Reviews and feature article
Analysis of the complete genome sequences of human rhinovirus

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Human rhinovirus (HRV) infection is the cause of about one half of asthma and chronic obstructive pulmonary disease exacerbations. With more than 100 serotypes in the HRV reference set, an effort was undertaken to sequence their complete genomes so as to understand the diversity, structural variation, and evolution of the virus. Analysis revealed conserved motifs, hypervariable regions, a potential fourth HRV species, within-serotype variation in field isolates, a nonscanning internal ribosome entry site, and evidence for HRV recombination. Techniques have now been developed using next-generation sequencing to generate complete genomes from patient isolates with high throughput, deep coverage, and low costs. Thus relationships can now be sought between obstructive lung phenotypes and variation in HRV genomes in infected patients and potential novel therapeutic strategies developed based on HRV sequence.

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HRV taxonomy and genomic features

The taxonomic classification of HRV has been recently revised (http://www.ictvonline.org). Previously, HRVs were placed in the family Picornaviridae, genus Rhinovirus, and each serotype was considered a species (eg, hrv-16 and hrv-28). The current taxonomy maintains HRV in the Picornaviridae family, but the genus is Enterovirus, with 3 species: HRV-A, HRV-B, and HRV-C. Within each species there are multiple HRVs (variably designated as “serotypes,” “types,” or “strains”). Of note, the original

Sequencing the reference set of HRVs

Our initial approach for sequencing the ATCC reference set of HRVs was to devise universal primers that might be suitable for PCR amplification along the genomes for all the HRVs from the ATCC, as well as field samples. The original primers were designed from the 8 full-genome sequences that were available at that time and from limited sequence fragments of other HRVs. However, this approach failed to produce conditions that consistently provided amplicons for sequencing, and therefore instead

Structure-based alignment of RNA and amino acid sequences

An accurate alignment of the RNA sequences and the translated amino acid sequences is necessary to begin to define the commonalities and differences between the serotypes. The alignment is also critical for ascertaining phylogenetic relationships. We thus chose to use a molecular structure–based approach for the alignments so as to maximize potential structure/function relationships and phylogenetic inferences. For the proteins, the initial fits were by means of superimposition of the amino

5′ UTR sequence analysis

A number of intriguing findings came from comparative analysis of the 5′ UTR sequences from the 99 HRV-A and HRV-B and 7 HRV-C complete genomes. As was expected, we found the 5′ terminal portion of the 5′ UTR modeled into a cloverleaf for each HRV (Fig 3, A). The configuration of the cloverleaf appeared to be quite similar between species. In contrast, a pyrimidine-rich spacer tract just 3′ to the cloverleaf had a different nucleotide sequence for each HRV serotype from the reference set, and

Coding sequence comparisons

The amino acid identities between any 2 hrvs is shown in matrix form in Fig 5. Readily apparent are the high degrees of identity between members of a given species (eg, orange and pink regions) and the lower identities when comparing across species (gray areas). It is also clear that there is heterogeneity within species, as represented by “islands” of discontinuity in the color scheme. This suggested that certain HRVs might cluster together in terms of relatedness and perhaps function.

Recombination between HRVs

The nature of the diversity of HRVs was further explored by examining the potential for recombination. Unexpectedly, we found highly statistically significant evidence for recombination, as shown in Fig 7. In this example hrv-53 and hrv-80 each contributed portions of their genomes to hrv-46. As can be seen, there is a high degree of identity between hrv-80 and hrv-46 in the 5′ portions of the genomes up to approximately 3,600 b, which represents most of the 5′ UTR, VP4, VP2, VP3, and VP1. The

Field isolates

Although we had full-genome sequences from only 10 field samples recently obtained from patients (nasal secretions), it was useful to see the variations between these genomes and the analogous HRVs represented by the ATCC samples, given that they differed by more than 30 years in collection times. From our phylogenetic analysis (Fig 6), it was readily apparent that each field isolate was identifiably close to one of the reference serotypes (see hrv-89-f09, hrv-89-f08, and hrv-89 at the bottom

Implications for obstructive lung disease and future directions

The results discussed in this review provide opportunities to explore basic questions about HRV biology and the link between HRV infection and asthma or COPD pathophysiology. First, we now have a scaffold (the alignments and phylogeny) to integrate additional strains as they are identified into the 3 (or 4) well-defined HRV species. Furthermore, the pipeline methods have been developed to rapidly sequence the complete genomes of HRVs obtained from patient samples. Therefore the diversity of HRV

Concluding remarks

In summary, the complete reference set of HRVs has now been sequenced at the full-genome level. The results provide new insights into HRV's evolution and diversity and suggest levels of relatedness that might provide for signatures for asthma or COPD exacerbations. Structural predictions from the genomes might provide for novel therapeutics targeted to subsets of HRVs based on specific sequences.

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      Finally, this study only incorporated the commonly sequenced 5’UTR and VP4/VP2 gene regions into the analysis. Translation of the approximately 7 kb RV genome produces a single polyprotein, which is cleaved to form mature capsid (VP1-4) and replication (2A-C, 3A-D) peptides (Palmenberg, Rathe et al. 2010). Studies have shown that the 5’UTR, which are common among species within the Enterovirus genus, have significant impacts on viral pathogenesis (Kawamura, Kohara et al. 1989; Guest, Pilipenko et al. 2004); however, differences in clinical manifestation are also attributed to other translatable peptides and genome regions, including the 3’UTR (Merkle, Van Ooij et al. 2002).

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    Series editors: Joshua A. Boyce, MD, Fred Finkelman, MD, William T. Shearer, MD, PhD, and Donata Vercelli, MD

    Supported by National Institutes of Health grants HL091490 and AI070503 and National Institute of Allergy and Infectious Diseases contract HHSN272200900009C.

    Terms in boldface and italics are defined in the glossary on page 1191.

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