果子狸源细小病毒分离鉴定及VP2基因遗传进化分析

【目的】探究果子狸源细小病毒流行特点及其VP2基因遗传变异情况,为细小病毒防治提供理论依据和技术支撑。【方法】采用猫肾细胞(CRFK细胞)对患有肠炎的果子狸肠道组织样品进行病毒分离,通过血凝试验、免疫荧光试验鉴定分离的病毒,并对病毒VP2基因进行PCR扩增和遗传进化分析。【结果】分离到的毒株在CRFK细胞中培养出现细胞脱落、崩解和破碎等典型细小病毒细胞病变效应(CPE);血凝试验结果显示,此病毒可凝集猪红细胞,血凝效价为1∶512;免疫荧光试验结果显示,在接种分离毒株的CRFK细胞内可观察到亮绿色荧光,而对照细胞没有荧光。将分离毒株命名为MPCPV-SX。VP2基因进化树分析发现,其与犬细小病毒处于同一分支,位于CPV-2和CPV-2a型之间,同时VP2氨基酸的87和101位显示为CPV-2型,而300和305位氨基酸则与CPV-2a型一致。【结论】本研究成功从果子狸肠道组织样品中分离出1株细小病毒MPCPV-SX,其VP2基因是介于CPV-2和CPV-2a之间的中间型,表明细小病毒通过果子狸等野生动物跨越宿主流行传播,可能在细小病毒遗传进化方面起到一定的过渡作用。

Isolation and Identification of Parvovirus from Palm Civets and Sequence Analysis of VP2 Gene

【Objective】 The purpose of the experiment was to explore the epidemic characteristics of Parvovirus from palm civets and the genetic variation of VP2 gene,so as to provide theoretical basis and technical support for Parvovirus control.【Method】 CRFK cells were used to isolate the virus from the intestines of palm civets with enteritis.The virus was identified by hemagglutination test and immunofluorescence test.The VP2 gene of the virus was amplified by PCR and its genetic variation was analyzed.【Result】 Typical cytopathic effects (CPE)of Parvovirus such as cell abscission,disintegration and fragmentation were observed in CRFK cells.The results of hemagglutination test showed that the virus could agglutinate porcine erythrocytes,and the hemagglutination titer was 1∶512.The results of immunofluorescence test showed that bright green fluorescence could be observed in the CRFK cells inoculated with the isolated strain,while no fluorescence was observed in the control cells.The isolated strain was named MPCPV-SX.Phylogenetic tree analysis of VP2 gene showed that it was in the same branch as Canine parvovirus,located between CPV-2 and CPV-2a.At the same time,the 87 and 101 amino acids of VP2 were CPV-2,while the 300 and 305 amino acids were consistent with CPV-2a.【Conclusion】 In this study,a strain of Parvovirus MPCPV-SX was successfully isolated from the intestines of palm civets.Its VP2 gene was an intermediate type between CPV-2 and CPV-2a,indicating that Parvovirus spreaded across the host through palm civets and other wild animals,which might play a transitional role in the genetic evolution of parvovirus.