Concise and broadly applicable method for determining the genomic sequences of North-American-type porcine reproductive and respiratory syndrome viruses in various clusters

J Vet Med Sci. 2014 Sep;76(9):1249-55. doi: 10.1292/jvms.14-0103. Epub 2014 Jun 10.

Abstract

We developed a concise and broadly applicable method for accurate genomic sequencing of North American genotype (NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomes high genetic variability of the viruses. The method, designated "combination of consensus oligonucleotide reverse transcription and multiple displacement amplification" (CORT-MDA), involves reverse-transcription of viral RNA followed by shotgun sequencing after amplification using only 11 degenerate oligonucleotide primers; these primers were designed against consensus regions within the open reading frames of the 124 NA-type PRRSV strains with reported full-length genomic sequences. Sequencing of the 192 shotgun clones generated per virus showed 80% to 94% coverage on the reported PRRSV genomic sequence, such that only 2 or 3 unread regions had to be resequenced after PCR amplification using custom primers. Direct sequencing of RT-PCR products confirmed absolute consistency between sequences determined by the CORT-MDA method and those from RT-PCR. These results suggest that our method is applicable to diverse NA-type viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Female
  • Genome, Viral / genetics*
  • North America
  • Phylogeny*
  • Porcine respiratory and reproductive syndrome virus / genetics*
  • RNA, Viral / chemistry
  • RNA, Viral / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA / methods*
  • Swine

Substances

  • RNA, Viral