Construction Of A CDNA Clone Of Porcine Reproductive And Respiratory Syndrome Virus And Mechanisms Of Its Interference With IFN-β Expression In MARC-145Cells

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease that has been affecting the swine industry worldwide and is characterized by both reproductive failure in pregnant sows and respiratory disease in nursery. PRRSV, a single-stranded positive sense RNA virus that causes PRRS, is classified in the family arteriviridae, genus arteriviridae along with equine arteritis virus (EAV), laetate dehydrogenase-elevating virus (LDV) of mice and simian hemorrhagic fever virus (SHFV). PRRSV genome is approximately15kb in length. The RNA viral genome is capped at the5’terminus and polyadenylated at the3’ terminus, and encodes12non-structural proteins (nsps) and6structural proteins. There are two untranslated regions (UTRs) located at the5’and3’ ends of the genome. The virus is. genetically, antigenically and pathogenically generogenous and PRRSV isolates are divided into North America type (represented by the strain VR2332) and European type (represented by the strain LV). The host immune system fails reportedly to mount effective innate immune response to PRRSV infection. Persistent infection is commonly seen in the field.PRRSV is highly variable and easily mutated. Genomic analysis of the epidemic strains could help us understand epidemiology of PRRS and adopt better prevention measures. The complete genomic sequence of a PRRSV strain JX-07was determined as being15338nt in length that comprises eight ORFs. Its genome has91.1%nucleotide identity with the North American prototype strain VR2332and95.8%nucleotide identity with the first Chinese PRRSV strain CHI a. The5’UTR region has188nt and3’UTR region150nt. The strain JX-07is conserved with some key amino acid residues in ORF5, NSP1and NSP2and some important nucleotides in the UTRs, though different from other strains to some extent.The full-length genomic cDNA of the PRRSV strain JX-07was assembled in pUC18and placed under the T7RNA polymerase promoter. A new restriction site was introduced at nucleotide position266of the genome to serve as a genetic marker. The recombinant cDNA clone was linearized and viral RNA was synthesized in vitro and transfected into BHK21cells, and the culture supernatant recovered from BHK21cells was used to infect MARC-145cells. A putative recombinant PRRSV was obtained. To study the replication and pathogenic mechanism,Secretion of interferon (EFN) is the first line of defense of the host to virus infections as part of the innate immunity. We confirmed some previous findings in other laboratories that expression of IFN-β was subdued in PRRSV infected MARC-145cells. However, the mechanisms have not been fully elucidated. We found that PRRSV suppresses IFN-P expression by decreasing phosphorylation, dimerization and nuclear translocation of IFN regulatory factor3(IRF-3) in MARC-145cells treated with poly(I:C) a synthetic double-stranded RNA agonist. These effects were independent of virus titers. Therefore, we tend to believe that PRRSV infection might not activate the signaling pathway for activation of type IFNs, but suppressed the expression of IFN-β by partial suppression of IRF-3activity along the IFN-β signaling pathway.The findings in the present may provide some basis for further research on the mechanisms of PRRSV pathogenicity and evasion from host innate immunity.