摘要
为调查内蒙古四子王旗某马场马流产是否由马疱疹病毒1型(EHV-1)感染导致,本研究采集该场马流产胎儿肝、脾等组织,将组织研磨上清液接种MDBK细胞,盲传5代,待出现细胞病变时收集细胞及培养上清,通过gB基因的PCR及透射电镜对分离病毒进行鉴定,采用2代测序技术对分离病毒全基因组测序,并进行分离病毒gB基因的同源性及遗传进化分析.采用SnapGene6.0.2软件分析分离病毒ORF30基因及其编码氨基酸序列.结果显示,组织上清液接种MDBK细胞并盲传5代后,细胞出现典型的圆缩、融合、脱落和聚堆成葡萄串状等细胞病变特征;EHV-1 gB基因的PCR检测结果呈阳性;电镜观察可见疱疹病毒典型的形态特征,表明分离到1株EHV-1,命名为EHV-1-NM2021.同源性分析结果显示:分离株EHV-1-NM2021 gB基因序列与国内外EHV-1参考株gB基因序列的同源性为97.7％～100％;构建gB基因的系统发育进化树,结果显示该基因与EHV-1德国株(RacL11)、澳大利亚株(438-77)处于同一进化分支,亲缘关系较近.此外,ORF30基因序列及氨基酸序列比对结果显示,EHV-1-NM2021 ORF30基因nt2254为鸟嘌呤(G),aa752为天冬氨酸(D),该分离株为神经致病型病毒.本研究首次在内蒙古鉴定并分离到神经致病型EHV-1,丰富了国内EHV-1的流行病学数据库,为EHV-1感染所导致马流产的防控奠定了研究基础.
Abstract
In order to identify whether an aborted mare was caused by Varicellovirus equid hepersvirus1(EHV-1)in a horse farm in Inner Mongolia.In this study,tissues such as liver and spleen were collected from aborted foetuses at the site,and the tissue grinding supernatant was inoculated into MDBK cells,blindly passed for 5 generations.Cells and culture supernatant were collected when cytopathic effects appeared,and the presence of EHV-1 was identified throug PCR and transmission electron microscopy followed by genetic evolutionary analysis.The results showed that the MDBK cells inoculated with the virus exhibited typical effects such as cell rounding,cell fusion,cell detachment and clustering into grape-like formations after the tissue supernatant blindly passing for five generations;Electron microscopy revealed the characteristic morphology of herpesvirus.The PCR detection of the gB gene was positive and it was named EHV-1-NM2021.Genetic evolutionary analyses showed that the gB gene sequence of the isolate strain EHV-1-NM2021 shares a nucleotide sequences homology of 97.7％to 100％with gB gene sequences of the domestic and international reference strains of EHV-1.By constructing a phylogenetic evolutionary tree for the gB gene,the results showed that this gene is in the same evolutionary branch as the German strain(RacL11)and the Australian strain(438-77),and that they are closely related.In addition,results of nucleotide sequence and amino acid sequence alignment showed that the EHV-1-NM2021 ORF30 gene has guanine(G)at 2254th position and aspartic acid(D)at 752th amino acid position,indicating that the isolate is a neuropathogenic strain.This study is the first to identify and isolate the neuro virulent EHV-1 in Inner Mongolia,enriching the epidemiological database of EHV-1 in China,and laying the epidemiological foundation for the prevention and control of equine abortion caused by EHV-1 infections.
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