RT-PCR ASSAYS USED TO IDENTIFY NEW HUMAN RHINOVIRUSES

Background: Human rhinoviruses (HRVs) commonly cause mild upper respiratory tract infections and have recently been associated with more severe lower respiratory tract disease, especially in young children.  Together with human enteroviruses (HEVs), HRVs are classified within the family Picornaviridae, and are further divided into two species (A and B) and 100 recognized serotypes.   Molecular typing of HRVs has become a practical alternative to type-specific neutralization and was recently used to describe a novel group of HRVs provisionally designated group “C”.   However, no fully validated standard method for genotyping HRVs has been described.  The strong correlation between serotype and VP1 capsid protein coding sequences has permitted successful molecular typing of HEVs.  The recent availability of full-length VP1 sequences for all HRV prototype strains offers the opportunity to develop similar assays for typing HRVs.    

Objectives: 1) To develop and validate molecular typing assays for HRVs based on RT-PCR and sequencing of a partial region of the VP1 coding region; 2) to use these assays to identify new HRVs. 

Methods:   Two RT-PCR assays based on COnsenseus DEgenerate Hybrid Oligo-nucleotide Primers (CODEHOP) designed to semi-conserved VP1 protein motifs of species A and B HRVs were developed.  Quantified RNA transcripts were prepared from representative HRV species A (1B) and B (14) serotypes for analytical sensitivity studies.

Amplification products from 65 recently acquired HRV field isolates, 88 respiratory specimens culture positive for HRV and 29 clinical specimens from asthma patients were sequenced and the predicted amino acid sequences aligned with those from published prototype strains using CLUSTAL W.  Phylograms were constructed using the Neighbor Joining distance method implemented in the program PAUP*. 

Results:  The RT-PCR assays amplified all HRV prototype strains and reproducibly detected 10 copies per reaction of representative HRV species A and B RNA transcripts. All field isolates and HRV positive clinical specimens were successfully amplified and sequenced. Three specimens showed evidence of coinfection between HRV A and B strians. Except for 10 HRVs, all others representing 65 types showed close genetic relationship with reference prototype strains (median amino acid sequence identity score 97.2%, range 88.3% to 100%).  Sequences from 2 species A viruses were highly divergent from their closest prototypes (73.0% and 78.2%, respectively), and likely represent new serotypes. Eight sequences formed a clade phylogenetically distinct from both HRV species A and B (38.5% to 49.8% and 29.3% to 35.8% to the closest matches from HRV A and B, respectively), that were closely related to a recently described group “C” HRVs, and showed substantial diversity within the group itself.

Conclusion:  Assays based on RT-PCR amplification and partial VP1 sequencing permit rapid and sensitive genotyping of HRVs and offer a practical tool for expanded study of HRVs in human disease.  Using these assays, we identified at least 2 new species A HRVs and confirmed identification of new group “C” HRVs, greatly expanding the number of HRVs predicted by traditional typing methods.