Evidence of recombination and genetic diversity in human rhinoviruses in children with acute respiratory infection

Abstract

Background: Human rhinoviruses (HRVs) are a highly prevalent cause of acute respiratory infection in children. They are classified into at least three species, HRV-A, HRV-B and HRV-C, which are characterized by sequencing the 5' untranslated region (UTR) or the VP4/VP2 region of the genome. Given the increased interest for novel HRV strain identification and their worldwide distribution, we have carried out clinical and molecular diagnosis of HRV strains in a 2-year study of children with acute respiratory infection visiting one district hospital in Shanghai.

Methodology/findings: We cloned and sequenced a 924-nt fragment that covered part of the 5'UTR and the VP4/VP2 capsid genes. Sixty-four HRV-infected outpatients were diagnosed amongst 827 children with acute low respiratory tract infection. Two samples were co-infected with HRV-A and HRV-B or HRV-C. By comparative analysis of the VP4/VP2 sequences of the 66 HRVs, we showed a high diversity of strains in HRV-A and HRV-B species, and a prevalence of 51.5% of strains that belonged to the recently identified HRV-C species. When analyzing a fragment of the 5' UTR, we characterized at least two subspecies of HRV-C: HRV-Cc, which clustered differently from HRV-A and HRV-B, and HRV-Ca, which resulted from previous recombination in this region with sequences related to HRV-A. The full-length sequence of one strain of each HRV-Ca and HRV-Cc subspecies was obtained for comparative analysis. We confirmed the close relationship of their structural proteins but showed apparent additional recombination events in the 2A gene and 3'UTR of the HRV-Ca strain. Double or triple infections with HRV-C and respiratory syncytial virus and/or bocavirus were diagnosed in 33.3% of the HRV-infected patients, but no correlation with severity of clinical outcome was observed.

Conclusion: Our study showed a high diversity of HRV strains that cause bronchitis and pneumonia in children. A predominance of HRV-C over HRV-A and HRV-B was observed, and two subspecies of HRV-C were identified, the diversity of which seemed to be related to recombination with former HRV-A strains. None of the HRV-C strains appeared to have a higher clinical impact than HRV-A or HRV-B on respiratory compromise.

Figure 1. Phylogenetic tree depicting relationships between known and novel HRVs based on VP4/VP2 gene analysis.

Grouping of HRV reference serotypes (designated R#), previously published novel HRV strains QPM from Australia (GenBank accession no. E186077), NAT001 and NAT045 from California (GenBank accession no. EF077237 and EF077281), and C024, C025, and C026 from Hong Kong (GenBank accession no. EF582385, EF582386, EF582387) designated by a pink triangle, and viral strains detected in our clinical samples designated N# and by open and filled circles, was based on 420 nt in the VP4/VP2 gene region. Strains fully sequenced in this study are designated by a pink star. HRV-A, HRV-B and HRV-C are drawn with red, blue and green colors, respectively. Strains classified in HRV-Cc and HRV-Ca subspecies are indicated by filled and open circles, respectively. Echo 11 and R87 prototypes are included as outgroups. Tree construction and bootstrap values indicated for each major branch in the tree were determined with PHYLIP package and SEQBOOT, with 1000 replicates.

Figure 2. Phylogenetic tree depicting relationships between known and novel HRVs based on 5’UTR analysis.

Grouping of HRV reference serotypes (designated R#), previously published novel HRV strains (designated by pink triangles), novel strains from Wisconsin (designated W#), and novel viral strains detected in our clinical samples (designated by N# and circles) was based on 285 nt in the 5’UTR. Strains fully sequenced in this study are designated by a pink star. See additional legend in Figure 1.

Figure 3. Analyses of recombination events in 5’UTR-VP2 partial sequences of HRVs.

Bootscanning analysis (A) and pairwise identity (B) of N25 strain (HRV-Ca) with other strains representative of HRV-A (R16) and HRV-B (R52) species, and HRV-Cc (N10) subspecies. C: Diagram of 5’UTR-VP2 sequences of strains from HRV-Ca subspecies indicating approximate sizes and sites of recombination between HRV-A (red) and HRV-Cc (black) strain sequences.

Figure 4. Analyses of recombination events in N4 isolate full-length genomic sequence.

A: Result of manual bootscanning of N4 genome against several viruses. B: Pairwise identity of N4 with several viruses. Grey boxes indicate possible recombination sites.

Figure 5. Phylogenetic tree based on the 3’ terminal part of the viral genomes (nt 6650 to end, according to N4 numbering).

Dark triangles represent HRV-A and HRV-B sequences clustering together.