Construction Of Reverse Genetic Systems And Reporter Viruses Of TGEV Virulent And Attenuated Strains

Transmissible gastroenteritis virus（TGEV）is one of the major pathogens causing swine viral diarrhea.The diseases caused by TGEV has high morbidity and mortality,sometimes it was up to 100%.Reverse genetic technology provides a scientific means for studying the replication characteristics,pathogenic characteristics,genetic characteristics of viruses and developing of new vaccines（chimeric and labeled vaccines）.Therefore,the construction of reverse genetic system of TGEV has important scientific significance.In order to successfully construct reverse genetic system of TGEV,the biological characteristics of Chinese classical vaccine strain H165 and newly isolated epidemic strain AH were analyzed firstly in this study.Animal pathogenicity test showed that Chinese classical vaccine strain H165 was not pathogenic to 1-week-old piglets and could not cause clinical symptoms such as diarrhea and vomiting in piglets,while its ancestral strain H16 and epidemic strain AH could cause piglets disease,with the performance of diarrhea,vomiting,severe watery diarrhea,and even cause piglets died（data not shown）;Whole genome sequence determination found that the epidemic strain AH and the Chinese classical vaccine strain H165 and its ancestors virulent strain H16 both had the genomic characteristics of Alpha-coronavirus,encoding 9 different ORFs in the order of 5’-la-lab-S-3a-3b-sM-M-N-7-3’;Molecular analysis showed that TGEV is divided into two large groups:one is similar to the Purdue strain,called Purdue like group,another was similar to the Miller strain and called Miller like group.The whole genome,structual and nonstructual genes of the classical vaccine strain H165 and its ancestral strain H16 were all in the Miller like group,indicating that they were Miller like strains.However,the S gene,N gene,1a gene of the newly isolated epidemic strain AH is similar to the Miller strain,but its E gene,M gene,1b gene,3a gene,3b gene and gene 7 were all derived from Purdue like group,suggesting that the AH strain is likely to be a recombinant strain in which the two large groups were slowly changing in the long-term evolution.The analysis of the amino acid level of the virulent and attenuated strains shows that the 3867P-R of ORF1a gene,48P-S,514L-P of S gene,23F-V,71I-L of E gene,55A-S of 3a gene,and 76T-I of gene 7 were maybe the key sites of a strain from virulent to attenuated.Analysis of strain H165,AH and PUR46-MAD strain shows that their 5’UTRs both had the same short ORF,and the pseudoknotted structure between ORF1a and ORF1b were also identical and can be formed smoothly and the-1 ribosomal fremashifing is completed.TRS_S2,TRS_3a,TRS_3b,TRS_M of Chinese classical vaccine strain H165 were not the same as those of PUR46-MAD which had been verified to be successfully worked in reverse genetic systme.To construct the full-length plasmid of TGEV based on BAC system,the BAC operating system was firstly optimized,and the methods of plasmid preparation,digestion,ligation,transformation and positive clone scanning were successfully established.Based on the analysis of the whole genome sequence of the virulent strain AH passage 5 and pBeloBAC 11 sequence,the strategy of dividing TGEV whole genome into A（5’）,B,C,D（3’）fragments was designed.Firstly,a BAC plasmid pBAC-AH-MCS containing NotI,BstBI,ClaI,NheI,XhoI,KasI,SacII polyclonal sites was constructed on the basis of pBeloBAC ll vector,and then the above four fragments were inserted into the plasmid pBAC-AH-MCS by fusion PCR,digestion and ligation,the△ClaI fragment（4418-9616）containing the toxic sequence was finally inserted,and then the full-length plasmid pBAC-TGEV（AH）-FL of the AH strain was obtained.The full-length plasmid contained the CMV promoter,HDV ribozyme（Rz）and BGH termination and polyadenylation sequences（BGH）.According to the analysis of the whole genome sequence of Chinese classical vaccine strain H165 and pBeloBAC 11 sequence,the whole genome of TGEV H165 strain was divided into 5’,A,B,C1,C2,3’fragments.Firstly,the intermidiate plasmid pBAC-TGEV（H165）5’-3’containing the 5’fragment and 3’fragment was constucted on the basis of the pBeloBAC ll vector,and the CMV promoter,HDV and BGH were also inserted.Also it contains the multiple cloning sites such as MluI,BbvCI,NheI,XhoI,KasI,SacII,which were required for subsequent cloning;And then,the four fragments of A,B,C1 and C2 were inserted into the intermediate plasmid pBAC-TGEV（H165）5’-3’;At last,the full-length plasmid pBAC-TGEV（H165）-FL of strain H165 was obtained by inserting the△ClaI fragment（4418-9616）which containing the toxic sequence.Finally,the plasmids pBAC-TGEV（AH）-△3-TRS_N-GFP and pBAC-TGEV（H165）-△3-TRS_N-GFP which deleted ORF3 gene and inserted GFP gene were also constructed.To rescue the virus successfully,two transfection rescue systems based on a cell line（BHK-pAPN）that stably expressing the TGEV receptor porcine aminopeptidase N（pAPN）were firstly established.Verified by the system for attenuated virus,the full-length plasmid pBAC-TGEV（H165）-FL of the Chinese classical vaccine strain could be successfully rescued,and the virulent strain AH could be rescued by the system for virulent virus.The 1360bp and 1131bp purpose bands could be amplified by TGEV N and M gene specific primers,separately.PK15 cells which infected with the resecued virus showed TGEV-specific cell swelling,aggregation and shedding.The indirect immunofluorescence assay（IFA）was positive based on the specific anti-TGEV-N protein monoclonal antibody.Molecular marker detection showed that the rescued strain completed the mutation of 18997T-C as expected for the H165 stain,and for the AH strain,18997T-C and 16930G-C were both completed.Electron microscopic observation showed that the rescued virus showed the specific morphological characteristics of the coronavirus,the virus particles was round or oval,the particle size is more uniform and the spikes were large and sparse and around the particles.The above showed that the Chinese classical vaccine strain H165 and virulent virus AH was both successfully rescued,named rTGEV（a）-H165 and rTGEV（v）-AH.The titer of the rescued viruses rTGEV（a）-H165 and rTGEV（v）-AH in different passage were also measured,and they were gradually improved with the increase of virus passage.The one-step growth curves of the rescued strains and their parent strains showed that they had similar growth characteristics.Repeated assay of the transfection test showed that as long as the plasmid purity(OD₂₆₀/OD₂₈₀)was between 1.8 and 2.0,and the plasmid concentration was 100-200ng/μl,the virus could be rescued and the reproducibility was very good and could reach 100%.At the end,the report virus stably expressing the GFP protein was rescued successfully,named rTGEV（H165）-GFP and rTGEV（AH）-GFP,indicating that the constructed systems could be used for expressing foreign proteins.