Isolation, Identification Of Porcine Reproductive And Respiratory Syndrome Virus CC-1 Isolate And Construction Of Full-length CDNA Clone Of PRRSV CC-1

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the causative agent of PRRS, a disease characterized by reproductive failure in sows, including early far rowing with stillborn piglets and late-term abortions, as well as respiratory distress in young pigs and an influenza-like disease in grow-finish swine. In this study, one porcine reproductive and respiratory syndrome virus (PRRSV) isolate named CC-1 was isolated and identified. The complete genome sequence of isolate CC-1 was obtained by RT-PCR and DNA sequencing. Then the molecular characteristics and phylogenetic of the PRRSV CC-1 isolate were analyzed by bioinformatics. Based on the complete genome sequencing the full length cDNA clone of PRRSV CC-1 isolate was constructed by utilizing reverse genetic manipulation technology. In the future the infectious of the full-length cDNA clone will be studied.PRRSV is a small enveloped virus with a single-stranded positive-sense RNA genome and is a member of the family Arteriviridae in the order Nidovirales. The 5′-capped and 3′-polyadenylated genome of PRRSV is 15.1 to 15.5 kb in length and consists of at least eight open reading frames (ORFs). ORF 1ab polyprotein that is proteolytically cleaved into products involved in virus transcription and replication. ORFs 2 to 7 code for PRRSV structural proteins. And it was generally believed that complete genome information of virus and full length cDNA clone would contribute to understanding the molecular characteristic of PRRSV and R&D of new vaccines and diagnosis techniques.Isolation and identification of the PRRSV CC-1 isolate. The lung and lymph node of piglets collected form one herd in Changchun region were PRRSV positive by RT-PCR. Inoculate the tissue lysate on monolayer of Marc-145 cell, 48h post-inoculate the obvious cytopathogenic effects were observed. The virus propagation in Marc-145 was determined by indirect fluorescent antibody assay (IFA). The virus specific fluorescent was observed in plasma of infected Marc-145 cell. The PRRSV-like particles about 55nm were observed in the infected cell culture lysate by electron microscopy.To obtain the full length sequence of PRRSV CC-1, six overlapping fragments with a size of 3616bp, 3757bp, 2731bp, 3458bp, 270bp and 2940bp of CC-1 genome were amplified with reverse transcription polymerase chain reaction (RT-PCR), 5′Rapid Amplification of cDNA Ends (5′RACE) and anchored-PCR. The PCR products were cloned into pMD18-T Vector and sequenced. The whole genome sequence of CC-1 (Genbank accession number: EF153486) was obtained by assemble each fragments sequence. The molecular characteristics of the PRRSV CC-1 isolate were analyzed by bioinformatics. The result showed that its genome consisted of 15411bp excluding the poly(A) tail, and contained nine ORFs (ORF1a, ORF1b, ORF2a, ORF2b-ORF7) that encoded proteins of 2503, 1457, 256, 73, 254, 178, 200, 174 and 123 aa.The phylogenetic tree constructed basing the full genomic sequence of 22 different PRRSV isolates confirmed that CC-1 belongs to North American-like strains and shared 99.4% and 60.8% nucleotide identity with the prototype North American strain VR-2332 and European strain Lelystad, respectively. Genetic analysis demonstrated that the ORF6 of CC-1 was the most conservative region, and shared approximately 95%-100% nucleotide identity and 97.1%-99.4% amino acid similarity to other sequenced North American-like PRRSV isolates. Three variable regions were identified, corresponding to ORF1a, ORF5 and ORF3.The putative N-linked glycosylation sites in GP3, GP4 and GP5 of CC-1 and other isolates (N44,N51) were well conserved. But the N34 in GP5 was variable in position and motif. This may associated with the virus infectivity and antigenicity.Sequencing and analysis of complete genome of porcine reproductive and respiratory syndrome virus isolate CC-1 have significance meaning for prevention PRRSV in Changchun region and contribute to understanding the rules of PRRSV epidemiology and variation in our country.Five pairs primers were designed according to the full-length genomic sequence of PRRSV CC-1. By using the RT-PCR, 5 overlapping cDNA fragments with a size of 3840bp, 4488bp, 2727bp, 3430bp and 2940bp covering the whole genome were amplified. Each fragment was cloned into pMD-18T vector, respectively. Then the full-length cDNA clone pMD-CC1 of PRRSV CC-1 was obtained when the 5 fragments were ligated in order and clone into pMD-18T vector. Unique restriction sites, which were absent from the viral sequence, were introduced at both end of the full-length cDNA clone. At the 5′end, the promoter sequence recognized by T7 RNA polymerase was introduced. A poly(A)30 sequence served as transcription terminate signal was introduced at 3′end of the full-length cDNA clone. A unique XbaI site in ORF1b was deleted by fusion-PCR served as a genetic marker of the full-length cDNA clone.This study would contribute to the gene functional study of PRRSV and desiging of new type vaccine.