Laboratory Detection of Rhinoviruses


This method is sensitive for the detection of many, but not all, RhV strains because some RhV genotypes will not grow well or at all in the cell lines normally used for cell culture in the clinical laboratory.1,32


As


enteroviruses grow on the same cell lines and produce similar cytopathic effects, differentiation between RhV and enteroviruses in cell culture is determined by optimal cultivation temperature (33°C for RhV versus 37°C for enteroviruses) and the acid lability test. RhV are acid labile; they are inactivated by transient exposure to pH 3 medium, while enteroviruses are not. However, these tests are not always accurate and misclassification can occur. Confirmation of RhV isolates by neutralization is not feasible due to the large number of serotypes and lack of a commercial source for hyperimmune sera. However, a recent paper described the identification of RhV isolates using two commercial fluorescent monoclonal antibody pools that were developed to identify enterovirus isolates.33


One antibody pool cross-reacted with RhV and


one did not. Staining suspected RhV infected cells with each antibody provided a rapid means to distinguish RhV from enterovirus isolates.


Serological testing of acute and convalescent sera has been used for epidemiologic studies of RhV infections34,35


but is not practical for


clinical laboratory detection. Similarly, antigen detection assays using fluorescent antibody (FA) methods or rapid immunochromatographic methods similar to those for influenza and respiratory syncytial virus, which can be performed more rapidly than cell culture, are not practical due to the large number and antigenic variability of RhV subtypes.7


RhV


do not contain a common group antigen that would be useful for generating diagnostic antibodies.36


Molecular Detection Methods


In the past few years, many nucleic acid amplification tests (NAAT), including conventional reverse transcription (RT)-polymerase chain reaction (PCR),5,27,37–48


realtime RT-PCR,49–57 sequence-based amplification (NASBA)58 and nucleic acid assays, have been developed


to detect the presence of RhV RNA in clinical specimens. These molecular methods, which detect RhV RNA instead of viable virus, not only provide results more quickly than culture, but are significantly more sensitive than cell culture.4,5,8,10–13,44,53,59


In fact, many RhV strains


present in clinical specimens were not previously detected by culture.1 The increased use of NAAT for the detection of RhV in clinical specimens has helped to elucidate the disease burden and epidemiology of RhV infections, revealing a much larger prevalence and range of RhV infections than previously recognized using cell culture methods.5,25,27,31


NAAT are being used with increasing frequency


by clinical diagnostic laboratories. The sensitivity and specificity of RhV detection by NAAT depends on careful primer and probe design and validation. Primer and probe design should include detection of all RhV genotypes including the newly identified RhV C subtypes. The challenge for the development of NAAT for RhV detection is to design primers that will specifically amplify all of the diverse RhV subtypes (groups A, B, and C) while exhibiting no cross-reactivity with other similar members of the picornavirus family. For this reason, most RhV primer sets target the viral 5´ non-coding region (NCR, 650–750 nucleotides), which contains the most sequence homology among RhV subtypes. However, this region is also highly conserved among the enteroviruses and differentiation of RhV from enteroviruses may also require an additional step after amplification to distinguish the amplicons.


US RESPIRATORY DISEASE


As RhV are RNA viruses, the first step in most molecular assays is reverse transcription of the viral RNA into complementary DNA (cDNA). For PCR assays, this can be performed as a separate reaction from the amplification (two-step method) or included with the amplification reaction (one-step method). The use of a commercial one-step RT-PCR master mix has advantages over using a two-step method in that it is easier and faster to use and reduces the risk of sample-to-sample contamination. The choice of master mix is important when evaluating new assays. Some commercial mixes perform better than others in combination with specific primer and probe sets.54,60


This is especially


true for RT-PCR reactions, in which the choice of reverse transcriptase enzyme may be important for efficient cDNA synthesis. The choice of a realtime sequence detection instrument is not as critical, and RT-PCR reactions previously evaluated on one instrument can usually be readily adapted to another instrument.


Conventional Reverse Transcription-polymerase Chain Reaction and Nucleic Acid Sequence-based Amplification There are many published examples of conventional, end-point RT-PCR35,37,39,40–43,48


and nested RT-PCR amplification assays5,38,45–47


for RhV


detection. Following reverse transcription and amplification, identification of amplicons has been accomplished gel electrophoresis to determine amplicon size5,37,38,46,47 polymorphism (RFLP) analysis,42 probes,35,40,41,43,45


sequencing,25,39,48 and use of mass tags.27,61


and restriction fragment length hybridization with specific Detection


using probes is usually more sensitive than by gel and increases the specificity of the assay. Amplicons can be hybridized to probes in solution or immobilized on a solid phase in microwell plates or microarrays. An example of RhV detection using a microarray is the Virochip, which is a panviral DNA chip platform using probes that have conserved sequences for all known viruses of humans, animals, plants, and microbes. Novel RhV genotypes were recently detected using this assay.62,63


amplicon detection has been reported.58


A NASBA assay using molecular beacon probes for The MassTag assay uses PCR


primers tagged with unique molecules to label the amplicons. After amplification, the molecules were released via a photolabile link and detected by mass spectrometry to identify 20 PCR products.61 This method has been useful for identifying novel RhV sequences.27,29 Sequencing of amplicons, especially those generated using primers targeting an RhV coding region, is a good method for determining RhV genotypes.


Realtime Reverse Transcription-polymerase Chain Reaction


Realtime RT-PCR, in which the amplicons are detected as they are synthesized, for RhV detection includes the use of TaqMan probes,49,51–53,55–57


molecular beacon probes,54 and SYBR Green.50 These


assays are usually more rapid than conventional PCR, especially those using a one-step RT-PCR method, and by combining amplification and amplicon detection in the same tube the risk of amplicon contamination during the post-PCR step is eliminated.


Commercial Multiplex Detection with Other Respiratory Viruses


No commercial assays are available for only the detection of RhV. However, molecular detection of RhV by the clinical laboratory is often


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