastle disease virus (NDV) vector (NDV/AI4-TFHN) was constructed with the replacement of the ectodomains of the fusion and hemagglutinin-neuraminidase proteins by those from avian paramyxovirus type 2. The chimeric virus induced high antibody response in chickens pre-immunized with NDV. A recombinant vaccine candidate, NDV/AI4-TFHN-H9, expressing the hemagglutinin of H9N2 avian influenza virus, was generated, on the basis of the chimeric NDV vector mentioned above. The NDV/AI4-TFHN-H9 vaccine elicited H9-specific hemagglutination inhibition antibodies in chickens pre-immunized with NDV vaccine, and reduced the numbers of chickens shedding virus after H9N2 challenge. NDV/AI4-TFHN-H9 could serve as an alternative vaccine for the prevention of H9N2 infection in commercial poultry flocks.

Long non-coding RNAs (lncRNAs) play multiple key regulatory roles in various biological processes. However, their function in influenza A virus (IAV) pathogenicity remains largely unexplored. Here, using next generation sequencing, we systemically compared the whole-transcriptome response of the mouse lung infected with either the highly pathogenic (A/Chicken/Jiangsu/k0402/2010, CK10) or the nonpathogenic (A/Goose/Jiangsu/k0403/2010, GS10) H5N1 virus. A total of 126 significantly differentially expressed (SDE) lncRNAs from three replicates were identified to be associated with the high virulence of CK10, whereas 94 SDE lncRNAs were related with GS10. Functional category analysis suggested that the SDE lncRNAs-coexpressed mRNAs regulated by CK10 were highly related with aberrant and uncontrolled inflammatory responses. Further canonical pathway analysis also confirmed that these targets were highly enriched for inflammatory-related pathways. Moreover, 9 lncRNAs and 17 lncRNAs-coexpressed mRNAs associated with a large number of targeted genes were successfully verified by qRT-PCR. One targeted lncRNA (NONMMUT011061) that was markedly activated and correlated with a great number of mRNAs was selected for further in-depth analysis, including predication of transcription factors, potential interacting proteins, genomic location, coding ability and construction of the secondary structure. More importantly, NONMMUT011061 was also distinctively stimulated during the highly pathogenic H5N8 virus infection in mice, suggesting a potential universal role of NONMMUT011061 in the pathogenesis of different H5 IAV. Altogether, these results provide a subset of lncRNAs that might play important roles in the pathogenesis of influenza virus and add the lncRNAs to the vast repertoire of host factors utilized by IAV for infection and persistence.

The emerged highly pathogenic avian influenza A (H7N9) (HPAI) viruses in China pose a dual challenge to public health and poultry industry. Thus H7N9 vaccines are in an urgent need. In this study, we constructed a Newcastle disease virus (NDV)-vectored vaccine (rLXHAF) expressing the hemagglutinin (HA) of H7N9 virus fused with the transmembrane/cytoplasmic tail domain of the NDV fusion protein. rLXHAF stably expressed the HA protein, exhibited similar growth kinetics and pathogenicity as the parental virus. rLXHAF induced positive NDV-specific hemagglutination inhibition (HI), virus neutralization (VN) and total IgY antibodies and completely protected chickens from NDV challenge. Unexpectedly, rLXHAF elicited undetectable HI and VN titers but high overall IgY antibody titers against H7N9 measured by ELISA. The vaccine provided 80% protection against HPAI H7N9 challenge. Virus shedding of NDV and H7N9 challenge strains was reduced. Our results suggest that rLXHAF is immunogenic and efficacious against HPAI H7N9 virus in chickens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.)

Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks.