内蒙古牛呼吸道合胞体病毒的分离鉴定及其全基因组分析

Isolation and identification of bovine respiratory syncytial virus from Inner Mongolia and complete genome analysis of the virus

为确定引起内蒙古地区3月龄~6月龄荷斯坦犊牛暴发呼吸系统疾病的病原,并分析其基因组序列,本研究对病死犊牛剖检并观察其各脏器的剖检病变,制备各组织病理切片,观察其组织病变。结果可见犊牛肺脏呈深红色有光泽的肌肉样实变,心脏肥大,肝脏表面散在细小的结节状灰白色病灶,肾脏表面散在细小深红色出血点;组织病理学观察可见典型间质性肺炎,多数各级细支气管腔和肺泡腔内巨噬细胞和淋巴细胞大量增生和浸润,偶见合胞体细胞,在各级细支气管部分黏膜上皮细胞浆内有大小不等的圆形红染的病毒包涵体;轻度纤维化和小坏死灶的变质性肝炎,急性炎性脾肿,多发性小出血灶的慢性硬化性肾小球肾炎。进一步进行RT-PCR检测,结果显示,扩增获得约600 bp的目的条带,测序后与GenBank中相关基因序列比对,结果显示目的基因与牛呼吸道合胞体病毒(BRSV)美国分离株USII/S1(KU159366)的同源性最高(98.42%),表明该地区某牧场犊牛感染了BRSV。将该病例肺组织研磨上清液接种牛肾细胞(MDBK)盲传3代出现典型的细胞病变,经RT-PCR检测确认为BRSV,进一步表明分离到1株BRSV,命名为IM BRSV-39。分段扩增IM BRSV-39株的全基因组序列,经测序后拼接获得长度为15130 bp的BRSV全基因组序列。采用MegAlign软件分析IM BRSV-39株的全基因组序列、G基因序列和F基因序列的同源性及突变位点;采用MEGA 11软件构建分离株全基因组进化树分析其遗传进化关系。结果显示,IM BRSV-39株的全基因组序列、G基因和F基因序列均与BRSVⅢ亚型分离株相应基因序列的同源性最高、且位于同一进化分支,证实IM BRSV-39株属于BRSVⅢ亚型;突变位点分析结果显示:与BRSVⅢ亚型参考株相比,IM BRSV-39株G基因序列存在7个突变位点,F基因序列存在12个突变位点;G蛋白氨基酸序列存在4个突变位点,其中L13H处在G蛋白的胞质结构域(aa1~aa37);Y^(127)H、I^(139)T、P^(177)L、A^(186)T处在G蛋白的胞外结构域(aa66~aa257);F蛋白氨基酸序列存在5个氨基酸突变位点,其中M^(4)T、M^(7)T、S^(14)C处在F蛋白氨基末端信号肽区(aa1~aa26)。本研究检测并分离到1株BSRV,揭示了该病毒基因组的遗传进化特征,为国内BRSV的深入研究奠定了基础。

In order to determine the pathogens responsible for the outbreak of respiratory disease in Holstein calves aged 3-6 months in Inner Mongolia and analyze its genome sequence.In this study,dead calve was dissected and observed for dissected lesions of each organ,and histopathological sections of each tissue were prepared and observed for histopathological lesions.The autopsy showed that the deep red shiny,muscular-like solid lesions in the lungs,hypertrophy of the heart,fine nodular greyish-white foci scattered on the surface of the liver,and fine deep red haemorrhagic dots scattered on the surface of the kidneys.Histopathological showed typical interstitial pneumonia with massive proliferation and infiltration of macrophages and lymphocytes in the lumen of most fine bronchioles at all levels and in the alveolar lumen,occasionally syncytiotrophoblast,reddish-stained virus inclusion bodies of varying sizes within the plasma of the epithelial cells of some of the mucous membranes of all bronchioles at all levels;metamorphic hepatitis with slight fibrosis and small necrotic foci,acute inflammatory splenomegaly,chronic sclerosing glomerulonephritis with multiple small haemorrhagic foci.RT-PCR results showed a target band of about 600bp,which was subjected to sequencing.Alignment of the target gene with the sequences available from GenBank showed that the highest homology(98.42%)is the BRSV US isolate USII/S1(KU159366),indicating the calf was infected with BRSV.The lung tissue grinding supernatants were inoculated into Madin-Darby Bovine Kidney(MDBK)cells,and a typical cytopathic effect was observed after three blind passages.BRSV was confirmed positive by RT-PCR detection,further confirming the successful isolation of BRSV,which was designated as IM BRSV-39.The complete genome sequence of IM BRSV-39 was amplified in segments,sequenced and spliced,revealing the full-length genome of the BRSV is 15130bp.The whole genome sequence,G and F gene sequence of IM BRSV-39 were analyzed for homology and mutation sites using MegAlign;the genetic evolutionary relationships of isolate were analyzed by constructing a genome-wide evolutionary tree using MEGA 11 software.The results showed that the whole genome sequence,G and F gene sequences of IM BRSV-39 had the highest homology and were in the same evolutionary branch with BRSV subtype III isolates,confirming that IM BRSV-39 belonged to BRSV subtype III.Compared with the reference BRSVⅢ,the IM BRSV-39 had 7 and 12 mutations in the G and F gene,respectively.In addition,four amino acid substitutions occurred in G protein,of which L13H was in the cytoplasmic structural domain of the G protein(aa1-aa37);Y^(127)H,I^(139)T,P^(177)L,A^(186)T are in the extracellular domain of G protein(aa66-aa257).Five amino acid substitutions were observed in F protein,in which M^(4)T,M^(7)T,and S^(14)C are in the signal peptide region of the N-terminus of F protein(aa1-aa26).In this study,we detected and isolated one strain of BSRV and revealed the genetic evolutionary characteristics of the viral genome,laying the foundatio.