A broad species tropism in vitro and a mouse model for Swine Acute Severe Diarrhea Syndrome Coronavirus infection

【作者】 杨永乐； 覃盼； 王斌； 朱书； 黄耀伟；

【Author】 Yong-Le Yang;Pan Qin;Bin Wang;Shu Zhu;Yao-Wei Huang;Department of Veterinary Medicine, Zhejiang University;

【机构】 Department of Veterinary Medicine, Zhejiang University；

【摘要】 Background:In February 2017,outbreaks of severe diarrhea of neonatal piglets in Guangdong,China resulted in isolation and discovery of a novel swine enteric alphacoronavirus（SeACoV）derived from the bat CoV HKU2 by our lab（Vet Microbiol,2017,211:15-21）.SeACoV was later referred as swine acute severe diarrhea syndrome（SADS）CoV by the other groups（Nature,2018,556:255-258）,although experimental infection of SADS-CoV（SeACoV）in pigs did not induce significantly clinical signs.It is believed that this HKU2-like CoV had’jumped’ from horseshoe bats to pig herds through cross-species transmission.AIM:In this manuscript,we compared differential cell lines susceptibility to the SADS-CoV providing the growth characteristics of this new virus.And we are trying to establish SADS-CoV infection in mice model for studying this emerging animal virus.Methods and Result:In the present study,we first demonstrated that SADS-CoV possess a broad range of species tropism as it is able to infect diverse cell lines from different species including bat,mouse,rat,hamster,pig,chicken,primates and human beings.With the observation that SADSCoV is capable of infecting various rodent cell lines,we next questioned whether mouse could be infected by SADS-CoV or not.Groups of C57 BL/6 mice were inoculated orally or intraperitoneally（i.p.）with 510⁵ TCID₅₀ units of SADS-CoV and tissue viral burdens and fecal viral shedding were determined by qRT-PCR.At 1 dpi,SADS-CoV replicates modestly in the intestinal tissues and viral titers declined at 3 dpi and completely cleared out by 7 dpi in both inoculation routes.The animals were more susceptible to i.p.infection because virus has better replication in the stomach,ileum,colon and induces delayed clearance in the stomach and cecum.More interestingly,SADS-CoV inoculation rendered persistent infection in the spleen of infected mice.We were able to localize the viral double-stranded RNA in the mantle zone lymphocytes on the edge of B cell rich germinal center using immunohistochemistry.Additionally,SADS-CoV N protein could be detected in almost 2%of total splenocytes at 3 dpi with intracellular staining by flowcytometry.Furthermore,splenocytes extracted from naive mice were infected with SADS-CoV ex vivo,displaying positive viral N protein expression in the cytoplasm of infected cells.The SADS-CoV genomic RNA peaked at 24 hpi and plateaued till 72 hpi.Conclusions:Our results suggest the possibility of a broad cross-species transmission of SADSCoV.The persistent infection of SADS-CoV in the spleen of experimentally infected mouse also implied the potential role of rodents as the intermediate host in the field.Furthermore,the utility of a widely accessible,genetically manipulable and reproducible murine model for the pathogenesis and immunology study of SADS-CoV is obviously advantageous.更多还原

【Abstract】 Background:In February 2017,outbreaks of severe diarrhea of neonatal piglets in Guangdong,China resulted in isolation and discovery of a novel swine enteric alphacoronavirus（SeACoV）derived from the bat CoV HKU2 by our lab（Vet Microbiol,2017,211:15-21）.SeACoV was later referred as swine acute severe diarrhea syndrome（SADS）CoV by the other groups（Nature,2018,556:255-258）,although experimental infection of SADS-CoV（SeACoV）in pigs did not induce significantly clinical signs.It is believed that this HKU2-like CoV had’jumped’ from horseshoe bats to pig herds through cross-species transmission.AIM:In this manuscript,we compared differential cell lines susceptibility to the SADS-CoV providing the growth characteristics of this new virus.And we are trying to establish SADS-CoV infection in mice model for studying this emerging animal virus.Methods and Result:In the present study,we first demonstrated that SADS-CoV possess a broad range of species tropism as it is able to infect diverse cell lines from different species including bat,mouse,rat,hamster,pig,chicken,primates and human beings.With the observation that SADSCoV is capable of infecting various rodent cell lines,we next questioned whether mouse could be infected by SADS-CoV or not.Groups of C57 BL/6 mice were inoculated orally or intraperitoneally（i.p.）with 510⁵ TCID₅₀ units of SADS-CoV and tissue viral burdens and fecal viral shedding were determined by qRT-PCR.At 1 dpi,SADS-CoV replicates modestly in the intestinal tissues and viral titers declined at 3 dpi and completely cleared out by 7 dpi in both inoculation routes.The animals were more susceptible to i.p.infection because virus has better replication in the stomach,ileum,colon and induces delayed clearance in the stomach and cecum.More interestingly,SADS-CoV inoculation rendered persistent infection in the spleen of infected mice.We were able to localize the viral double-stranded RNA in the mantle zone lymphocytes on the edge of B cell rich germinal center using immunohistochemistry.Additionally,SADS-CoV N protein could be detected in almost 2%of total splenocytes at 3 dpi with intracellular staining by flowcytometry.Furthermore,splenocytes extracted from naive mice were infected with SADS-CoV ex vivo,displaying positive viral N protein expression in the cytoplasm of infected cells.The SADS-CoV genomic RNA peaked at 24 hpi and plateaued till 72 hpi.Conclusions:Our results suggest the possibility of a broad cross-species transmission of SADSCoV.The persistent infection of SADS-CoV in the spleen of experimentally infected mouse also implied the potential role of rodents as the intermediate host in the field.Furthermore,the utility of a widely accessible,genetically manipulable and reproducible murine model for the pathogenesis and immunology study of SADS-CoV is obviously advantageous.更多还原