Study On Genetic Evolution Of HP-PRRSV Epidemic Strains In Guangxi Province

To investigate the variation and genetic characteristics of porcine reproductive and respiratory syndrome virus (PRRSV) strains in Guangxi, tissues from suspected dead pigs in the field were collected, freezed-and-thawed and inoculated into Marc-145cells to isolate PRRSV. After appearing cytopathic effect (CPE), the supernatant was used to identify PRRSV by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunoflueorescent assay (IFA). The results showed that three PRRSV strains were isolated and named as GX1001, GX1002and GX1003, respectively. The full genome sequence of three PRRSV strains was acquired through using RT-PCR amplification, clone, sequencing, and splicing technologies. The results showed that the full genome sequences of GX1001, GX1002and GX1003strains were15329,15318and15320nucleotides (nt) in length except the poly (A) tail, respectively. Full-length sequence analysis revealed that GX1001, GX1002and GX1003strains shared99.3%～99.4%nucleotide homology and99.5%～99.7%amino acid homology with each other,84.9%～99.5%nucleotide homology and74.9%～99.5%amino acid homology with North American (NA) type isolates,61.5%～61.9%nucleotide homology and51.0%～51.5%amino acid homology with European (EU) type isolates, respectively. Sequence analysis demonstrated that GX1001, GX1002and GX1003strains had discontinuous deletion of30amino acid (aa) in481aa and533～561aa of NSP2region compared with that of the NA prototype VR-2332, which was considered as the genetic marker of highly pathogenic PRRSV (HP-PRRSV), and had new multiple genomic variations such as mutation, deletion and insertion distributing in different regions. Phylogenetic analysis showed that all the NA type isolates could be divided into four subgroups. GX1001, GX1002and GX1003strains belonged to the4subgroup representing by the highly pathogenic JXA1strain. The results indicated that GX1001, GX1002and GX1003strains from Guangxi province belonged to HP-PRRSV with new characteristic variations in different regions. This study provided valuable genomic data for further surveillance of HP-PRRSV.To investigate the genetic evolution of HP-PRRSV strains in the field under immune pressure, serum and tissue samples were collected quarterly from four scale pig farms in Guangxi during2011-2012, of which two farms were vaccinated with PRRSV vaccines and the other tow farms were not vaccinated with PRRSV vaccines, to detect PRRSV antibody and pathogens. The results showed that the PRRSV antibody levels (s/p mean value) in the four pig farms were over the threshold of positive values each quarter, and the positive rates of serum samples were45.7%-96.7%. Through RT-PCR and cloning technology,91Nsp2,98ORF5,98ORF6and98ORF7gene sequences were obtained, respectively. Sequence analysis revealed that all the gene sequences belonged to North American (NA) type and the Nsp2, ORF5, ORF6and ORF7gene sequences shared90.1%-100%nucleotide homology and91.6%-100%amino acid homology,83.3%-100%nucleotide homology and81.1%-100%amino acid homology,96.6%-100%nucleotide homology and96.0%-100%amino acid homology,86.0%-100%nucleotide homology and87.9%-100%amino acid homology with each other, respectively. The mean substitution rates of the Nsp2, ORF5, ORF6and ORF7genes of the epidemic strains in four farms were4.30×10-4,4.09×10-5,2.65×10-6and4.89×10-6substitutions/site/day, respectively, indicating that Nsp2gene is the most easily mutated, followed by ORF5gene, then by ORF7gene, and ORF6gene is the most stable among the four genes. The mean substitution rates of Nsp2, ORF5, ORF6and ORF7gene of PRRSV strains in the non-vaccinated pig farms were4.18×10-4,3.98×10-5,2.59×10-6and4.84×10-6substitutions/site/day, respectively, while the mean substitution rates of Nsp2, ORF5, ORF6and ORF7gene of PRRSV strains in the vaccinated pig farms were4.42X10"4,4.20×10-5,2.71×10-6and4.93×10-6substitutions/site/day, respectively, indicating that the mean substitution rates of Nsp2, ORF5, ORF6and ORF7gene of PRRSV strains in the vacinizaed pig farms were higher than those in the non-vaccinated pig farms. We analyzed the genetic evolution of HP-PRRSV epidemic strains in the field under immune pressure, which could supply basic data and theoretical basis for HP-PRRSV molecular epidemiology and prevention and control of this disease as well.