Improved Detection of Rhinoviruses by Nucleic Acid Sequence-Based Amplification after Nucleotide Sequence Determination of the 5′ Noncoding Regions of Additional Rhinovirus Strains

Abstract

The isothermal nucleic acid sequence-based amplification (NASBA) system was applied for the detection of rhinoviruses using primers targeted at the 5 noncoding region (5 NCR) of the viral genome. The nucleotide sequence of the 5 NCRs of 34 rhinovirus isolates was determined to map the most conserved regions and design more appropriate primers and probes. The assay amplified RNA extracted from 30 rhinovirus reference strains and 88 rhinovirus isolates, it did not amplify RNA from 49 enterovirus isolates and other respiratory viruses. The assay allows one to discriminate between group A and B rhinoviruses. Sensitivities for the detection of group B and group A rhinoviruses was 20 and 200 50% tissue culture infective doses, respectively.Rhinoviruses (RVs) are the most frequent cause of acute upper respiratory tract infections in humans and are usually associated with the common cold (22,24). However, they can also cause lower respiratory tract infections resulting in severe disease in children, in the elderly (17,19,23,26), and in immunosuppressed patients (11,43 P1006, 1999). RVs have been isolated in association with an underlying disease, such as cystic fibrosis (37), and in cases of otitis media (3) and sinusitis (31). In addition, RV infections may exacerbate chronic bronchitis and asthma (9,15,16,28).Andries et al., through evaluation of capsid-binding compounds, classified the RVs into two groups: A and B (2). This classification correlates with sequence similarities.Detection of RVs by tissue culture is slow and cumbersome due to the specific culture conditions required. This limits diagnostic capabilities to a few reference laboratories (29). Serologic diagnosis is virtually impossible because of the existence of more than 100 serotypes.Several studies have shown RT-PCR to be more sensitive than culture for the detection of RVs (1,10,14,15,16,31,33). These assays are based on known sequences of the RV serovars.Nucleic acid sequence-based amplification (NASBA) (Organon Teknika, Boxtel, The Netherlands) might offer an interesting alternative to reverse transcriptase PCR (RT-PCR), because it directly amplifies RNA. It uses the simultaneous enzymatic activities of avian myeloblastosis virus (AMV) RT, RNase H, and T7 RNA polymerase under isothermal conditions. The NASBA technique has already been successfully applied for the detection of human immunodeficiency virus type 1 (HIV-1) RNA (18) We applied NASBA for the detection of RVs. Because the sensitivity of the assay based on the known sequences of RVs was unsatisfactory, we determined the sequence of the 5Ј nocoding regions (NCRs) of 34 additional RV strains, selected new primers and probes, and assessed their specificities and sensitivities for RV strains. MATERIALS AND METHODSVirus strains. The 30 RV reference strains (group A: 4, 6, 13, 14, 17, 26, 27, 43, 45, 52, 69, 70, 72, 84, 86, and 91; group B: 2, 15, 29, 30, 31, 39, 41, 44, 51, 56, 59, 63, 85, 89) were kindly provided by K. Andries, Janssen Research Foundation, Beerse, Belgium (2). RVs were cult...

Detection of Rhinoviruses by Tissue Culture and Two Independent Amplification Techniques, Nucleic Acid Sequence-Based Amplification and Reverse Transcription-PCR, in Children with Acute Respiratory Infections during a Winter Season

“…Similar observations have been reported by others (19). The presence of mismatches between the target RNA and the primers and probes causes an inefficient amplification and detection, as reported previously (8,9,15,26,27). Gobbers et al (15) reported that single point mutations near the 3Ј end of the primers, closely followed by a second mismatch, seem to hamper NASBA amplification.…”

“…In earlier studies, group A RVs were also detected less frequently than group B RVs (1,2,24,26,32). Although at present, a higher number of group B RVs is known than group A RVs, analysis of epidemiological data indicated that group B RVs produced more than twice as many clinical infections per serotype as group A RVs did.…”

“…The primers and probes were described previously (26). One primer mix composed of EG 59 and pd 906 amplified group B RVs, and the amplicons were detected by ECL with the probe combination pd 910, pd 908, and pd 1147.…”

“…Two NAATs are presently available for the detection of RV: nucleic acid sequence-based amplification (NASBA) (26,42) and reverse transcription-PCR (RT-PCR) (13). Both NASBA and RT-PCR have their advantages.…”

Currently used nucleic acid amplification tests for the detection of viruses and atypicals in acute respiratory infections

“…In addition, RNA is the genomic material of numerous respiratory viruses. The application of an RNA-based amplification technique offers potential advantages com- Kidd et al (1996) VA region, variable, subgenera A + RFLP Morris et al (1996) Hexon, 300 bp/243 bp, subgenus C N-A types Tiemessen and Nel (1996) Long-fiber gene, 152 bp, subgenus F A-H Avellon et al (2001) Hexon, 168 bp, polyvalent A Allard et al (2001) Hexon, 301 bp/171, typing N-A-RFLP Rhinovirus Gamma et al (1989) 5 -Non-coding region A Hyypia et al (1989) 5 -Non-coding region A + H Arruda and Hayden (1993) 5 -Non-coding region A Johnston et al (1993) 5 -Non-coding region, 900 bp A + H Santti et al (1997) 5 -Non-coding region, 126/96/533 bp A Samuelson et al (1998) 5 -Non-coding region NASBA, ECL Andeweg et al (1999) 5 -Non-coding region-VP4 N-A-H Steininger et al (2001) 5 -Non-coding region, 106/93 bp N-A Billaud et al (2003) 5 -Non-coding region, rhino v N-A Loens et al (2003a) 5 -Non-coding region NASBA-ECL Deffernez et al (2004) 5 -Non-coding region A pared to a DNA-based amplification technique: no additional reverse transcriptase step is required, thus saving time and reducing the risk of contamination. The specificity of the reactions might, however, be lower because the enzymes used are not thermostable, so that the reaction temperature may not exceed 42 • C without compromising the reaction.…”