Influenza A virus(IAV) has a wide host range,including wild birds,poultry,various mammals,and even humans(Xu et al.2024).Currently,two subtypes of canine influenza virus(CIV),H3N8 and H3N2,are primarily circulating in...Influenza A virus(IAV) has a wide host range,including wild birds,poultry,various mammals,and even humans(Xu et al.2024).Currently,two subtypes of canine influenza virus(CIV),H3N8 and H3N2,are primarily circulating in dogs.The H3N8 CIV was introduced from horses into dogs in 2004(Crawford et al.2005),while the H3N2 CIV originated from chickens in Asia in 2007(Song et al.2008).In China,H3N2 is the predominant CIV subtype,with a prevalence rate of up to 5.63% in the canine population,as reported by Chen et al.(2023).CIV infection typically manifests with symptoms such as coughing,sneezing,runny nose,and fever but is rarely fatal.However,co-infection with other pathogens(e.g.,Streptococcus,Mycoplasma or canine parainfluenza virus) can exacerbate symptoms and lead to lethal outcomes(Yondo et al.2023).展开更多
A型流感病毒(influenza A virus,IAV)极易发生突变和重组,导致现有疫苗和抗流感药物失效。因此,开发有效的广谱抗流感病毒疫苗和药物是我国乃至全球的重要需求。2025年1月9日,中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全...A型流感病毒(influenza A virus,IAV)极易发生突变和重组,导致现有疫苗和抗流感药物失效。因此,开发有效的广谱抗流感病毒疫苗和药物是我国乃至全球的重要需求。2025年1月9日,中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院朱启运研究员团队在Nature Communications杂志发表了题为“Influenza A Virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection”的研究论文。该研究分离并纯化了一株特异性识别A型流感病毒血凝素(HA)的纳米抗体E10,该抗体对不同亚型流感病毒具有跨组中和和体内保护效应。研究鉴定E10靶向HA头部结构域的保守氨基酸位点(K166/S167),并且含有E10表位的多肽分子能够诱导交叉反应性抗体,能够对小鼠提供部分交叉免疫保护。该研究不仅为流感防治提供了新的手段,也为广谱流感疫苗研发提供了新的靶点和希望。展开更多
【目的】构建A型流感病毒(Influenza A virus,IAV)聚合酶酸性蛋白(polymerase acidic protein,PA)的特异性纳米抗体(nanobody,Nb)噬菌体展示文库,获得广谱特异性识别PA蛋白的纳米抗体,为IAV基础研究和应用研究提供生物材料。【方法】构...【目的】构建A型流感病毒(Influenza A virus,IAV)聚合酶酸性蛋白(polymerase acidic protein,PA)的特异性纳米抗体(nanobody,Nb)噬菌体展示文库,获得广谱特异性识别PA蛋白的纳米抗体,为IAV基础研究和应用研究提供生物材料。【方法】构建真核表达IAV PA的重组质粒,使用Flag标签抗原特异抗体亲和层析法获得纯化蛋白,并以此作为免疫原与弗氏佐剂混合,对羊驼进行免疫。第5次免疫14 d后,采用间接ELISA检测羊驼血清中针对PA蛋白的抗体效价。从羊驼外周血中分离出淋巴细胞(peripheral blood lymphocyte,PBL),提取总RNA后反转录成cDNA,利用巢式PCR技术扩增重链抗体的重链可变区(variable domain of heavy chain of heavy-chain antibody,VHH)基因,构建pComb-VHH噬菌体重组质粒并电转化大肠杆菌SS320感受态细胞,构建纳米抗体噬菌体展示文库,同时测定该文库库容量及多样性。使用PA蛋白包被酶标板,淘选获得与PA蛋白反应的纳米抗体阳性克隆。对淘选获得的阳性克隆进行测序及真核表达,通过Western blotting、间接免疫荧光试验鉴定纳米抗体特异性。【结果】经测序验证,获得序列正确的pcDNA3.1-Flag-PA真核表达重组质粒,表达并纯化获得PA蛋白作为免疫原对羊驼进行免疫。经5次免疫后,羊驼血清中针对PA蛋白的抗体效价达到1∶64000;成功扩增出VHH基因并构建重组率为100%、序列多样性为90%、库容量为6.5×107 CFU/mL的纳米抗体噬菌体展示文库。经过3轮富集淘选,获得6株功能区序列不同的PA蛋白纳米抗体,分别为Nb13、Nb14、Nb16、Nb20、Nb42和Nb72,其中纳米抗体Nb16可广谱特异性识别不同亚型IAV的PA蛋白。【结论】本研究成功构建IAV PA蛋白纳米抗体噬菌体展示文库,筛选获得1株广谱特异性识别IAV PA蛋白的纳米抗体。展开更多
Artemisia annua L.is a medicinal herb with multiple therapeutic applications,whereas its antiinfluenza A virus(IAV)efficiency and mechanism of action are still unclear.Here,we investigated the inhibition activity and ...Artemisia annua L.is a medicinal herb with multiple therapeutic applications,whereas its antiinfluenza A virus(IAV)efficiency and mechanism of action are still unclear.Here,we investigated the inhibition activity and mechanism of A.annua leaf methanol extracts(AALME)against IAV in vitro and in vivo.Our results revealed that AALME exhibits potent anti-IAV activity by interacting with IAV particles.Mechanistically,AALME directly targets the IAV nucleoprotein(NP)protein and abolishes the nuclear import of IAV NP.AALME profoundly suppresses IAV-induced mitochondrial apoptosis via suppressing ROS-mediated AIF-dependent pathways.More importantly,we found that AALME plays a crucial role in protecting mice from IAV infection and mitigating IAV pathogenicity.This current work provides mechanistic insight into the mechanism by which AALME controls IAV infection in vitro and in vivo,potentially contributing to the development of antiviral treatments for IAV infection.展开更多
Influenza A virus(IAV)remains a global public health concern,causing influenza-like illness and severe respiratory tract infections.Two major subtypes,A/pdm09 H1N1 and A/H3N2,circulate globally,and their epidemics are...Influenza A virus(IAV)remains a global public health concern,causing influenza-like illness and severe respiratory tract infections.Two major subtypes,A/pdm09 H1N1 and A/H3N2,circulate globally,and their epidemics are influenced by multiple factors,especially during the COVID-19 pandemic.Based on data from the National Influenza Surveillance Program in China,we analyzed the epidemiological and genomic data in Tianjin collected from 2017 to 2025.A total of 77,473 throat swabs were collected,of which 9144 were IAV-positive.The A/pdm09 H1N1 and A/H3N2 lineages exhibited distinct epidemics across different influenza seasons,with a decline in cases observed during the COVID-19 pandemic.We sequenced the genomes of 128 A/pdm09 H1N1 and 113 A/H3N2 clinical isolates and characterized their temporal evolution and genetic diversity using time-scaled phylogenetic analysis.Additionally,we conducted a genetic risk evaluation of the hemagglutinin and neuraminidase segments,identifying key amino acid residues associated with viral adaptation,transmissibility,virulence,and drug resistance.Moreover,no antigenic variants were found in clinical isolates during the recent influenza seasons,though reduced sensitivity to oseltamivir and zanamivir was observed in individual strains.Our surveillance highlights the epidemiology and evolution of IAV before and after the COVID-19 pandemic in Tianjin.展开更多
Influenza A viruses(IAVs)are single-stranded negative-sense RNA viruses that continually challenge animal and human health.In IAV-infected cells,host RNA-binding proteins play key roles in the life cycle of IAV by dir...Influenza A viruses(IAVs)are single-stranded negative-sense RNA viruses that continually challenge animal and human health.In IAV-infected cells,host RNA-binding proteins play key roles in the life cycle of IAV by directly binding to viral RNA.Here,we examined the role of the host RNA-binding protein nucleophosmin-1(NPM1)in IAV replication.We found that,as a nucleolar phosphoprotein,NPM1 directly binds to viral RNA(vRNA)and inhibits the replication of various subtypes of IAV.NPM1 binding to vRNA competitively reduces the assembly of the viral ribonucleoprotein complex and the viral polymerase activity,thereby reducing the generation of progeny viral RNA and virions.The RNA-binding activity of NPM1,with the key residues T199,T219,T234,and T237,is essential for its anti-influenza function.Taken together,our findings demonstrate that NPM1 acts as an RNA-binding protein and interacts with IAV vRNA to suppress viral replication.展开更多
Influenza A viruses(IAVs)possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health.The control of IAV epidemics desperately necessitates an effic...Influenza A viruses(IAVs)possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health.The control of IAV epidemics desperately necessitates an efficient platform for screening antiviral compounds and evaluating vaccine efficacy.In this study,we utilized the H9N2 subtype IAV as the working model.An 11-amino-acid HiBiT tag,derived from NanoLuc luciferase,was incorporated into the flexible linker region of the NS1 protein.Subsequently,the recombinant HiBiT-tagged virus was rescued.The recombinant virus exhibited high genetic stability and similar virological characteristics to the parental virus,both in vitro and in vivo.Particularly importantly,the replication profile of the HiBiT-tagged virus can be easily measured using the Nano-Glo assay system,achieving an efficient screening platform.Based on this platform,we have developed assays with both convenience and efficiency for screening antiviral reagents,evaluating immunization efficacy,and measuring neutralizing antibodies.展开更多
流行性感冒(简称“流感”)是由流感病毒引起的急性呼吸道传染疾病,据世界卫生组织统计,流感每年可导致300万~500万严重病例,其中29万~65万病例死亡,给社会带来沉重的经济负担,是一个世界性的公共卫生难题。研究发现宿主细胞中存在多条...流行性感冒(简称“流感”)是由流感病毒引起的急性呼吸道传染疾病,据世界卫生组织统计,流感每年可导致300万~500万严重病例,其中29万~65万病例死亡,给社会带来沉重的经济负担,是一个世界性的公共卫生难题。研究发现宿主细胞中存在多条信号通路参与对流感病毒感染的应答,越来越多的研究表明宿主miRNAs通过直接或间接的方式,在流感病毒感染、复制的不同阶段发挥着重要调控作用。本文综合分析了目前关于宿主细胞miRNA对流感病毒复制调控的研究进展,对不同的miRNA具体的调控机制进行系统地归类总结后发现:甲型流感病毒(Influenza A virus,IAV)的PB1、PB2、NA、NP、M1基因是宿主miRNA直接抑制病毒复制的主要靶基因,而在间接调控过程中宿主miRNA主要作用在RIG-I样受体信号通路,Jak-STAT信号通路和Toll样受体信号通路三条流感病毒应答信号途径中,以上发现将更有助于全面理解宿主miRNA对于流感病毒调控网络和宿主细胞与流感病毒的互作机制。展开更多
基金supported by the National Key Research and Development Program of China (2021YFD1800200)the National Natural Science Foundation of China (32170539)。
文摘Influenza A virus(IAV) has a wide host range,including wild birds,poultry,various mammals,and even humans(Xu et al.2024).Currently,two subtypes of canine influenza virus(CIV),H3N8 and H3N2,are primarily circulating in dogs.The H3N8 CIV was introduced from horses into dogs in 2004(Crawford et al.2005),while the H3N2 CIV originated from chickens in Asia in 2007(Song et al.2008).In China,H3N2 is the predominant CIV subtype,with a prevalence rate of up to 5.63% in the canine population,as reported by Chen et al.(2023).CIV infection typically manifests with symptoms such as coughing,sneezing,runny nose,and fever but is rarely fatal.However,co-infection with other pathogens(e.g.,Streptococcus,Mycoplasma or canine parainfluenza virus) can exacerbate symptoms and lead to lethal outcomes(Yondo et al.2023).
文摘A型流感病毒(influenza A virus,IAV)极易发生突变和重组,导致现有疫苗和抗流感药物失效。因此,开发有效的广谱抗流感病毒疫苗和药物是我国乃至全球的重要需求。2025年1月9日,中国农业科学院兰州兽医研究所/兰州大学动物医学与生物安全学院朱启运研究员团队在Nature Communications杂志发表了题为“Influenza A Virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection”的研究论文。该研究分离并纯化了一株特异性识别A型流感病毒血凝素(HA)的纳米抗体E10,该抗体对不同亚型流感病毒具有跨组中和和体内保护效应。研究鉴定E10靶向HA头部结构域的保守氨基酸位点(K166/S167),并且含有E10表位的多肽分子能够诱导交叉反应性抗体,能够对小鼠提供部分交叉免疫保护。该研究不仅为流感防治提供了新的手段,也为广谱流感疫苗研发提供了新的靶点和希望。
文摘【目的】构建A型流感病毒(Influenza A virus,IAV)聚合酶酸性蛋白(polymerase acidic protein,PA)的特异性纳米抗体(nanobody,Nb)噬菌体展示文库,获得广谱特异性识别PA蛋白的纳米抗体,为IAV基础研究和应用研究提供生物材料。【方法】构建真核表达IAV PA的重组质粒,使用Flag标签抗原特异抗体亲和层析法获得纯化蛋白,并以此作为免疫原与弗氏佐剂混合,对羊驼进行免疫。第5次免疫14 d后,采用间接ELISA检测羊驼血清中针对PA蛋白的抗体效价。从羊驼外周血中分离出淋巴细胞(peripheral blood lymphocyte,PBL),提取总RNA后反转录成cDNA,利用巢式PCR技术扩增重链抗体的重链可变区(variable domain of heavy chain of heavy-chain antibody,VHH)基因,构建pComb-VHH噬菌体重组质粒并电转化大肠杆菌SS320感受态细胞,构建纳米抗体噬菌体展示文库,同时测定该文库库容量及多样性。使用PA蛋白包被酶标板,淘选获得与PA蛋白反应的纳米抗体阳性克隆。对淘选获得的阳性克隆进行测序及真核表达,通过Western blotting、间接免疫荧光试验鉴定纳米抗体特异性。【结果】经测序验证,获得序列正确的pcDNA3.1-Flag-PA真核表达重组质粒,表达并纯化获得PA蛋白作为免疫原对羊驼进行免疫。经5次免疫后,羊驼血清中针对PA蛋白的抗体效价达到1∶64000;成功扩增出VHH基因并构建重组率为100%、序列多样性为90%、库容量为6.5×107 CFU/mL的纳米抗体噬菌体展示文库。经过3轮富集淘选,获得6株功能区序列不同的PA蛋白纳米抗体,分别为Nb13、Nb14、Nb16、Nb20、Nb42和Nb72,其中纳米抗体Nb16可广谱特异性识别不同亚型IAV的PA蛋白。【结论】本研究成功构建IAV PA蛋白纳米抗体噬菌体展示文库,筛选获得1株广谱特异性识别IAV PA蛋白的纳米抗体。
基金supported by grants from the National Natural Science Foundation of China(32170937)Shenzhen Medical Research Fund(SMRF,A2303015)+3 种基金Pearl River Talent Project of Guangdong Province(2021QN02Y426)Shenzhen Peacock Plan Project(827/000655)to Liang Yethe Yunnan Provincial Key Areas Science and Technology Plan Project(202303AC100025)Yunnan Scholar of Yunling(YNWRYLXZ-2019-019)to Rongping Zhang.
文摘Artemisia annua L.is a medicinal herb with multiple therapeutic applications,whereas its antiinfluenza A virus(IAV)efficiency and mechanism of action are still unclear.Here,we investigated the inhibition activity and mechanism of A.annua leaf methanol extracts(AALME)against IAV in vitro and in vivo.Our results revealed that AALME exhibits potent anti-IAV activity by interacting with IAV particles.Mechanistically,AALME directly targets the IAV nucleoprotein(NP)protein and abolishes the nuclear import of IAV NP.AALME profoundly suppresses IAV-induced mitochondrial apoptosis via suppressing ROS-mediated AIF-dependent pathways.More importantly,we found that AALME plays a crucial role in protecting mice from IAV infection and mitigating IAV pathogenicity.This current work provides mechanistic insight into the mechanism by which AALME controls IAV infection in vitro and in vivo,potentially contributing to the development of antiviral treatments for IAV infection.
基金supported by Tianjin Municipal Health Commission(TJWJ2022ZD010)Tianjin Key Medical Discipline(Specialty)Construction Project.
文摘Influenza A virus(IAV)remains a global public health concern,causing influenza-like illness and severe respiratory tract infections.Two major subtypes,A/pdm09 H1N1 and A/H3N2,circulate globally,and their epidemics are influenced by multiple factors,especially during the COVID-19 pandemic.Based on data from the National Influenza Surveillance Program in China,we analyzed the epidemiological and genomic data in Tianjin collected from 2017 to 2025.A total of 77,473 throat swabs were collected,of which 9144 were IAV-positive.The A/pdm09 H1N1 and A/H3N2 lineages exhibited distinct epidemics across different influenza seasons,with a decline in cases observed during the COVID-19 pandemic.We sequenced the genomes of 128 A/pdm09 H1N1 and 113 A/H3N2 clinical isolates and characterized their temporal evolution and genetic diversity using time-scaled phylogenetic analysis.Additionally,we conducted a genetic risk evaluation of the hemagglutinin and neuraminidase segments,identifying key amino acid residues associated with viral adaptation,transmissibility,virulence,and drug resistance.Moreover,no antigenic variants were found in clinical isolates during the recent influenza seasons,though reduced sensitivity to oseltamivir and zanamivir was observed in individual strains.Our surveillance highlights the epidemiology and evolution of IAV before and after the COVID-19 pandemic in Tianjin.
基金supported by funding from the National Natural Science Foundation of China(U23A20243 and 32272972 to QZ,32172820 to SX)the Major Science and Technology Project of Gansu Province(22ZD6NA001 to SX)+1 种基金the Youth Innovation Program(Y2023QC30)the Agricultural Science and Technology Innovation Program(CAAS-ASTIP-JBGS-20210102 to SX)of the Chinese Academy of Agricultural Sciences.
文摘Influenza A viruses(IAVs)are single-stranded negative-sense RNA viruses that continually challenge animal and human health.In IAV-infected cells,host RNA-binding proteins play key roles in the life cycle of IAV by directly binding to viral RNA.Here,we examined the role of the host RNA-binding protein nucleophosmin-1(NPM1)in IAV replication.We found that,as a nucleolar phosphoprotein,NPM1 directly binds to viral RNA(vRNA)and inhibits the replication of various subtypes of IAV.NPM1 binding to vRNA competitively reduces the assembly of the viral ribonucleoprotein complex and the viral polymerase activity,thereby reducing the generation of progeny viral RNA and virions.The RNA-binding activity of NPM1,with the key residues T199,T219,T234,and T237,is essential for its anti-influenza function.Taken together,our findings demonstrate that NPM1 acts as an RNA-binding protein and interacts with IAV vRNA to suppress viral replication.
基金the Animal Ethics Committee of the Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences(SYXK-2020-0010).
文摘Influenza A viruses(IAVs)possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health.The control of IAV epidemics desperately necessitates an efficient platform for screening antiviral compounds and evaluating vaccine efficacy.In this study,we utilized the H9N2 subtype IAV as the working model.An 11-amino-acid HiBiT tag,derived from NanoLuc luciferase,was incorporated into the flexible linker region of the NS1 protein.Subsequently,the recombinant HiBiT-tagged virus was rescued.The recombinant virus exhibited high genetic stability and similar virological characteristics to the parental virus,both in vitro and in vivo.Particularly importantly,the replication profile of the HiBiT-tagged virus can be easily measured using the Nano-Glo assay system,achieving an efficient screening platform.Based on this platform,we have developed assays with both convenience and efficiency for screening antiviral reagents,evaluating immunization efficacy,and measuring neutralizing antibodies.
文摘流行性感冒(简称“流感”)是由流感病毒引起的急性呼吸道传染疾病,据世界卫生组织统计,流感每年可导致300万~500万严重病例,其中29万~65万病例死亡,给社会带来沉重的经济负担,是一个世界性的公共卫生难题。研究发现宿主细胞中存在多条信号通路参与对流感病毒感染的应答,越来越多的研究表明宿主miRNAs通过直接或间接的方式,在流感病毒感染、复制的不同阶段发挥着重要调控作用。本文综合分析了目前关于宿主细胞miRNA对流感病毒复制调控的研究进展,对不同的miRNA具体的调控机制进行系统地归类总结后发现:甲型流感病毒(Influenza A virus,IAV)的PB1、PB2、NA、NP、M1基因是宿主miRNA直接抑制病毒复制的主要靶基因,而在间接调控过程中宿主miRNA主要作用在RIG-I样受体信号通路,Jak-STAT信号通路和Toll样受体信号通路三条流感病毒应答信号途径中,以上发现将更有助于全面理解宿主miRNA对于流感病毒调控网络和宿主细胞与流感病毒的互作机制。
