一株H1N1亚型猪流感病毒的遗传演化分析及在雪貂体内复制和飞沫传播能力评估.pdf

中国预防兽医学报Chinese Journal of Preventive Veterinary Medicine第 45 卷 第 10 期2023 年 10 月Vol.45，No.10Oct.2023doi院10.3969/j.issn.1008-0589.202303008一株H1N1亚型猪流感病毒的遗传演化分析及在雪貂体内复制和飞沫传播能力评估张奕杰覮，孟飞覮，杨焕良，宋祖晨，陈艳，陈化兰*(中国农业科学院哈尔滨兽医研究所 动物疫病防控全国重点实验室/农业农村部动物流感重点开放实验室，黑龙江 哈尔滨 150069)摘要：本实验室于2016年在天津分离到一株猪流感病毒(SIV)，命名为A/swine/Tianjin/312/2016(H1N1)(简称为TJ312株)。为了解该株病毒的生物学特性及遗传演化特征，本研究对经PCR分段扩增该病毒全基因组并测序，采用SeqMan软件拼接成全基因组序列经BLAST比对，得到与该SIV各基因节段序列同源性最高的病毒株，采用mafft分析其各基因节段编码氨基酸序列的分子特征，利用MEGA5.1软件分别构建该病毒8个基因节段的进化树。结果显示，SIV TJ312株各基因节段与2016年分离自天津的H1N1 SIV相应各基因节段的同源性在99%100%。进化树结果显示该病毒株HA、NA和M基因均来自欧亚类禽 H1N1(EA H1N1)SIV谱系；PB2、PB1、PA和NP基因来自2009/H1N1流感病毒谱系；NS基因来自北美三重配H1N2 SIV谱系。蛋白分子特征分析结果显示，HA蛋白碱性氨基酸裂解位点序列为PSIQSR引G，符合低致病性流感病毒分子特征；受体结合位点符合人源唾液酸受体(琢-2，6唾液酸受体)的分子特性；NA蛋白aa275为H、aa295为N，且该蛋白头部区域未缺失，表明该病毒对奥司他韦类药物敏感；PA蛋白356R为病毒复制增强和对哺乳动物致病性增强的分子特征；NP蛋白存在41I以及210E病毒聚合酶活性增强的分子特征；M2蛋白的31N表示该SIV对金刚烷胺类药物耐药。将该病毒以106EID50/只经鼻腔感染2只雪貂，感染后96 h剖杀，采集各脏器组织观察其剖检病变并对出现病变的肺脏制备病理切片观察其组织病变，采用免疫组化试验检测肺脏组织中的病毒抗原。将采集的所有脏器处理后接种鸡胚检测脏器内的病毒滴度评估该病毒在雪貂体内的复制能力；将该株病毒以上述剂量经鼻腔感染3只雪貂分别放入3个铁笼内作为感染组，24 h后在相邻3个铁笼内分别放入1只未感染雪貂作为传播组。所有雪貂自感染组接种病毒后2 d 起，隔天采集鼻洗液至第14 d，通过检测雪貂鼻洗液内的病毒滴度与感染后21 d血清HI抗体效价评估病毒在雪貂间的飞沫传播能力。结果显示，TJ312株感染后引起雪貂肺脏明显的剖检病变，其余脏器均无肉眼可见病变。肺脏病理切片及免疫组化结果显示，肺脏组织出现了一定的病理损伤，且支气管上皮细胞中出现大量棕黄色和深棕色颗粒，即检测到了病毒抗原。病毒滴定结果显示，TJ312株在雪貂呼吸道内复制良好，在其鼻甲中复制能力最强，平均病毒滴度6.13 log10EID50/mL，在其肺右下叶中的复制能力次之，平均病毒滴度可达6 log10EID50/mL。在脾脏、肾脏、肝脏中均未检出病毒。但在1只雪貂的脑中检出微量病毒，滴度为0.63 log10EID50/mL。飞沫传播试验中，感染组3只雪貂的鼻洗液中均检测到高滴度的病毒，病毒滴度最高达5.50 Log10EID50/mL，血清中均检测到了高水平的HI抗体效价(1颐1 280、1颐1 280、1颐2 560)；传播组3只雪貂中有1只鼻洗液病毒滴定结果呈阳性(最高达5.75 Log10EID50/mL)且其血清中检出较高的HI抗体效价(1颐2 560)。上述结果表明，TJ312株可能是由2016年天津其他H1N1 SIV间的基因节段重组而来，且该株病毒存在部分耐药性及对哺乳动物致病性增强的氨基酸分子特征。病毒在雪貂的呼吸道中复制水平较高并对其肺脏造成损害，且可以通过飞沫在雪貂间传播。本研究为进一步了解EA H1N1 SIV的生物学特性及探究其感染机制提供一定的参考依据。关键词：猪流感病毒；欧亚类禽 H1N1；遗传演化；飞沫传播中图分类号：S852.65文献标识码：A文章编号：1008-0589(2023)10-0987-07收稿日期：2023-03-20基金项目：十四五国家重点研发计划(2021YFD1800200)覮 共同第一作者：张奕杰(1998-)，男，四川成都人，硕士研究生，主要从事动物流感病毒分子生物学方向研究；孟飞(1990-)，男，山东济宁人，博士后，主要从事动物流感病毒流行病学及分子生物学方向研究.*通信作者：E-mail：*Corresponding author;覮Equal contributorsGenetic evolution analysis of a H1N1 sultype swine influenza virusand evaluation of its replication and droplet transmissibility in ferretsZHANG Yi-jie覮,MENG Fei覮,YANG Huan-liang,SONG Zu-chen,CHEN Yan,CHEN Hua-lan*(Animal Influenza Key Laboratory of the Ministry of Agriculture and Rural Affairs,State Key Laboratory of Animal disease control andprevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Science,Harbin 150069,China)Abstract:In order to investigate the genetic evolution background and biological characteristics of the swine influenza virus(SIV)A/swine/Tianjin/312/2016(H1N1)isolated from Tianjin Province in 2016,the whole genome of the virus was amplified byPCR and sequenced in this study,and the complete genome sequence was spliced by SeqMan software and compared by BLAST toobtain the virus strain with the highest homology with each gene segment sequence.Mafft was used to analyze the molecularcharacteristics of amino acid sequences encoded by each gene segment and MEGA5.1 software was used to construct theevolutionary tree of 8 gene segments of the virus.The results showed that all gene segments of SIV TJ312 strain share 99%-100%homology to the corresponding gene segments of H1N1 SIVs isolated from Tianjin in 2016.The results of the evolutionary treeshowed that the HA,NA and M genes of this strain came from the Eurasian avian H1N1(EA H1N1)SIV lineage.PB2,PB1,PAand NP genes were derived from 2009/H1N1 influenza virus lineage.The NS gene was derived from the North American triplereassortment H1N2 SIV lineage.The protein molecular characteristics analysis results showed that the sequence of the basic aminoacid cleavage site of HA protein was PSIQSR引G,which was consistent with the molecular characteristics of low pathogenicinfluenza virus.The receptor binding sites correspond to the molecular properties of human sialic acid receptors(琢-2,6 sialic acidreceptors).The NA protein aa275 is H and aa295 is N,and the head region of this protein is not missing,indicating that the strainis sensitive to oseltamivir drugs.PA356R was a molecular characteristic of increased viral replication and pathogenicity in mammals.NP protein41I and210E could enhance the activity of viral polymerase.31N of M2 protein is a molecular characteristic of resistanceto amantadines.In this study,the ability of TJ312 strain to replicate and spread by droplets in ferrets was further evaluated.Twoferrets were infected through the nasal cavity at 106EID50/ferret doses.The ferrets were killed 96 hours after infection.The nasalturbinate,soft palate,trachea,lung,tonsil,spleen,kidney,liver and brain were collected.Pathological sections of the diseasedlungs were prepared to observe the pathological changes,and the virus antigen in the lung tissues was detected byimmunohistochemical test.All the collected organs were treated and inoculated with chicken embryos to detect the virus titer in theorgans and evaluate the replication ability of the virus in ferrets.Three ferrets were infected intranasally with the same dose ofTJ312 strain,and three naive ferrets were placed in adjacent cages as exposed group 24 hours after infection.The nasal washeswere collected seven times every other day from the second day to the 14th day.Droplet virus transmission between ferrets wasevaluated by detecting virus titers in nasal washes and serum HI antibody titers from ferrets in the exposed group.The resultsshowed that the infection of TJ312 strain caused significant pulmonary lesions in ferrets and no visible lesions in other organs.Pathological lung sections and immunohistochemical results showed some pathological injuries in the lung tissue,and a largenumber of brown-yellow and dark brown particles appeared in the bronchial epithelial cells,meaning viral antigens were detected.The virus titration results of various organs showed that TJ312 strain replicated well in the respiratory tract of ferrets and had thehighest replication capacity in turbinate with an average viral titer of 6.13log10EID50/mL,followed by the replication capacity in theright caudal of lung.The mean virus titer was 6log10EID50/mL.No virus was detected in the spleen,kidney or liver.However,tracesof the viruses were detected in the brain of one ferret with a titer of 0.63log10EID50/mL.In the 14-day droplet transmission test,ahigh titer of the virus was detected in the nasal wash of the 3 ferrets in the infected group,the highest titer of virus was5.50Log10EID50/mL,and high levels of HI antibody titer(1颐280,1颐280,1颐2560)were detected in the serum of the 3 ferrets.One ofthe 3 ferrets in the transmission group was positive for viral titration(up to 5.75Log10EID50/mL)and had a high HI antibody titer(1颐2560)in the serum.These results suggest that the SIV TJ312 strain may be derived from inter-segment gene recombination ofother H1N1 SIV strains that existed in Tianjin in 2016,and this strain has partial drug resistance and amino acid characteristics thatenhance the pathogenicity in mammals.The virus can replicate at high levels in ferrets respiratory tracts and damage their lungs.It中 国 预 防 兽 医 学 报2023 年988can be transmitted from ferret to ferret by droplets.This study provides some reference for further understanding the biologicalcharacteristics of EA H1N1 SIV and exploring its infection mechanism.Key words:swine influenza virus;Eurasian avian-like H1N1;genetic and evolution;droplet transsmission多种不同亚型的A型流感病毒均可以感染猪，目前从世界各地的猪中分离到的猪流感病毒(SIV)包括H1、H2、H3、H4、H5、H7和H9等亚型1，但长久以来只有3种亚型在猪群中稳定存在，其中以H1N1亚型流行最广泛，其次是H1N2和H3N2亚型2。猪群中存在的H1N1亚型SIV又可分为多个谱系，主要包含经典H1N1 SIV(CS H1N1 SIV)谱系和欧亚类禽H1N1(Eurasian avian-like H1N1，EA H1N1)SIV谱系，它们分别自1918年和1979年在猪群中传播，其中EA H1N1 SIV在比利时猪中首次分离，此后在猪群中广泛感染并迅速传遍欧洲，抗原性分析及测序结果表明其来源于鸭的H1N1禽流感病毒3-4。EAH1N1 SIV在欧洲和中国的猪群中广泛传播，与北美三重配H1N2(North Amercian triple reassortant H1N2)SIV、2009/H1N1等流感病毒株重组演化出多种基因型的EA H1N1 SIV，并已在多个国家引起人的感染。对哺乳动物致病性呈日益增强趋势，许多病毒株已经具备了在雪貂间通过飞沫传播的能力，抗原性也与目前人类所使用的疫苗株存在较大差异5，种种迹象提示EA H1N1 SIV具有在人与人之间大流行的潜在风险。飞沫传播指易感者通过吸入感染原(如流感患者)产生的含病毒粒子的飞沫而感染。实际上，由咳嗽、喷嚏和说话所产生的飞沫直径在0.1 mm0.2 mm，喷出的距离常小于1 m。因此，飞沫传播是流感近距离传播的一种主要形式6。雪貂作为探究流感病毒在哺乳动物间传播的模式动物之一，具有对流感病毒天然易感，上呼吸道受体与人相似等特点，被广泛用于流感病毒致病性及传播能力的研究6-7。本研究对国家禽流感参考实验室于2016年在天津猪流感流行病学调查中分离到的A/swine/Tian-jin/312/2016(H1N1)株(简称为TJ312株)进行了基因序列同源性、分子特征及遗传演化分析，并利用雪貂模型评估该SIV在哺乳动物体内复制及飞沫传播能力，为了解EA H1N1 SIV对公共卫生安全带来的风险及对其的监测和SI防控策略的制定提供了数据支撑。1材料与方法1.1病毒株及实验动物SIV TJ312株为国家禽流感参考实验室于2016年分离自天津。10日龄SPF鸡胚和SPF鸡血均购自国家禽类实验动物资源库；雪貂购自无锡珊瑚礁生物科技有限公司。1.2主要试剂RNA提取试剂盒购自天根生化科技(北京)有限公司；M-MLV反转录试剂盒购自东洋纺(上海)生物科技有限公司；Primer STAR MAX扩增酶购自TaKaRa公司；测序所用试剂盒Big DyeTer-minator3.1购自美国ABI公司；免疫组化所用一抗为流感病毒鼠源NP蛋白单克隆抗体(MAb)购自北京义翘神州科技股份有限公司；HRP标记的山羊抗鼠IgG(IgG-HRP)购自丹麦Dako公司。1.3病毒全基因组测序与序列分析利用RNA提取试剂盒提取分离SIV的总RNA，并利用12 bp流感病毒反转录通用引物(5-AGCAAAAGCAGG-3)将其反转录为cDNA，参考文献中的各基因节段特异性引物对TJ312株基因组8节段分别经PCR扩增8，经琼脂糖凝胶电泳鉴定条带大小正确后胶回收纯化，按照Big DyeTerminator测序试剂盒说明书测序，利用SeqMan软件拼接各基因节段完整序列的全基因组。并在GISAID网站经BLAST比对，得到与各基因节段序列同源性最高的病毒株，采用Mafft软件分析各基因节段编码氨基酸序列的分子特征，利用MEGA5.1软件分别构建该病毒8个基因节段的进化树。1.4病毒在雪貂体内的复制水平的检测将病毒液经鼻腔感染2只雪貂(106EID50/只，250 滋L/鼻孔)，感染后96 h剖杀，采集其鼻甲、软腭、气管、肺脏(左上叶、左中叶、左下叶、右上叶、右中叶、右下叶)、扁桃体、脾脏、肾脏、肝脏、脑，观察剖检病变并对出现剖检病变的肺脏组织制备病理切片经HE染色后观察其组织病变；以鼠源NP蛋白MAb(1颐200)为一抗，以山羊抗鼠IgG-HRP(原液)为二抗，经免疫组化试验检测肺脏组织中的病毒抗原。分别称等质量的所有脏器样品后加入1 mL含青霉素(1 000 U/mL)和链霉素(1 000 U/mL)的灭菌PBS，充分研磨、离心张奕杰，等.一株 H1N1 亚型猪流感病毒的遗传演化分析及在雪貂体内复制和飞沫传播能力评估第 10 期989后取其上清液，10倍倍比稀释后经尿囊腔途径接种3枚10日龄鸡胚，37 孵化60 h后，测定鸡胚尿囊液的血凝活性，利用Reed-Muench法计算病毒滴度。1.5病毒在雪貂间飞沫传播能力的评估将6只雌性雪貂随机分为2组，每组3只，一组为感染组，一组为传播组。感染组雪貂以滴鼻途径接种病毒液(106EID50/500滋L，250 滋L/鼻孔)后分别放入3个笼子，24 h后在对应相邻3个笼子内各放入1只健康雪貂作为传播组。环境条件设定为24 和30%40%的相对湿度，隔离器内气流为水平方向从感染组到传播组，速度为0.1 m/s。6只雪貂自感染组接种病毒后2 d，隔天采集鼻洗液至感染后14 d，共采集7次，鼻洗液加入含青霉素(1 000 U/mL)和链霉素(1 000 U/mL)的灭菌PBS 10倍倍比稀释，按照1.4中的方法接种鸡胚测定并计算病毒滴度。感染后21 d采集所有雪貂的血清，56 灭活30 min，加入4倍体积经钙盐溶液(1 g CaCl2、9 g NaCl、1.2 g H3BO3，0.052 gNa2B4O7 10H2O溶于1 L ddH2O)20倍稀释的受体破坏酶(RDE)37 水浴18 h后加入5倍体积的1.5%柠檬酸钠溶液，56 灭活30 min后，通过血凝抑制试验测定各组雪貂血清中的HI抗体效价。2结果2.1病毒全基因组测序及序列同源性分析提取SIV TJ312株总RNA反转录为cDNA后作为模板，采用PCR分别扩增病毒8个基因节段。结果显示，均扩增到与预期相符的目的片段(图略)。采用Big DyeTe-rminator测序试剂盒测序，利用SeqMan软件拼接各基因节段完整序列的全基因组序列。各基因节段测序结果在GISAID网站进行BLAST基因序列同源性比对，得到与各基因节段序列同源性最高的病毒株。SIV TJ312株各基因节段与2016年分离自天津的H1N1SIV相应各基因节段的同源性在99%100%(表1)。表明，2016年分离自天津的N1N1 SIV TJ312株各基因节段均与天津2016年分离的N1N1 SIV株的各基因节段同源性最高，该分离株可能是由这些病毒株重组而来。2.2病毒HA、NA基因编码氨基酸序列分子特征及基因遗传演化分析对SIV TJ312株的HA、NA基因编码氨基酸序列进行分子特征及遗传演化分析。结果显示，HA基因编码区全长1 701 nt，共编码566个氨基酸，其碱性氨基酸裂解位点为PSIQSR引G，符合低致病性流感病毒分子特征。受体结合位点分子特征为190D、225E、226Q、228G，符合与人源唾液酸受体(琢-2，6唾液酸受体)结合的分子特性。HA氨基酸序 列 存 在26NNS28、27NST29、39NVT41、211NCT213、290NCT292、497NGT499、556NGS5587个潜在糖基化位点。HA基因进化树显示其位于EA H1N1 SIV谱系(图 1A)。SIV TJ312株NA基因编码区全长1 410 nt，编码469个氨基酸，有7个潜在糖基化位点44NQS46、58NNT60、63NQT65、68NVS70、88NSS90、146NGT148、235NGS237，aa275为H、aa295为N，该蛋白头部区域未缺失，表明该病毒株仍对奥司他韦类药物敏感。NA基因进化树显示其位于EA H1N1 SIV谱系(图1B)。上述结果表明，该SIV分离株HA、NA基因节 段 与A/swine/Tianjin/848/2016(H1N1)株同源性高达100%，可能由其提供TJ312株表面蛋白编码基因节段，且HA蛋白具有结合人源唾液酸受体的特征。2.3病毒内部基因分子特征及遗传演化分析内部基因进化树结果显示，SIV TJ312株PB2、PB1、PA、NP基因均属于2009/H1N1流感病毒谱系。M、NS基因 分 别 属 于 EA H1N1 SIV 谱 系 和 North Americiantriple H1N2 SIV谱系(图2)，所有与TJ312株高度同源的病毒均为H1N1 SIV。该株病毒的PB2蛋白氨基酸未发生E627K或D701N对哺乳动物致病性增强的突变，PA蛋白氨基酸出现了356R为病毒复制和对哺乳动物致病性增强的分子特征，NP蛋白存在41I以及210E的分子特征，是病毒聚合酶活性增强的分子标记，M2蛋白31N，是对金刚烷胺类药物耐药的分子标记，NS1蛋白172E，未发生E172K对哺乳动物致病性增强的突变。以上结果表明TJ312株在形成过程中可能发生了不同H1N1 SIV病毒株间的基因节段重组，且该株病毒存在部分耐药性及对哺乳动物致病性增强的氨基酸分子特征。表1与SIV TJ312株各基因节段同源性最高的病毒株基因GenesPB2PB1PAHANPNAMNS同源性最高的病毒株The highest homologous strainsA/swine/Tianjin/212/2016(H1N1)A/swine/Tianjin/212/2016(H1N1)A/swine/Tianjin/212/2016(H1N1)A/swine/Tianjin/848/2016(H1N1)A/swine/Tianjin/848/2016(H1N1)A/swine/Tianjin/848/2016(H1N1)A/swine/Tianjin/947/2016(H1N1)A/swine/Tianjin/848/2016(H1N1)同源性(%)Homology(%)99%99%99%100%100%100%100%100%基因登录号Accession No.EPI2049418EPI2049419EPI2049417EPI2049701EPI2049694EPI2049700EPI2049704EPI2049695中 国 预 防 兽 医 学 报2023 年990张奕杰，等.一株 H1N1 亚型猪流感病毒的遗传演化分析及在雪貂体内复制和飞沫传播能力评估第 10 期9912.4病毒在雪貂体内复制水平的检测结果将TJ312株经鼻腔感染2只雪貂，96 h后剖杀，无菌采集 鼻 甲(Turbinates)、软 腭(Soft palate)、气 管(Tra-chea)、肺脏包括左肺及右肺的上叶(Cranial)、中叶(Middle)及 下 叶(Caudal)、扁 桃 体(Tonsil)、脾 脏(Spleen)、肾脏(Kidney)、脑(Brain)并观察剖检病变。结果显示，仅肺脏肉眼观察到可见病变，右下叶出现明显的局灶性暗红色实变(图3A)，其他脏器均未观察到肉眼可见病变。因此选取肺脏右下叶病变部位制备病理切片经HE染色观察，结果显示，肺泡上皮细胞广泛性增生，肺泡膈轻度增宽，细支气管及血管周围水肿，炎性细胞浸润，细支气管上皮细胞注：红色字体为本研究鉴定的病毒，后同。Notes:The red font represents the virus identified in this seudy,the same as below.图1TJ312病毒株HA(A)和NA(B)基因进化树A/swine/Tianjin/821/2016(H1N1)EPI2049693A/swine/Heilongjiang/44/2009(H1N1)EPI353606A/Sichuan/1/2009(H1N1)EPI180217A/California/04/2009(H1N1)EPI1859607A/swine/Tianjin/212/2016(H1N1)EPI2049421A/swine/HongKong/813/1993(H1N1)EPI310981A/swine/Shanghai/2/2005(H1N1)EPI222855A/swine/Guangdong/6/2010(H1N1)EPI407853A/swine/Guangdong/306/2013(H1N1)EPI580096A/swine/Heilongjiang/30/2012(H1N1)EPI912997A/swine/Fujian/204/2007(H1N1)EPI190597A/swine/Shaanxi/s2/2012(H1N1)EPI398465A/swine/Liaoning/777/2018(H1N1)EPI2048973A/Jiangsu/1/2011(H1N1)EPI476438A/Jiangsu/ALS1/2011(H1N1)EPI327050A/swine/Liaoning/940/2018(H1N1)EPI2048981A/swine/Liaoning/1152/2018(H1N1)EPI2048845A/swine/Liaoning/1100/2018(H1N1)EPI2048837A/swine/Liaoning/438/2019(H1N1)EPI2048941A/swine/Liaoning/217/2019(H1N1)EPI2048861A/swine/Liaoning/337/2019(H1N1)EPI2048909A/swine/Hebei/165/2017(H1N1)EPI2048061A/swine/Hebei/726/2017(H1N1)EPI2048181A/swine/Guizhou/828/2016(H1N1)EPI1987749A/swine/Tianjin/947/2016(H1N1)EPI2049709A/swine/Tianjin/396/2016(H1N1)EPI2049501A/swine/Tianjin/848/2016(H1N1)EPI2049701A/swine/Tianjin/312/2016(H1N1)0.080.090.020.020.090.05A/swine/Tianjin/396/2016(H1N1)EPI2049500A/swine/Tianjin/312/2016(H1N1)A/swine/Tianjin/821/2016(H1N1)EPI2049692A/swine/Tianjin/212/2016(H1N1)EPI2049420A/swine/Tianjin/848/2016(H1N1)EPI2049700A/swine/Heilongjiang/44/2009(H1N1)EPI353608A/Sichuan/1/2009(H1N1)EPI506040A/California/04/2009(H1N1)EPI604486A/swine/HongKong/813/1993(H1N1)EPI311015A/swine/Shanghai/2/2005(H1N1)EPI222860A/swine/Guangdong/6/2010(H1N1)EPI407855A/swine/Guangdong/306/2013(H1N1)EPI580098A/swine/Heilongjiang/30/2012(H1N1)EPI912999A/swine/Tianjin/217/2007(H1N1)EPI2049428A/swine/Shaanxi/s2/2012(H1N1)EPI398466A/swine/Liaoning/777/2018(H1N1)EPI2048972A/swine/Liaoning/940/2018(H1N1)EPI2048980A/swine/Liaoning/1152/2018(H1N1)EPI2048844A/swine/Liaoning/1100/2018EPI2048836A/swine/Liaoning/423/2019(H1N1)EPI2048932A/swine/Liaoning/217/2019(H1N1)EPI2048860A/swine/Liaoning/721/2019(H1N1)EPI2048964A/swine/Liaoning/DL1007/2020(H1N1)EPI2143550A/swine/Guizhou/828/2016(H1N1)EPI1987751A/swine/Tianjin/947/2016(H1N1)EPI2049708A/swine/Liaoning/DL1022/2020(H1N1)EPI2143551AB图2基于内部基因的SIV TJ312株的进化树A/swine/Guangxi/NNXD2023/2013(H1N1)EPI1081676A/swine/Guangxi/1080/2013(H1N1)EPI2046728A/swine/Guangxi/683/2013(H1N1)EPI2047898A/swine/Tianjin/312/2016(H1N1)A/swine/Guangxi/717/2019(H1N1)EPI2047914A/swine/Guangxi/1170/2013(H1N1)EPI2048354A/swine/Guangxi/148/2014(H1N1)EPI2046826A/swine/Chongqing/88/2017(H1N1)EPI2046582A/swine/Liaoning/PJ89/2014(H1N1)EPI1788085A/swine/Liaoning/CY102/2014(H1N1)EPI1788005A/swine/Shandong/S269/2014(H1N1)EPI743543A/swine/Anhui/156/2013(H1N1)EPI2046389A/swine/Ningjin/03/2014(H1N1)EPI778351A/swine/Tianjin/212/2016(H1N1)EPI1