Infectious cDNA clone of the RA27/3 vaccine strain of Rubella virus

Rubella virus (RUB), a small plus-strand RNA virus, is a significant human pathogen. The RA27/3 vaccine strain of RUB is one of the most successful li ve attenuated vaccines developed. In this article, we report the constructi on of an RA27/3 infectious clone, a complete cDNA copy of the RA27/3 genome that can be transcribed in vitro to generate infectious RNA molecules. Vir us generated from such in vitro transcripts was phenotypically similar to R A27/3 virus. To investigate the attenuation of the RA27/3 strain, a series of chimeras was made by the insertion of different fragments of the RA27/3 genome into an infectious clone based on the Therien wild-type strain of RU B. Analysis of the resulting chimeric viruses revealed that the pattern of RA27/3 attenuation in cell culture is complex: attenuating elements in the RA27/3 genome were found in the 5' untranslated region (UTR), a region of t he nonstructural proteins containing the protease motif and the capsid gene . Within the 5' UTR, the attenuation determinant was mapped to nt 7 Surpris ingly, these analyses also revealed a potentiating mutation at nt 164 of th e RA27/3 genome. Although this determinant was within the coding sequences of the nonstructural proteins, the encoded amino acid had no effect on cell culture phenotype and thus the determinant may operate at the level of RNA structure. In addition to investigation of the mechanisms of RA27/3 attenu ation, the availability of the RA27/3 infectious clone offers the opportuni ty for strict genetic control over RUB vaccine manufacturing, for developme nt of novel DNA-based vaccines against RUB, and for development of recombin ant RUB vaccines that also target other diseases. (C) 2000 Academic Press.