Chapter 5. Virus isolation and identification of measles and rubella in cell culture

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Overview

Although rarely used for diagnosis of measles or rubella due to the lengthy procedure for virus isolation, the ability to successfully produce a viable virus stock from clinical specimens collected from cases of measles or rubella is important for additional testing, including sequence analysis. Occasionally, the viral RNA extracted directly from the clinical specimen provides a positive signal by RT-PCR that is useful for case confirmation (chapter 6) but subsequent efforts to utilize the directly extracted RNA to sequence the region necessary for identification of the genotype prove to be unsuccessful. In addition to providing a source of viral RNA for genotyping, studies such as virus neutralization assays could not be undertaken without the availability of live virus.

The ability to recover virus from clinical specimens and maintain viable virus stocks is also important since a virus sample should be submitted to the measles or rubella virus strain bank if a novel genotype is identified. Also, larger quantities of viral RNA may be required for molecular epidemiologic investigations of outbreaks, particularly those that involve additional gene targets or extended sequencing.

The successful isolation of live virus from clinical specimens depends on the timing and type of specimens as well as other variables that have little or no impact on the ability to detect RNA. For example, a well-timed specimen that was positive by RT-PCR may be unsuitable for virus growth due to bacterial contamination or the presence of substances that are toxic to the cell culture. The optimal timing of collection for the different types of samples for virus isolation is described in detail in chapter 3. In general, clinical specimens that are appropriate for RT-PCR testing for measles and rubella can serve as good sources of virus. Oropharyngeal or nasopharyngeal swabs are recommended for both purposes, and the availability of a urine sample has proven useful as a back-up sample. However, both measles and rubella viruses are difficult to culture from oral fluid (OF). Other types of clinical specimens (e.g., throat swabs) that provide a reasonably good opportunity for successful virus isolation should be collected in addition to OF from representative cases in a chain of transmission.

Note: The abbreviation, RT-PCR, may be used herein to refer to both conventional (end-point) reverse transcription PCR and to real-time (kinetic) RT-PCR, when describing characteristics of RT-PCR in general. Because real-time RT-PCR can be used for quantification of RNA in clinical specimens, real-time RT-PCR is sometimes referred to as quantitative real-time RT-PCR and abbreviated as RT-qPCR. However, in the context of the diagnostic use of RNA detection from measles and rubella samples specifically employing the use of real-time RT-PCR protocols, the term real-time RT-PCR will be used.