大鼠细小病毒KRV/QD/2022株的分离鉴定及VP2基因遗传进化分析

Isolation and Identification of Rat Parvovirus KRV Strain and Genetic Evolution Analysis of VP2 Gene

为了解山东地区Kilham细小病毒(kilham rat virus,KRV)的流行情况并探究其分子生物学特征,本试验从某疑似感染KRV的大鼠养殖场分离病毒并对其致病性进行研究。通过观察患病大鼠的临床症状和病理变化并使用PCR方法检测病料确定是否为KRV病毒感染。将阳性病料接种到大鼠神经胶质瘤细胞C6上分离KRV并对其VP2全长测序并构建进化树,通过病毒TCID 50测定、血凝和血凝抑制试验及动物回归试验确定分离株的致病性。结果显示养殖场中流产母鼠子宫体积变小,子宫内胎儿全部死亡或部分死亡。将阳性病料接种于C6细胞上,盲传三代后出现明显病变,对分离株VP2基因进行测序发现该分离株与LG/HN/CHN/2016株相似度最高为98.51%,与其他目前已知分离株的VP2同源性在95.20%~97.31%之间。分离株传到7代后的TCID 50为105.56/0.1 mL,血凝效价为1∶1280,血凝抑制效价为27,证明分离株确为KRV,将其命名为KRV/QD/2022。动物回归试验证明该分离株能感染大鼠并导致大鼠出现流产、繁殖障碍等特征性病变。上述结果显示,本研究成功分离出了一株KRV细小病毒并进行了系统的研究,为该病毒的诊断和预防提供了一定的依据。

To comprehend the prevalence of kilham rat virus(KRV)in the Shandong region and explore its molecular characteristics,in the present study,we isolated the virus from a rat farm suspected of KRV infection and investigated its pathogenicity.KRV infection was confirmed through observation of the rats′clinical symptoms and pathological changes,coupled with PCR detection.Positive samples inoculated onto rat glioma cells C6 to observe lesions,and VP2 of isolation strain fully amplified and sequenced,virus TCID 50,hemagglutination and hemagglutination inhibition tests were conducted to determine whether the virus was KRV.The isolation strain′s pathogenicity was confirmed by animal regression test.The results exhibited smaller uterine volume in the miscarried mother rats at the breeding farm,all or some of the fetuses in the uterus were dead.Noteworthy pathological changes appeared in C6 cells after three passages of the positive materials;sequencing of the isolated strain VP2 showed a maximum similarity of 98.51%with the LG/HN/CHN/2016 strain and homology between 95.20%to 97.31%with other known isolated strains.After seven generations,the TCID 50 of the isolated strain was 105.56/0.1 mL,hemagglutination titer was 1:1280,and hemagglutination inhibition was 27,confirming the strain as KRV and naming it KRV/QD/2022.Animal back-passaging experiments confirmed that this isolated strain could infect rats and cause characteristic pathological lesions such as abortion and reproductive disorders.In summary,this study successfully isolated a strain of KRV and conducted systematic research,laying a foundation for the diagnosis and prevention of KRV.