The picornavirus 5′ untranslated region (5′ UTR) has proven the most suitable genomic target for molecular detection (8,9). At the time of this study, only 5 AiV complete genomes were available in GenBank, complicating selection of reliable oligonucleotides for universal detection. A nested RT-PCR encompassing the AiV 5′ UTR was developed (Table 1) and used for screening of stool samples collected in northern Germany from outpatients with gastroenteritis. The first subcohort of this collection consisted of 499 patients with gastroenteritis; samples were collected evenly from January through December 2004 in a prospective study on acute community-acquired diarrhea by 47 general practitioners in Bremen, northern Germany (10). The second subcohort consisted of 39 control patients without symptoms of gastroenteritis seen by the same physicians. The third subcohort consisted of 118 patients with diarrhea linked to outbreak scenarios involving canteen food (n = 36), kindergartens (n = 54), or retirement homes (n = 28).

We purified viral RNA from stool samples by using the QIAGEN Viral RNA and DNA Stool Mini Kits (QIAGEN, Hilden, Germany) as described (8); 9 samples were positive for AiV by nested RT-PCR. The 5′ UTR sequences of these viruses were determined and deposited in GenBank (accession nos. GQ927704–GQ927712). With additionally available sequence data, real-time RT-PCR targeting conserved regions of the viral 5′ UTR was developed (Table 1). Assay sensitivity was determined to be ≈1.5 copies per reaction by using photometrically quantified in vitro cRNA transcripts, as described (9).

Retesting of all samples with this highly sensitive assay increased the AiV detection rate, yielding 10 positive samples. All case-patients were part of the subcohort of symptomatic outpatients seen by general practitioners (Figure 1). In this cohort, AiV was detected at a 2.0% rate (10/499 patients). No patients from the control group (n = 39) or from foodborne outbreaks (n = 118) had positive test results for AiV. However, this difference was not statistically significant for any group comparison (Fisher exact test, 2-tailed p>0.05 for all). As shown in Table 2, all AiV-positive patients had abdominal pain, diarrhea, or nausea. No other virus commonly associated with diarrhea was detected in any patient, including norovirus, rotavirus, adenovirus, astrovirus, parechovirus, or enterovirus. A bacterial cause of disease was ruled out by using standard culture methods (10). No clear association with a foodborne etiology, sociologic risk factor (including travel history), or contact with animals was observed. As shown in Table 2, high RNA copy numbers of up to 1.32 × 10¹² per gram of stool (mean 1.32 × 10¹¹, median = 1.82 × 10⁷) were found in patients with positive test results.

Although samples had been collected throughout 2004, all AiV-positive cases occurred during 8 weeks from October to December and originated from a geographically restricted area within the city of Bremen. To verify if this temporal and geographic accumulation of cases represented a point-source outbreak, we amplified and sequenced the entire viral protein (VP) 1 gene, which is commonly used for picornavirus typing, from 9/10 samples (GenBank accession nos. GQ927704–GQ927712). Failure of VP1 amplification in sample D/VI2591 was probably caused by low virus concentration. As shown in Figure 2, a total of 8 samples formed a distinct phylogenetic cluster within AiV genotype B. The first 3 strains, sampled from October 19 through November 10 (Table 2), were almost identical in VP1, with only 1 strain (D/VI2244) diverging by 2 synonymous substitutions. All other samples (November 16–December 7) showed a VP1 nucleotide diversity of up to 0.8% (2–7/864 nt) and an amino acid diversity of up to 1.4% (1–4/288 residues) in comparison to the 3 initially sampled specimens and to each other. Strain D/VI2287, sampled November 8, belonged to AiV genotype A, with nucleotide and amino acid differences of up to 13.2% and 5.4% from the genotype B strains.

For many of the recently described picornaviruses, human pathogenicity is still under study. With the advent of metagenomics, the description of multiple novel viruses can be expected. Although it is generally difficult to generate sufficiently large and appropriately sampled control groups in studies on respiratory and enteric diseases, quantitative data on virus shedding and proof of monocausality can contribute to confirm the link of novel viruses to human disease. For AiV, the high concentrations found in several samples in this study provide support for viral replication in humans. However, virus shedding appeared unrelated to the severity of symptoms, and clinical presentations were generally mild. Contrary to the findings of previous studies from France and Tunisia (5,6), no food association could be observed in this study. In agreement with some, but not all, published reports (5,11), no case of AiV-associated gastroenteritis from this study had apparent co-infections, further supporting AiV pathogenicity in humans. The overall 2.0% detection rate of AiV in stool samples from outpatients with gastroenteritis is compatible with detection rates in recent studies from several European and Asian countries (5,12). Surprisingly, AiV infection affected all age groups. This was in sharp contrast to parechoviruses and cardioviruses detected predominantly in patients <6 years of age in the same study cohorts (8,9) and indicated different modes of transmission and maintenance of these genetically related picornaviruses at the population level. Similarly, the lower AiV infection rate described here was consistent with the 51.0% seroprevalence rate described in German infants <2 years of age (13) compared with >75.0% described for cardioviruses and parechoviruses (14,15).

The geographic and temporal accumulation of cases, together with the observed sequence variation, supports locally and temporally restricted circulation of AiV with human-to-human transmission, rather than a point-source epidemic pattern as observed in foodborne infections. Fecal–oral human-to-human transmission would be facilitated by the high fecal virus concentrations in some patients. Our data indicate that AiV can be considered an authentic human pathogen that can be transmitted from human to human.