| Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to undergo both homologous and nonhomologous RNA recombination. A recombinant virus was identified from nucleotide sequencing of RT-PCR products from a panel of fifty PRRSV field isolates, indicating that PRRSV RNA recombination occurs in nature. Co-infection of MA-104 cells with two North American PRRSV strains resulted in recombinant viruses containing chimeric ORF 3 and ORF 4 proteins. Nucleotide sequence analysis of cloned recombinant PCR products, encompassing 1182 bases of the 15.4 kb viral genome, revealed six independent recombination events. Our results demonstrated that PRRSV undergoes homologous RNA recombination in vitro and in nature, thus playing a major role in virus evolution and virus replication. Furthermore, PRRSV was shown to undergo nonhomologous RNA recombination thereby producing subgenomic RNAs that contain ORF 1a nucleotides. The unique properties of these novel subgenomic RNAs, designated as heteroclite (meaning deviating from common rules and forms) RNAs, include (a) apparent association with normal virus infection, (b) packaging into virions, (c) short, heterogeneous nucleotide direct repeats which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (e) little apparent interference with parental virus replication. In summary, PRRSV readily undergoes non-homologous RNA recombination to generate heteroclite subgenomic RNAs. Northern blot analysis demonstrated that heteroclite RNAs are packaged into virions purified by sucrose cushion centrifugation. RT-PCR amplication of the viral RNAs from three virion bands, collected after sucrose density gradient centrifugation, demonstrated that heteroclite RNAs are co-packaged with genomic RNAs. A survey of the nucleotide sequence of eight packaged heteroclite RNAs indicated that the packaging signal of the PRRSV genomic RNA resides in the first 278 nt at the 5' end of the ORF 1a coding region. Together, I discovered that PRRSV readily undergoes RNA recombination, which opens new avenues for further dissection of PRRSV replication and evolution. |
