一株重配型H1N2猪流感病毒的进化分析及对小鼠的致病性
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摘要
为了进一步了解我国辽宁省猪流感(SI)的流行情况及病原的进化规律,对2016年年末在辽宁省某生猪屠宰场进行SI病原学监测时分离到的一株H1N2亚型猪流感病毒(SIV)A/swine/Liaoning/FX575/2016(简称FX575)进行全基因组序列测定,通过对其8个基因片段的基因来源进行分析,确定基因型;构建系统进化树;分析相关分子特征;进一步以6周龄雌性BALB/c小鼠为感染模型,通过测定感染后小鼠的体重变化和脏器病毒含量评估病毒的致病性。结果显示:FX575为一株三重重配型的H1N2病毒,遗传进化分析结果显示病毒的HA基因来自于欧亚类禽型H1N1(EA H1N1)分支的SIV,NA基因来自于北美三重配型H1N2分支的SIV,6个内部基因(PB2、PB1、PA、NP、M和NS)均来自于2009/H1N1分支的病毒。以10~6EID_(50)的剂量经鼻腔途径感染小鼠后,能够引起小鼠出现明显的体重下降,其中有1只小鼠在感染后第7天体重下降的比率超过25%,判为死亡;感染后第3天在小鼠肺及鼻甲骨中均检测到较高滴度的病毒,含量分别为4.82、4.20 log_(10)EID_(50)·mL~(-1)。结果表明SIV在不断流行的过程中,不同基因型病毒之间发生重组导致出现基因三重配的病毒,病毒对小鼠呈现中等致病力特征,提示应进一步加强对SI的主动监测,从而降低病毒对兽医及公共卫生学风险。
The objective of this study was to investigate the epidemiological situation of swine influenza and viral molecule characteristics in North-eastern China. One H1 N2 subtype swine influenza virus, designated as A/swine/Liaoning/FX575/2016(H1 N2)(FX575), was isolated from a slaughterhouse in Liaoning province during an active surveillance in 2016. The complete genomic sequence of the strain was sequenced and subjected to phylogenetic analysis, molecular characteristic analysis and genotypic analysis. Then, the viral pathogenesis was carried out by intranasal infecting the six-week-old female BALB/c mice and evaluated by measuring body weight loss of mice and virus replication titer. FX575 was a triple reassortant H1 N2 virus, which had the nearest genetic relationship with the H1 N2 influenza viruses previously isolated from China. Phylogenetic analysis results demonstrated that HA gene of the isolate belonged to the Eurasian avian-like swine H1 N1 lineage, the NA gene was clustered into the North American swine H1 N2 lineage, and the six internal genes fell into the pandemic 2009/H1 N1 virus lineage, respectively. The virus FX575 could cause significant weight losses of mice within one week after infection, and one mouse with its weight loss exceeding 25 percent was considered dead. The viral replications occurred both in lungs and nasal turbinates, with the titers of 4.82 and 4.20 log_(10)EID_(50)·mL~(-1), respectively. The results indicated that continuous reassortments of different genotypes of influenza viruses occurred in the swine population, and the novel reassortant virus showed moderate pathogenicity to mice, which further suggested that the active monitoring of SIV should be strengthened in the future.
The objective of this study was to investigate the epidemiological situation of swine influenza and viral molecule characteristics in North-eastern China. One H1 N2 subtype swine influenza virus, designated as A/swine/Liaoning/FX575/2016(H1 N2)(FX575), was isolated from a slaughterhouse in Liaoning province during an active surveillance in 2016. The complete genomic sequence of the strain was sequenced and subjected to phylogenetic analysis, molecular characteristic analysis and genotypic analysis. Then, the viral pathogenesis was carried out by intranasal infecting the six-week-old female BALB/c mice and evaluated by measuring body weight loss of mice and virus replication titer. FX575 was a triple reassortant H1 N2 virus, which had the nearest genetic relationship with the H1 N2 influenza viruses previously isolated from China. Phylogenetic analysis results demonstrated that HA gene of the isolate belonged to the Eurasian avian-like swine H1 N1 lineage, the NA gene was clustered into the North American swine H1 N2 lineage, and the six internal genes fell into the pandemic 2009/H1 N1 virus lineage, respectively. The virus FX575 could cause significant weight losses of mice within one week after infection, and one mouse with its weight loss exceeding 25 percent was considered dead. The viral replications occurred both in lungs and nasal turbinates, with the titers of 4.82 and 4.20 log_(10)EID_(50)·mL~(-1), respectively. The results indicated that continuous reassortments of different genotypes of influenza viruses occurred in the swine population, and the novel reassortant virus showed moderate pathogenicity to mice, which further suggested that the active monitoring of SIV should be strengthened in the future.
引文
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[22] DENG Y M,SPIRASON N,IANNELLO P,et al.A simplified sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses[J].J Clin Virol,2015,68:43-48.
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[25] GU R X,LIU L A,WANG Y H,et al.Structural comparison of the wild-type and drug-resistant mutants of the influenza a M2 proton channel by molecular dynamics simulations[J].J Phys Chem B,2013,117(20):6042-6051.
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[27] LIANG H Y,LAM T T Y,FAN X H,et al.Expansion of genotypic diversity and establishment of 2009 H1N1 pandemic-origin internal genes in pigs in China[J].J Virol,2014,88(18):10864-10874.
[28] YANG H,CHEN Y,QIAO C,et al.Two different genotypes of H1N2 swine Influenza virus isolated in northern China and their pathogenicity in animals[J].Vet Microbiol,2015,175(2-4):224-231.
[29] KONG W L,HUANG L Z,QI H T,et al.Genetic characterization of H1N2 influenza a virus isolated from sick pigs in southern China in 2010[J].Virol J,2011,8(1):469.
[30] KAMAL R P,KATZ J M,YORK I A.Molecular determinants of influenza virus pathogenesis in mice[J].Curr Top Microbiol Immunol,2014,385:243-274.
[31] MATROSOVICH M,TUZIKOV A,BOVIN N,et al.Early alterations of the receptor-binding properties of H1,H2,and H3 Avian influenza virus hemagglutinins after their introduction into mammals[J].J Virol,2000,74(18):8502-8512.
[32] WANG Z,YANG H,CHEN Y,et al.A single-amino-acid substitution at position 225 in Hemagglutinin alters the transmissibility of Eurasian avian-like H1N1 swine influenza virus in Guinea Pigs[J].J Virol,2017,91:e00800-17.
[33] SHI J Z,DENG G H,MA S J,et al.Rapid evolution of H7N9 highly pathogenic viruses that emerged in China in 2017[J].Cell Host Microbe,2018,24(4):558-568.
[34] MEHLE A,DOUDNA J A.Adaptive strategies of the influenza virus polymerase for replication in humans[J].Proc Natl Acad Sci U S A,2009,106(50):21312-21316.
[35] MA M J,WANG G L,ANDERSON B D,et al.Evidence for cross-species influenza A virus transmission within swine farms,China:a one health,prospective cohort study[J].Clin Infect Dis,2018,66(4):533-540.
[36] MA M,ANDERSON B D,WANG T,et al.Serological evidence and risk factors for swine influenza infections among Chinese swine workers in Guangdong province[J].PLoS One,2015,10(5):e0128479.
[37] HE L,WU Q W,JIANG K J,et al.Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 Influenza A viruses from humans and swine[J].Arch Virol,2014,159(7):1743-1754.
[38] ABENTE E J,KITIKOON P,LAGER K M,et al.A highly pathogenic avian-derived influenza virus H5N1 with 2009 pandemic H1N1 internal genes demonstrates increased replication and transmission in pigs[J].J Gen Virol,2017,98(1):18-30.
[2] GARTEN R J,DAVIS C T,RUSSELL C A,et al.Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans[J].Science,2009,325(5937):197-201.
[3] WONG F Y K,DONATO C,DENG Y M,et al.Divergent human-origin influenza viruses detected in Australian swine populations[J].J Virol,2018,92(16):e00316-18.
[4] YANG H L,QIAO C L,TANG X,et al.Human infection from avian-like influenza A (H1N1) viruses in pigs,China[J].Emerg Infect Dis,2012,18(7):1144-1146.
[5] MYERS K P,OLSEN C W,GRAY G C.Cases of swine influenza in humans:a review of the literature[J].Clin Infect Dis,2007,44(8):1084-1088.
[6] ANDERSON T K,NELSON M I,KITIKOON P,et al.Population dynamics of cocirculating swine influenza A viruses in the United States from 2009 to 2012[J].Influenza Other Respir Viruses,2013,7(Suppl 4):42-51.
[7] RAJ?O D S,GAUGER P C,ANDERSON T K,et al.Novel reassortant human-like H3N2 and H3N1 influenza a viruses detected in pigs are virulent and antigenically distinct from swine viruses endemic to the United States[J].J Virol,2015,89(22):11213-11222.
[8] ABENTE E J,GAUGER P C,WALIA R R,et al.Detection and characterization of an H4N6 avian-lineage influenza a virus in pigs in the midwestern United States[J].Virology,2017,511:56-65.
[9] VINCENT A,AWADA L,BROWN I,et al.Review of influenza A virus in swine worldwide:a call for increased surveillance and research[J].Zoonoses Public Health,2014,61(1):4-17.
[10] YANG H L,CHEN Y,QIAO C L,et al.Prevalence,genetics,and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses[J].Proc Natl Acad Sci U S A,2016,113(2):392-397.
[11] SUGIMURA T,YONEMOCHI H,OGAWA T,et al.Isolation of a recombinant influenza virus (Hsw1N2) from swine in Japan[J].Arch Virol,1980,66(3):271-274.
[12] JUNG K,CHAE C.First outbreak of respiratory disease associated with swine influenza H1N2 virus in pigs in Korea[J].J Vet Diagn Invest,2005,17(2):176-178.
[13] BROWN I H,CHAKRAVERTY P,HARRIS P A,et al.Disease outbreaks in pigs in Great Britain due to an influenza A virus of H1N2 subtype[J].Vet Rec,1995,136(13):328-329.
[14] KARASIN A I,LANDGRAF J,SWENSON S,et al.Genetic characterization of H1N2 influenza A viruses isolated from pigs throughout the United States[J].J Clin Microbiol,2002,40(3):1073-1079.
[15] QI X,LU C P.Genetic Characterization of novel reassortant H1N2 influenza A viruses isolated from pigs in southeastern China[J].Arch Virol,2006,151(11):2289-2299.
[16] 陈义祥,蒙雪琼,刘棋,等.中国“人-猪-禽”基因重排H1N2亚型猪流感病毒全基因克隆及遗传进化的研究[J].微生物学报,2008,48(4):466-472.CHEN Y X,MENG X Q,LIU Q,et al.Phylogenetic analysis of human/swine/avian gene reassortant H1N2 influenza A virus isolated from a pig in China[J].Acta Microbiologica Sinica,2008,48(4):466-472.(in Chinese)
[17] YU H,ZHANG P C,ZHOU Y J,et al.Isolation and genetic characterization of avian-like H1N1 and novel ressortant H1N2 influenza viruses from pigs in China[J].Biochem Biophys Res Commun,2009,386(2):278-283.
[18] ALI A,KHATRI M,WANG L Y,et al.Identification of swine H1N2/pandemic H1N1 reassortant influenza virus in pigs,United States[J].Vet Microbiol,2012,158(1-2):60-68.
[19] QIAO C L,LIU L P,YANG H L,et al.Novel triple reassortant H1N2 influenza viruses bearing six internal genes of the pandemic 2009/H1N1 influenza virus were detected in pigs in China[J].J Clin Virol,2014,61(4):529-534.
[20] 中华人民共和国农业部.GB/T 27536-2011 猪流感病毒分离与鉴定方法[S].北京:中国标准出版社,2012.Ministry of Agriculture of the People’s Republic of China.GB/T 27536-2011 Isolation and identification of swine influenza virus[S].Beijing:China Standard Press,2012.(in Chinese)
[21] 杨焕良,乔传玲,陈艳,等.猪流感病毒H1N1、H1N2和H3N2亚型多重RT-PCR诊断方法的建立[J].中国预防兽医学报,2007,29(9):714-718.YANG H L,QIAO C L,CHEN Y,et al.Subtyping of H1N1,H1N2 and H3N2 swine influenza viruses by two multiplex RT-PCR[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(9):714-718.(in Chinese)
[22] DENG Y M,SPIRASON N,IANNELLO P,et al.A simplified sanger sequencing method for full genome sequencing of multiple subtypes of human influenza A viruses[J].J Clin Virol,2015,68:43-48.
[23] CATON A J,BROWNLEE G G,YEWDELL J W,et al.The antigenic structure of the influenza virus A/PR/8/34 Hemagglutinin (H1 Subtype)[J].Cell,1982,31(2 Pt 1):417-427.
[24] PRACHANRONARONG K L,?ZEN A,THAYER K M,et al.Molecular basis for differential patterns of drug resistance in influenza N1 and N2 Neuraminidase[J].J Chem Theory Comput,2016,12(12):6098-6108.
[25] GU R X,LIU L A,WANG Y H,et al.Structural comparison of the wild-type and drug-resistant mutants of the influenza a M2 proton channel by molecular dynamics simulations[J].J Phys Chem B,2013,117(20):6042-6051.
[26] 刘慧敏,李一经.一株pH1N1/2009猪流感病毒的进化分析与生物学特性研究[J].中国预防兽医学报,2017,39(2):94-97.LIU H M,LI Y J.Evolution analysis and biological characteristics of a pandemic H1N1 swine influenza virus[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(2):94-97.(in Chinese)
[27] LIANG H Y,LAM T T Y,FAN X H,et al.Expansion of genotypic diversity and establishment of 2009 H1N1 pandemic-origin internal genes in pigs in China[J].J Virol,2014,88(18):10864-10874.
[28] YANG H,CHEN Y,QIAO C,et al.Two different genotypes of H1N2 swine Influenza virus isolated in northern China and their pathogenicity in animals[J].Vet Microbiol,2015,175(2-4):224-231.
[29] KONG W L,HUANG L Z,QI H T,et al.Genetic characterization of H1N2 influenza a virus isolated from sick pigs in southern China in 2010[J].Virol J,2011,8(1):469.
[30] KAMAL R P,KATZ J M,YORK I A.Molecular determinants of influenza virus pathogenesis in mice[J].Curr Top Microbiol Immunol,2014,385:243-274.
[31] MATROSOVICH M,TUZIKOV A,BOVIN N,et al.Early alterations of the receptor-binding properties of H1,H2,and H3 Avian influenza virus hemagglutinins after their introduction into mammals[J].J Virol,2000,74(18):8502-8512.
[32] WANG Z,YANG H,CHEN Y,et al.A single-amino-acid substitution at position 225 in Hemagglutinin alters the transmissibility of Eurasian avian-like H1N1 swine influenza virus in Guinea Pigs[J].J Virol,2017,91:e00800-17.
[33] SHI J Z,DENG G H,MA S J,et al.Rapid evolution of H7N9 highly pathogenic viruses that emerged in China in 2017[J].Cell Host Microbe,2018,24(4):558-568.
[34] MEHLE A,DOUDNA J A.Adaptive strategies of the influenza virus polymerase for replication in humans[J].Proc Natl Acad Sci U S A,2009,106(50):21312-21316.
[35] MA M J,WANG G L,ANDERSON B D,et al.Evidence for cross-species influenza A virus transmission within swine farms,China:a one health,prospective cohort study[J].Clin Infect Dis,2018,66(4):533-540.
[36] MA M,ANDERSON B D,WANG T,et al.Serological evidence and risk factors for swine influenza infections among Chinese swine workers in Guangdong province[J].PLoS One,2015,10(5):e0128479.
[37] HE L,WU Q W,JIANG K J,et al.Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 Influenza A viruses from humans and swine[J].Arch Virol,2014,159(7):1743-1754.
[38] ABENTE E J,KITIKOON P,LAGER K M,et al.A highly pathogenic avian-derived influenza virus H5N1 with 2009 pandemic H1N1 internal genes demonstrates increased replication and transmission in pigs[J].J Gen Virol,2017,98(1):18-30.
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