Studies on receptor-independent infection of coronaviruses and its implication in the pathogenesis

冠状病毒的受体非依赖性感染及其在发病机制中的意义的研究

基本信息

批准号：
19390135
负责人：
TAGUCHI Fumihiro
金额：
$ 11.56万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2007
资助国家：
日本
起止时间：
2007 至 2009
项目状态：
已结题

项目摘要

Murine coronavirus wild type (wt) JHM strain infects in a receptor-independent fashion (receptor independent infection, RII in short), while a mutant isolated from wild type wt JHM, called srr7, lacks this activity, when examined at 24 hours after infection of mixed neural cell culture. However, variant viruses with RII activity were isolated from srr7 infected mixed cells, when infection was maintained for 3 days. Wt JHM with RII activity showed high neurovirulence for mice, killing those mice within 2-3 days after infection. Srr7 also killed mice when higher dose was inoculated after longer survival time compared with that of wt JHM. Wt JHM antigen was detected in a variety of neural cells, which may be attributed to the RII in mouse brain. However, srr7 antigen was not widely distributed. These results suggest the possibility that high virulence of JHM is attributed to the RII activity. We also tried to obtain porcine coronavirus that grows and shows the virulence for mice to see whether coronaviruses other than JHM display RII activity. After passage of Vero-cell adapted porcine epidemic diarrhea virus (PEDV) through suckling mouse brain, we obtained variant with higher virulence for mice, which shows enhanced cell-cell fusion activity compared with original, non-adapted virus. The mutant showed 4 amino acids mutation in the S protein which is believed to be critical for cell-cell fusion. However, it is not understood whether those mutations in the S could be responsible for higher virulence. In order to pursue this issue, we have to establish the reverse genetics system of PEDV

鼠冠状病毒野生型（WT）JHM菌株以与受体无关的方式感染（受体独立感染，RII，RII），而从野生型WT JHM（称为SRR7）中分离出的突变体缺乏这种活性，当在感染混合神经细胞培养后24小时检查。然而，当维持3天的感染时，从SRR7感染的混合细胞中分离出具有RII活性的变异病毒。具有RII活性的WT JHM对小鼠的神经动力率高，在感染后2-3天内杀死了这些小鼠。与WT JHM相比，在生存时间更长的生存时间后接种较高剂量时，SRR7还杀死了小鼠。在多种神经细胞中检测到WT JHM抗原，这可能归因于小鼠脑中的RII。但是，SRR7抗原并未被广泛分布。这些结果表明，JHM的高毒力归因于RII活性。我们还试图获得猪冠状病毒，该猪冠状病毒生长并显示了小鼠的毒力，以查看JHM以外的冠状病毒是否显示RII活性。在通过哺乳小鼠脑的Vero细胞改编的猪流行腹泻病毒（PEDV）通过后，我们获得了小鼠的毒力较高的变体，与原始的非适应性病毒相比，这表明细胞细胞融合活性增强。突变体在S蛋白中显示4种氨基酸突变，这被认为对细胞 - 细胞融合至关重要。但是，尚不清楚S中的这些突变是否可能导致更高的毒力。为了解决这个问题，我们必须建立PEDV的反向遗传系统