云南省家鼠鼠疫疫源地鼠形动物戊型肝炎病毒感染及基因多态性研究

Hepatitis E virus infection and gene polymorphism in murine-shaped animals of plague foci in Yunnan Province

目的了解云南省家鼠鼠疫疫源地鼠形动物戊型肝炎病毒(hepatitis E virus,HEV)感染情况及基因多态性。方法2019年7-8月,在云南省家鼠鼠疫疫源地的16个自然村(弥勒市4个、芒市6个、梁河县6个)周围,采用夹夜法收集鼠形动物,提取肝脏RNA,应用一步法荧光定量PCR检测鼠HEV目的基因,计算鼠HEV阳性率;采用PCR法对阳性样本进行目的基因扩增及克隆测序,应用MegAlign和MEGA 7.0软件对测序样本序列与GenBank注册的HEV基因序列进行序列对比分析,构建系统进化树。结果共捕获鼠形动物491只,隶属3目、5科、8属、15种;鼠HEV阳性率为4.89%(24/491),其中黄胸鼠和针毛鼠的阳性率分别为9.39%(23/245)和1/3,其他鼠形动物未检出。不同生境鼠形动物鼠HEV阳性率比较差异有统计学意义(P=0.014),且民居附近生境鼠HEV阳性率高于其他生境(P均<0.05)。序列对比分析结果显示,来源于梁河县的P018样本基因序列与来自香港首例感染鼠HEV患者的样本MG813927.1序列同源性为100.0%,且与MG813927.1及来源于鼠的样本LC549185.1序列聚为同一分支,为HEV C型;来源于芒市的G024样本与其他病毒株的序列同源性为20.7%~31.5%,与来源于鸟类的样本AY535004.1序列聚为同一分支。结论云南省家鼠鼠疫疫源地鼠形动物存在鼠HEV感染,可能存在基因多态性,且感染差异与生境有关。

Objective To learn about the infection and gene polymorphisms of hepatitis E virus(HEV)in murine-shaped animals of plague foci in Yunnan Province.Methods From July to August 2019,around 16 natural villages(4 in Mile City,6 in Mangshi and 6 in Lianghe County),which were the foci of domestic plague in Yunnan Province,the murine-shaped animals were captured by the night-time method.The liver RNA was extracted,and the target gene of rat HEV was detected by one-step real-time fluorescence quantitative PCR,and the positive rate of rat HEV was calculated.The rat HEV positive samples were amplified by PCR for further clone sequencing,and the resulting sequences were compared with the HEV sequences registered in the GenBank and a phylogenetic tree was constructed by using MegAlign and MEGA 7.0.ResultsA total of 491 murine-shaped animals were captured from 3 orders,5 families,8 genera,15 species,and the positive rate of rat HEV was 4.89%(24/491).Among them,the positive rate of Rattus tanezumi and Niviventer fulvescens was 9.39%(23/245)and 1/3,respectively;and other species were negative.There was a statistically significant difference in the positive rate of rat HEV between different habitats(P=0.014),and the positive rate of rat HEV in the habitats near the dwellings was higher than that in other habitats(P<0.05).The sequence comparison analysis showed that the gene sequence of P018 from Lianghe County was 100.0%homologous to the MG813927.1 sequence of the first patient with rat HEV in Hongkong,and it was clustered into the same branch with the sequences of MG813927.1 and LC549185.1 from rat,was the type HEV C.G024 from Mangshi shared a low homology(20.7%-31.5%)with other virus strains,and it was clustered into the same branch with a HEV sequence from an avian(AY535004.1).Conclusions Rat HEV is prevalent in murine-shaped animals of plague foci of Yunnan Province,and there may be gene polymorphisms of rat HEV.In addition,the difference infection rate may be related to the habitats.