A Diverse Group of Previously Unrecognized Human Rhinoviruses Are Common Causes of Respiratory Illnesses in Infants

Human rhinoviruses (HRVs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious.To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples). Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group ("C"). None of these new strains could be cultured in traditional cell lines.By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group

Identification of culturable HRV clinical isolates by phylogenetic tree reconstruction.

a)<p>Four isolates clustered with P1-P2 reference sequence with highly significant bootstrap values (96%), and one with lower but still significant bootstrap value (62%).</p

Phylogenetic tree depicting the relationships among all 101 HRV serotypes in the P1-P2 region of 5'NCR.

<p>This tree was constructed using the neighbor joining method according to the distances (divergences) between all pairs of sequences in a multiple alignment. The confidence of sequence clustering was evaluated by bootstrapping (1000 replicates). Only significant bootstrap values (>500) were shown. The scale bar (top left) represents the genetic distance (nucleotide substitutions per site). The 101 HRV serotypes (R) clustered into 2 previously defined groups: HRVA and HRVB with a perfect bootstrap value (1000). HRVA group had 76 serotypes and HRVB, 25 serotypes.</p

Detection of known HRV serotypes in clinical specimens.

a)<p>The first 19 serotypes are HRVA. HRV14, 27, 52 and 83, are HRVB.</p>b)<p>Three additional HRVs clustered with HRV89 reference sequence with insignificant bootstrap values (42–50%).</p

Phylogenetic tree depicting the relationships among 101 HRV serotypes (R) and 26 new strains (W) in the P1-P2 region of 5'NCR.

<p>This tree was generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000966#pone-0000966-g003" target="_blank">Figure 3</a>. None of the new strains clustered with HRVB viruses. Seventeen new strains (blue) belonged to HRVA group, and 9 strains (red) cluster into a new group (“C”) that is separate from groups A and B.</p

Distribution of pairwise nucleotide divergence values between 101 HRV serotypes.

<p>The horizontal axis shows the value of divergence (%) in pairwise comparisons and the column height indicates the frequency of observations. Divergence values were calculated as distance value×100%.</p

Schematic representation of the first 1100 base of a HRV genome showing the locations of the highly conserved regions (P1, P2 and P3) and variable region between P1 and P2 (P1-P2 in red) at the 5'NCR and the PCR fragments used in this study.

<p>P1, P2 and P3 are located at bases 163–181, 443–463 and 535–551, respectively in HRV16 genome. PCR fragment A (about 900bps) was used to determine the 5'NCR sequences of all 101 HRV serotypes. It was amplified using pan-HRV PCR forward primer P1-1, which anneals to conserved region P1, and a serotype-specific reverse primer annealed to the 5' end of VP2 gene (between base# 1000 and 1100). PCR fragment B (about 390 bps) was generated with pan-HRV PCR forward primer P1-1 and reverse primer P3-1. PCR fragment C (about 300 bps) was generated with forward primer P1-1 and an equimolar mixture of reverse primers P2-1, P2-2 and P2-3. The variable sequences of P1-P2 were used for the molecular typing assay.</p

Phylogenetic tree depicting the relationships between 9 HRVC strains (W in red) and all known HEV (n = 74) in the P1-P2 region of 5'NCR. HEV include polioviruses (PV), coxsackieviruses A (CA), coxsackieviruses B (CB), echoviruses (E) and the newer numbered EVs.

<p>For 5 serotypes (CA16, CA24, CB4, CB5 and EV71), 2 different P1-P2 sequences were found in GenBank. This tree was generated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000966#pone-0000966-g003" target="_blank">Figure 3</a>. HRVC and HEV clustered into 2 different groups.</p

Detection of novel HRV strains in clinical specimens.

<p>Detection of novel HRV strains in clinical specimens.</p