ARS | Publication request: Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine

Submitted to: Virus Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 27, 2011
Publication Date: November 1, 2011
Citation: Guo, B., Vorwald, A.C., Alt, D.P., Lager, K.M., Bayles, D.O., Faaberg, K.S. 2011. Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine. Virus Research. 161(2):162-169.

Interpretive Summary: Porcine reproductive and respiratory syndrom virus (PRRSV) is the foremost disease of swine in the United States. In this report, the first method for generating full-length PRRSV sequences using a new technology was detailed. Furthermore, the nucleotide sequences of 15 PRRSV genomes were generated simultaneously using the technology. The 15 genomes were used to assess what changes occurred in the PRRSV genome after deletion of parts of a replicase protein followed by growth in swine. The research show that although the deletion mutant viruses had deficient growth properties in swine, there were no other deletions or insertions seen in the viral genome, an issue of concern for marker vaccine development. This report describes the first PRRSV genome study using a new sequencing technology. Furthermore, the results of this new PRRSV sequencing method provides additional information to scientists and vaccine companies who seek to rationally design improved vaccines against porcine reproductive and respiratory syndrome virus.

Technical Abstract: Genomes from fifteen porcine reproductive and respiratory syndrome virus (PRRSV) isolates were derived simultaneously using 454 pyrosequencing technology. The viral isolates sequenced were from a recent swine study, in which engineered Type 2 prototype PRRSV strain VR-2332 mutants, with 87, 184, 200, and 403 amino acid deletions in the second hypervariable region of nsp2, were found to be stable in the nsp2 coding region after in vivo infection (Faaberg et al., 2010). To further assess the stability of the engineered viruses elsewhere in their respective genomes, all sequenced genomes were compared and contrasted. No specific mutations occurred in all nsp2 deletion mutant genomes that were not also seen in the parent genome of Type 2 PRRSV strain VR-2332. In addition, second site (non-nsp2) deletions and/or insertions were not evident after replication in swine. The number of mutations seen increased with deletion size, but even the largest deletion (403 aa) had very few consensus mutations. Thus, our findings provide further substantiation that the nsp2 deletion mutant genomes were genetically stable after in vivo passage.