Assay for 5' noncoding region analysis of all human rhinovirus prototype strains

Abstract

Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5' noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).

FIG. 1.

Phylogenetic tree of HRV prototype strains based on analysis of the 5′ NCR. HRVs cluster into genogroups A and B. HRV 87, which is more closely related to enteroviruses, demonstrates closer relationship to ECHO 11 (the outgroup) than other HRVs.

FIG. 2.

Pairwise nucleotide divergence betweeen HRV prototype strains. Percent divergence in increments of 10% was plotted versus the frequency of occurrence. The range was from 0.3 to 63.3%.

FIG. 3.

Phylogenetic tree of HRV group A prototype strains and clinical viral isolates based on 5′ NCR analysis. ECHO 11 was defined as the outgroup. T06-3575 and T05-1169 were more closely related to enterovirus 71 in the 5′ NCR and were identified as enteroviruses.

FIG. 4.

Phylogenetic tree of HRV group B prototype strains and clinical viral isolates based on 5′ NCR analysis. ECHO 11 was defined as an outgroup.

FIG. 5.

Phylogenetic tree of all 101 HRV prototype strains clustering in group A (shown in blue), group B (green), and group C (purple) and clinical viral isolates (red) based on 5′ NCR analysis. Group C strains include T07-1639, T07-2385, T07-0049, and T07-2387 (this study) and strain W37 from Wisconsin (GenBank accession no. E126788). HRVA2 strains (highlighted by an orange arrow) include strain X1 from UCSF (EF077279), strain 026 from Hong Kong (EF582387), strain QPM from Australia (EF186077), and T07-1643 (this study). GAC1 strains (highlighted by a brown arrow) include strain 003 from Hong Kong (EF582386), strain X2 from UCSF (EF077280), and T07-4473 and T07-2103 (this study). GAC2 strains (highlighted by a pink arrow) include strain W38 from Wisconsin (E126789) and T07-4480 (this study). ECHO 11 was defined as an outgroup.