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Abstract：
Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.
摘要：
冠状病毒（CoV）反复从野生动物宿主和受感染的人类和家畜动物中出现，导致具有显著发病率和死亡率的流行病。CoV感染各种器官,包括呼吸系统和肠道系统，例如新出现的严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）。导致肠道疾病发展的病毒因子群仍然难以捉摸。这里,我们研究了CoV干扰素拮抗剂对肠道发病机制的贡献。利用肠道冠状病毒的感染性克隆，猪流行性腹泻病毒(icPEDV),我们产生了干扰素拮抗剂非结构蛋白1（nsp1）的非活性形式的病毒，nsp15和nsp16单独或组合到一个病毒称为icPEDV-mut4。与野生型icPEDV感染相比，干扰素反应性PK1细胞被这些病毒感染并产生更高水平的干扰素反应。icPEDV-mut4引起强烈的干扰素反应，并在PK1细胞中的复制严重受损。为了评估病毒的发病机制，用icPEDV或icPEDV-mut4感染仔猪。虽然感染icPEDV的仔猪表现出临床疾病，感染icPEDV-mut4的仔猪在感染后第2天没有临床症状，肠道病理正常。icPEDV-mut4在肠道中复制，通过检测粪便拭子中的病毒RNA，序列分析记录了输入菌株的遗传稳定性。重要的是,icPEDV-mut4感染引起IgG和对PEDV的中和抗体应答。这些结果鉴定nsp1、nsp15和nsp16为有助于猪中PEDV诱导的腹泻发展的毒力因子。灭活这些CoV干扰素拮抗剂是产生候选疫苗以预防疾病和肠道CoV传播的合理方法。包括SARS-CoV-2.重要性新兴冠状病毒，包括SARS-CoV-2和猪CoV,可以感染肠细胞，引起腹泻,被扔在粪便里.需要新的方法来了解肠道发病机理并开发疫苗和疗法以防止这些病毒的传播。这里,我们利用了肠道病毒的反向遗传系统，猪流行性腹泻病毒(PEDV),并概述了一种基因灭活高度保守的病毒因子的方法，这些病毒因子已知会限制宿主对感染的先天免疫反应。我们的报告显示，用三种病毒干扰素拮抗剂的非活性形式产生PEDV，非结构蛋白1、15和16可产生高度减毒的病毒，该病毒不会在动物中引起腹泻，并在病毒感染的动物中引起中和抗体反应。该策略可用于产生可预防肠道CoV疾病和粪便传播的减毒活疫苗候选物。包括SARS-CoV-2.
