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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

Astrovirus open reading frame (ORF) 1b alignments for design of pan-astrovirus primers. Astrovirus RNA polymerase sequences (ORF1b) were aligned at the amino acid level to define the conserved regions used for the design of primers SF0073 (A) and SF0076 (B). The numbers to the right of the sequences indicate the position of the last amino acid within each ORF1b sequence. Red boxes represent the specific regions that were reverse translated into the corresponding nucleic acid sequences used for the design of SF0073 (C) and SF0076 (D). Red sequences shown in the nucleotide alignments are the actual primer sequences.
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Figure 1: Astrovirus open reading frame (ORF) 1b alignments for design of pan-astrovirus primers. Astrovirus RNA polymerase sequences (ORF1b) were aligned at the amino acid level to define the conserved regions used for the design of primers SF0073 (A) and SF0076 (B). The numbers to the right of the sequences indicate the position of the last amino acid within each ORF1b sequence. Red boxes represent the specific regions that were reverse translated into the corresponding nucleic acid sequences used for the design of SF0073 (C) and SF0076 (D). Red sequences shown in the nucleotide alignments are the actual primer sequences.

Mentions: We identified conserved regions by using multiple sequence alignments of AstV-MLB1 amino acid sequences to all fully sequenced astrovirus genomes (Figure 1, panels A and B). The corresponding nucleotide sequences for these regions were then aligned to define the most highly conserved regions (Figure 1, panels C and D). Two regions within open reading frame (ORF) 1b were identified that yielded primers SF0073 (5′-GATTGGACTCGATTTGATGG-3′) and SF0076 (5′-CTGGCTTAACCCACATTCC-3′), which are predicted to generate an ≈409-bp product. Control experiments validated this primer pair could detect AstV-MLB1, as well as human astrovirus 1 (Appendix Figure). Given that some of the canonical human astroviruses are identical in the primer binding sites, these data suggest that at least some of the canonical human astroviruses can be detected by the primer pair SF0073/SF0076. In theory, under appropriate experimental conditions, these primers also may be able to detect all other known human and animal astroviruses, although that remains to be experimentally tested. These primers were used with the QIAGEN One-Step RT-PCR Kit (QIAGEN, Valencia, CA, USA) by using the following cycling conditions: 30 min RT step, 94ºC for 10 min, followed by 40 cycles of 94ºC for 30 s, 52ºC for 30 s, and 72ºC for 50 s.


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)

Astrovirus open reading frame (ORF) 1b alignments for design of pan-astrovirus primers. Astrovirus RNA polymerase sequences (ORF1b) were aligned at the amino acid level to define the conserved regions used for the design of primers SF0073 (A) and SF0076 (B). The numbers to the right of the sequences indicate the position of the last amino acid within each ORF1b sequence. Red boxes represent the specific regions that were reverse translated into the corresponding nucleic acid sequences used for the design of SF0073 (C) and SF0076 (D). Red sequences shown in the nucleotide alignments are the actual primer sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666294&req=5

Figure 1: Astrovirus open reading frame (ORF) 1b alignments for design of pan-astrovirus primers. Astrovirus RNA polymerase sequences (ORF1b) were aligned at the amino acid level to define the conserved regions used for the design of primers SF0073 (A) and SF0076 (B). The numbers to the right of the sequences indicate the position of the last amino acid within each ORF1b sequence. Red boxes represent the specific regions that were reverse translated into the corresponding nucleic acid sequences used for the design of SF0073 (C) and SF0076 (D). Red sequences shown in the nucleotide alignments are the actual primer sequences.
Mentions: We identified conserved regions by using multiple sequence alignments of AstV-MLB1 amino acid sequences to all fully sequenced astrovirus genomes (Figure 1, panels A and B). The corresponding nucleotide sequences for these regions were then aligned to define the most highly conserved regions (Figure 1, panels C and D). Two regions within open reading frame (ORF) 1b were identified that yielded primers SF0073 (5′-GATTGGACTCGATTTGATGG-3′) and SF0076 (5′-CTGGCTTAACCCACATTCC-3′), which are predicted to generate an ≈409-bp product. Control experiments validated this primer pair could detect AstV-MLB1, as well as human astrovirus 1 (Appendix Figure). Given that some of the canonical human astroviruses are identical in the primer binding sites, these data suggest that at least some of the canonical human astroviruses can be detected by the primer pair SF0073/SF0076. In theory, under appropriate experimental conditions, these primers also may be able to detect all other known human and animal astroviruses, although that remains to be experimentally tested. These primers were used with the QIAGEN One-Step RT-PCR Kit (QIAGEN, Valencia, CA, USA) by using the following cycling conditions: 30 min RT step, 94ºC for 10 min, followed by 40 cycles of 94ºC for 30 s, 52ºC for 30 s, and 72ºC for 50 s.

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