Construction Of Infectious Clone Of Porcine Transmissible Gastroenteritis Virus And Study On The Location And Function Of Accessory Protein 3 (ORF3)

Porcine transmissible gastroenteritis virus(TGEV)belongs to the Nidoviridae order,coronaviridae,α Coronavirus which mainly infects newborn piglets,causing diarrhea,vomiting,dehydration,and death in piglets.Clinically,it is often co-infected with other diarrhea related viruses such as porcine epidemic diarrhea virus(PEDV)and porcine rotavirus(PRo V),causing huge economic losses to the world’s pig industry.In addition to encoding structural proteins and non-structural proteins involved in viral genome transcription and replication,coronaviruses also encode a series of unique accessory proteins.Current research indicates that most accessory proteins are not essential for virus replication,but accessory proteins may affect the pathogenicity of coronavirus.TGEV has been identified to encode two types of accessory protein 3(ORF3)and accessory protein 7(ORF7).Existing studies have shown that ORF3 is involved in the regulation of the host’s natural immune response,but its role in virus replication and pathogenesis in vitro is still unclear.The reverse genetic system can provide material basis and technical support for the research of viral protein function,the identification of virulence factor,the development of new vaccines and the screening of antiviral drugs.Based on the above situation,this study aims to establish a reverse genetic system forTGEV TH-98 strain;explore the role of TGEV ORF3 in virus replication in vitro;determine whether ORF3 is a structural protein of TGEV and whether it can induce immune response in the body and lay the foundation for constructing recombinant virus vaccines using TGEV as the skeleton and ORF3 as the insertion site.The main research results are as follows:1.Successfully established a reverse genetic operation system forTGEV TH-98 strainThe full length genome of TGEV TH-98 strain was divided into six(A-F)segments with homologous arms,and the segments were cloned in p Blunt vectors.Subsequently,TGEV full-length cDNA clones were obtained in the A-F-D-E-B-C construction orders.TGEV full-length cDNA clone were transfected to ST cells to rescue TGEV.Comparative analysis of the rescue virus rTH-98 and the TGEV parent virus TH-98 showed that the entire genome sequences of both viruses were identical except for their molecular markers,which could produce the same cytopathic effects on ST cells.Western Blot and indirect immunofluorescence assay(IFA)could detect the expression of TGEV N protein.The biological characteristics of rescue virus rTH-98 were studied,and it was found that the size of the plaque produced by its parent virus was similar to TH-98 on ST cells,and the growth kinetics were similar in ST cells and IPEC-J2 cells.The characteristics of classical coronavirus were observed under electron microscopy.The above results indicate that we have successfully established a TGEV reverse genetic system,laying a material foundation for subsequent research.2.Rescue the recombinant virus rTH-98-Δ3-COE-HA and revealed the role of TGEV ORF3 in virus replicationTo investigate whether the absence of ORF3 affects virus replication,we replaced the ORF3 gene in the TGEV TH-98 genome with the PEDV COE gene and introduced a HA tag at its C-terminal,rescuing the recombinant virus rTH-98-Δ3-COE-HA.The analysis of the biological characteristics of the recombinant virus rTH-98-Δ3-COE-HA indicates that the foreign gene in the genome of the recombinant virus can be stably passage and effectively expressed,indicating that the recombinant virus has been successfully rescued and TGEV can still replicate in vitro after deletion of accessory protein 3,indicating that ORF3 is not necessary forTGEV replication.The biological characteristics of the recombinant virus after infecting ST cells,such as plaque size,viral titer,and growth curve,are similar to those of its parent strain,but the replication efficiency of the recombinant virus rTH-98-Δ3-COE-HA is significantly reduced,indicating that TGEV ORF3 may play a key role in promoting viral replication.3.Explored the location of ORF3 in TGEVIn order to further analyze whetherTGEV ORF3 exists on mature viral particles,Western Blot analysis was performed on the purified recombinant viral particles,and no COE-HA protein was detected in the purified viral particles.It was preliminarily proved that ORF3 does not exist in mature viral particles.Subsequently,the purified recombinant virus was subcutaneously immunized into mice,and the immunized mouse serum was collected.The specific Ig G of the mouse anti TGEV N protein could be detected,but the specific Ig G of the mouse anti COE protein could not be detected,further proving that the ORF3 was not present in mature viral particles,indicating that the ORF3 was a non-structural protein.4.Explored the level of expression of inflammatory factors in IPEC-J2 cells induced by recombinant virusesIn order to study the expression level of inflammatory factors in recombinant virus rTH-98-Δ3-COEHA infected IPEC-J2 cells,the expression level of inflammatory factors in the cell samples were detected by RT-q PCR.The results showed that the recombinant virus could induce the expression of inflammatory cytokines IL-1β,TNF-α,IL-6 and IL-8 in IPEC-J2 cells,but the expression level was significantly lower than that of the parental virus TH-98 and parental rescue virus rTH-98.This result suggests that TGEV ORF3 may play a key role in inducing host cell immune response.5.Immunogenicity of recombinant virus expressing exogenous proteins in miceTo determine whether the recombinant virus rTH-98-Δ3-COE-HA expresses exogenous proteins with immunogenicity,mice were subcutaneously immunized with recombinant virus culture medium.After immunization,mouse serum was collected,and TGEV N protein and PEDV COE protein were used as coating antigens.Indirect ELISA was used to detect the expression level of specific Ig G in mouse serum.The results showed that the levels of anti-TGEV specific Ig G and anti-PEDV COE specific Ig G in mice were significantly increased.The titer of TGEV neutralizing antibody in serum is 1:16.The neutralizing antibody titer of PEDV is 1:4.The above results indicate that the recombinant virus can induce a specific immune response in the body targeting the expressed foreign protein.In summary,this study successfully constructed a reverse genetic operation system forTGEV TH-98 strain and rescued the recombinant virus rTH-98 based on this system-Δ3-COE-HA.It is proved that ORF3 is a non-structural protein and does not exist in mature virion.This study demonstrates that ORF3 is not necessary forTGEV replication,but it promotes TGEV replication.Compared to the parent virus,the expression level of inflammatory factors in IPEC-J2 cells of the recombinant virus is significantly reduced,and the expressed exogenous protein can induce specific immune response in immunized mice.The results of this study provide important clues to reveal the role of ORF3 in virus replication and pathogenesis,and lay the theoretical and material foundation for the construction of recombinant viral multiplex vaccines.