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Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing.

Agoti CN, Otieno JR, Munywoki PK, Mwihuri AG, Cane PA, Nokes DJ, Kellam P, Cotten M - J. Virol. (2015)

Bottom Line: The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement.The new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development.Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

View Article: PubMed Central - PubMed

Affiliation: KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya.

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(A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.
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Figure 1: (A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.

Mentions: Two sets of reverse transcription and PCR primers were selected from all available RSVA and RSVB genomic sequence data based on frequency, location, and predicted PCR function (see Table 1 for further details). The general pattern of primer sites and the locations of primer targets in RSVA and RSVB genomes are shown in Fig. 1A. Actual PCR results are shown in Fig. 1B for RSVA and RSVB samples, with PCR products of the expected size obtained for all 6 amplicons. These primers were used as part of a deep-sequencing process for RSV combining the full cDNA preparation and genome amplification, deep sequencing with Illumina MiSeq, and de novo assembly (Fig. 1C) to generate 27 complete or nearly complete genomes (11 group A and 16 group B; median length, 14,990 nt; range, 14,666 to 15,232 nt). An additional number of samples yielded RSV contigs of >5,000 nt in length, and these were also retained for further analysis. A summary of the genomic sequences in this study is provided in Table 2.


Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing.

Agoti CN, Otieno JR, Munywoki PK, Mwihuri AG, Cane PA, Nokes DJ, Kellam P, Cotten M - J. Virol. (2015)

(A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403408&req=5

Figure 1: (A) PCR primer target sites in RSVA and RSVB. The primer target sequences in representative RSVA (left) and RSVB (right) viruses were determined. Circular markers indicate positions of primer target sites in the test genome color-coded by number of mismatches with the primer; gray bars indicate lengths and positions of the predicted products. (B) Two examples of reverse transcription-PCR function. The DNA products of reverse transcription and PCR amplification of two samples were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining. Sizes of some of the molecular size markers (in base pairs) are indicated to left of the gel. Lane m, molecular size markers; lanes 1 to 6, individual 2- to 3-kb RSV amplicons 1 to 6, respectively. (C) Flowchart of the RSV sequencing process.
Mentions: Two sets of reverse transcription and PCR primers were selected from all available RSVA and RSVB genomic sequence data based on frequency, location, and predicted PCR function (see Table 1 for further details). The general pattern of primer sites and the locations of primer targets in RSVA and RSVB genomes are shown in Fig. 1A. Actual PCR results are shown in Fig. 1B for RSVA and RSVB samples, with PCR products of the expected size obtained for all 6 amplicons. These primers were used as part of a deep-sequencing process for RSV combining the full cDNA preparation and genome amplification, deep sequencing with Illumina MiSeq, and de novo assembly (Fig. 1C) to generate 27 complete or nearly complete genomes (11 group A and 16 group B; median length, 14,990 nt; range, 14,666 to 15,232 nt). An additional number of samples yielded RSV contigs of >5,000 nt in length, and these were also retained for further analysis. A summary of the genomic sequences in this study is provided in Table 2.

Bottom Line: The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement.The new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development.Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

View Article: PubMed Central - PubMed

Affiliation: KEMRI-Wellcome Trust Research Programme, Kilifi, Kenya.

Show MeSH
Related in: MedlinePlus