Determination of alphaviral titers.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Alphaviruses have also been applied in vaccine development and gene therapy. Before use in vitro or in vivo, it is essential to determine the titer of the generated alphaviral particles. Because defective alphaviruses do not produce plaques, their titers cannot be determined by conventional methods. However, viral titers can be determined readily in cases where the recombinant viruses express reporter genes such as green fluorescent protein or β-galactosidase, as well as indirectly by immunofluorescence methods. The potency of viral stocks can also be evaluated by light microscopic analysis. Alphavirus-infected cells show a dramatic decrease in growth and can be easily distinguished from noninfected control cells through their rounded morphology.