Ⅷ群神经致病型马疱疹病毒1型的分离及鉴定

Isolation and identification of neuropathogenic equid herpesvirus 1 within group Ⅷ

为研究伊犁马是否存在Ⅷ群神经致病型马疱疹病毒1型(EHV-1)的感染,并分析其遗传进化特征,本研究采集新疆伊犁昭苏县某马场93份伊犁马流产胎儿肺组织样品,经PCR鉴定为EHV-1阳性后,将部分EHV-1阳性的肺组织研磨上清液接种MDBK细胞,盲传3代,待出现细胞病变时收集细胞及培养上清,通过PCR及透射电镜对EHV-1进行鉴定,并对其进行不同基因的遗传进化分析。结果显示,93份临床样品中有43份样品呈EHV-1阳性。将其中11份阳性样品接种MDBK细胞盲传3代后,有4份出现细胞病变,电镜观察可见疱疹病毒典型的形态特征,且均经PCR检测到EHV-1 g B基因,表明分离到4株EHV-1,分别命名为XJ-YLZS-21、XJYLZS-22、XJ-YLZS-29和XJ-YLZS-31。同源性分析结果显示,4株EHV-1分离株gB基因序列与EHV-1参考株Ab4相应基因序列的同源性为100%。ORF30基因序列分析结果显示,4株分离株第2 254位均为鸟嘌呤(G),编码天冬氨酸(D),表明4株EHV-1分离株为神经致病型EHV。ORF68基因及其编码氨基酸序列的遗传进化分析结果显示,4株EHV-1分离株的核苷酸及氨基酸序列同源性均为100%,并处于独立的进化分支,将其划分为Ⅷ群神经致病型EHV-1。本研究首次在国内鉴定并分离到Ⅷ群神经致病型EHV-1,丰富了EHV-1的流行病学数据库,为EHV-1感染所致马流产的防控奠定了流行病学基础。

The aim of this study was to isolate and identify neuropathogenic equid herpesvirus 1(EHV-1) of Ⅷ group and analyze its genetic characteristics. Ninety-three aborted fetal lung tissues of Yili horses, collected from six horse farms in Zhaosu country, Yili, Xinjiang, were detected EHV-1 by PCR. Some of the EHV-1 positive lung tissue grinding supernatants were inoculated into bovine kidney cells(MDBK) for three generations. The virus was collected when cytopathy(CPE) occurred and identified by PCR and transmission electron microscope. PCR results showed that 43 samples were positive for EHV-1. Four of eleven positive samples showed CPE, and the EHV-1 glycoprotein B(gB) gene was amplified. The virus particles with a diameter of about 120 nm were observed by transmission electron microscope. Four EHV-1 isolates were named XJ-YLZS-21, XJ-YLZS-22,XJ-YLZS-29, and XJ-YLZS-31, and g B gene sequences of those isolates and EHV-1 reference strain Ab4 shared 100% nucleotide identity. Our findings indicated that four EHV-1 strains were successfully isolated. In addition, we found that the ORF30 gene sequence of isolates was guanine(G) at 2254 position and encoded aspartic acid(D), indicating that those viruses were neuropathogenic EHV-1. The ORF68 gene sequence analysis showed that four isolates had identical nucleotides. Next, a phylogenetic tree was constructed based on ORF68 gene sequences, revealing that four EHV-1 isolates in this study formed an independent branch and were clustered as group Ⅷ EHV-1. In this study, we firstly isolated and identified neuropathogenic EHV-1within group Ⅷ, which enriches the epidemiological database of EHV-1 and lays an epidemiological foundation for the prevention and control of horse abortion caused by EHV-1.