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Investigating intra-host and intra-herd sequence diversity of foot-and-mouth disease virus ☆

View Article: PubMed Central - PubMed

ABSTRACT

Due to the poor-fidelity of the enzymes involved in RNA genome replication, foot-and-mouth disease (FMD) virus samples comprise of unique polymorphic populations. In this study, deep sequencing was utilised to characterise the diversity of FMD virus (FMDV) populations in 6 infected cattle present on a single farm during the series of outbreaks in the UK in 2007. A novel RT–PCR method was developed to amplify a 7.6 kb nucleotide fragment encompassing the polyprotein coding region of the FMDV genome. Illumina sequencing of each sample identified the fine polymorphic structures at each nucleotide position, from consensus level changes to variants present at a 0.24% frequency. These data were used to investigate population dynamics of FMDV at both herd and host levels, evaluate the impact of host on the viral swarm structure and to identify transmission links with viruses recovered from other farms in the same series of outbreaks. In 7 samples, from 6 different animals, a total of 5 consensus level variants were identified, in addition to 104 sub-consensus variants of which 22 were shared between 2 or more animals. Further analysis revealed differences in swarm structures from samples derived from the same animal suggesting the presence of distinct viral populations evolving independently at different lesion sites within the same infected animal.

No MeSH data available.


The average coverage distribution for the sequenced samples. For both PCR duplicate sets (with filtered, trimmed reads), average coverage values were between 5.79 × 102 and 1.00 × 104 nucleotides/site, with the mean of all samples being 6.44 × 103 nucleotides/site. Each sample is indicated by a different colour. Sample 147 – red; sample 161A – orange; sample 004 – yellow; sample 241 – green; sample 161B – dark blue; sample 341 – black, and sample 238 – grey.
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f0005: The average coverage distribution for the sequenced samples. For both PCR duplicate sets (with filtered, trimmed reads), average coverage values were between 5.79 × 102 and 1.00 × 104 nucleotides/site, with the mean of all samples being 6.44 × 103 nucleotides/site. Each sample is indicated by a different colour. Sample 147 – red; sample 161A – orange; sample 004 – yellow; sample 241 – green; sample 161B – dark blue; sample 341 – black, and sample 238 – grey.

Mentions: FMDV quantified by qRT–PCR in samples ranged between 1.19 × 105 (sample 341) and 2.50 × 107 (sample 161b) viral copies/μl (median of 8.51 × 106). Comparison of the qRT–PCR results with the estimated lesion age revealed an inverse correlation, with samples with a lesion age of 6 days having an average of 3.03 × 105 viral copies/μl and samples with a lesion age of 2 or 3 days having an average of 1.35 × 107 viral copies/μl (Table 1). The MiSeq produced a total of 3.18 × 106 paired end reads for all samples, with an average of 4.55 × 105 paired end reads generated for each duplicated sample. Approximately 14% of these reads were eliminated by quality filtering. Of the remaining reads, between 78% (sample 147) and 99.99% (sample 341) mapped to the FMDV O1BFS reference genome (mean = 96%). The average coverage depth at each genome position across the amplified fragment ranged from 5.79 × 102 to 1.00 × 104 reads (mean = 6.44 × 103) (Table 1; Fig. 1).


Investigating intra-host and intra-herd sequence diversity of foot-and-mouth disease virus ☆
The average coverage distribution for the sequenced samples. For both PCR duplicate sets (with filtered, trimmed reads), average coverage values were between 5.79 × 102 and 1.00 × 104 nucleotides/site, with the mean of all samples being 6.44 × 103 nucleotides/site. Each sample is indicated by a different colour. Sample 147 – red; sample 161A – orange; sample 004 – yellow; sample 241 – green; sample 161B – dark blue; sample 341 – black, and sample 238 – grey.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036933&req=5

f0005: The average coverage distribution for the sequenced samples. For both PCR duplicate sets (with filtered, trimmed reads), average coverage values were between 5.79 × 102 and 1.00 × 104 nucleotides/site, with the mean of all samples being 6.44 × 103 nucleotides/site. Each sample is indicated by a different colour. Sample 147 – red; sample 161A – orange; sample 004 – yellow; sample 241 – green; sample 161B – dark blue; sample 341 – black, and sample 238 – grey.
Mentions: FMDV quantified by qRT–PCR in samples ranged between 1.19 × 105 (sample 341) and 2.50 × 107 (sample 161b) viral copies/μl (median of 8.51 × 106). Comparison of the qRT–PCR results with the estimated lesion age revealed an inverse correlation, with samples with a lesion age of 6 days having an average of 3.03 × 105 viral copies/μl and samples with a lesion age of 2 or 3 days having an average of 1.35 × 107 viral copies/μl (Table 1). The MiSeq produced a total of 3.18 × 106 paired end reads for all samples, with an average of 4.55 × 105 paired end reads generated for each duplicated sample. Approximately 14% of these reads were eliminated by quality filtering. Of the remaining reads, between 78% (sample 147) and 99.99% (sample 341) mapped to the FMDV O1BFS reference genome (mean = 96%). The average coverage depth at each genome position across the amplified fragment ranged from 5.79 × 102 to 1.00 × 104 reads (mean = 6.44 × 103) (Table 1; Fig. 1).

View Article: PubMed Central - PubMed

ABSTRACT

Due to the poor-fidelity of the enzymes involved in RNA genome replication, foot-and-mouth disease (FMD) virus samples comprise of unique polymorphic populations. In this study, deep sequencing was utilised to characterise the diversity of FMD virus (FMDV) populations in 6 infected cattle present on a single farm during the series of outbreaks in the UK in 2007. A novel RT–PCR method was developed to amplify a 7.6 kb nucleotide fragment encompassing the polyprotein coding region of the FMDV genome. Illumina sequencing of each sample identified the fine polymorphic structures at each nucleotide position, from consensus level changes to variants present at a 0.24% frequency. These data were used to investigate population dynamics of FMDV at both herd and host levels, evaluate the impact of host on the viral swarm structure and to identify transmission links with viruses recovered from other farms in the same series of outbreaks. In 7 samples, from 6 different animals, a total of 5 consensus level variants were identified, in addition to 104 sub-consensus variants of which 22 were shared between 2 or more animals. Further analysis revealed differences in swarm structures from samples derived from the same animal suggesting the presence of distinct viral populations evolving independently at different lesion sites within the same infected animal.

No MeSH data available.