Complete coding sequence characterization and comparative analysis of the putative novel human rhinovirus (HRV) species C and B

Abstract

Background: Human Rhinoviruses (HRVs) are well recognized viral pathogens associated with acute respiratory tract illnesses (RTIs) abundant worldwide. Although recent studies have phylogenetically identified the new HRV species (HRV-C), data on molecular epidemiology, genetic diversity, and clinical manifestation have been limited.

Result: To gain new insight into HRV genetic diversity, we determined the complete coding sequences of putative new members of HRV species C (HRV-CU072 with 1% prevalence) and HRV-B (HRV-CU211) identified from clinical specimens collected from pediatric patients diagnosed with a symptom of acute lower RTI. Complete coding sequence and phylogenetic analysis revealed that the HRV-CU072 strain shared a recent common ancestor with most closely related Chinese strain (N4). Comparative analysis at the protein level showed that HRV-CU072 might accumulate substitutional mutations in structural proteins, as well as nonstructural proteins 3C and 3 D. Comparative analysis of all available HRVs and HEVs indicated that HRV-C contains a relatively high G+C content and is more closely related to HEV-D. This might be correlated to their replication and capability to adapt to the high temperature environment of the human lower respiratory tract. We herein report an infrequently occurring intra-species recombination event in HRV-B species (HRV-CU211) with a crossing over having taken place at the boundary of VP2 and VP3 genes. Moreover, we observed phylogenetic compatibility in all HRV species and suggest that dynamic mechanisms for HRV evolution seem to be related to recombination events. These findings indicated that the elementary units shaping the genetic diversity of HRV-C could be found in the nonstructural 2A and 3D genes.

Conclusion: This study provides information for understanding HRV genetic diversity and insight into the role of selection pressure and recombination mechanisms influencing HRV evolution.

Figure 1

Phylogenetic analysis illustrating genetic relationships between HRV species based on sequence alignment of 6 complete coding sequences amplified from our study (black triangle) compared with all known HRV prototypes. The neighbor-joining phylogenetic tree was constructed using Kimura's two-parameter with 1,000 bootstrap replicates using the MEGA4 program. Evolutionary distance was represented by the scale bar in the unit of nucleotide substitutions per site. The selected HRV strain name in this study refers to number of specimen and patient's admission month and year.

Figure 2

Complete coding sequence similarity plot illustrating pairwise sequence identity between HRV-CU072 compared with the most closely related Chinese strain (N4; green line) and other HRV members (HRV-C024; yellow line, HRV-76; blue line, HRV-35; gray line). Constructed using SimPlot v3.2 with Jukes-Cantor parameter, window size of 400 bp and a step size of 20 bp, and 1,000 bootstrap replicates.

Figure 3

A Bootscanning plot of recombination between the daughter strain HRV-CU211 and major (HRV-35) or minor (HRV-69) parental strains. Recombination breakpoint was predicted to occur at the ORF's nucleotide positions 766-1,590 covering partial VP2 and VP3 capsid encoding genes. Bootstrapping support value was computed using the RDP3 program with a window size of 200 bp, step size of 10 bp, and 1,000 bootstrap replicates.

Figure 4

Phylogenetic compatibility matrices of HRV species A, B, and C. Multiple sequence alignments of all known HRV prototypes including 6 identified sequences derived from our study were individually performed using TreeOrderScan program (Simmonds and Smith, 1999). The numbers of phylogeny violation are color coded corresponding to an incompatibility frequency score of pairwise fragment comparison.

Figure 5

Phylogenetic analysis based on deduced amino acid sequences of VP1-3 and 3D viral proteins of 6 identified strains compared with previously published prototypes. Two new strains, HRV-CU072 and HRV-CU211, derived from our study are denoted by a black arrow. CU211 resulted from recombination between HRV-35 (major parent) and HRV-69 (minor parent). Tree constructions based on neighbor-joining method with 1,000 replicates.

Figure 6

Average G+C composition percentage along the ORF of HRVs and HEV. Each viral gene was depicted in relation to ORF arrangement. Average values were computed from multiple sequence alignments of representative serotypes or strains with each species by using 500 bp sliding window and 10 bp increment size. Standard deviation (SD) value of each species' representative data was represented by the shaded area.