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

小鼠冠状病毒野生型（wt） JHM毒株以受体独立的方式感染（受体独立感染，简称RII），而从野生型wt JHM分离的突变体srr7在混合神经细胞培养物感染后24小时检测时缺乏这种活性。然而，从srr7感染的混合细胞中分离出具有RII活性的变异病毒，感染维持3天。具有RII活性的Wt JHM对小鼠具有较高的神经毒力，感染后2-3天内小鼠死亡。Srr7接种剂量越大，小鼠存活时间越长。在多种神经细胞中检测到Wt JHM抗原，这可能与小鼠大脑的RII有关。但srr7抗原分布并不广泛。这些结果表明，JHM的高毒力可能归因于RII活性。我们还试图获得对小鼠生长并显示毒力的猪冠状病毒，以观察除JHM以外的冠状病毒是否显示RII活性。将猪流行性腹泻病毒（PEDV）的Vero-cell适应型病毒经哺乳小鼠脑传代后，获得了对小鼠具有较高毒力的变异株，该变异株的细胞-细胞融合活性较原始的非适应型病毒增强。突变体S蛋白出现4个氨基酸突变，这被认为是细胞-细胞融合的关键。然而，尚不清楚S中的这些突变是否会导致更高的毒力。为了解决这一问题，我们必须建立PEDV的反向遗传系统

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