Isolation, Identification And Cloning Of MN Gene Of Porcine Reproductive And Respiratory Syndrome (PRRS) Virus In Sichuan

Porcine Reproductive and Respiratory Syndrome(PRRS) Virus belongs to the family Arteriviridae. PRRS is one of the most important diseases in the female pigs.lt has caused great damages since its being recognized. It's characteristic is rapid transmission and great damage.In this study, Porcine Reproductive and Respiratory Syndrome Virus was successfully isolated from serum of female pigs from Sichuan province.The isolate was designated SCMS. MARC-145 cells were used for the replication and isolation of the virus.After three passages ,the isolate could produce obvious cytopathic effects(CPE).Tipical globular PRRSV particles,40-80nm in diameter ,were seen with direct electron microscope.They could not be inhibited by BUDR on MARC-145 cells culture.The TCID50 of the isolate is 10-4.46/0.05ml.The neutralisation index is 100.According to the reported complete nucleotide sequences of ATCC VR-2332 in GenBank,a pair of primers were designed to amplify MN gene of SCMS with Oligo 6.0 software.The fragments of coplete MN gene of SCMS were obtained from RT-PCR using RNA extracted from the virus as template.The product of RT-PCR named MN is approximate 1.0Kb in lengh.The MN gene was cloned into pMD-T vector and sequenced.The results of sequencing showed that SCMS isolate complete MN gene was 886bp and had complete open reding frame of M and N gene which encoded 297 amino acides.The sequence was analyzed homologously in comparison with the MN nucleotide sequences of two reference Pocine Reproductive and Respiratory Syndrome Virus , ATCC VR-2332 and LV strain. Nucleotide sequences comparison indicated that SCMS shared 99.7 % and 61.6% with ATCC VR-2332 and LV strain respectively.The deduced ammo acid sequences according to M gene were98.9% and 78.7% correspondingly. The deduced animo acid sequences according to N gene were 100% and 52.8 % correspondingly.The result showed that SCMS isolate is more similar to ATCC VR-2332 strain than LV in gene type.