Construction Of Infectious Clones Of TGEV/FCoV And The Effect Of Spike Protein In Viral Pathogenictiy

Coronavirus is the largest single-stranded,positive-sense RNA virus in the genome.Coronaviruses are closely related to human and animal life activities,especially some animals that are closely related to human life in nature.With the global pandemic of human novel coronavirus,more and more people know about coronavirus,but the pathogenic mechanism of coronavirus still needs further study.Spike(S)protein of coronavirus can recognize and infect host cells,and it is also the main target of host to produce neutralizing antibodies.However,the influence of S protein on the pathogenicity of coronaviruses is still unclear,and whether other genes other than S protein affect the virulence of the virus needs to be verified urgently.Coronavirus reverse genetics system is an important molecular biology method for the study of coronavirus infections.Through this method,RNA at the coronavirus genome level can be manipulated to quickly carry out antiviral drug screening,virulence gene identification,pathogenic mechanism and the other related research.In this project,TGEV and FCoV in ?-coronavirus are the research objects.Through the reverse genetics of the coronavirus and animal experiments,the pathogenic mechanism of the?-coronavirus is initially explored.The specific content is as follows:1.The construction of TGEV infectious clone and the study of the influence of the N-terminus of TGEV S gene on virus virulence.We firstly used the bacterial artificial chromosome(BAC)as a vector to construct the TGEV full-length genomic cDNA(ORF3 region inserted into the EGFP gene for later observation)and transcriptional regulatory elements into the BAC vector,and successfully constructed the TGEV infectious clone p BAC-TGEV-GFP(corresponding to the recombinant virus TGEV-GFP).In order to research the effect of the N terminal domain(NTD)of the TGEV S gene on the virulence of the virus,we compared the S gene of porcine respiratory coronavirus(PRCV)and TGEV,and found that the PRCV S gene is missing 224 amino acids when compared with TGEV S gene N-terminus;therefore,we constructed an infectious clone p BAC-TGEV-GFP-?S?NTD(corresponding to the recombinant virus TGEV-GFP-?S?NTD)with 224 amino acids missing from the N-terminus of the TGEV S gene.Newborn piglets were infected with recombinant viruses TGEV-GFP and TGEV-GFP-?S?NTD,and it was found that TGEV-GFP-?S?NTD lacking the N-terminus of the S gene can also kill the piglets;above experimental results indicate that the N-terminus of the TGEV S gene is not determinance for TGEV virulence.2.Construction of full-length cDNA of wild-type and cell-adapted FCoV strains and corresponding viruses rescue.We firstly collected samples the from disease cat that was naturally infected and diagnosed as FIP,extracted the FIPV RNA from samples,determined the viral genome sequence,and finally obtained the complete genome sequence of a wild-type FIPV strain(named as QS);At the same time,we also determined the complete genome sequence of the cell-adapted FCoV79-1146 strain(named as 79-1146?CA).Using BAC as a vector,we constructed full-length cDNA clones of QS and 79-1146?CA strains(corresponding to plasmids p BAC-QS and p BAC-79-1146?CA,respectively).The full-length cDNA clone was transfected into CRFK cells for virus rescue.It was found that only p BAC-79-1146?CA could be rescued to produce the infectious recombinant virus(the recombinant virus was named as r79-1146?CA).To further rescue the QS strain,we replaced the extracellular domain of the S gene of 79-1146?CA into p BAC-QS(the mutant plasmid was named as p BAC-QS-79),transfected p BAC-QS-79 into CRFK cells,and found the S gene related chimeric recombinant virus rQS-79(corresponding to plasmid p BAC-QS-79)can proliferate well in cells.3.The establishment of an animal model of FCoV oral infection and the effect of FCoV S gene on virus virulence.The oral infection animal experiment of r79-1146?CA and rQS-79 indicated that rQS-79 could effectively infect and maintain high pathogenicity.rQS-79 can cause the diseased cats to show typical FIP signs and be 100% lethal;while r79-1146?CA only caused slight fever in individual cats,and there were no obvious clinical signs.The study obtained 100% fatal feline coronavirus for the first time,which also shows that the FCoV type II S gene does not determine the virulence of the virus.4.The application of an animal model of oral infection with the virulent and attenuated FCoV strains.We further infected cats immunized with r79-1146?CA with the rQS-79 strain,and found that although high-titer neutralizing antibodies can relieve the cat's FIP signs and prolong the life time,it does not produce immune protection.However,after we used the drug candidate GS-441524 to treat cats infected with rQS-79,we found that the signs of the sick cats quickly alleviated.The study shows that the current type II FIPV neutralizing antibody is not effective in the protection of FIP,and antiviral drugs can be considered for the treatment of FIP.In conclusion,this project takes TGEV,a typical representative of ?-coronavirus,as the research object.Through reverse genetics and animal experiments,it has been shown that N-terminal deletion of the S gene cannot completely attenuate the virus.At the same time,taking FCoV as the research object,through reverse genetics and recombinant virus(r79-1146?CA,rQS-79)oral infection animal experiments,it was found that the S gene of FCoV does not determine the virus virulence,and it was the first time to obtain virulent and attenuated FCoV infectious clone.The construction of infectious clones of TGEV and FCoV and animal infection models provide a solid technical platform for coronavirus antiviral drug screening,vaccine development,and pathogenic mechanism research.