Detection of Newly Described Astrovirus MLB1 in Stool Samples from Children

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.


The Study
Pediatric stool specimens sent for bacterial culture to the clinical microbiology laboratory at the St. Louis Children's Hospital were analyzed for AstV-MLB1. The Human Research Protection Offi ce of Washington University in St. Louis approved this study. Samples were collected during January through May 2008. Stools were diluted in phosphate-buffered saline at a 1:6 ratio (wt/vol), and total nucleic acid was extracted from 200 μL of each stool sus-pension by the MagNAPure LC Automated Nucleic Acid Extraction System (Roche, Indianapolis, IN, USA).
Previously described astrovirus primers Mon269 and Mon270 (11) frequently have been used for detecting human astrovirus serotypes 1-8 in clinical stool specimens. However, the extensive divergence of AstV-MLB1 to the known human astroviruses rendered these primers unable to amplify AstV-MLB1 (data not shown). Because AstV-MLB1 might represent a new grouping of astroviruses that could include multiple subtypes, we designed primers to conserved regions of the AstV-MLB1 genome to maximize the likelihood of detecting any AstV-MLB1 variant viruses or even other novel astroviruses.
We identifi ed 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 defi ne the most highly conserved regions ( Figure 1, panels C and D). Two regions within open reading frame (ORF) 1b were identifi ed 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 (online Appendix Figure, available from www.cdc.gov/EID/content/15/3/441-appF. htm). 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.
Samples that tested positive with primers SF0073 and SF0076 were then tested in a second round of screening with 2 different primer sets in parallel to determine whether the samples contained canonical human astrovirus serotypes 1-8 or AstV-MLB1. The previously reported Mon269 (5′-CAACTCAGGAAACAGGGTGT-3′) and Mon270 (5′-TCAGATGCATTGTCATTGGT-3′) primers, which generate a 449-bp amplicon, were used to detect canonical human astroviruses (11). Another set of primers, SF0053 (5′-CTGTAGCTCGTGTTAGTCTTAACA-3′) and SF0061 (5′-GTTCATTGGCACCATCAGAAC-3′), was designed to exclusively detect AstV-MLB1 and produce a 402-bp PCR product. These primers target a region of the capsid gene. The second round of screening with both sets of primer pairs was performed as described above except that an annealing temperature of 56ºC was used.
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 amplifi ed using AstV-MLB1-specifi c 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 Clust-alX 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).
Patients with AstV-MLB1-positive stools ranged in age from ≈4 months to 4 years ( Table 2). All patients had symptoms of diarrhea at stool collection, except the patient with isolate WD0016, who reported having diarrhea 2 days before stool collection. All specimens were tested for Escherichia coli, Campylobacter spp., Yersinia spp., Shigella spp., and Salmonella spp. by standard bacterial culture. The WD0227 sample tested positive for E. coli O157:H7; the other samples were negative for all bacterial cultures. A pan-viral microarray, the ViroChip (GEO platform GPL 3429; National Center for Biotechnology Information, Bethesda, MD, USA) (13), was used to examine whether other viruses were present in the stool of 3 (WD0055, WD0104, and WD0227) of the 4 AstV-MLB1-positive samples for which enough material remained for analysis. WD0055 and WD0104 were negative by array, but WD0227 was positive for rotavirus as determined by the ViroChip.

Conclusions
The newly identifi ed AstV-MLB1 virus was discovered in a stool specimen collected in Melbourne, Victoria, Australia, in 1999. In this study, we describe the detection of AstV-MLB1 in a cohort from St. Louis collected in 2008. This observation provides evidence of AstV-MLB1 outside Australia and suggests that AstV-MLB1 is likely to be globally widespread. In addition, these data demonstrate that AstV-MLB1 is circulating in the human population. The sequence divergence of ≈8% at the nucleotide level between the reference AstV-MLB1 genome and the viruses detected in this study suggests substantial sequence heterogeneity within the AstV-MLB1 group of viruses. Multiple serotypes or subtypes of AstV-MLB1 might exist, as with the canonical human astroviruses. More extensive screening of stool samples with PCR primers targeted toward detection of AstV-MLB1, such as those described here, may provide insight into the true diversity and prevalence of AstV-MLB1-like viruses. Finally, a critical direction for future investigation is determining whether AstV-MLB1, like the canonical astrovirus serotypes 1-8, is a causal agent of human diarrhea, and if so, assessing the extent and severity of disease associated with this virus. Further epidemiologic studies, including both case-control prevalence studies and seroprevalence assays, and efforts to fulfi ll Koch's postulates should be pursued.