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Detection of newly described astrovirus MLB1 in stool samples from children.

Finkbeiner SR, Le BM, Holtz LR, Storch GA, Wang D - Emerging Infect. Dis. (2009)

Bottom Line: The prevalence of the recently identified astrovirus MLB1 in a cohort of children with diarrhea in St. Louis, Missouri, USA, was defined by reverse transcription-PCR.Of 254 stool specimens collected in 2008, 4 were positive for astrovirus MLB1.These results show that astrovirus MLB1 is circulating in North America.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The prevalence of the recently identified astrovirus MLB1 in a cohort of children with diarrhea in St. Louis, Missouri, USA, was defined by reverse transcription-PCR. Of 254 stool specimens collected in 2008, 4 were positive for astrovirus MLB1. These results show that astrovirus MLB1 is circulating in North America.

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Related in: MedlinePlus

Phylogenetic analysis of astrovirus MLB1 (AstV-MLB1) isolates. A region of the serine protease (A) and the capsid (B) of each virus detected by the AstV-MLB1–specific primers was amplified and sequenced. Multiple sequence alignments were then generated with these sequences and the corresponding regions of known astroviruses using ClustalX (www.clustal.org). PAUP* (Sinauer Associates, Sunderland, MA, USA) was used to generate phylogenetic trees; bootstrap values (>700) from 1,000 replicates are shown. The previously identified AstV-MLB1 isolate (9,10) and the isolates from this study are shown in boldface. Scale bars indicate number of amino acid substitutions per site.
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Figure 2: Phylogenetic analysis of astrovirus MLB1 (AstV-MLB1) isolates. A region of the serine protease (A) and the capsid (B) of each virus detected by the AstV-MLB1–specific primers was amplified and sequenced. Multiple sequence alignments were then generated with these sequences and the corresponding regions of known astroviruses using ClustalX (www.clustal.org). PAUP* (Sinauer Associates, Sunderland, MA, USA) was used to generate phylogenetic trees; bootstrap values (>700) from 1,000 replicates are shown. The previously identified AstV-MLB1 isolate (9,10) and the isolates from this study are shown in boldface. Scale bars indicate number of amino acid substitutions per site.

Mentions: Of 254 stool specimens screened, 9 (3.5%) tested positive in the initial round of screening that used the newly designed pan-astrovirus primers, SF0073 and SF0076. Secondary screening showed that 5 (2% of all samples) were canonical human astroviruses. This probably underestimates the prevalence of the astrovirus serotype 1–8 in the cohort because the initial screening primers were biased toward detection of AstV-MLB1. The remaining 4 (1.6% of all samples) were positive for AstV-MLB1 using primers SF0053 and SF0061. For each of the 4 samples positive for AstV-MLB1, 2 additional fragments were generated by RT-PCR for phylogenetic analysis. A 1,228-bp fragment of ORF1a, which encodes the serine protease, and a 920-bp fragment of ORF2, which encodes the capsid proteins, were amplified using AstV-MLB1–specific primers from each of the 4 samples, designated WD0016, WD0055, WD0104, and WD0227. The primers used for the ORF1a fragment are SF0080 (5′-AAGGATAGTGCTGGTAAAGTAGTTCAGA-3′) and SF0094 (5′-CAAGAGCCTTATCAACAACGTA-3′) and the primers used for the ORF2 fragment are SF0064 (5′-GTAAGCATGGTTCTTGTGGAC-3′) and SF0098 (5′-TGCATACATTTATGCTGGAAGA-3′). The ORF1a fragments (GenBank accession nos. FJ227120–FJ227123) from these samples all shared ≈92% nt identity to the reference astrovirus MLB1 sequence (GenBank accession no.: FJ222451) and 99% aa identity, indicating that most mutations were synonymous. The ORF2 fragments (GenBank accession nos. FJ227124–FJ227127) shared ≈91%–92% nt identity and 95%–96% aa identity to the reference astrovirus MLB1 sequence. The 4 positive St. Louis samples shared ≈99% nt identity to each other. The ORF1a and ORF2 sequences were aligned to other astroviruses for which full genome sequences were available using ClustalX version 1.83 (www.clustal.org); maximum-parsimony trees were generated using PAUP with 1,000 bootstrap replicates (12) (Figure 2). The entire genome of one of the isolates, WD0016 (GenBank accession no. FJ402983), was sequenced and had 92.6% identity overall to that of AstV-MLB1 on the basis of a pairwise nucleotide alignment (Table 1).


Detection of newly described astrovirus MLB1 in stool samples from children.

Finkbeiner SR, Le BM, Holtz LR, Storch GA, Wang D - Emerging Infect. Dis. (2009)

Phylogenetic analysis of astrovirus MLB1 (AstV-MLB1) isolates. A region of the serine protease (A) and the capsid (B) of each virus detected by the AstV-MLB1–specific primers was amplified and sequenced. Multiple sequence alignments were then generated with these sequences and the corresponding regions of known astroviruses using ClustalX (www.clustal.org). PAUP* (Sinauer Associates, Sunderland, MA, USA) was used to generate phylogenetic trees; bootstrap values (>700) from 1,000 replicates are shown. The previously identified AstV-MLB1 isolate (9,10) and the isolates from this study are shown in boldface. Scale bars indicate number of amino acid substitutions per site.
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Related In: Results  -  Collection

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Figure 2: Phylogenetic analysis of astrovirus MLB1 (AstV-MLB1) isolates. A region of the serine protease (A) and the capsid (B) of each virus detected by the AstV-MLB1–specific primers was amplified and sequenced. Multiple sequence alignments were then generated with these sequences and the corresponding regions of known astroviruses using ClustalX (www.clustal.org). PAUP* (Sinauer Associates, Sunderland, MA, USA) was used to generate phylogenetic trees; bootstrap values (>700) from 1,000 replicates are shown. The previously identified AstV-MLB1 isolate (9,10) and the isolates from this study are shown in boldface. Scale bars indicate number of amino acid substitutions per site.
Mentions: Of 254 stool specimens screened, 9 (3.5%) tested positive in the initial round of screening that used the newly designed pan-astrovirus primers, SF0073 and SF0076. Secondary screening showed that 5 (2% of all samples) were canonical human astroviruses. This probably underestimates the prevalence of the astrovirus serotype 1–8 in the cohort because the initial screening primers were biased toward detection of AstV-MLB1. The remaining 4 (1.6% of all samples) were positive for AstV-MLB1 using primers SF0053 and SF0061. For each of the 4 samples positive for AstV-MLB1, 2 additional fragments were generated by RT-PCR for phylogenetic analysis. A 1,228-bp fragment of ORF1a, which encodes the serine protease, and a 920-bp fragment of ORF2, which encodes the capsid proteins, were amplified using AstV-MLB1–specific primers from each of the 4 samples, designated WD0016, WD0055, WD0104, and WD0227. The primers used for the ORF1a fragment are SF0080 (5′-AAGGATAGTGCTGGTAAAGTAGTTCAGA-3′) and SF0094 (5′-CAAGAGCCTTATCAACAACGTA-3′) and the primers used for the ORF2 fragment are SF0064 (5′-GTAAGCATGGTTCTTGTGGAC-3′) and SF0098 (5′-TGCATACATTTATGCTGGAAGA-3′). The ORF1a fragments (GenBank accession nos. FJ227120–FJ227123) from these samples all shared ≈92% nt identity to the reference astrovirus MLB1 sequence (GenBank accession no.: FJ222451) and 99% aa identity, indicating that most mutations were synonymous. The ORF2 fragments (GenBank accession nos. FJ227124–FJ227127) shared ≈91%–92% nt identity and 95%–96% aa identity to the reference astrovirus MLB1 sequence. The 4 positive St. Louis samples shared ≈99% nt identity to each other. The ORF1a and ORF2 sequences were aligned to other astroviruses for which full genome sequences were available using ClustalX version 1.83 (www.clustal.org); maximum-parsimony trees were generated using PAUP with 1,000 bootstrap replicates (12) (Figure 2). The entire genome of one of the isolates, WD0016 (GenBank accession no. FJ402983), was sequenced and had 92.6% identity overall to that of AstV-MLB1 on the basis of a pairwise nucleotide alignment (Table 1).

Bottom Line: The prevalence of the recently identified astrovirus MLB1 in a cohort of children with diarrhea in St. Louis, Missouri, USA, was defined by reverse transcription-PCR.Of 254 stool specimens collected in 2008, 4 were positive for astrovirus MLB1.These results show that astrovirus MLB1 is circulating in North America.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology and Microbial Pathogenesis Program, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The prevalence of the recently identified astrovirus MLB1 in a cohort of children with diarrhea in St. Louis, Missouri, USA, was defined by reverse transcription-PCR. Of 254 stool specimens collected in 2008, 4 were positive for astrovirus MLB1. These results show that astrovirus MLB1 is circulating in North America.

Show MeSH
Related in: MedlinePlus