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↓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(α-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106 EID50/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∶1 280、1∶1 280、1∶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∶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.
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 by PCR and sequenced in this study,and the complete genome sequence was spliced by SeqMan software and compared by BLAST to obtain the virus strain with the highest homology with each gene segment sequence.Mafft was used to analyze the molecular characteristics of amino acid sequences encoded by each gene segment and MEGA5.1 software was used to construct the evolutionary 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 tree showed that the HA,NA and M genes of this strain came from the Eurasian avian H1N1(EA H1N1)SIV lineage.PB2,PB1,PA and NP genes were derived from 2009/H1N1 influenza virus lineage.The NS gene was derived from the North American triple reassortment H1N2 SIV lineage.The protein molecular characteristics analysis results showed that the sequence of the basic amino acid cleavage site of HA protein was PSIQSR↓G,which was consistent with the molecular characteristics of low pathogenic influenza virus.The receptor binding sites correspond to the molecular properties of human sialic acid receptors(α-2,6 sialic acid receptors).The NA protein aa275 is H and aa295 is N,and the head region of this protein is not missing,indicating that the strain is sensitive to oseltamivir drugs.PA 356R was a molecular characteristic of increased viral replication and pathogenicity in mammals.NP protein 41I and 210E could enhance the activity of viral polymerase.31N of M2 protein is a molecular characteristic of resistance to amantadines.In this study,the ability of TJ312 strain to replicate and spread by droplets in ferrets was further evaluated.Two ferrets were infected through the nasal cavity at 106EID50/ferret doses.The ferrets were killed 96 hours after infection.The nasal turbinate,soft palate,trachea,lung,tonsil,spleen,kidney,liver and brain were collected.Pathological sections of the diseased lungs were prepared to observe the pathological changes,and the virus antigen in the lung tissues was detected by immunohistochemical test.All the collected organs were treated and inoculated with chicken embryos to detect the virus titer in the organs and evaluate the replication ability of the virus in ferrets.Three ferrets were infected intranasally with the same dose of TJ312 strain,and three naive ferrets were placed in adjacent cages as exposed group 24 hours after infection.The nasal washes were collected seven times every other day from the second day to the 14th day.Droplet virus transmission between ferrets was evaluated by detecting virus titers in nasal washes and serum HI antibody titers from ferrets in the exposed group.The results showed 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 large number 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 the highest replication capacity in turbinate with an average viral titer of 6.13log10EID50/mL,followed by the replication capacity in the right caudal of lung.The mean virus titer was 6log10EID50/mL.No virus was detected in the spleen,kidney or liver.However,traces of the viruses were detected in the brain of one ferret with a titer of 0.63log10EID50/mL.In the 14-day droplet transmission test,a high titer of the virus was detected in the nasal wash of the 3 ferrets in the infected group,the highest titer of virus was 5.50Log10EID50/mL,and high levels of HI antibody titer(1∶280,1∶280,1∶2560)were detected in the serum of the 3 ferrets.One of the 3 ferrets in the transmission group was positive for viral titration(up to 5.75Log10EID50/mL)and had a high HI antibody titer(1∶2560)in the serum.These results suggest that the SIV TJ312 strain may be derived from inter-segment gene recombination of other H1N1 SIV strains that existed in Tianjin in 2016,and this strain has partial drug resistance and amino acid characteristics that enhance the pathogenicity in mammals.The virus can replicate at high levels in ferrets'respiratory tracts and damage their lungs.It can be transmitted from ferret to ferret by droplets.This study provides some reference for further understanding the biological characteristics of EA H1N1 SIV and exploring its infection mechanism.