Screening respiratory samples for detection of human rhinoviruses (HRVs) and enteroviruses: comprehensive VP4-VP2 typing reveals high incidence and genetic diversity of HRV species C

Abstract

Rhinovirus infections are the most common cause of viral illness in humans, and there is increasing evidence of their etiological role in severe acute respiratory tract infections (ARTIs). Human rhinoviruses (HRVs) are classified into two species, species A and B, which contain over 100 serotypes, and a recently discovered genetically heterogeneous third species (HRV species C). To investigate their diversity and population turnover, screening for the detection and the genetic characterization of HRV variants in diagnostic respiratory samples was performed by using nested primers for the efficient amplification of the VP4-VP2 region of HRV (and enterovirus) species and serotype identification. HRV species A, B, and C variants were detected in 14%, 1.8%, and 6.8%, respectively, of 456 diagnostic respiratory samples from 345 subjects (6 samples also contained enteroviruses), predominantly among children under age 10 years. HRV species A and B variants were remarkably heterogeneous, with 22 and 6 different serotypes, respectively, detected among 73 positive samples. Similarly, by using a pairwise distance threshold of 0.1, species C variants occurring worldwide were provisionally assigned to 47 different types, of which 15 were present among samples from Edinburgh, United Kingdom. There was a rapid turnover of variants, with only 5 of 43 serotypes detected during both sampling periods. By using divergence thresholds and phylogenetic analysis, several species A and C variants could provisionally be assigned to new types. An initial investigation of the clinical differences between rhinovirus species found HRV species C to be nearly twice as frequently associated with ARTIs than other rhinovirus species, which matches the frequencies of detection of respiratory syncytial virus. The study demonstrates the extraordinary genetic diversity of HRVs, their rapid population turnover, and their extensive involvement in childhood respiratory disease.

FIG. 1.

Diversity of HRV variants detected in the study. The phylogeny of the VP4-VP2 sequences amplified from strains from the study subjects and the results of a comparison of those sequences with those from reference strains of species A and B or with other published sequences from species C of currently unassigned types are shown (for clarity, sequences showing less than 0.5% divergence in the analyzed region were excluded from the tree). For phylogeny estimation, neighbor-joining trees were constructed by using maximum-composite-likelihood distances estimated between sequences in the amplified region (positions 629 to 1063 numbered according to the HRV-B14 reference sequence [GenBank accession number NC_001490]). Data were bootstrap resampled 100 times to assess the robustness of the branches; values of 70% or greater are shown. All trees were drawn to the same scale; for species A and C variants, HRV-B (GenBank accession number NC_001490) was used to root the trees; sequence HRVACG (serotype 1B; GenBank accession number D00329) was used to root the species B tree.

FIG. 2.

VP4-VP2 and VP1 phylogenies of putative new serotypes and the most closely related HRV-A serotypes. A comparison of the phylogenies in the VP4-VP2 and VP1 regions of the putative new serotypes, NS1 and NS2, and the most closely related HRV-A serotypes is shown. The symbols correspond to those used in Fig. 1.

FIG. 3.

Pairwise distances between and within HRV-A and HRV-B serotypes and identification of the divergence threshold in species C. The distributions of the pairwise distances between the available VP4-VP2 sequences (between positions 629 and 958) of HRV-A (n = 447), HRV-B (n = 141), and HRV-C (n = 220) variants are shown (sequences were downloaded from the GenBank database on 12 May 2009). The inter- and intraserotype (shaded light and dark gray, respectively) distance ranges for species A and B were calculated by using reference sequences of assigned serotypes (14, 25, 29, 36). The dotted line shows the upper limit of the intraspecies divergence in HRV-A and HRV-B and a provisionally assigned threshold of 0.1 for the currently unclassified HRV-C variants. For all three species, the y-axis scale for intraserotype comparisons (inferred in the case of HRV-C) was expanded fivefold for clarity.

FIG. 4.

Epidemiological characteristics of HRV- and HEV-infected subjects. Frequencies of detection of HRV and HEV in the two sampling months (proportion of samples) (A), between males and females (proportion of subjects; the sex of one study subject was unknown) (B), and in different age bands (proportion of subjects) (C) are shown. The y axis depicts the proportion of samples or subjects positive from the total in each category. m, months.

FIG. 5.

Proportion of subjects under the age of 10 years infected with HRV-A, HRV-B, HRV-C, RSV, and AdV with recorded symptoms or diagnoses of acute respiratory disease or those treated on critical care wards (intensive therapy, high-dependency, and neonatal units). The numbers at the tops of the bars are the total number of subjects in each category; these were used as the denominators to calculate the HRV detection frequencies (recorded on the y axis).