Evidence of Recombination and Genetic Diversity in Human Rhinoviruses in Children with Acute Respiratory Infection

International audienceOur 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

High permissiveness for genetic exchanges between enteroviruses of species A, including enterovirus 71, favours evolution through intertypic recombination in Madagascar

International audienceHuman enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains

Characterization of 66 clinical HRV strains in clinical specimens based on partial VP4/VP2 gene sequences.

a<p>Subspecies classification based on local 5′UTR sequence variation (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-t003" target="_blank">Table 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g002" target="_blank">2</a>).</p>b<p>Strains closely related with more than 96% identity.</p>c<p>Strains isolated within 3 months.</p

Characterization of 66 HRV strains in clinical specimens based on sequences in the 5′UTR.

a<p>Subspecies based on phylogenetic analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g002" target="_blank">2</a>) and on local recombination in 5′UTR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g003" target="_blank">Fig. 3</a>).</p>b<p>Strains closely related with more than 93% identity.</p>c<p>These strains clustered differently when based on VP4/VP2 sequences (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g002" target="_blank">2</a>).</p

Comparative study of HRV genomes and individual proteins.

a<p>Strains of this study.</p><p>nt: nucleotides; aa: amino acids; ORF: open reading frame; UTR: untranslated region.</p

Degenerate and specific primers used for RNA amplification from clinical samples.

a<p>Nucleotide position relative to HRV16.</p><p>W = (A/T),H = (A/T/C),D = (G/A/T),Y = (C/T),K = (G/T), R = (A/G),N = (A/G/C/T).</p><p>I:Inosine.</p><p><i>Id</i>: this study.</p

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

<p>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.</p

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

<p>Grouping of HRV reference serotypes (designated R#), previously published novel HRV strains (designated by pink triangles), novel strains from Wisconsin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone.0006355-Lee1" target="_blank">[15]</a> (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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006355#pone-0006355-g001" target="_blank">Figure 1</a>.</p