Study Of The Infectious Clone And Replicon Vector Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a serious infectious disease of swine. It is characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the United States in 1987, PRRS has already been one of the most economically important diseases of the global swine industry. PRRSV was first isolated in 1996 in our country. Since June 2006, highly pathogenic PRRSV has been outbreaked in South China's Hubei, Hunan, Jiangxi and Anhui provinces; At least more than 200 million pigs have been infected and more than 40 million pigs died from this disease, resulted in serious economic losses. It was confirmed that the highly pathogenic PRRSV mutants were the major pathogens of the"high fever"syndrome by lots of research. Positive-strand RNA virus reverse genetics system is an useful tool in viral replication, pathogenesis research, screening antiviral drugs and new vaccines, vector-oriented areas such as the preparation. In this study, we concntrates on genomic clone, sequencing, phylogenetic analysis, infectious clone and replicon vector of prrsv. The main research was described as follows.1. Genomic clone, sequencing and phylogenetic analysis of reproductive and respiratory syndrome virus (PRRSV) Clone20 strainTo porcine reproductive and respiratory syndrome virus (PRRSV) Clone20 strain for the materials, reference to GeneBank PRRSV VR2332 strain (AY150564) gene sequences, we designed four primers points by RT-PCR amplification of the PRRSV Clone20 strain genome. Obtained cDNA sequence was cloned into the pMD18-T vector for sequencing. Sequencing results showed that: whole-genome overlapping cDNA fragments A, B, C and D followed by the size of 3920bp, 5715bp, 3783bp and 3323bp is correct by sequencing analysis, and complete genome sequence is obtained by splicing sequence of four overlapping cDNA fragments using the DNASTAR software. It showed that the genome of PRRSV Clone20 was 15412bp in length (non-poly (A) tail) and had been submitted to GenBank, accession number for the sequence: FJ899592.Genetic analysis of the phylogenetic tree shows that the epidemic strain in China can be divided into two subgroups: CH-1a JXA1, HUN4 as the representative of the highly pathogenic strain belonging to subgroup 1; VR2332 represented containing a small amount of strains isolated belong to subgroup 2. Clone20 of this study are American-type strain in the phylogenetic tree Clone20 and CC-1, MLV, and BJ-4 strain in the same small branch, with the American type reference strains (VR-2332 strain) belong to subgroup 2. After 25 PRRSV sequence homology analysis was carry out by using ClustalX2 software, the sequence similarity analysis and BOOTSCAN analysis was further run by Simplot software; when the analysis was carried out for strain HB-1, two different recombination points (detected at positions 12665, and 14105 of alignment) and two putative parentallike strains (CH-1a and JX143) were observed. Homologous recombination generates genetic variation and is considered playing an important role in evolution of porcine reproductive and respiratory syndrome virus (PRRSV).2. Construction of infectious clone of reproductive and respiratory syndrome virus (PRRSV) Clone20 strainIn this study, carried out using high-fidelity DNA polymerase gene amplification, each fragment of the positive clones were sequenced for 3 times; and sequencing included the same sequence in GenBank for comparison, as far as possible to minimize the introduction of mutations in PCR to obtain a more reliable cDNA sequence of the genome. 4 gene fragment will be A, B, C, D sequence was cloned into low copy plasmid in pOK-12, at the same time in the whole genome 5 'end of the introduction of CMV promoter sequence, in the 3' end poly (A), respectively, after the introduction of ribozyme HDV sequence and the BGH termination sequence, the final full-length cDNA clone strain pOK-Clone20. The plasmid with the quality pOK-Clone20 Liposome transfection BHK-21 cells, 24 hours after transfection the supernatant against Marc-145 cells cultured for 3 generations, spread to the third generation of significant cytopathic; extended by RT-PCR by sgmRNA7, size and sequence in line with expectations, confirming there was the recovery of infectious virus, indicating a successful build with full-length infectious cDNA clone of PRRSV. For future research in the cDNA level of the PRRSV viral replication, pathogenesis research, screening antiviral drugs and new vaccines, vector-oriented aspects of the preparation, such as the provision of technical platform.3. Porcine reproductive and respiratory syndrome virus (PRRSV) replicon vector researchAs a reporter gene construct containing the porcine reproductive and respiratory syndrome virus subgenomic replication system, we first infected in the cloning strategy to build on the basis of the method using PCR fragments C and D fragment containing the ORF2-ORF6 deletion of the gene , to retain the structural gene and the porcine reproductive and respiratory syndrome virus replication would like to the N protein sequences, including upstream and downstream TRS7 the sequence of the 3 'end non-coding cis-activation sequence, while the use of PCR method to introduce the reporter gene EGFP. In addition, as the construction of infectious clone, in replicon 5 'end of the introduction of CMV promoter sequence, in the 3' end poly (A), respectively, after the introduction of HDV ribozyme sequence and the BGH termination sequence. Then build a strategy for cloning infectious sequence of each fragment was cloned into low copy plasmid pOK-12, the final report containing EGFP gene plasmid replication subsystem pOK-Clone20-rep. The plasmid pOK-Clone20-rep with body fat mass transfected BHK-21 cells, by observing EGFP fluorescence, flow analysis and RT-PCR, the stability of test and other methods of identification replicon. The results showed that, pOK-Clone20-rep in BHK-21 cells can be stably expressing EGFP, in BHK-21 cells and N sgmRNA detected EGFP sgmRNA, flow analysis showed that in pOK-Clone20-rep-transfected BHK -21 cells in the issue of 9.64% fluorescent cells. The results show that, PRRSV replicon in BHK-21 cells to normal reproduction and self-expression. Built by the Institute to purchase a reporter gene containing the PRRSV subgenomic replication system in BHK-21 cells were the expression of a stable, successful replicon construct its further development into a vaccine vector for the study of virus replication, pathogenicity mechanisms and antiviral drugs laid the foundation for screening.In order to further explore the PRRSV replicon vector as the possibility of vaccine to mice as a model animal, the replicon vector pOK-Clone20-rep to mice immunized with different doses (100μg and 10μg). The experimental results show that, regardless of humoral immunity or cellular immunity, replicon vector can be a higher level of humoral immunity and cellular immunity, has a good immune effect. Replicon at the same time also on the N protein can stimulate the humoral immunity, indicating that PRRSV replicon vector can be used as a new type of multi-gene expression vector vaccines candidate.