Analysis on Heredity and Variation of the ORF_5 Gene of Prevalence Strains Porcine Reproductive and Respiratory Syndrome Virus

[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproductive and respiratory syndrome virus published by the GenBank, the primers were designed and synthesized. ORF5 gene sequences of seven prevalence strains were amplified by RT-PCR. The sequences of ORF5 genes were analyzed by DNAStar and compared with those of ATCC VR-2332, CH-1 a, B J-4, LV-M96262 and MLV vaccine strains, phylogenetic tree among isolates was analyzed. [Result] Analysis of nucleotide sequence showed that the homology was 88.1% - 98.8%, 89.9% -95.2%, 85.6% -98.7% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, BJ-4, and the homology was 54.7% -56.9% between ORF5 genes and LV. Analysis of amino acid sequence showed that the homology was 88.1% -96.8%, 88.1% - 94.5%, 86.1% -96.5% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, bBJ-4, the homology was 54.7% -56.2% between the ORF5 genes and LV.[ Conclusion] The variation of prevalence strains was great in the ORF5 gene region, the homology of ORF5 gene sequence was higher and genetic relationship was nearer during prevalence strains in the same region, or was far in different regions.

Genetic Variation of the ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus in East China during 2008-2010

[Objective] A total of 260 swine samples of dead or sick pigs collected from 7 provinces （municipalities） Jiangsu, Anhui, Shanghai, Shandong, Zhejiang, Fujian and Jiangxi of China during 2008-2010 were detected for porcine reproductive and respiratory syndrome virus （PRRSV）. And the ORF5 genes of some isolates were amplified and sequenced for understanding the molecule epidemiology and the genetic evolution of PRRSV in East China. [Method] Using RT-PCR method, PRRSV was detected by RT-PCR from samples. The complete ORF5 genes of 36 PRRSV positive samples was amplified, sequenced and analyzed with other 15 strains available on GenBank. [Result] PRRSV was detected in 118/260 of the clinical samples, with a positive rate was 45.4%. Sequence analysis showed that the 36 isolates of this study belonged to the North American-type PRRSV strains and were closely related to the highly pathogenic PRRSV （HP-PRRSV） with 94.6%-100% amino acid sequence identities. The sequence analysis combined with the phylogenetic analysis indicated that all these North American-type PRRSV strains in East China were further divided into five subgenotypes, subgenotype Ⅲ showed closer identity with HPPRRSV; almost all subgenotypes were found to be variable in the primary neutralizing epitope; subgenotypes Ⅲ and IV had more glycosylation sites than others. Although these 36 isolates were collected from different provinces in East China, there were no obvious relations between the distribution of PRRSV and the region. [Conclusion] The PRRSV infection was widespread and HP-PRRSV was the popular strain in East China during 2008-2010. However some different genetic characteristics appeared in the genomes, the genetic evolution was relatively stable. There exists a cross-cutting phenomenon on the genetic relationship of PRRSV isolates obtained from different provinces. Subgenotypes IV and V only appeared in some provinces, but the distribution of PRRSV did not show apparent geographical characteristics.

Attenuation of Virulent Porcine Reproductive and Respiratory Syndrome Virus Strain CH-1a and Genetic Variation of ORF5 Gene

To develop a modified live vaccine （MLV） against porcine reproductive and respiratory syndrome virus （PRRSV）, virulent CH-Ia strain was attenuated by serial passages up to 130 passage （P130） in Marc-145 cells. The virulence and immune efficacy of the attenuated CH-1 a were evaluated in pigs. The results showed that animals inoculated with P130 did not develop any clinical sign of the disease, but produced rapid and effective humoral immune responses against PRRSV challenge, indicating that attenuated CH-1 a P 130 is the candidate as the effective vaccine against PRRSV. To define the potential mutations in the attenuated CH-la genome, we sequenced and analyzed the ORF5 gene of CH-la strain of different passages （P39, P55, P65, P70, P85, P100, P115, P120, P125, and P130） and found that three mutations （C5Y, H38Q and L146Q） which may be related with the attenuation of CH-la. In addition, we also found a unique restriction enzyme site （TspEI） in the ORF5 gene of attenuated CH-la, which can be used as a genetic marker to distinguish original and attenuated CH- 1 a.