ORF3蛋白促进猪流行性腹泻病毒在Vero细胞上的增殖

ORF3 protein promotes the proliferation of porcine epidemic diarrhea virus on Vero cells

摘要:

【背景】猪流行性腹泻(Porcine epidemic diarrhea，PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)感染猪而引起的一种急性肠道传染病，常导致病猪水样腹泻、呕吐、脱水。自2010年起，其大规模的暴发给养猪业造成巨大的经济损失。由于对PEDV免疫机理及侵入机制知之甚少，至今仍缺乏有效的PED防治措施。【目的】研究orf3对PEDV体外增殖的影响。【方法】利用基于RNA同源重组的PEDV反向遗传学操作技术拯救一系列携带不同orf3基因及orf3基因缺失的重组PEDV；将获得的重组PEDV以MOI 0.1感染Vero细胞，分别于感染的第8、16、24、32、40、48 h测定其TCID50并绘制病毒生长曲线；分别在感染25 h和36 h利用全自动细胞计数分析仪对6孔板内的细胞进行计数，并于感染后的第12、24、36、48 h用CCK-8试剂盒对其细胞活力进行测定。【结果】RT-PCR结果及细胞病变观察证明成功拯救到了携带不同orf3基因或orf3基因缺失的重组PEDV；进一步的免疫组化分析结果证实PEDV的ORF3蛋白可以在Vero细胞中合成。SPSS软件分析表明携带orf3基因的重组PEDV的滴度(TCID50)显著高于缺失orf3基因的重组PEDV的滴度；带有orf3基因的重组PEDV感染Vero细胞25 h和36 h时的活细胞数显著高于缺失orf3基因的重组病毒感染相同时间时的活细胞数；而且重组PEDV感染Vero细胞24 h后，带有orf3基因的重组PEDV的细胞活性显著高于缺失orf3基因的重组病毒。【结论】ORF3蛋白对于PEDV在Vero细胞中的增殖具有促进作用，该作用是通过延缓或减少感染细胞的死亡实现的。本研究为揭示PEDV orf3基因的功能和PEDV复制机制的研究提供理论基础。

Abstract:

[Background] Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is an acute intestinal infectious disease of pigs that is characterized by diarrhea, vomiting, and dehydration. Its massive outbreak has caused huge losses to the pig industry since 2010. Due to the poor understanding of the immune mechanism and invasion mechanism of PEDV, there is still no effective prevention and treatment about PED. [Objective] The main aim of this study is to explore the effects of ORF3 protein on the proliferation of PEDV in vitro. [Methods] Recombinant PEDV DR13 (attenuated) strains with orf3 genes from different origins or without orf3 gene were rescued by using reverse genetics operating system based on targeted RNA recombination. Then the Vero cells were infected with the acquired recombinant PEDV strains at a multiplicity of infection (MOI) of 0.1. The viral titers (TCID50) at 8, 16, 24, 32, 40, 48 hours post infection (hpi) were measured and the growth curve of each recombinant PEDV was drawn. The intact cells at 25 and 36 hpi were counted by using an automatic cell counting analyzer. Meanwhile, the cell viability were measured at 12, 24, 36, 48 hpi using Cell Counting Kit-8. [Results] Successful rescue of recombinant PEDV strains with or without orf3 was verified by using RT-PCR and observing the cytopathic effect (CPE). The results of immunohistochemistry assay indicated that orf3 gene of recombinant PEDV was expressed in Vero cells. The viral titers of recombinant PEDV strains with orf3 gene were significantly higher than that without orf3 gene. Furthermore, the statistical analysis results using SPSS showed that the numbers of intact cells left in the flasks infected with the recombinant PEDV strain with orf3 gene at both 25 and 36 hpi were significantly higher than that infected with the strain without orf3 gene and viability of the cells infected by recombinant PEDV strain with orf3 gene were significantly higher at 24, 36 and 48 hpi than that infected with the strain without orf3 gene. [Conclusion] PEDV ORF3 protein promotes the proliferation of PEDV on Vero cells, the mechanism of which might be the time delay of death of infected cells by the ORF3 protein. These results provided information on the function of PEDV orf3 gene and were helpful in understanding the replication mechanisms of PEDV.