Improved molecular typing assay for rhinovirus species A, B, and C

Abstract

Human rhinoviruses (RVs), comprising three species (A, B, and C) of the genus Enterovirus, are responsible for the majority of upper respiratory tract infections and are associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types necessitate regular updating of the diagnostic assays for the accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5' untranslated region were improved to increase assay sensitivity and to eliminate nonspecific amplification of human sequences, which are observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes for seminested PCR assays provided higher specificity and sensitivity for virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignments for RV-C isolates, based on phylogenetic analysis of partial VP4/VP2 coding sequences. The improved assay was evaluated by typing RVs in >3,800 clinical samples. RVs were successfully detected and typed in 99% of the samples that were RV positive in multiplex diagnostic assays.

FIG 1

Locations of primers used for detection and typing of RV. (A) Schematic representation of the previously published (9) and modified diagnostic primers in the virus genome. (B) Secondary structure of the 5′ UTR and cis-acting replication element (cre) of RV-C15 (GenBank accession number GU219984) predicted using the Mfold web server (38) with mapped primer annealing sites. Major stem-loop domains are numbered 1 to 6 below the structure, and the open reading frame (ORF) start codon (position 608) is indicated. Published primers are shaded, and modified primer annealing sites are underlined, boxed (position 529), or shown in bold (VP2-C252-5r). (+), forward primer; (−), reverse primer.

FIG 2

Nonspecific amplification product (A) and consensus sequences of RV-A, RV-B, and RV-C reference strains and clinical isolates in the primer annealing sites in 5′ UTR (B) and VP2 (C). Alignment positions correspond to the RV-C15 sequence (GenBank accession number GU219984). RV-C 5′-UTR sequences are divided into Ca and Cc subgroups. Human RNAB2/Chr6 sequences (GenBank accession numbers GQ497714 and NT007592) matching RV sequences in the 5′ UTR are shaded. Degenerate nucleotide positions are designated by the standard ambiguity code and shown in bold. Reverse primers are shown as reverse complementary sequences to match the alignments. Annealing sites of the original primers targeting the 5′ UTR (B) and novel primers targeting the 5′ UTR and VP2 (C) are boxed.

FIG 3

Elimination of nonspecific amplification of human sequences in the RV 5′-UTR assay. (A) Effect of exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT) treatment of the first PCR products on nonspecific amplification of human sequences, using the original (P3) or modified (5′UTR-rev) primers in the first and/or second PCR. (B) Effect of the reverse primer in the first PCR on detection of RV-A, RV-B, and RV-C isolates and nonspecific amplification of human sequences (−). (C and D) Comparison of the original and modified reverse primers in the first PCR in testing of a set of clinical samples. A total of 17 clinical samples with high human RNA concentrations were amplified with P3 (C) generating nonspecific product or 5′UTR-rev (D) primers in the first PCR. Products of the second PCR using the modified primers are shown in panels B to D. L, 1-kb Plus DNA ladder (Life Technologies).

FIG 4

Sensitivity and specificity of the VP4/VP2 PCR assay for detection and molecular typing of RV-C. (A) Amplicons obtained after the first (left) or second (right) PCR with serial 10-fold dilutions (10⁵ PFU to 10⁻¹ PFU equivalents) of purified RV-C15. (B) Test of primer specificity using clinical samples containing RV-A, RV-B, or RV-C isolates. (C) PCR amplification of a larger set of RV-C and novel RV-A isolates from clinical samples confirms VP4/VP2 assay sensitivity and specificity. *, potentially novel RV-A types (W22, W48, and W49 isolates) that do not match any of the reference types. L, 1-kb Plus DNA ladder (Life Technologies). Products of the second PCR are shown in panels B and C.

FIG 5

Neighbor-joining phylogenetic trees based on partial 5′UTR and VP4/VP2 nucleotide sequences of selected RV-C reference types and newly identified or putative recombinant Wisconsin (W) types, constructed using MEGA 5.1 software. All major nodes are labeled with bootstrap values (% of 500 replicates). Branch lengths are proportional to nucleotide similarity (p-distance). RV-A16 and RV-B14 were included to represent RV-A and RV-B clusters, respectively, and poliovirus (PV-3l) was included as an outgroup. Accession numbers for the 52 W types have been published (6, 9); GenBank accession numbers for the newly identified W types (shown in bold) are KF695389 to KF695402. W type numbers are followed by corresponding RV-C type designations (in parentheses) based on VP4/VP2 partial sequences (http://www.picornastudygroup.com/types/enterovirus/hrv-c.htm) except for the four putative recombinant W types (underlined), which are followed by the closest reference 5′-UTR sequence type in the VP4/VP2 tree and by the closest reference VP4/VP2 sequence type in the 5′-UTR tree, to show incongruent clustering.

FIG 6

Nucleotide sequence alignments of three recent RV isolates (A to C) and the W14 type (D) with putative artificial recombinant 5′-UTR sequences and parental reference strains. Potential recombination sites are boxed and shaded.

FIG 7

Workflow for the modified RV molecular typing assay. Novel RV isolates required VP1 or full-genome sequencing for definitive type assignment.