Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses
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摘要
The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.
The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.
The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.
引文
[1]Influenza Palese P.Old and new threats.Nat Med 2004;10:S82–7.
[2]Li C,Chen H.Enhancement of influenza virus transmission by gene reassortment.Curr Top Microbiol Immunol 2014;385:185–204.
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[17]Imai M,Watanabe T,Hatta M,et al.Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1virus in ferrets.Nature 2012;486:420–8.
[18]Zhang Y,Zhang QY,Kong HH,et al.H5N1 hybrid viruses bearing 2009/H1N1virus genes transmit in guinea pigs by respiratory droplet.Science2013;340:1459–63.
[19]Zhu H,Wang D,Kelvin DJ,et al.Infectivity,transmission,and pathology of human-isolated H7N9 influenza virus in ferrets and pigs.Science2013;341:183–6.
[20]Li XY,Shi JZ,Guo J,et al.Genetics,receptor binding property,and transmissibility in mammals of naturally isolated H9N2 avian influenza viruses.PLo S Pathog 2014;10:e1004508.
[21]Breen M,Nogales A,Baker SF,et al.Replication-competent influenza A viruses expressing reporter genes.Viruses 2016;8:E179.
[22]Fujii Y,Goto H,Watanabe T,et al.Selective incorporation of influenza virus RNA segments into virions.Proc Natl Acad Sci USA 2003;100:2002–7.
[23]Watanabe T,Watanabe S,Noda T,et al.Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes.J Virol 2003;77:10575–83.
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[26]Muramoto Y,Takada A,Fujii K,et al.Hierarchy among viral RNA(v RNA)segments in their role in v RNA incorporation into influenza A virions.J Virol2006;80:2318–25.
[27]Marsh GA,Hatami R,Palese P.Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions.J Virol 2007;81:9727–36.
[28]Ozawa M,Fujii K,Muramoto Y,et al.Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication.J Virol2007;81:30–41.
[29]Liang YH,Huang TY,Ly H,et al.Mutational analyses of packaging signals in influenza virus PA,PB1,and PB2 genomic RNA segments.J Virol2008;82:229–36.
[30]Kittel C,Sereinig S,Ferko B,et al.Rescue of influenza virus expressing GFP from the NS1 reading frame.Virology 2004;324:67–73.
[31]Li F,Feng LQ,Pan WQ,et al.Generation of replication-competent recombinant influenza A viruses carrying a reporter gene harbored in the neuraminidase segment.J Virol 2010;84:12075–81.
[32]Manicassamy B,Manicassamy S,Belicha-Villanueva A,et al.Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus.Proc Natl Acad Sci USA 2010;107:11531–6.
[33]Heaton NS,Leyva-Grado VH,Tan GS,et al.In vivo bioluminescent imaging of influenza A virus infection and characterization of novel cross-protective monoclonal antibodies.J Virol 2013;87:8272–81.
[34]Munier S,Rolland T,Diot C,et al.Exploration of binary virus-host interactions using an infectious protein complementation assay.Mol Cell Proteomics2013;12:2845–55.
[35]Pan WQ,Dong ZY,Li F,et al.Visualizing influenza virus infection in living mice.Nat Commun 2013;4:2369.
[36]Pena L,Sutton T,Chockalingam A,et al.Influenza viruses with rearranged genomes as live-attenuated vaccines.J Virol 2013;87:5118–27.
[37]Tran V,Moser LA,Poole DS,et al.Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.J Virol2013;87:13321–9.
[38]Eckert N,Wrensch F,Gartner S,et al.Influenza A virus encoding secreted gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.PLo S One 2014;9:e97695.
[39]Sutton TC,Obadan A,Lavigne J,et al.Genome rearrangement of influenza virus for anti-viral drug screening.Virus Res 2014;189:14–23.
[40]Fukuyama S,Katsura H,Zhao DM,et al.Multi-spectral fluorescent reporter influenza viruses(color-flu)as powerful tools for in vivo studies.Nat Commun2015;6:6600.
[41]Karlsson EA,Meliopoulos VA,Savage C,et al.Visualizing real-time influenza virus infection,transmission and protection in ferrets.Nat Commun2015;6:6378.
[42]Nogales A,Baker SF,Martinez-Sobrido L.Replication-competent influenza A viruses expressing a red fluorescent protein.Virology 2015;476:206–16.
[43]Spronken MI,Short KR,Herfst S,et al.Optimisations and challenges involved in the creation of various bioluminescent and fluorescent influenza A virus strains for in vitro and in vivo applications.PLo S One 2015;10:e0133888.
[44]Yan D,Weisshaar M,Lamb K,et al.Replication-competent influenza virus and respiratory syncytial virus luciferase reporter strains engineered for coinfections identify antiviral compounds in combination screens.Biochemistry 2015;54:5589–604.
[45]Neumann G,Watanabe T,Ito H,et al.Generation of influenza A viruses entirely from cloned c DNAs.Proc Natl Acad Sci USA 1999;96:9345–50.
[46]Szymczak-Workman AL,Vignali KM,Vignali DA.Design and construction of 2a peptide-linked multicistronic vectors.Cold Spring Harb Protoc2012;2012:199–204.
[47]Reed LJMH.A simple method of estimating fifty percent endpoints.Am J Hygiene 1938;27:493–7.
[48]Rekas A,Alattia JR,Nagai T,et al.Crystal structure of venus,a yellow fluorescent protein with improved maturation and reduced environmental sensitivity.J Biol Chem 2002;277:50573–8.
[49]Astrahan P,Arkin IT.Resistance characteristics of influenza to aminoadamantyls.Biochim Biophys Acta 2011;1808:547–53.
[50]Takeda M,Pekosz A,Shuck K,et al.Influenza A virus M2 ion channel activity is essential for efficient replication in tissue culture.J Virol 2002;76:1391–9.
[51]Hao L,Sakurai A,Watanabe T,et al.Drosophila RNAi screen identifies host genes important for influenza virus replication.Nature 2008;454:890–3.
[52]Konig R,Stertz S,Zhou Y,et al.Human host factors required for influenza virus replication.Nature 2010;463:813–7.
[53]Read EKC,Digard P.Individual influenza A virus m RNAs show differential dependence on cellular NXF1/TAP for their nuclear export.J Gen Virol2010;91:1290–301.
[54]Hai R,Krammer F,Tan GS,et al.Influenza viruses expressing chimeric hemagglutinins:Globular head and stalk domains derived from different subtypes.J Virol 2012;86:5774–81.
[2]Li C,Chen H.Enhancement of influenza virus transmission by gene reassortment.Curr Top Microbiol Immunol 2014;385:185–204.
[3]Chen H,Deng G,Li Z,et al.The evolution of H5N1 influenza viruses in ducks in Southern China.Proc Natl Acad Sci USA 2004;101:10452–7.
[4]Swayne DE.Impact of vaccines and vaccination on global control of avian influenza.Avian Dis 2012;56:818–28.
[5]Li CJ,Bu ZG,Chen HL.Avian influenza vaccines against H5N1’bird flu’.Trends Biotechnol 2014;32:147–56.
[6]Su S,Bi YH,Wong G,et al.Epidemiology,evolution,and recent outbreaks of avian influenza virus in China.J Virol 2015;89:8671–6.
[7]Jhung MA,Nelson DI.Outbreaks of avian influenza A(H5N2),(H5N8),and(H5N1)among birds–United states,December 2014-January 2015.MmwrMorbid Mortal W 2015;64:111.
[8]WHO.Influenza at the human-animal interface,http://www.Who.Int/influenza/human_animal_interface/influenza_summary_ira_ha_interface_07_25_2017.Pdf.2017.
[9]Gao RB,Cao B,Hu YW,et al.Human infection with a novel avian-origin influenza A(H7N9)virus.New Engl J Med 2013;368:1888–97.
[10]Zhang QY,Shi JZ,Deng GH,et al.H7N9 influenza viruses are transmissible in ferrets by respiratory droplet.Science 2013;341:410–4.
[11]Ke C,Mok CKP,Zhu W,et al.Human infection with highly pathogenic avian influenza A(H7N9)virus,China.Emergy Infect Dis 2017;23:1332–40.
[12]Wang XR,Gu LL,Shi JZ,et al.Development of a real-time RT-PCR method for the detection of newly emerged highly pathogenic H7N9 influenza viruses.J Integr Agr 2017;16:2055–61.
[13]Zhou L,Tan Y,Kang M,et al.Preliminary epidemiology of human infections with highly pathogenic avian influenza A(H7N9)virus,China,2017.Emergy Infect Dis 2017;23:1355–9.
[14]Jin Y,Yu D,Ren HG,et al.Phylogeography of avian influenza A H9N2 in China.BMC Genom 2014;15:1110.
[15]Sun Y,Liu J.H9N2 influenza virus in China:a cause of concern.Protein Cell2015;6:18–25.
[16]Herfst S,Schrauwen EJA,Linster M,et al.Airborne transmission of influenza A/H5N1 virus between ferrets.Science 2012;336:1534–41.
[17]Imai M,Watanabe T,Hatta M,et al.Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1virus in ferrets.Nature 2012;486:420–8.
[18]Zhang Y,Zhang QY,Kong HH,et al.H5N1 hybrid viruses bearing 2009/H1N1virus genes transmit in guinea pigs by respiratory droplet.Science2013;340:1459–63.
[19]Zhu H,Wang D,Kelvin DJ,et al.Infectivity,transmission,and pathology of human-isolated H7N9 influenza virus in ferrets and pigs.Science2013;341:183–6.
[20]Li XY,Shi JZ,Guo J,et al.Genetics,receptor binding property,and transmissibility in mammals of naturally isolated H9N2 avian influenza viruses.PLo S Pathog 2014;10:e1004508.
[21]Breen M,Nogales A,Baker SF,et al.Replication-competent influenza A viruses expressing reporter genes.Viruses 2016;8:E179.
[22]Fujii Y,Goto H,Watanabe T,et al.Selective incorporation of influenza virus RNA segments into virions.Proc Natl Acad Sci USA 2003;100:2002–7.
[23]Watanabe T,Watanabe S,Noda T,et al.Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes.J Virol 2003;77:10575–83.
[24]Fujii K,Fujii Y,Noda T,et al.Importance of both the coding and the segmentspecific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions.J Virol 2005;79:3766–74.
[25]Liang YY,Hong Y,Parslow TG.Cis-acting packaging signals in the influenza virus PB1,PB2,and PA genomic RNA segments.J Virol 2005;79:10348–55.
[26]Muramoto Y,Takada A,Fujii K,et al.Hierarchy among viral RNA(v RNA)segments in their role in v RNA incorporation into influenza A virions.J Virol2006;80:2318–25.
[27]Marsh GA,Hatami R,Palese P.Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions.J Virol 2007;81:9727–36.
[28]Ozawa M,Fujii K,Muramoto Y,et al.Contributions of two nuclear localization signals of influenza A virus nucleoprotein to viral replication.J Virol2007;81:30–41.
[29]Liang YH,Huang TY,Ly H,et al.Mutational analyses of packaging signals in influenza virus PA,PB1,and PB2 genomic RNA segments.J Virol2008;82:229–36.
[30]Kittel C,Sereinig S,Ferko B,et al.Rescue of influenza virus expressing GFP from the NS1 reading frame.Virology 2004;324:67–73.
[31]Li F,Feng LQ,Pan WQ,et al.Generation of replication-competent recombinant influenza A viruses carrying a reporter gene harbored in the neuraminidase segment.J Virol 2010;84:12075–81.
[32]Manicassamy B,Manicassamy S,Belicha-Villanueva A,et al.Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus.Proc Natl Acad Sci USA 2010;107:11531–6.
[33]Heaton NS,Leyva-Grado VH,Tan GS,et al.In vivo bioluminescent imaging of influenza A virus infection and characterization of novel cross-protective monoclonal antibodies.J Virol 2013;87:8272–81.
[34]Munier S,Rolland T,Diot C,et al.Exploration of binary virus-host interactions using an infectious protein complementation assay.Mol Cell Proteomics2013;12:2845–55.
[35]Pan WQ,Dong ZY,Li F,et al.Visualizing influenza virus infection in living mice.Nat Commun 2013;4:2369.
[36]Pena L,Sutton T,Chockalingam A,et al.Influenza viruses with rearranged genomes as live-attenuated vaccines.J Virol 2013;87:5118–27.
[37]Tran V,Moser LA,Poole DS,et al.Highly sensitive real-time in vivo imaging of an influenza reporter virus reveals dynamics of replication and spread.J Virol2013;87:13321–9.
[38]Eckert N,Wrensch F,Gartner S,et al.Influenza A virus encoding secreted gaussia luciferase as useful tool to analyze viral replication and its inhibition by antiviral compounds and cellular proteins.PLo S One 2014;9:e97695.
[39]Sutton TC,Obadan A,Lavigne J,et al.Genome rearrangement of influenza virus for anti-viral drug screening.Virus Res 2014;189:14–23.
[40]Fukuyama S,Katsura H,Zhao DM,et al.Multi-spectral fluorescent reporter influenza viruses(color-flu)as powerful tools for in vivo studies.Nat Commun2015;6:6600.
[41]Karlsson EA,Meliopoulos VA,Savage C,et al.Visualizing real-time influenza virus infection,transmission and protection in ferrets.Nat Commun2015;6:6378.
[42]Nogales A,Baker SF,Martinez-Sobrido L.Replication-competent influenza A viruses expressing a red fluorescent protein.Virology 2015;476:206–16.
[43]Spronken MI,Short KR,Herfst S,et al.Optimisations and challenges involved in the creation of various bioluminescent and fluorescent influenza A virus strains for in vitro and in vivo applications.PLo S One 2015;10:e0133888.
[44]Yan D,Weisshaar M,Lamb K,et al.Replication-competent influenza virus and respiratory syncytial virus luciferase reporter strains engineered for coinfections identify antiviral compounds in combination screens.Biochemistry 2015;54:5589–604.
[45]Neumann G,Watanabe T,Ito H,et al.Generation of influenza A viruses entirely from cloned c DNAs.Proc Natl Acad Sci USA 1999;96:9345–50.
[46]Szymczak-Workman AL,Vignali KM,Vignali DA.Design and construction of 2a peptide-linked multicistronic vectors.Cold Spring Harb Protoc2012;2012:199–204.
[47]Reed LJMH.A simple method of estimating fifty percent endpoints.Am J Hygiene 1938;27:493–7.
[48]Rekas A,Alattia JR,Nagai T,et al.Crystal structure of venus,a yellow fluorescent protein with improved maturation and reduced environmental sensitivity.J Biol Chem 2002;277:50573–8.
[49]Astrahan P,Arkin IT.Resistance characteristics of influenza to aminoadamantyls.Biochim Biophys Acta 2011;1808:547–53.
[50]Takeda M,Pekosz A,Shuck K,et al.Influenza A virus M2 ion channel activity is essential for efficient replication in tissue culture.J Virol 2002;76:1391–9.
[51]Hao L,Sakurai A,Watanabe T,et al.Drosophila RNAi screen identifies host genes important for influenza virus replication.Nature 2008;454:890–3.
[52]Konig R,Stertz S,Zhou Y,et al.Human host factors required for influenza virus replication.Nature 2010;463:813–7.
[53]Read EKC,Digard P.Individual influenza A virus m RNAs show differential dependence on cellular NXF1/TAP for their nuclear export.J Gen Virol2010;91:1290–301.
[54]Hai R,Krammer F,Tan GS,et al.Influenza viruses expressing chimeric hemagglutinins:Globular head and stalk domains derived from different subtypes.J Virol 2012;86:5774–81.