Study On Detection, Isolation And Characteristics For PRRSV Variants

The Porcine Reproductive and Respiratory Syndrome(PRRS) is a new infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV). Since the disease broke out first in America in 1987,then the disease has rapidly spread the worldwide.From May 2006,a previously unknown severe disease design-nated "high fever" occurred in several pig farms and subsequently overwhelmed almost Pig-farming areas of China,great damages have took place in pig industry. The PRRSV variants were confirmed one of significant etiological agent for swine "high fever disease".Based on the deletion information of PRRSV PRRSV variants genome,3 oligo-nucleotide primers were designed and synthesized.Specific and sensitive reverse transcription-PCR(RT-PCR) assays was developed for the detection of high virulent PRRSV.By means of sensitive and specific assays and clinical application showed that this RT-PCR assays proved to be an accurate differential detection method for PRRSV variant and normal PRRSV strains with the characteristics of rapidity, sensitivity and specificity,and has a strong clinical relevance.Used Marc-145 cells to isolate virus from tissue of pig that 36 PRRSV variants clinical samples and five variant-PRRSV field isolates were isolated form them after passaged three times,named ZZ-7,YY-8,CS06,CD06 and XT07.ZZ-7 isolates has higher-title in Marc-145 cells,TCID₅₀was 10^-4.63/0.1mL.By gene sequenced of five field isolates,the Nsp2 gene deletion information were coincident with other PRRSV isolates in our country,Gp5 gene has variation,sequence homology is 98.7%-99.7%, amino acid sequence homology is 98.5%-100%.And the five isolates belong to one branch,from we can conjecture they root in the same one probably.According to characteristics of DI RNA of EAV,oligonucleotide primer were designed and synthesized in Nsp1 and Nsp11 gene of PRRSV respectively.380-1100bp cDNA sequence were amplified by RT-nest PCR in different PRRSV genome and sequenced after being directly cloned into pMD18-T vector,the result showed that the PCR products were small fragment of PRRSV genome.We inferred that the small fragments were DI RNA genome of PRRSV and the different DI RNA genome in different virus was recognized.The deletion region of different DI RNA genome was mainly in the Nsp1 3'end/Nsp2 5'end to Nsp10 5'end of parental genome.Part of Nsp2 and Nsp10 gene and whole Nsp3 to Nsp9 gene were deleted.Further PRRSV RNA hybridized to a DIG-labeled oligonucleotide complementary to the Nsp1 gene of PRRSV.By Northern blotting we observed,a new PRRSV RNA species,which found under the 15.4-kb genome(RNA1) in denaturing agarose gels,the size of this new RNA(DI RNA) was estimated to be 6.4 to 7-kb and concluded that the DI RNA composition of PRRSV DI RNA gene including 5'UTR to Nsp1 3'end/Nsp2 5'end and Nsp10 3'end to 3'-terminal of parental genome.In this paper we first confirmed that the DI RNA exist in PRRSV,and lay a foundation for further study on propa-gation, virulence,evolution of PRRSV.