Study On Correlation Between PRRSV Nsp4 And Replication Of PRRSV

Porcine reproductive and respiratory syndrome(PRRS) is an infectious disease which is caused by Porcine reproductive and respiratory syndrome virus(PRRSV), and damage sows and sucking pigs seriously. PRRSV is a member of the Arteriviridae family of enveloped viruses with positive-sense (+) RNA genome. The genome contains 9 open reading frames(ORFs). Translation of ORF1 (ORF1a and ORF1b) yield polyproteins pp1a and pp1ab, involving in the replication and transcription of virus. ORF2a,ORF3-5 code glycosylated membrane proteins GP2a,GP3,GP4 and GP5, ORF2b and ORF6 code non-glycosylated membrane protein E and M, ORF7 codes the nucleocapsid protein(N). The polyproteins pp1a and pp1ab are processed by viral proteases to release 14 non-structural proteins(Nsp). Nsp4 is characterized by 3C-like serine protease and utilizes conserved His39-Asp64-Ser118 catalytic triad, which is responsible for cleaving pp1a and pp1a into several Nsps(Nsp3-12). Those Nsps compose the replicase and transcriptase complex(RTC) to complish the replication and proliferation of virus.In order to study the enzyme kinetics of different PRRSV strains, the Nsp4 gene of HuN4 strain and CH-1a strain were cloned into the pGEX-6P-1 vector. SDS-PAGE analysis showed the restructured plasmids HuN4-pGEX and CH-1a-pGEX were expressed correctly. The soluble fusion proteins were purified by Glutathione-Sepharose 4B. Western Blot indicated the purified proteins could be specifically recognized by PRRSV positive serum, suggesting the expressed proteins have a good immunogenicity. To research the enzyme kinetics, the Nsp4 was reacted with synthetic substrate peptide Dabcyl-KTAYFQLE↓GRHFE-Edans (including connected sites of Nsp11-12) and the fluorescence values were detected. The results showed the expressed Nsp4 could cleave the peptide, Km and Kcat of HuN4 Nsp4 were 0.1431±0.009718 mM and 0.2126±0.000569 min^-1 respectively; Km and Kcat of CH-1a Nsp4 were 0.1170±0.009223 mM and Kcat=0.3323±0.009672 min^-1 respectively, suggesting catalytic efficiency of CH-1a was significant higher than HuN4'in vitro(P<0.05).In order to confirm the gene expression of PRRSV Nsp4, a fluorogenic quantitative RT-PCR assay of the PRRSV was established using specific primers and Taq Man fluorogenetic probe based on the Nsp4 gene of PRRSV. The assay had a good linear range of the 10¹ï½ž10⁸copies/μL and could detect 10 copies of cDNA. It showed no cross reaction with classical swine fever virus (CSFV), porcine parvovirus (PPV), porcine circovirus (PCV) and pseudorabies virus (PRV). The coefficient of variation (CV) of three different samples of PRRSV was 0.81% to 1.36% in intra-assay and 1.77% to 2.56% in inter-assay respectively. The method had the advantages of specificity sensibility and stability.The Marc-145 cell and PAM were incubated by PRRSV HuN4 strains and CH-1a strains. Its Nsp4 mRNA copies of different times were detected. The results showed at 12 h to 96 h, Nsp4 mRNA copies of HuN4 were 1.06¹.14 and 1.21¹.36 times more than CH-1a'in Marc-145 cell and PAM respectively, indicating Nsp4 mRNA copies of HuN4 were significant higher than CH-1a'at 12 h to 96 h after the cells were incubated with PRRSV(P<0.01). Those studies conclude it is because the level of HuN4 Nsp4 mRNA is higher than CH-1a Nsp4'catalytic efficiency that leads to quicker proliferation of PRRSV HuN4 strains.