猪伪狂犬病病毒SX-2018变异株的分离鉴定及其致病性研究

Isolation and identification of porcine pseudorabies virus variant strain of SX-2018 and its pathogenicity to mouse

本研究从疑似暴发伪狂犬病(pseudorabies,PR)猪场死亡的仔猪中分离到1株病毒,经组织病理学剖检、PCR试验、Western-blot分析,将其鉴定为伪狂犬病病毒(pseudorabies virus,PRV),遂被命名为SX-2018,该病毒在MDCK细胞上具有较强的适应性。将SX-2018株gE基因的序列与国内外21株PRV参考序列进行同源性比较,结果显示,SX-2018株gE基因与国内外参考毒株的核苷酸序列同源性为97.1%~99.9%,氨基酸同源性为94.5%~100%。遗传进化分析显示,SX-2012株与我国近几年新分离的猪伪狂犬变异毒株处于同一分支;并且与经典株相比,gE蛋白在第48位和第492位各有1个天冬氨酸的插入。为了进一步探讨SX-2018毒株的致病性,将SX-2018株和Min-A株分别感染BALB/c小鼠,分析两组小鼠的发病特征和组织损伤情况;结果显示,SX-2018组的小鼠死亡时间早于Min-A组;并且Min-A组小鼠的肺脏主要表现为间质性肺炎,而SX-2018组的小鼠以出血性肺炎为主。荧光定量PCR分别检测两组小鼠肺脏内PRV gI基因和炎症相关因子的变化;结果显示,SX-2018组小鼠肺组织内的gI基因以及炎症相关因子的mRNA水平显著地高于Min-A组小鼠。以上结果表明,本文成功分离到1株PRV变异毒株,并且与PRV经典株Min-A株相比,新分离的变异毒株可以诱导不同程度的病理损伤和炎症反应。

In the present study,a virus strain was isolated from suspicious pseudorabies virus(PRV)samples collected from dead piglets,the virus was demonstrated to be PRV via histopathological examination,PCR and Western blot,and was termed as SX-2018 strain,it has good adaptability in MDCK cells.The genetic analyses compared with another 21 PRV references at home and abroad,the nucleotide sequence homology was 97.1%—99.9%,and the amino acid homogy was 94.5%—100%.Phylogenetic analyse of gE nucleotide sequence showed SX-2018 strain clustered together with another home PRV variantsisolated from the recent years with a relatively independent branch.Compared with classical PRV strains,there was an amino acid insertion(amino acid D)at the side 48 and 496 in gE.To further explore the pathogenicity1 of SX-2018 strain,BALB/c mice were challenged with SX-2018 and Min-A,respectively,and then compared the pathogenetic characteristics and tissue damages of mice in the two group.The results showed that SX-2018 innoculated mice died sooner than Min-A innoculted mice,histopathological observation suggested that the lungs of mice in the Min-A group were mainly charaterized by interstitial pneumonia,while,the mice in the SX-2018 group mainly showed homarragic pneumonia.Subsequently,the changes of PRV gI gene and inflammation-related factors in lungs of mice in the two groups were detected by fluorescence quantitative PCR,and the results demonstrated that mRNA levels of gI gene and inflammation-related factors in the lungs of mice infected with SX-2018 were significantly higher than those of mice innoculated with Min-A and DMEM.The above results indicated that a variant strain of PRV was successfully isolated in this study,the isolate could induce different degree of pathological injury and inflammatory response compared with the classical strain of PRV Min-A.