title: Concise and broadly applicable method for determining the genomic sequences of North-American-type porcine reproductive and respiratory syndrome viruses in various clusters.
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.
authors.affiliation: Animal Research Division, Institute of Japan Association for Techno-innovation in Agriculture, Forestry and Fisheries, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan.
authors.forename: Takeya
authors.initials: T
authors.lastname: Morozumi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Iseki
authors.forename: Daisuke
authors.initials: D
authors.lastname: Toki
authors.forename: Michihiro
authors.initials: M
authors.lastname: Takagi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsunemitsu
authors.forename: Hirohide
authors.initials: H
authors.lastname: Uenishi
mesh_terms: D000818:Animals; D005260:Female; D016679:Genome, Viral / Q000235:genetics*; D009656:North America; D010802:Phylogeny; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013552:Swine
chemical_list: D012367:RNA, Viral