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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...