Real-time RT-PCR and semi-nested RT-PCR assays were developed, targeting regions in ORF1b and ORF2 of AstV-VA1, respectively. All six samples were tested with both assays (Table ). Four independent nucleic acid extractions of each sample were prepared. Each extraction of samples B, C, and F was unequivocally positive in both assays, with threshold cycle (Ct) values in the real-time RT-PCR assay ranging from 18 to 20, suggesting that a high copy number of Ast-VA1 was present in those samples. The other three samples were intermittently weakly positive in the semi-nested RT-PCR assay (A, 1/4 extractions; D, 3/4 extractions; E, 1/3 extractions) and in the real-time RT-PCR assay (A, 1/4 extractions; D and E, 3/4 extractions). For the real-time RT-PCR assay, in the instances where these three samples were positive, the Ct values were near the limit of detection, ranging from 34 to 42. These results suggest that samples A, D, and E may contain very low copy numbers of AstV-VA1 RNA, which may explain the variation in results for the four independent nucleic acid extractions. Negative controls included on each run were all negative. The 250-bp amplicon generated by the semi-nested PCR assay was confirmed as AstV-VA1 in all samples by sequencing.
Despite the availability of improved molecular diagnostic methods for an increasing panel of gastroenteritis agents in humans, the etiology of 12 to 41% of the outbreaks of gastroenteritis remains unexplained (13
). In this study, we identified a novel astrovirus (AstV-VA1) in fecal samples from an outbreak of acute gastroenteritis. Complete genome sequencing and phylogenetic analysis demonstrated that AstV-VA1 was highly divergent from all previously described astroviruses, including the eight human astrovirus serotypes and the recently described astrovirus MLB1 (AstV-MLB1) (6
). The discovery of AstV-VA1 following the recent identification of AstV-MLB1 clearly demonstrates that a much greater diversity of astroviruses exists in humans than was previously recognized.
The detection of AstV-VA1 at high copy numbers in three out of six samples (and potentially at very low levels in the other three samples) from this outbreak suggests a potential association between AstV-VA1 and diarrheal illness. However, because of the limited number of samples available for analysis in this cluster, further studies defining the frequency of detection of AstV-VA1 in samples from individuals with and without acute gastroenteritis are needed to define the role of AstV-VA1 in human diarrheal disease.