Analysis of the complete genome sequences of human rhinovirus

Abstract

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.

Figure 1

Organization of the HRV genome. The genome consists of 5′- and 3′UTRs, and a single open reading frame encoding one protein, which is subsequently cleaved to form 11 viral proteins which act to form the capsid (VP4-VP1) or are required for viral replication.

Figure 2

Representative HRV sequence coverage by the sequence-independent random primer method. Each horizontal line represents contiguous sequence, ranging in size from ~50 to ~1000 bases. The vertical component represents the extent of redundancy for any given nucleotide or genome segment. The horizontal component represents the extent of the coverage of the genome.

Figure 3

Variability of the 5′UTR pyrimidine-rich spacer tract in the HRV genome. Shown are representative modelings of the 5′UTR from the first base to ~130 bases, revealing a cloverleaf secondary structure motif followed by the spacer tract (A). This spacer tract, which is immediately 3′ to the cloverleaf, is highly variable between the serotypes (B). As indicated by the red boxes which compares the reference hrv-52 to the field sample hrv-52-f10, the tract is also variable within-serotype.

Figure 4

Internal ribosome entry sites (IRES) for HRVs. Minimal energy secondary structure models were constructed for 5′UTRs of each HRV sequence. The 3′ region of each IRES (bases ~100 to ~625) forms an unbranched stem of varying length and composition. However, the statistically lowest energy conformation invariably pairs the last AUG of the IRES (light green box) with the ORF AUG (dark green box), as part of an HRV-specific mechanism to facilitate ribosome entry. Shown are representative stems from the three recognized species (HRV-A, -B, and -C) and clade D.

Figure 5

Amino acid identity heat map of HRVs. Shown are comparisons between all the reference HRV-A and -B, and five HRV-C. For clarity, the specific HRVs are not shown. The scale of identity ranges from ~45% (dark grey) to 100% (yellow). The diagonal set of yellow blocks represents the comparison of the same two HRVs. The dashed line shows the sequence identity comparison between hrv-32 and all other HRVs. Heterogeneity within-species, shown by blocks and islands of discontinuity in the identities, suggests that certain HRVs are clustered based on composition. See also the phylogenetic tree of Fig 6.

Figure 6

Phylogeny of HRV as illustrated in a Neighbor-Joining tree. Three human enteroviruses (HEV-C: coxackie virus-a21 and -a13, and poliovirus-1m) were utilized for outgroup rooting of the tree. Individual serotypes with an “f” designation represent a field isolate obtained from a patient. The numbers at various branch points are bootstrap values indicating the confidence of the branching (100 is maximal).

Figure 7

HRV recombination has resulted in emergence of a third HRV. Shown is an example where hrv-53 and hrv-80 (parental strains) contributed sequence to generate the daughter strain hrv-46. The Y-axis represents the identity between a given pair of indicated hrvs at any nucleotide (X-axis) along the genome.