The HN protein of Newcastle disease virus induces cell apoptosis through the induction of lysosomal membrane permeabilization.

Lysosomes are acidic organelles that mediate the degradation and recycling of cellular waste materials. Damage to lysosomes can cause lysosomal membrane permeabilization (LMP) and trigger different types of cell death, including apoptosis. Newcastle disease virus (NDV) can naturally infect most birds. Additionally, it serves as a promising oncolytic virus known for its effective infection of tumor cells and induction of intensive apoptotic responses. However, the involvement of lysosomes in NDV-induced apoptosis remains poorly understood. Here, we demonstrate that NDV infection profoundly triggers LMP, leading to the translocation of cathepsin B and D and subsequent mitochondria-dependent apoptosis in various tumor and avian cells. Notably, the released cathepsin B and D exacerbate NDV-induced LMP by inducing the generation of reactive oxygen species. Additionally, we uncover that the viral Hemagglutinin neuraminidase (HN) protein induces the deglycosylation and degradation of lysosome-associated membrane protein 1 (LAMP1) and LAMP2 dependent on its sialidase activity, which finally contributes to NDV-induced LMP and cellular apoptosis. Overall, our findings elucidate the role of LMP in NDV-induced cell apoptosis and provide novel insights into the function of HN during NDV-induced LMP, which provide innovative approaches for the development of NDV-based oncolytic agents.

The NP protein of Newcastle disease virus dictates its oncolytic activity by regulating viral mRNA translation efficiency.

Newcastle disease virus (NDV) has been extensively studied as a promising oncolytic virus for killing tumor cells in vitro and in vivo in clinical trials. However, the viral components that regulate the oncolytic activity of NDV remain incompletely understood. In this study, we systematically compared the replication ability of different NDV genotypes in various tumor cells and identified NP protein determines the oncolytic activity of NDV. On the one hand, NDV strains with phenylalanine (F) at the 450th amino acid position of the NP protein (450th-F-NP) exhibit a loss of oncolytic activity. This phenotype is predominantly associated with genotype VII NDVs. In contrast, the NP protein with a leucine amino acid at this site in other genotypes (450th-L-NP) can facilitate the loading of viral mRNA onto ribosomes more effectively than 450th-F-NP. On the other hand, the NP protein from NDV strains that exhibit strong oncogenicity interacts with eIF4A1 within its 366-489 amino acid region, leading to the inhibition of cellular mRNA translation with a complex 5' UTR structure. Our study provide mechanistic insights into how highly oncolytic NDV strains selectively promote the translation of viral mRNA and will also facilitate the screening of oncolytic strains for oncolytic therapy.

Cellular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein.

The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Current status and future direction of duck hepatitis A virus vaccines.

Duck viral hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a highly fatal and rapidly spreading infectious disease of young ducklings that seriously jeopardizes the duck industry worldwide. DHAV type 1 (DHAV-1) is the main genotype responsible for disease outbreaks since 1945, and the disease situation is complicated by the emergence and dissemination of a novel genotype (DHAV-3) in some countries in Asia and Africa. Live attenuated DHAV vaccines are widely used to induce a considerable degree of protection in ducklings. Breeder ducks are immunized with inactivated or/and live DHAV vaccines to achieve satisfactory levels of passive immunity in progeny. In addition, novel characteristics of virus transmission, pathogenicity and pathogenesis of DHAV were recently characterized, necessitating the development of new vaccines and effective vaccination programmes against DVH. Therefore, a systematic dissection of the profiles, strengths and shortcomings of the available DHAV vaccines is essential. Moreover, to further increase the efficiency of vaccine production and administration, the development of next-generation DHAV vaccines using cutting-edge technologies is also required. In this review, based on a comprehensive summary of the research advances in the epidemiology, pathogenicity, and genomic features of DHAV, we focus on reviewing and analysing the features of the commercial and experimental DHAV vaccines. We also propose perspectives for disease control based on the specific disease situations in different countries. This review provides essential information for vaccine development and disease control of DVH.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses.

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.

Current situation and future direction of Newcastle disease vaccines.

Newcastle disease (ND) is one of the most economically devastating infectious diseases affecting the poultry industry. Virulent Newcastle disease virus (NDV) can cause high mortality and severe tissue lesions in the respiratory, gastrointestinal, neurological, reproductive and immune systems of poultry. Tremendous progress has been made in preventing morbidity and mortality caused by ND based on strict biosecurity and wide vaccine application. In recent decades, the continual evolution of NDV has resulted in a total of twenty genotypes, and genetic variation may be associated with disease outbreaks in vaccinated chickens. In some countries, the administration of genotype-matched novel vaccines in poultry successfully suppresses the circulation of virulent NDV strains in the field. However, virulent NDV is still endemic in many regions of the world, especially in low- and middle-income countries, impacting the livelihood of millions of people dependent on poultry for food. In ND-endemic countries, although vaccination is implemented for disease control, the lack of genotype-matched vaccines that can reduce virus infection and transmission as well as the inadequate administration of vaccines in the field undermines the effectiveness of vaccination. Dissection of the profiles of existing ND vaccines is fundamental for establishing proper vaccination regimes and developing next-generation vaccines. Therefore, in this article, we provide a broad review of commercial and experimental ND vaccines and promising new platforms for the development of next-generation vaccines.

Foreign gene expression attenuates a virulent Newcastle disease virus in chickens.

Newcastle disease virus (NDV) is an important pathogen for poultry and is used as a vector for developing novel poultry vaccines. Previous studies showed that foreign gene insertion in NDV vector decreases virulence determined by in vitro assays; however, the impact of foreign gene expression on the pathogenicity of NDV in susceptible chickens is not fully investigated. In this study, a recombinant NDV based on a velogenic strain carrying the orange fluorescent protein (OFP) gene between the phosphoprotein (P) and matrix (M) genes was generated using reverse genetics. Biological characteristics, including virus replication, virulence, and OFP expression, and the pathogenicity in chickens were evaluated. The recombinant NDV showed comparable replication capacity in eggs and cells as the parental virus, whereas OFP insertion resulted in a mild impairment of virulence, evidenced by longer mean death time in embryos. High OFP expression was detected in the cells inoculated with the recombinant NDV. In addition, the recombinant NDV induced delayed onset of disease, lower severity of clinical signs, and lower mortality in chickens compared to the parental virus. Moreover, high titers of the parental virus were detected in the spleen, lung, and intestinal tract, while no recombinant NDV was recovered from these tissues. Our findings suggest that in vitro characteristics related to the insertion of the OFP gene in a virulent NDV do not correlate to alteration of the pathogenicity in chickens. Our results provided new information regarding assessment of the impact of foreign gene expression on the pathogenicity of NDV.

Characterization of antibody response to an epitope spanning the haemagglutinin cleavage site of H7N9 subtype avian influenza virus for the differentiation of infected and vaccinated chickens.

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.

Matrix metalloproteinase-14 regulates collagen degradation and migration of mononuclear cells during infection with genotype VII Newcastle disease virus.

Upregulation of matrix metalloproteinase (MMP)-14, a major driven force of extracellular-matrix (ECM) remodelling and cell migration, correlates with ECM breakdown and pathologic manifestation of genotype VII Newcastle disease virus (NDV) in chickens. However, the functional relevance between MMP-14 and pathogenesis of genotype VII NDV remains to be investigated. In this study, expression, biofunction and regulation of MMP-14 induced by genotype VII NDV were analysed in chicken peripheral blood mononuclear cells (PBMCs). The results showed that JS5/05 significantly increased expression and membrane accumulation of MMP-14 in PBMCs, correlating to enhanced collagen degradation and cell migration. Specific MMP-14 inhibition significantly impaired collagen degradation and migration of JS5/05-infected cells, suggesting dependence of these features on MMP-14. In addition, MMP-14 upregulation correlated with activation of the extracellular signal-regulated kinase (ERK) pathway upon JS5/05 infection, and blockage of the ERK signalling significantly suppressed MMP-14-mediated collagen degradation and migration of JS5/05-infected cells. Using a panel of chimeric NDVs derived from gene exchange between genotype VII and IV NDV, the fusion and haemagglutinin-neuraminidase genes were identified as the major viral determinants for MMP-14 expression and activity. In conclusion, MMP-14 was defined as a critical regulator of collagen degradation and cell migration of chicken PBMCs infected with genotype VII NDV, which may contribute to pathology of the virus. Our findings add novel information to the body of knowledge regarding virus-host biology and NDV pathogenesis.

Activation of the extracellular signal-regulated kinase pathway is required for replication of Newcastle disease virus.

Replication of Newcastle disease virus (NDV) is regulated by various host mechanisms, but the role of the extracellular signal-regulated kinase (ERK) pathway in regulating NDV replication is an open question. In this study, the relationship between the ERK pathway and NDV replication was investigated. NDV activated the ERK signaling in chicken embryo fibroblasts at the late stage of infection, correlating to expression of viral proteins. Specific blockage of the ERK pathway activation significantly decreased the transcription and translation levels of viral genes as well as virus replication and the cytopathogenic effect caused by NDV. Our results demonstrate that activation of the ERK pathway is required for NDV replication.

Recombinant baculovirus vaccine expressing hemagglutinin of H7N9 avian influenza virus confers full protection against lethal highly pathogenic H7N9 virus infection in chickens.

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.

Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity.

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.

The virulence factor PA protein of highly pathogenic H5N1 avian influenza virus inhibits NF-κB transcription in vitro.

Nuclear factor kappa B (NF-κB) plays a crucial role in inflammation and immune responses. Our previous studies have demonstrated that the innate immune response affect H5N1 virus virulence in mice. In this study, we first showed that the PA protein of the highly pathogenic avian influenza virus strain CK10 had the strongest inhibitory effect on NF-κB activation when compared with other genes, and that it acted in a dose independent-manner. We then determined the critical amino acids of PA that contribute to this effect. Furthermore, PA also inhibited NF-κB-regulated inflammatory factors, including IL-6, IL-2, Nos-2 and TNF-α. However, the inhibitory effect on NF-κB activation mediated by PA was not associated with nuclear translocation of p65.

PA-X decreases the pathogenicity of highly pathogenic H5N1 influenza A virus in avian species by inhibiting virus replication and host response.

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.

The M, F and HN genes of genotype VIId Newcastle disease virus are associated with the severe pathological changes in the spleen of chickens.

BACKGROUND: The strains of the genotype VIId Newcastle disease virus (NDV) induce more severe tissue damage in lymphoid organs than other virulent strains. The underlying molecular mechanisms are poorly understood. METHODS: Genotype IV NDV Herts/33 and genotype VIId NDV JS5/05 have a distinctive pathological profile in the spleen. These two strains of viruses were selected as parental viruses to generate a panel of chimeric viruses by replacing the M, F and HN genes of Herts/33 individually or in combination with the corresponding genes of JS5/05 using reverse genetic. Virulence and in vitro characteristics of the recombinant viruses were assessed. In addition, pathological changes, virus load, and transcriptional cytokine response in the spleen of chickens infected with these recombinant viruses were also analyzed. RESULTS: Pathogenicity test showed that all chimeric viruses are virulent. In vitro characterization revealed that gene replacement did not change growth kinetics and HN expression on cell surface of the recombinant viruses. However, replacement of the M, F and HN genes resulted in apparent changes in the fusion activity. Moreover, pathological studies revealed that only inclusion of the homologous M, F and HN genes of JS5/05 in Herts/33 backbone resulted in severe pathological changes characterized by extensive necrosis in the spleen, similar to that induced by JS5/05. In addition, this gene replacement significantly increased virus replication and the levels of transcriptional cytokine response, compared to Herts/33. Conversely, inclusion of the M, F and HN genes of Herts/33 into JS5/05 backbone resulted in Herts/33-specific pathological changes and significantly decreased virus load and the expression levels of cytokine genes, compared to JS5/05. CONCLUSIONS: The M, F and HN genes are related to the severe pathological changes in the spleen of chickens infected with genotype VIId NDV.

High levels of virus replication and an intense inflammatory response contribute to the severe pathology in lymphoid tissues caused by Newcastle disease virus genotype VIId.

Some strains of Newcastle disease virus (NDV) genotype VIId cause more-severe tissue damage in lymphoid organs compared to other virulent strains. In this study, we aim to define the mechanism of this distinct pathological manifestation of genotype VII viruses. Pathology, virus replication, and the innate immune response in lymphoid tissues of chickens infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV F48E8 and genotype IV NDV Herts/33, were compared. Histopathologic examination showed that JS5/05 and JS3/05 produced more-severe lesions in the spleen and thymus, but these four virulent strains caused comparable mild lesions in the bursa. In addition, JS3/05 and JS5/05 replicated at significantly higher levels in the lymphatic organs than F48E8 and Herts/33. A microarray assay performed on the spleens of chickens infected with JS5/05 or Herts/33 revealed that JS5/05 elicited a more potent inflammatory response by increasing the number and expression levels of activated genes. Moreover, cytokine gene expression profiling showed that JS5/05 and JS3/05 induced a stronger cytokine response in lymphoid tissues compared to F48E8 and Herts/33. Taken together, our results indicate that the severe pathology in immune organs caused by genotype VIId NDV strains is associated with high levels of virus replication and an intense inflammatory response.

The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice.

Highly pathogenic H5N1 influenza A virus remains a substantial threat to public health. To understand the molecular basis and host mechanism for the high virulence of H5N1 viruses in mammals, we compared two H5N1 isolates which have similar genetic backgrounds but greatly differ in their virulence in mice. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is nonpathogenic. We first showed that CK10 elicited a more potent innate immune response than did GS10 in mouse lungs by increasing the number and expression levels of activated genes. We then generated a series of reassortants between the two viruses and evaluated their virulence in mice. Inclusion of the CK10 PA gene in the GS10 background resulted in a dramatic increase in virulence. Conversely, expression of the GS10 PA gene in the CK10 background significantly attenuated the virulence. These results demonstrated that the PA gene mainly determines the pathogenicity discrepancy between CK10 and GS10 in mice. We further determined that arginine (R) at position 353 of the PA gene contributes to the high virulence of CK10 in mice. The reciprocal substitution at position 353 in PA or the exchange of the entire PA gene largely caused the transfer of viral phenotypes, including virus replication, polymerase activity, and manipulation of the innate response, between CK10 and GS10. We therefore defined a novel molecular marker associated with the high virulence of H5N1 influenza viruses, providing further insights into the pathogenesis of H5N1 viruses in mammals.

The PA and HA gene-mediated high viral load and intense innate immune response in the brain contribute to the high pathogenicity of H5N1 avian influenza virus in mallard ducks.

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.

Mutations in the FPIV motif of Newcastle disease virus matrix protein attenuate virus replication and reduce virus budding.

The FPIV-like late domains identified in the matrix (M) proteins of parainfluenza virus 5 and mumps virus have been demonstrated to be critical for virus budding. In this study, we found that the same FPIV sequence motif is present in the N-terminus of the Newcastle disease virus (NDV) M protein. Mutagenesis experiments demonstrated that mutation of either phenylalanine (F) or proline (P) to alanine led to a more obvious decrease in viral virulence and replication and resulted in poor budding of the mutant viruses. Additionally, evidence for the involvement of cellular multivesicular body (MVB) proteins was obtained, since NDV production was inhibited upon expression of dominant-negative versions of the VPS4A-E228Q protein. Together, these results demonstrate that the FPIV motif, especially the residues F and P, within the NDV M protein, plays a critical role in NDV replication and budding, and this budding process likely involves the cellular MVB pathway.

Dual N-linked glycosylation at residues 133 and 158 in the hemagglutinin are essential for the efficacy of H7N9 avian influenza virus like particle vaccine in chickens and mice.

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.