Submitted to: Research Workers in Animal Diseases Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: November 16, 2009
Publication Date: December 4, 2009
Citation: Guo, B., Faaberg, K.S., Lager, K.M., Kehrli, Jr., M.E. 2009. Large-Scale parallel pyrosequencing technology reveals PRRSV VR-2332 Nsp2 deletion mutants stable in swine [abstract]. International PRRS Symposium, Chicago, IL. Paper No. 34. Technical Abstract: PRRSV nonstructural protein 2 (nsp2) is cleaved from the replicase polyprotein (ORF1ab) and encodes a cysteine protease (PL2) domain at the N-terminus, a middle hypervariable region and four transmembrane domains near the C-terminus. Studies have demonstrated the hypervariable region is nonessential for replication in vitro, and up to 403 amino acids could be deleted without affecting the viability of type 2 strain VR-2332 (Han et al., J. Virol, 2007). Swine inoculation studies of 4 PRRSV nsp2 deletion mutants along with wild-type (wt) recombinant virus have shown the selected nsp2 deletion mutants can also replicate in vivo as demonstrated by virus isolation, qRT-PCR, and antibody responses. However, different nsp2 deletion mutants manifested different phenotypes in terms of lymphadenopathy and interferon gamma production by the pig's immune system. The entire predicted nsp2 gene was sequenced from viruses in day 35 serum of two animals of four included in the study, which revealed that the mutants were genetically stable in the entire nsp2 region. Next, to understand whether other compensatory mutations occurred during in vivo replication, full genome nucleotide sequence analysis of the study viruses and pig passage of one mutant was completed using the Roche 454 pyrosequencing technology. Our results demonstrated that appropriate methods would generate a contiguous sequence encompassing all or nearly all of the full-genome in a single run, although the 5' and 3' termini were often not revealed and occasional bases were absent when compared to the parent sequence. Sanger sequencing was employed to determine the sequence of the few remaining genome gaps. The combined sequencing results revealed that, although sporadic mutations were identified throughout each of genomes causing silent mutations as well as conserved and non-conserved single amino acid mutations, the genomic sequences of the animal passaged nsp2 mutant viruses and wt recombinant viruses were quite stable.