Isolation, Identification And Cloning Of M Gene Of Porcine Reproductive And Respiratory Syndrome Virus From Chonqing

Isolation ,identification and cloning of M gene of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV) from Chongqing were performed in this study. One virus strain was successfully isolated from the tissues of pigs which were preliminarily diagnosed to be infected with PRRSV, designated C14-2. The isolate can proliferated on Marc-145 cell line. After three passages, apparent CPE were observed in Marc-145 cell. Further experiments were done to identify the isolate. A large number of global particles (40-60nm in diameter) were observed under electron microscope. The TCID₅₀ was 10^-5.70/0.1ml. The virus could not be inhibited by BUDR on Marc-145 cell culture. When the virus was treated at 56 ℃ for 20-30 min ,the virus lost the ability to infect Marc-145 cell line. The virus can be neutralized by the serum of PRRSV American strain. The neutralization index is 1:27. Slight improvement of body temperature and respiratory syndrome appeared when 40-day-old piglets were inoculated with the isolate. Above experiments proved the isolate to be PRRSV.According to the nucleotide sequence of PRRSV VR2332, a pair of primers was designed to amplify M gene of C14-2 with template RNA extracted from the virus. The fragments obtained from RT-PCR was approximate 550bp. The M gene was cloned into PMD18-T and sequenced. The result of sequencing showed the M gene of C14-2 was 525bp, which encoded 174 amino acids. The nucleotide sequence and deduced amino acid sequence of M gene of C14-2 were aligned with other reference strains .It displayed 99.8% and 99.4% homology in nucleotide and amino acid sequence with American strain VR2332 However it displayed 68.6% and 78.2% homology with European strain LV. So the result showed that C14-2 is more similar to American strains in gene type.