Construction Of Infectious CDNA Clone Of European Type Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is a servere swine infectious disease, characterized by either reproductive failure in pregnant sows, or respiratory tract distress particularly in suckling pigs. The causative agent, PRRS virus (PRRSV) was first isolated in Europe and that isolate (Lelystad virus), together with the first North American isolate (VR2332), now classified as the two recognized genotypes of PRRSV: European (EU genotype, type I) and North American (NA genotype, type II). As a member of the Arteriviridae family in the order Nidovirales, PRRSV is an enveloped virus with a positive-sense, single-stranded RNA genome of approximately 15.5 kb in length, which contains nine open reading frames (ORFs). In China, since the first outbreak of PRRS in 1995, this disease has occurred in almost all provinces and economically has been one of the most severe viral diseases for pig farms. Based on the numerous complete or partial genome sequences deposited in the GenBank database, almost all of the Chinese PRRSV isolates belong to the NA genotype. However, it was recently reported that EU genotype PRRSV was positive by RT-PCR from submitted clinical samples suggestive of PRRS.In addition, EU genotype PRRSV (AMERVAC-PRRS/A3) attenuated vaccine had been once applied extensively in China. To understand the relation between (AMERVAC-PRRS/A3) attenuated vaccine and the EU genotype PRRSV which form clinical tissue samples, we amplified the complete genome of the vaccine strain PRRSV by five segments, which were cloned into pCR BluntII-TOPO -vector and sequenced. The result shows that the complete genome of AMERVAC-PRRS/A3 stain PRRSV is 15098bp in length, excluding poly(A) tail, and has 100% identity with ORF7 gene of Ningbo42 strain PRRSV (EF473137) and 98% identity with nsp2 gene of FJ0603 strain PRRSV (EF592535). The data suggest that, the EU genotype PRRSV attenuated vaccine is the most possible source of EU genotype PRRSV detected in clinical samples.To further study the molecular biology via reverse gentic manipulation, and infectious cDNA clone was developed for AMERVAC-PRRS/A3 strain PRRSV. The full-length genome was assembled as a full-length cDNA clone, under the control of the prokaryotic T7 promoter. Transfection of cells with the RNA transcripts of the cDNA clone induced cytopathic effects and produced infectious progeny. The reconstituted virus was stable and displayed similar virological characteristics with these of the parental virus in cultured cells, demonstrating that the PRRSV infectious cDNA clone was constructed successfully. The development of reverse genetics systems for EU genotype PRRSV will be helpful to us for better unsteranding the moleculer mechanism of the virus.