A novel bocavirus associated with acute gastroenteritis in Australian children

Abstract

Acute gastroenteritis (AGE) is a common illness affecting all age groups worldwide, causing an estimated three million deaths annually. Viruses such as rotavirus, adenovirus, and caliciviruses are a major cause of AGE, but in many patients a causal agent cannot be found despite extensive diagnostic testing. Proposing that novel viruses are the reason for this diagnostic gap, we used molecular screening to investigate a cluster of undiagnosed cases that were part of a larger case control study into the etiology of pediatric AGE. Degenerate oligonucleotide primed (DOP) PCR was used to non-specifically amplify viral DNA from fecal specimens. The amplified DNA was then cloned and sequenced for analysis. A novel virus was detected. Elucidation and analysis of the genome indicates it is a member of the Bocavirus genus of the Parvovirinae, 23% variant at the nucleotide level from its closest formally recognized relative, the Human Bocavirus (HBoV), and similar to the very recently proposed second species of Bocavirus (HBoV2). Fecal samples collected from case control pairs during 2001 for the AGE study were tested with a bocavirus-specific PCR, and HBoV2 (sequence confirmed) was detected in 32 of 186 cases with AGE (prevalence 17.2%) compared with only 15 controls (8.1%). In this same group of children, HBoV2 prevalence was exceeded only by rotavirus (39.2%) and astrovirus (21.5%) and was more prevalent than norovirus genogroup 2 (13.4%) and adenovirus (4.8%). In a univariate analysis of the matched pairs (McNemar's Test), the odds ratio for the association of AGE with HBoV2 infection was 2.6 (95% confidence interval 1.2-5.7); P = 0.007. During the course of this screening, a second novel bocavirus was detected which we have designated HBoV species 3 (HBoV3). The prevalence of HBoV3 was low (2.7%), and it was not associated with AGE. HBoV2 and HBoV3 are newly discovered bocaviruses, of which HBoV2 is the thirdmost-prevalent virus, after rotavirus and astrovirus, associated with pediatric AGE in this study.

Figure 1. Phylogenetic analysis of parvoviruses: Maximum likelihood dendrogram (bootstrapped, 1,000 replicates) based on full-length parvovirus genome sequence (nucleotide) alignments.

Approximately 100–200 nucleotides are absent from each termini of the HBoV2, HBoV3, and HBoV sequences.

Figure 2. The HBoV2 and HBoV3 genomes.

Diagrammatic representation of HBoV2 (HBoV2-W153, 5204 nucleotides) and HBoV3 (HBoV3-W471, 5242 nucleotides) sequence showing the position of open reading frames for NS1, NP1, VP1, and VP2, compared with other bocaviruses: bovine parvovirus 1 (BPV 1), minute virus of canines (MVC), and human bocavirus (HBoV). For both HBoV2 and HBoV3, as with HBoV, the NP1 gene is in an alternate reading frame to VP1 and overlaps the start of VP1 by 13 nucleotides. Similarly, VP2 is collinear to VP1 and results from initiation of translation at a downstream ATG and co-terminates.

Figure 3. Genome similarity plot of HBoV3-W471 against HBoV (black line, DQ000495) and HBoV2-W153 (grey line) showing percentage similarity along the aligned genomes (SimPlot 3.2, 2-parameter (Kimura) distance model, window size is 300 bp, step size is 30 bp).

Approximate recombination sites are indicated by the crossover of the sequence most similar to HBoV3-W471 at positions 200 and 3,000 nucleotides, just upstream of NS1 and VP1/2 ORFs; highlighted by the two arrows. Note the high level of conservation between all three genomes toward the 3′ terminus of NS1 and the 5′ terminus of VP1/2 ORFs, presumably the result of functional constraint.

Figure 4. Maximum likelihood dendrogram (bootstrapped, 1,000 replicates) of HBoV, HBoV2, and HBoV3 partial NS1 sequences.