置换弱毒C株非编码区的嵌合猪瘟病毒对猪的致病性研究

Pathogenicity to Pigs of a Chimeric Classical Swine Fever Virus with Substitution of the Untranslated Regions from C-strain

猪瘟(CSF)是由猪瘟病毒(CSFV)引起的一种高度接触性传染病。研究表明,CSFV的非编码区(UTR)与病毒复制和毒力相关。为了分析置换CSFV弱毒疫苗C株UTR的嵌合毒株对病毒复制和猪致病性的影响,以构建的C株和QZ14(CSFV 2.1 d型毒株)感染性克隆质粒pA-C和pA-QZ14为基础,扩增pA-C的UTR区和pA-QZ14除UTR以外的基因组序列,利用同源重组技术构建重组质粒pA-QZ14-CUTR;将体外转录的病毒基因组RNA转染PK-15细胞,连续传代以拯救嵌合病毒株QZ14-CUTR;将其接种PK-15细胞,通过免疫荧光和测序鉴定以确定拯救是否成功;RT-qPCR和毒价滴定法测定病毒生长趋势,分析其细胞适应性;将嵌合病毒以1×10^(5)TCID 50剂量肌肉接种试验猪以观察其致病性。结果表明,QZ14-CUTR拯救成功,基因组中稳定携带C株UTR序列;生长曲线显示,QZ14-CUTR病毒基因组拷贝数和滴度均低于亲本毒株QZ14;相较于QZ14株,QZ14-CUTR仅引起猪轻微的临床症状,不显著影响猪白细胞和淋巴细胞数;病理变化程度、排毒阳性率和组织中的病毒核酸阳性率均低于QZ14株;QZ14-CUTR能诱导特异性CSFV E2抗体,且抗体水平持续上升。综上所述,CSFV流行毒株QZ14的UTR被弱毒C株UTR置换后,其在PK-15细胞中的复制能力有所下降,对猪的致病力降低。

Classical swine fever(CSF)is a highly contagious disease caused by classical swine fever virus(CSFV).The untranslated regions(UTR)of CSFV are reported to correlates with replication and virulence of the virus.In order to study the effect of UTR of CSFV strain C on the pathogenicity of a field strain QZ14,an infectious clone pA-QZ14-CUTR was constructed by homologous recombination based on the plasmids pA-C of the C-strain and pA-QZ14 of the QZ14 strain maintained in our laboratory.The chimeric viral RNA in vitro transcribed from pA-QZ14-CUTR was transfected into PK-15 cells to rescue the chimeric virus QZ14-CUTR,which was then used to infect PK-15 cells for identification by immunofluorescence assay(IFA)and sequencing.Virus growth kinetics was examined by RT-qPCR and virus titration to analyze its adaptability in PK-15 cells.The chimeric virus was inoculated intramuscularly into pigs at a dose of 1×10^(5)TCID 50 to examine its pathogenicity to pigs.IFA and sequencing confirmed that the chimeric virus QZ14-CUTR was successfully rescued from the transfected cells with the C-strain UTR stable in the viral genome.Growth kinetics revealed that the virus QZ14-CUTR showed slower growth in PK-15 cells than its parental QZ14.QZ14-CUTR caused only slight clinical signs,and did not affect the leukocyte and lymphocyte counts,as compared with the QZ14 strain.Gross and microscopic histopathological changes were less pronounced,and viral secretion from fecal and nasal discharges as well as viral recovery in tissues from pigs inoculated with QZ14-CUTR were lower than in those infected with QZ14 inoculation.QZ14-CUTR could induce specific CSFV E2 antibody with steady increase over time.In summary,replacement of the QZ14 strain UTRs with those from the C-strain decreases its adaptability in PK-15 cells and reduces its pathogenicity to pigs.