Prevalence, types, and RNA concentrations of human parechoviruses, including a sixth parechovirus type, in stool samples from patients with acute enteritis

Abstract

Parechovirus epidemiology and disease association are not fully understood. Real-time reverse transcriptase PCR (RT-PCR) for all human parechoviruses (HPeV) was applied on stool samples from two groups of patients. Both groups contained patients with acute enteritis of all age groups, seen during one full year. Patients with norovirus, adenovirus, enterovirus, astrovirus, or rotavirus infections were excluded. In 118 patients from outbreak and hospital settings, no HPeV was detected. In a prospective study group of 499 nonhospitalized patients, the detection rate was 1.6%. One virus-positive patient was detected from 39 control patients. Positive samples occurred only in summer and autumn. Only one patient had accompanying respiratory symptoms. An association with travel or animal contact was not found. All positive patients except one were <2 years of age, with a neutral gender ratio. In children <2 years of age, the detection rate was 11.6% (7 of 60 children). The range of viral loads was 3,170 to 503,377,290 copies per gram or milliliter of stool. One of the highest viral loads occurred in a control child without symptoms at the time of testing. Phylogenetic analysis showed mainly contemporary HPeV1 strains in our patients but also showed a separate new lineage of HPeV1 in evolutionary transition from the historical prototype strain. Moreover, a novel sixth HPeV type was identified. Full genome analysis of the two viruses revealed recombination between HPeV1 and -3 in one and HPeV6 and -1 in another. HPeV seems relevant in children <2 years and specific RT-PCR for HPeV should be included in enteritis screening.

FIG. 1.

Nucleic acid alignment of the hybridization sites of diagnostic real-time RT-PCR oligonucleotides. Oligonucleotides are shown below the alignment panel. The base count in the top line is based on HPeV1 reference strain Harris, which also serves as the comparison sequence in the alignment. Dots represent identical bases in compared sequences; deviations are spelled out. A slash (/) represents a gap in the alignment; (rc) means that the reverse complementary sequence is shown for the antisense primer. Note that almost all nucleotide mismatches in this design are stable G:T non-Watson-Crick base pairs (23, 32). FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine.

FIG. 2.

Age distribution of patient cohorts tested in this study. The y axis in each panel shows patient age, the x axis shows patient count. (A) Patients tested in the routine diagnostic laboratory. Children in this cohort were mostly hospitalized; adults were mostly tested in the context of investigations of food-borne enteritis outbreaks or in outbreaks in retirement homes. (B) Outpatients seen for enteritis by practitioners. Samples in both cohorts were pretested negative for astro-, rota-, adeno-, and noroviruses. Arrows point to patients tested positive for parechovirus by real-time RT-PCR. (C) Nonenteritis control patients for the study cohort shown in panel B.

FIG. 3.

Phylogenetic analysis of P1 protein regions (amino acids 76 to 773; isolate Harris), analyzed using the p-distance substitution model. VP1-based HPeV types are shown next to clades on the right margin. Analysis was conducted in MEGA4 (27). The evolutionary histories were inferred using the neighbor-joining method (24). Relevant bootstrap values from 500 replicate trees are shown next to the branches (10). The scale shows the evolutionary distance from each root (trees are drawn to scale). GenBank accession numbers: BNI-67, EU022171; BNI-R90, EU024630; BNIR4, EU024631; BNI-R9, EU024632; BNI-R15, EU024633; BNI-R21, EU024634; BNI-R30, EU024635; and BNI-R32, EU024636. Strain BNI-788st (EF051629) is not described in this study and will be described in more detail separately.

FIG. 4.

Recombination analysis carried out with SimPlot software (19), using a 600-bp sliding window. Window's middle position is given on the x axis. Prototype strains used for comparison are shown in the inserts in each window. (A) Similarity plot analysis. The y axis shows percent nucleic acid identities in the sliding analysis window. (B) Bootscan analysis. Bootstrapped phylogenesis is performed in a sliding window. The y axis shows for each of the prototype strains the percentage of permuted trees in which the prototype strain clusters with strain BNI-67.