猪繁殖与呼吸综合征病毒强/弱毒感染PAM细胞生物学特性比较分析

GROWTH DYNAMICS OF HIGH/LOW VIRULENT PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IN PORCINE ALVEOLAR MACROPAGES

为了解猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)强、弱毒株在PAM细胞上的增殖特性,本研究在体外分离培养了健康猪肺泡巨噬细胞(porcine alveolar macrophages,PAM),然后分别用高致病性PRRSV强毒HuN4株和弱毒疫苗HuN4-F112株感染PAM细胞,细胞病变观察和间接免疫荧光检测结果显示,二者在体外均可以感染PAM细胞,其中强毒HuN4株感染PAM细胞CPE较为明显。在两个毒株感染PAM细胞后12、24、36、48、60h分别收获病毒感染的细胞,利用抗PRRSV N蛋白单抗进行Western blot分析检测病毒核蛋白在不同时间表达量的变化,结果表明,强毒株在感染PAM细胞的早期,N蛋白合成表达量明显高于弱毒疫苗株,而弱毒疫苗株在感染Macr-145细胞早期,N蛋白的合成量则明显优于强毒株。比较HuN4株与HuN4-F112株在PAM细胞和Marc-145细胞的生长曲线,结果显示强、弱毒在PAM细胞和Marc-145细胞生长趋势存在明显差异,其中强毒HuN4株在PAM细胞上增殖能力明显强于弱毒株,而弱毒HuN4-F112株在Marc-145细胞上的增殖能力明显强于其在PAM细胞上的增殖能力,表明PRRSV强毒株对靶细胞PAM的感染能力较强,弱毒疫苗株对其感染能力相对较弱。本研究为深入了解PRRSV强毒株与弱毒疫苗株的致病性差异提供了理论依据。

Abstract： To better understand the growth properties of high/low-virulent Porcine reproductive and respiratory syndrome viruses （PRRSVs）, highly virulent strain HuN4 and attenuated vaccine strain HuN4-F112 were used to infect primary porcine alveolar macrophages （PAMs）. The cytopathic effect （CPE） in HuN4 infected PAMs was more severe than in HuN4-F112 infected PAMs although both strains were able to infect PAMs as determined by the formation of CPE and immunofluorescence assay. The viruses were harvested at 12, 24, 36, 48 and 60 hours post infection in PAMs and then the amounts of N protein were examined in Western blot. The results showed that the early expression of N protein was significantly higher in highly virulent HuN4 strain than attenuated HuN4-F 112 strain. However, similar experiment in Marc-145 cells yielded opposite conclusion. These results suggested that the cell tropism and replication ability of PRRSV have changed during repeated passages in continuous cell line Marc-145. The present study provided useful informationfor further investigation into the relationship between the virulence and growth properties of highly virulent and attenuated PRRSV strains.