New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses

Abstract

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

Figure 1

5′ untranslated region (A), capsid protein VP1 (B), and complete genome (C) phylogeny of the virus clades studied. Trees were produced by condensing the full phylogeny shown in Technical Appendix 2 Figure 1, panels A, B, and D. Human rhinovirus C′ (HRV-C′) includes the divergent rhinoviruses described in 2007 (13) and a related clinical strain (CL-Fnp5). HRV-C includes the new clade described since 2006 (–14,16). Enterovirus 104 (EV-104) and the related strain CL-1231094 refer to a previously unknown enterovirus clade described in this study. In panel C, HRV-C′ is shown in brackets to indicate its expected location (based on VP1 and 3D sequences). Simian picornavirus 1 (SV2) was used as an outgroup. HEV, human enterovirus. Bootstrap support values <50 are not shown in the trees. New viruses are shown in boldface.

Figure 2

Full genome phylogenetic tree of enterovirus 104 (EV-104), representative strain CL-1231094, and members of the human enterovirus C (HEV-C) species. Human rhinovirus A (HRV-A) (GenBank accession no. DQ473509) was used as outgroup. Coxsackievirus A1 (CV-A1) (AF499635), CV-A21 (AF546702), CV-A20 (AF499642), CV-A17 (AF499639), CV-A13 (AF499637), CV-A11 (AF499636), CV-A19 (AF499641), CV-A22 (AF499643), CV-A24 (D90457), poliovirus 1 (PV-1) (V01148), PV-2 (X00595), and PV-3 (X00925) sequences were obtained from GenBank.

Figure 3

Nearest-neighbor relatedness of rhinovirus CL-013775 (and CL-073908) along the 5′ untranslated region/VP4/VP2 region (A), and nearest-neighbor relatedness of rhinovirus CL-135587 along the complete genome (B), identified by bootscanning. At each position of a sliding window, the solid circles indicate the closest relative within a defined threshold of the phylogenetic distance to CL-013775 (A) and CL-135587 (B). Both panels show phylogenetic trees of analyzed serotypes over the entire scanned region. Human rhinovirus 7 (HRV-7), -9, -10, -11, -24, -32 (accession nos. EU096019, AF343584), -36, -39, -44, -56, -58 (EU096045, AY040236), -59, -67 (EU096054, AF343603, and DQ473505), -76, -88, and -89 sequences were obtained from GenBank (see Technical Appendix 2 Figure 1, for full-length genome accession numbers).