High prevalence of human enterovirus a infections in natural circulation of human enteroviruses

Abstract

Human enterovirus (HEV) infections can be asymptomatic or cause only mild illness; recent evidence may implicate HEV infection in type 1 diabetes mellitus and myocarditis. Here, we report the molecular characterization of HEV obtained in serial monthly collections from healthy Norwegian infants. A total of 1,255 fecal samples were collected from 113 healthy infants beginning at age 3 months and continuing to 28 months. The samples were analyzed for HEV nucleic acid by real-time PCR. Fifty-eight children (51.3%) had HEV infections. One hundred forty-five positive samples were typed directly by nucleotide sequencing of the VP1 region. HEV-A was detected most frequently, with an overall prevalence of 6.8%. HEV-B was present in 4.8% of the samples and HEV-C in only 0.2% of the samples. No poliovirus or HEV-D group viruses were detected. Twenty-two different serotypes were detected in the study period: the most common were EV71 (14.5%), CAV6 (10.5%), CAV4 (8.9%), E18 (8.9%), and CBV3 (7.3%). These findings suggest that the prevalence of HEV infections in general, and HEV-A infections in particular, has been underestimated in epidemiological studies based on virus culture.

FIG. 1.

Rooted phylogenetic trees of CAV4 (A), CAV9 (B), and E18 (C) 3′-end VP1 sequences and available reference sequences and prototypes from GenBank for each of the serotypes. The evolutionary distances were calculated using the Kimura two-parameter model as a model of nucleotide substitution and the neighbor-joining method to reconstruct the phylogenetic tree (MEGA version 3.0 software package). The bootstrap values (the percentage of 1,000 pseudoreplicate data sets) supporting each cluster are shown at the nodes; for clarity, only values of >60% are shown. The outgroup taxa are the prototype strains of the closest serotypes. Sequence names for field strains (in boldface) are constructed as follows: municipality number (starting with 1, in Hordaland county; starting with 0, in Akershus county), month and year of sampling, child identifier, sample number, and serotype. The reference sequences have GenBank accession numbers. The scale bar represents the genetic distance (the number of nucleotide substitutions per site).

FIG. 2.

Three-month moving average of the age-specific prevalences of HEV-A- and HEV-B-positive samples for ages 3 to 23 months (n = 1,237 person-months). The curve was truncated at 24 months, due to few observations (18 stool samples) from 24 to 28 months of age. A logistic regression model was applied to adjust for the potential mutual confounding of age and season or year. Adjusting for season or year indicated that confounding by these factors was minimal or did not change the effect of age in the logistic-regression model.