Deep sequencing of PRRSV isolates: rapid and large-scale characterization of viral genomes

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single stranded, positive sense RNA virus with a genome size of approximately 15 kb. Much of the genetic characterization or viral genotyping of PRRSV isolates is limited to one or two viral genes only (ORF5 and/or ORF7) for a number of reasons, for example: (1) characterizing one or two ORFs is sufficient for diagnostics; (2) genome characterization is laborious because traditional (Sanger) sequencing yields only a single sequence of 800-1000 bases per reaction; (3) large-scale genome characterization is time-consuming and costly. Collectively, this hinders the study of PRRSV genomic evolution at different levels (host, regional, and global). We demonstrate here the use of 454 technology to rapidly sequence PRRSV genomic nucleic acid from different sources (cell culture and swine tissue), genotypes (type 1 and type 2), and genome structure (non-deletion vs. deletion variants). Samples (n=16) were multiplexed to bring down cost per genome sequence. Assembly of sample specific reads resulted in a single contig in almost all instances (15 out of 16). Average genome coverage was 96.7% with reference to prototype isolates (Lelystad virus for type 1 and ATCC VR2332 for type 2). Average sequence depth was 405 reads per nucleotide position. This high sequence depth allowed characterization of variants from quasispecies that occurred at frequencies even lower than 1%. In summary, next generation sequencing technology offers unparalleled opportunity to quickly and efficiently characterize near complete length PRRSV genomes in an economical manner. This allows experiments to be designed with considerations to viral genomic evolution rather than those with limited insights from select viral genes only.