RESUMO
H7N9 subtype avian influenza viruses (AIVs) cause 1567 human infections and have high mortality, posing a significant threat to public health. Previously, we reported that two avian-derived H7N9 isolates (A/chicken/Eastern China/JTC4/2013 and A/chicken/Eastern China/JTC11/2013) exhibit different pathogenicities in mice. To understand the genetic basis for the differences in virulence, we constructed a series of mutant viruses based on reverse genetics. We found that the PB2-E627K mutation alone was not sufficient to increase the virulence of H7N9 in mice, despite its ability to enhance polymerase activity in mammalian cells. However, combinations with PB1-V719M and/or PA-N444D mutations significantly enhanced H7N9 virulence. Additionally, these combined mutations augmented polymerase activity, thereby intensifying virus replication, inflammatory cytokine expression, and lung injury, ultimately increasing pathogenicity in mice. Overall, this study revealed that virulence in H7N9 is a polygenic trait and identified novel virulence-related residues (PB2-627K combined with PB1-719M and/or PA-444D) in viral ribonucleoprotein (vRNP) complexes. These findings provide new insights into the molecular mechanisms underlying AIV pathogenesis in mammals, with implications for pandemic preparedness and intervention strategies.
Assuntos
Subtipo H7N9 do Vírus da Influenza A , Mutação , Infecções por Orthomyxoviridae , Proteínas Virais , Animais , Camundongos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/veterinária , Virulência , Feminino , Proteínas Virais/genética , Proteínas Virais/metabolismo , Camundongos Endogâmicos BALB C , Replicação ViralRESUMO
The haemagglutinin-neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.
Assuntos
Apoptose , Proteína HN , NF-kappa B , Doença de Newcastle , Vírus da Doença de Newcastle , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/patogenicidade , Animais , Proteína HN/genética , Proteína HN/metabolismo , Doença de Newcastle/virologia , NF-kappa B/metabolismo , Doenças das Aves Domésticas/virologia , Galinhas , Embrião de GalinhaRESUMO
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.
Assuntos
Vírus da Doença de Newcastle , Vírus Oncolíticos , Animais , Vírus da Doença de Newcastle/genética , Aminoácidos , Leucina , Vírus Oncolíticos/genética , RNA Mensageiro/genética , Biossíntese de ProteínasRESUMO
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.
Assuntos
Proteína HN , Vírus da Doença de Newcastle , Animais , Vírus da Doença de Newcastle/metabolismo , Proteína HN/metabolismo , Catepsina B , Apoptose , Lisossomos/metabolismoRESUMO
Influenza A virus (IAV) continues to pose a pandemic threat to public health, resulting a high mortality rate annually and during pandemic years. Posttranslational modification of viral protein plays a substantial role in regulating IAV infection. Here, based on immunoprecipitation (IP)-based mass spectrometry (MS) and purified virus-coupled MS, a total of 89 phosphorylation sites distributed among 10 encoded viral proteins of IAV were identified, including 60 novel phosphorylation sites. Additionally, for the first time, we provide evidence that PB2 can also be acetylated at site K187. Notably, the PB2 S181 phosphorylation site was consistently identified in both IP-based MS and purified virus-based MS. Both S181 and K187 are exposed on the surface of the PB2 protein and are highly conserved in various IAV strains, suggesting their fundamental importance in the IAV life cycle. Bioinformatic analysis results demonstrated that S181E/A and K187Q/R mimic mutations do not significantly alter the PB2 protein structure. While continuous phosphorylation mimicked by the PB2 S181E mutation substantially decreases viral fitness in mice, PB2 K187Q mimetic acetylation slightly enhances viral virulence in mice. Mechanistically, PB2 S181E substantially impairs viral polymerase activity and viral replication, remarkably dampens protein stability and nuclear accumulation of PB2, and significantly weakens IAV-induced inflammatory responses. Therefore, our study further enriches the database of phosphorylation and acetylation sites of influenza viral proteins, laying a foundation for subsequent mechanistic studies. Meanwhile, the unraveled antiviral effect of PB2 S181E mimetic phosphorylation may provide a new target for the subsequent study of antiviral drugs.
Assuntos
Virus da Influenza A Subtipo H5N1 , Influenza Humana , RNA Polimerase Dependente de RNA , Proteínas Virais , Animais , Humanos , Camundongos , Virus da Influenza A Subtipo H5N1/fisiologia , Fosforilação , Proteínas Virais/metabolismo , Virulência , Replicação Viral , RNA Polimerase Dependente de RNA/metabolismoRESUMO
H9N2 subtype avian influenza virus (AIV) can transmit by direct as well as airborne contacts. It has been widespread in poultry and continued to contribute to zoonotic spillover events by providing its six internal genes for the reassortment of novel influenza viruses (eg, H7N9) that infect poultry and humans. Compared to H7N9, H9N2 virus displays an efficient airborne transmissibility in poultry, but the mechanisms of transmission difference have been insufficiently studied. The Hemagglutinin (HA) and viral polymerase acidic protein (PA) have been implicated in the airborne transmission of influenza A viruses. Accordingly, we generated the reassortant viruses of circulating airborne transmissible H9N2 and non-airborne transmissible H7N9 viruses carrying HA and/or PA gene. The introduction of the PA gene from H7N9 into the genome of H9N2 virus resulted in a reduction in airborne transmission among chickens, while the isolated introduction of the HA gene segment completely eliminated airborne transmission among chickens. We further showed that introduction of HA gene of non-transmissible H7N9 did not influence the HA/NA balance of H9N2 virus, but increased the threshold for membrane fusion and decreased the acid stability. Thus, our results indicate that HA protein plays a key role in replication, stability, and airborne transmission of the H9N2 subtype AIV.
Assuntos
Subtipo H7N9 do Vírus da Influenza A , Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Influenza Humana , Humanos , Animais , Galinhas , Hemaglutininas , Subtipo H7N9 do Vírus da Influenza A/genética , Aerossóis e Gotículas Respiratórios , Aves Domésticas , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírus Reordenados/genética , Vírus Reordenados/metabolismo , FilogeniaRESUMO
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.
RESUMO
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.
Assuntos
Hemaglutininas , Subtipo H7N9 do Vírus da Influenza A , Influenza Aviária , Influenza Humana , Animais , Humanos , Galinhas , Subtipo H7N9 do Vírus da Influenza A/genética , Mutação , Aves Domésticas , Hemaglutininas/genética , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genéticaRESUMO
IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.
Assuntos
Galinhas , Infecções por Herpesviridae , Herpesvirus Galináceo 1 , Interleucina-2 , Células Matadoras Naturais , Linfócitos T Citotóxicos , Vacinas Virais , Animais , Administração Oral , Galinhas/imunologia , Galinhas/virologia , Túnica Conjuntiva/virologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/virologia , Herpesvirus Galináceo 1/imunologia , Interleucina-2/administração & dosagem , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/virologia , Doenças Respiratórias/imunologia , Doenças Respiratórias/prevenção & controle , Doenças Respiratórias/veterinária , Doenças Respiratórias/virologia , Linfócitos T Citotóxicos/imunologia , Traqueia/virologia , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/efeitos adversos , Vacinas Virais/biossíntese , Vacinas Virais/imunologiaRESUMO
The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.
RESUMO
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 LCâMS/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.
Assuntos
Doença de Newcastle , Vírus da Doença de Newcastle , Animais , Vírus da Doença de Newcastle/fisiologia , Proteína HN/genética , Vimentina/genética , Cromatografia Líquida/veterinária , Espectrometria de Massas em Tandem/veterinária , GalinhasRESUMO
Clade 2.3.4.4 H5N6 avian influenza virus (AIV) has been predominant in poultry in China, and the circulating haemagglutinin (HA) gene has changed from clade 2.3.4.4h to clade 2.3.4.4b in recent years. In 2021, we isolated four H5N6 viruses from ducks during the routine surveillance of AIV in China. The whole-genome sequencing results demonstrated that the four isolates all belonged to the currently prevalent clade 2.3.4.4b but had different internal gene constellations, which could be divided into G1 and G2 genotypes. Specifically, G1 possessed H9-like PB2 and PB1 genes on the H5-like genetic backbone while G2 owned an H3-like PB1 gene and the H5-like remaining internal genes. By determining the characteristics of H5N6 viruses, including growth performance on different cells, plaque-formation ability, virus attachment ability, and pathogenicity and transmission in different animal models, we found that G1 strains were more conducive to replication in mammalian cells (MDCK and A549) and BALB/c mice than G2 strains. However, G2 strains were more advantageously replicated in avian cells (CEF and DF-1) and slightly more transmissible in waterfowls (mallards) than G1 strains. This study enriched the epidemiological data of H5 subtype AIV to further understand its dynamic evolution, and laid the foundation for further research on the mechanism of low pathogenic AIV internal genes in generating novel H5 subtype reassortants.
Assuntos
Patos , Vírus da Influenza A , Animais , Camundongos , Virulência/genética , China/epidemiologia , Genótipo , Vírus da Influenza A/genética , Camundongos Endogâmicos BALB C , MamíferosRESUMO
H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.
Assuntos
Vírus da Influenza A Subtipo H9N2 , Influenza Aviária , Animais , Influenza Aviária/epidemiologia , Hemaglutininas/genética , Deriva e Deslocamento Antigênicos , Teorema de Bayes , Galinhas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Aves Domésticas , China/epidemiologia , FilogeniaRESUMO
Long-term evolution of Newcastle disease virus (NDV) results in substantial alteration in viral pathogenesis. NDVs of genotype VII, a late genotype, show marked tropism to lymphoid tissues, especially to macrophages in chickens. However, the role of macrophages in the pathogenesis of genotype VII NDV is still unclear. Herein, NDV infectivity in macrophages and the role of macrophages in the pathogenesis of genotype VII NDV in chickens were investigated. We reported that NDV strains of genotype VII (JS5/05) and IV (Herts/33) can replicate in the adherent (predominantly macrophages) and non-adherent cells (predominantly lymphocytes) derived from chicken peripheral blood mononuclear cells (PBMCs), and significantly higher virus gene copy was detected in the adherent cells. In addition, JS5/05 had significantly higher infectivity in PBMC-derived adherent cells than Herts/33, correlating with its enhanced tropism to macrophages in the spleen of chickens. Interestingly, the depletion of 68% of macrophages exerted no significant impact on clinical signs, mortality and the systematic replication of JS5/05 in chickens, which may be associated with the contribution of non-depleted macrophages and other virus-supportive cells to virus replication. Macrophage depletion resulted in a marked exacerbation of tissue damage and apoptosis in the spleen caused by JS5/05. These findings indicated that macrophages play a critical role in alleviating tissue damage caused by genotype VII NDV in chickens. Our results unveiled new roles of macrophages in NDV pathogenesis in chickens.
RESUMO
Emerging evidence has demonstrated the critical role of long noncoding RNAs (lncRNAs) in regulating gene expression. However, the functional significance and mechanisms underlying influenza A virus (IAV)-host lncRNA interactions are still elusive. Here, we identified a functional lncRNA, LncRNA#61, as a broad anti-IAV factor. LncRNA#61 is highly upregulated by different subtypes of IAV, including human H1N1 virus and avian H5N1 and H7N9 viruses. Furthermore, nuclear-enriched LncRNA#61 can translocate from the nucleus to the cytoplasm soon after IAV infection. Forced LncRNA#61 expression dramatically impedes viral replication of various subtypes of IAV, including human H1N1 virus and avian H3N2/N8, H4N6, H5N1, H6N2/N8, H7N9, H8N4, H10N3, H11N2/N6/N9 viruses. Conversely, abolishing LncRNA#61 expression substantially favored viral replication. More importantly, LncRNA#61 delivered by the lipid nanoparticle (LNP)-encapsulated strategy shows good performance in restraining viral replication in mice. Interestingly, LncRNA#61 is involved in multiple steps of the viral replication cycle, including virus entry, viral RNA synthesis and the virus release period. Mechanistically, the four long ring arms of LncRNA#61 mainly mediate its broad antiviral effect and contribute to its inhibition of viral polymerase activity and nuclear aggregation of key polymerase components. Therefore, we defined LncRNA#61 as a potential broad-spectrum antiviral factor for IAV. Our study further extends our understanding of the stunning and unanticipated biology of lncRNAs as well as their close interaction with IAV, providing valuable clues for developing novel broad anti-IAV therapeutics targeting host lncRNAs.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Subtipo H7N9 do Vírus da Influenza A , Influenza Humana , RNA Longo não Codificante , Animais , Humanos , Camundongos , Antivirais/farmacologia , Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/farmacologia , Replicação ViralRESUMO
Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: ⢠A broad-spectrum mAb against various subtypes and sources of IAV was developed ⢠The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays ⢠The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Humana , Animais , Humanos , Camundongos , Suínos , Anticorpos Monoclonais , Nucleoproteínas , Epitopos de Linfócito B , Vírus da Influenza A Subtipo H3N2 , Anticorpos AntiviraisRESUMO
Highly pathogenic (HP) avian influenza A H7N9 virus has emerged in China since 2016. In recent years, it has been most prevalent in northern China. However, several strains of HP H7N9 reappeared in southwestern China (Yunnan Province) in 2021. As a result, we are wondering if these viruses have re-emerged in situ or been reintroduced. Here, we present phylogenetic evidence that the HP H7N9 viruses isolated in Yunnan emigrated from northern to southwestern China in 2020. The northern subregion of China has become a novel epicenter in HP H7N9 dissemination. Meanwhile, a cleavage motif re-emerged due to the T341I mutation, implying a parallel evolution. This cross-region transmission, which originated in non-adjacent provinces and traveled a great geographic distance in an unknown way, indicates that HP H7N9 dissemination did not halt in 2020, even under the shadow of the COVID-19 pandemic. Additional surveillance studies in poultry are required to determine the HP H7N9 virus's geographic distribution and spread.
Assuntos
COVID-19 , Subtipo H7N9 do Vírus da Influenza A , Influenza Aviária , Influenza Humana , Animais , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Filogenia , Pandemias , China/epidemiologia , COVID-19/epidemiologiaRESUMO
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.
Assuntos
Subtipo H7N9 do Vírus da Influenza A , Influenza Aviária , Doença de Newcastle , Vacinas Virais , Animais , Camundongos , Vírus da Doença de Newcastle/genética , Influenza Aviária/prevenção & controle , Galinhas , Proteínas Recombinantes/genética , Anticorpos , Doença de Newcastle/prevenção & controle , Vacinas Virais/genética , Anticorpos AntiviraisRESUMO
As a multifunctional protein, the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is involved in various biological functions. A velogenic genotype III NDV JS/7/05/Ch evolving from the mesogenic vaccine strain Mukteswar showed major amino acid (aa) mutations in the HN protein. However, the precise biological significance of the mutant HN protein remains unclear. This study sought to investigate the effects of the mutant HN protein on biological activities in vitro and in vivo. The mutant HN protein (JS/7/05/Ch-type HN) significantly enhanced the hemadsorption (HAd) and fusion promotion activities but impaired the neuraminidase (NA) activity compared with the original HN protein (Mukteswar-type HN). Notably, A494D and E495K in HN exhibited a synergistic role in regulating biological activities. Moreover, the mutant HN protein, especially A494D and E495K in HN, enhanced the F protein cleavage level, which can contribute to the activation of the F protein. In vitro infection assays further showed that NDVs bearing A494D and E495K in HN markedly impaired the cell viability. Simultaneously, A494D and E495K in HN enhanced virus replication levels at the early stage of infection but weakened later in infection, which might be associated with the attenuated NA activity and cell viability. Furthermore, the animal experiments showed that A494D and E495K in HN enhanced case fatality rates, virus shedding, virus circulation, and histopathological damages in NDV-infected chickens. Overall, these findings highlight the importance of crucial aa mutations in HN in regulating biological activities of NDV and expand the understanding of the enhanced pathogenicity of the genotype III NDV.
Assuntos
Proteína HN , Vírus da Doença de Newcastle , Animais , Proteína HN/química , Neuraminidase/genética , Neuraminidase/metabolismo , Hemaglutininas , Galinhas , Genótipo , MutaçãoRESUMO
The zoonotic H7N9 avian influenza virus emerged with the H9N2-origin internal gene cassette. Previous studies have reported that genetic reassortments with H9N2 were common in the first five human H7N9 epidemic waves. However, our latest work found that the circulating high pathogenicity H7N9 virus has established a dominant internal gene cassette and has decreased the frequency of reassortment with H9N2 since 2018. This dominant cassette of H7N9 was distinct from the cocirculating H9N2, although they shared a common ancestor. As a result, we suppose that this dominant cassette may benefit the viral population fitness and promote its continuous circulation in chickens.