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

Morozumi T, Iseki H, Toki D, Takagi M, Tsunemitsu H, Uenishi H - J. Vet. Med. Sci. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

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Related in: MedlinePlus

Phylogenetic relationships among Japanese isolates of NA-type PRRSVs. Thephylogenetic analysis was performed according to the neighbor-joining method by usingthe nucleotide sequences of ORF5 [11]determined according to ClustalW [2]. Scale forlengths of branches is shown at the bottom. Bootstrap trials were conducted 1,000times, and values exceeding 80% are shown at nodes. Closed circles indicate thestrains sequenced by the CORT-MDA method in this study.
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fig_002: Phylogenetic relationships among Japanese isolates of NA-type PRRSVs. Thephylogenetic analysis was performed according to the neighbor-joining method by usingthe nucleotide sequences of ORF5 [11]determined according to ClustalW [2]. Scale forlengths of branches is shown at the bottom. Bootstrap trials were conducted 1,000times, and values exceeding 80% are shown at nodes. Closed circles indicate thestrains sequenced by the CORT-MDA method in this study.

Mentions: Preparation of genomic RNA and cDNA of viruses: Viruses sequenced in thecurrent study were strains EDRD-1 (an NA-type reference strain in Japan [15, 29], clusterIII), Nagasaki 11-14 (isolated in 2011, cluster I), Jam2 (Aomori 00; isolated in 2000,cluster II), Yamagata 10-7 (isolated in 2010, cluster III) and Aomori 10-5 (isolated in2010, cluster III) (Fig. 2Fig. 2.


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

Morozumi T, Iseki H, Toki D, Takagi M, Tsunemitsu H, Uenishi H - J. Vet. Med. Sci. (2014)

Phylogenetic relationships among Japanese isolates of NA-type PRRSVs. Thephylogenetic analysis was performed according to the neighbor-joining method by usingthe nucleotide sequences of ORF5 [11]determined according to ClustalW [2]. Scale forlengths of branches is shown at the bottom. Bootstrap trials were conducted 1,000times, and values exceeding 80% are shown at nodes. Closed circles indicate thestrains sequenced by the CORT-MDA method in this study.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197153&req=5

fig_002: Phylogenetic relationships among Japanese isolates of NA-type PRRSVs. Thephylogenetic analysis was performed according to the neighbor-joining method by usingthe nucleotide sequences of ORF5 [11]determined according to ClustalW [2]. Scale forlengths of branches is shown at the bottom. Bootstrap trials were conducted 1,000times, and values exceeding 80% are shown at nodes. Closed circles indicate thestrains sequenced by the CORT-MDA method in this study.
Mentions: Preparation of genomic RNA and cDNA of viruses: Viruses sequenced in thecurrent study were strains EDRD-1 (an NA-type reference strain in Japan [15, 29], clusterIII), Nagasaki 11-14 (isolated in 2011, cluster I), Jam2 (Aomori 00; isolated in 2000,cluster II), Yamagata 10-7 (isolated in 2010, cluster III) and Aomori 10-5 (isolated in2010, cluster III) (Fig. 2Fig. 2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus