Genetic Variation And Phylogenetic Analysis Of Reproductive And Respiratory Syndrome Virus (PRRSV) And The Research Of The Rule Of Immune Body Increased And Decreased

The porcine reproductive and respiratory syndrome (PRRS) is a commercially very important swine disease, with infected sows suffering from severe, and sometimes fatal respiratory disease and reproductive failure. The disease is caused by porcine reproductive and respiratory syndrome virus (PRRSV). Since the disease was discoveried, which created the huge economic loss in our country, in the last few years, most districts of our country, the high fever disease was appeared in summer and autumn, in 2007, the high fever main cause of disease was the variation of the porcine reproductive and respiratory syndrome, which determined by the Ministry of Agriculture of our country.To explore possible mechanism of genetic variation of highly pathogenic PRRSV, the ORF5 gene and the Nsp2 gene porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Henan region of China between 2006～2008 year were analyzed using RT-PCR and sequencing. The pig's blood samples and sickness materials were collected by the way of delivery randomly, which were disposaled by sterilitas and vaccinated the Marc-145 cell. The ORF5 gene of 16 porcine reproductive and respiratory syndrome virus (PRRSV) isolates and the partical Nsp2 gene of 4 porcine reproductive and respiratory syndrome virus (PRRSV) isolates were obtained from the positive organization materials from Henan region of China. The PCR products were extracted from agarose gel as manufacture's recommendation and cloned into pTG19-T vector which were identified by restriction endonucleases BamHI and sequencing using T7 promoter primer (boshang Bioengineering Corporation, shanghai, China). The depletion of the Nsp2 were all existed. The depletion positions of Hn-4, Hn-8 and Hn-14 PRRSV strains were the same as the PRRSV variation strains, which were used with this experimental analysis, there name were the HUB1,HUB2,HEB,Jx0612,JXA1和Jxwn06. 3 flaw position spots and other 86 flaw position spots, which were not continual, but the 86 flaw position spots were actually continual, but HB-2 strain and Hn-2 which islated in this experiment both had 3 nucleotide flaws, but other sequences, such as 16244B, BJ-4, CH-1a, CC-1, RespRRS MLV and the VR-2332, which used in our study did not have the flaw. The ORF5 gene of 16 porcine reproductive and respiratory syndrome virus (PRRSV) isolates'lengthes were all 603bp. And the partical of Nsp2 gene of 4 PRRSV isolates'lengthes were 2027bp.Sequence analyses revealed that these acute PRRSV isolates shared 85.6%-99.5% identities to each other, 88.1-98.7% identities with other North American PRRSV isolates. Phylogenetic analysis revealed that all acute PRRSV isolates are clustered within the North American genotype and could be assigned to two clusters. while otherwise isolate Hn-3 from a non-vaccinated swine herd in cluster Subgroup2 has 99.2% identities with RespPRRS MLV. the Nsp2 gene was amplified by RT-PCR and sequenced. The nucleotide sequence were 80.0%~99.7%; the results indicated that identity of Nap2 gene between our 4 strains and classical strain VR-2332 strains was order nucleotide the similarity was 80.6%～82.2%, with high pathogenicity fever variation in 2006to 2007 were 96.0%～99.3%. Reference the GenBank database accession numbers for the sequences of PRRSV isolates, a phylogenetic tree based on the nucleotide sequence of the ORF5 gene and Nsp2 gene of PRRSV. The tree was constructed with the acid of the Meg4. 16 isolates in cluster Subgroup1 has 96-99.8% identity with the highly pathogenic PRRSV strain JXA1, HEB1,JA142 HUB2,Jx0612,HUB1 emerged in 2006, while otherwise isolate Hn-3 from a non-vaccinated swine herd in cluster Subgroup2 has 99.2% identities with RespPRRS MLV. The 16 isolates in This research institute and the vaccine VR2332CH-1a, RespPRRS MLV did not belong to the same identical branch, so the heredity was far; but the 16 isolates with the domestic separation's variation had the near genetic, the result indicated that this 16 separates isolates were possibly variation, but were not evoluted from the vaccine strain.Some materials reported that the PRRSV which had flaw spots of the Nsp2 gene as characteristic appeared recently was newly high infectious, to clarify a poison pathogenicity which we separated, the two strains of Hn-2 and Hn-4, which had 3 flaws continuously and had 89 continual flaws infected the newborn pig artificialy, and the health comparison by the Marc-145 cell bubble nose infection newborn pig, after post-inoculation, at different tine the sera and leucocytes were separated aseptically, the virus and the anti-PRRSV immune body level and the neutralizing antibody level were to be detected, and compared with virus and immune body level relations at different time. The study results suggested that the two experimental groups both had not death, after the pig infected Hn-2 and Hn-4 strains, at the 7and 14d post-inoculation, the PRRSV could be detected, and anti-PRRSV N protein immune body also could be detected after 7d post-inoculation and had maintained the high level, after infection 41d and 50d, the low level neutralizing antibody could be detected respectivly, after that the neutralizing antibody titre elevated gradually.