PRRSV强弱毒感染PAM和Marc-145细胞入胞途径差异的研究

Analysis of the Differences of the Entry Pathway of PAM and Marc-145 Infected with Highly Pathogenic and Attenuated PRRSVs

猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的猪场最重要传染病之一,给全世界养猪业造成了持续的经济损害。减毒活疫苗免疫接种作为防控PRRS的有效手段在全世界范围内得到广泛应用。减毒活疫苗毒株是在猴源细胞系上连续传代获得的,导致其与亲本毒株在生物学特性上存在巨大差异,同时本实验室前期结果也提示HP-PRRSV野毒株和疫苗毒株在入胞途径上可能存在差异。Na^(+)/H^(+)交换的依赖是巨胞饮的标志性特征,阿米洛利(EIPA)是Na^(+)/H^(+)交换的特异性抑制剂,细胞骨架重排在病毒的入胞过程中发挥重要作用,细胞松弛素D(Cyt D)是肌动蛋白聚合的特异性抑制剂。分别利用EIPA和Cyt D预处理PAM和Marc-145细胞后接毒,对比强弱毒在两种细胞内病毒拷贝数、蛋白表达水平及子代病毒的释放上与空白对照组的差异。结果表明,PRRSV强弱毒均不利用巨胞饮途径感染Marc-145或者PAM细胞,但强弱毒在感染不同细胞时对肌动蛋白的依赖性存在明显的区别。本研究结果初步揭示了PRRSV野毒株和疫苗毒株入胞过程的差异,为进一步揭示PRRSV疫苗毒株致弱机制提供了前期基础。

Porcine reproductive and respiratory syndrome(PRRS),which caused by Porcine reproductive and respiratory syndrome virus(PRRSV) is considered as the most important infectious disease to the pig industry.Immunization is considered as an effective way to control PRRS all over the world.Live attenuated vaccine which obtained by continuous generation on Marc-145 cells was considered as the most effective vaccine,the generation on allogenic cell line resulting huge biological difference compaired with their parents strains including growth characteristic.Our previous results also showed that PRRSV field strain and vaccine strain might have differences in the pathway of entry the cell.Na^(+)/H^(+) exchange is the key characteristic of macropinocytosis,Amiloride(EIPA) is the specific inhibitors of Na^(+)/H^(+) exchange,Cytochalasin D(Cyt D) was widely used to inhibited actin polymerzation.The PAM and Marc-145 cells were pretreated with EIPA and Cyt D respectively,and then inoculated with the virus.Compare the differences between HuN4-F5 and HuN4-F112 in the copy number,protein expression level and the release of progeny viruses in the two cells with the blank control group.These results showed that PRRSV do not utilize macropinocytosis pathway entry Marc-145 and PAM cells.Instead,actin is important in PRRSV entry and the usage of actin on Mrac-145 and PAM cells is the main differenct of PRRSV field strain HuN4-F5 and vaccine strain HuN4-F112 during the cell entrance of infection.