Molecular Characteristics Of Complete Genome Of Porcine Reproductive And Respiratory Sydrome Virus

In this study, the two porcine reproductive and respiratory syndrome virus (PRRSV) isolates were obtained from serum samples isolated from PRRSV infection and morbid pig farms by inoculating serum samples into SPF porcine alveolar macrophages(PAMs), and their propagation in PAMs were identified by RT-PCR, IFA and immune electron microscopy (IBM). The all cDNA fragments of the two isolates were amplified and cloned by RT-PCR and 5' Full RACE, and the complete genomes were obtained by assembling these sequence. Sequence comparison suggested the two isolates belong to American type, then the molecular characteristics of HB-l(sh)/2002 and HB-2(sh)/2002 were analyzed by bioinformatics.1. The serum samples that were PRRSV positive by RT-PCR were collected and used to inoculate porcine alveolar macrophages (PAMs) lavaged from fifty-day SPF piglet. The two isolates were obtained, which were designated as HB-1(sh)/2002 and HB-2(sh)/2002. The obvious cytopathogenic effects were observed after 48 hours postinfection, and the virus propagation in PAMs were determined by RT-PCR. The specific fluorescence was observed in plasma by IFA using PRRSV positive serum. The TCID50 of passage 2 virus cultures in PAMs were 10~75 /0.1ml and 10 -6.5/0.1ml respectively. The cell cultures of HB-1(sh)/2002 and HB-2(sh)/2002 were condensed by differential centrifugation, then the virus particles about 60nm were observed by immune electron microscopy (IBM).2.The cDNA fragments covered full genomes of HB-l(sh)/2002 and HB-2(sh)/2002 were amplified from total RNA isolated from passage 2 virus cultures by employing 5' Full RACE method and RT-PCR, and were cloned into pGEM-Teasy vector respectively. Then the complete genomes sequence of the two isolates were obtained by sequencing and assembling these fragments.3. Sequence comparison showed that the complete nucleotide sequence of HB-1(sh)/2002 and HB-2(sh)/2002 shared 89.1%~96.0% and 88.7%~95.1% identity with American type isolates respectively, but only 54.7% and 54.3% with European prototype LV, which suggested HB-l(sh)/2002 artd HB-2(sh)/2002 belong to American type in genotype. In order to well understand the genetic relations of American type isolates, a phylogenetic tree was generated based on Nsp2 and ORF5. Results from the evolutionary analysis indicates that American type isolates can be divided into two subgroups based on Nsp2 and five subgroups based on ORF5 genes. PRRSV isolates of China mainland seem belong to different subgroup. HB-l(sh)/2002, HB-2(sh)/2002 and CH-1a belong to one subgroup, and BJ-4, SI and Jl belong to another subgroup. The secondary structure and epitopes analysis of structural proteins showed some differences exist among different isolates.