Study On The Cell Receptor And The Virulence Factors Of Porcine Enteric Coronavirus

Coronavirus(CoV)has a wide range of natural hosts,including humans,livestock,poultry and other vertebrates,which has the ability of interspecies transmission.Porcine enteric CoVs mainly include transmissible gastroenteritis virus(TGEV),porcine epidemic diarrhea virus(PEDV),swine enteric alphacoronavirus(SeACoV)from the alphacoronavirus and porcine deltacoronavirus(PDCoV)from the deltacoronavirus.At present,PEDV and PDCoV are the most pathogenic porcine enteric Co Vs in the swine industry.This study focused on the virus identification from the porcine diarrhea samples,virus isolation and the cell receptor usage for virus entry,as well as establishing reverse genetic system to explore the key pathogenic genes of the virus.This study had a comprehensive understanding of the invasion and the pathogenic mechanism of the porcine enteric CoVs,which would provide references for the disease prevention and vaccine development.In this study,the pathogen of clinical diarrhea samples from a pig farm in Zhejiang province was identified.The results showed that only the nucleic acid of PDCoV was positive in the samples while TGEV,PEDV and SeACoV were all negative detected by RT-PCR with specific detection primers for porcine intestinal CoVs.Subsequently,the virus was successfully isolated from the diarrhea samples by passaging on the LLC-PK1 cells.The virus was confirmed as PDCoV by complete genome sequencing and alignment,as well as IFA identification with PDCoV structural protein antibodies.The phylogenetic analysis indicated that the PDCoV obtained in this study was a Chinese strain.The S gene has the highest homology with the strain of CH-WH-2017(MK040451).Whole genome recombination analysis revealed that the non-structural protein of this strain may come from the recombination of the strains of CHJXNI2/15(KR131621.1)and CHN-HG-2017(MF095123.1).What's more,this study investigated the cell receptors for PDCoV entry.The S1 domain of PDCoV S protein could bind to PDCoV susceptible cell membranes through porcine aminopeptidase N(pAPN),and soluble pAPN protein could block this binding.PDCoV S1 protein could interact with pAPN protein confirmed by in vivo and in vitro experiments.The exogenous expression of pAPN protein could mediate the invasion of PDCoV into non-susceptible cells,and the progeny viruses were also infective.The PDCoV infection was reduced when silencing pAPN,indicating that pAPN can act as a receptor for PDCoV invasion.The loss of pAPN protein almost but not completely block the PDCoV infection,indicating that there may be other receptor proteins in addition to pAPN for PDCoV infection.In order to better understand the pathogenicity of porcine enteric CoVs,this study tried to establish a reverse genetic system of PDCoV virus.We successfully constructed a PDCoV infectious clone plasmid by amplifying the full-length genome of PDCoV in segments and inserting the full-length virus genome into the vector by homologous recombination using the bacterial artificial chromosome(BAC)as a vector.By transfecting the cells with the plasmid,we detected the expression of viral structural proteins both in the transfected cells and the first-generation infected cells.This study successfully established the PDCoV reverse genetic system.Using the same method of the infectious clone construction and virus rescue,this study successfully established the reverse genetic system of PEDV.The gene of PEDV ORF3 and the 7 amino acids at the C-terminal of the S protein were both or separately deleted with this system.The pathogenicity changes of these mutant viruses were studied through the piglet challenge assay.The results showed that the deletion of ORF3 and the 7 amino acids at the C-terminal of S protein could attenuate the infection of PEDV to some extent,and the attenuation effect was more significant when deleting both genes.In addition,we constructed a chimeric PEDV which the S gene of high-virulent genogroup ? was replaced with the low-virulent genogroup ?.Animal experiments indicated that PEDV S gene is a key gene that affects the virus virulence and other genes except for S also play a role in PEDV virulence.In summary,we systematically studied the cell invasion and pathogenic mechanism of porcine enteric Co Vs in this study.We successfully isolated a porcine intestinal CoV-PDCoV and identified its entry receptor.We also confirmed that the S gene is the main factor affecting the PEDV virulence based on the reverse genetic system.