23] Cloning of viral double-stranded RNA genomes by single primer amplification

Abstract

This chapter describes the cloning of viral double-stranded RNA genomes by single primer amplification. Several cloning strategies have been devised for viruses that possess segmented double-stranded (ds) RNA genomes. The chapter illustrates a circumstance that occurred in a laboratory during the characterization of a virus obtained from a fatal case of gastroenteritis in Bristol, England: the causative agent was later identified as a group C rotavirus. Several groups of rotaviruses are currently recognized based on the antigenic differences in the major inner shell protein and on the characteristic banding pattern of their 11 dsRNA genome segments. The single-primer amplification of viral dsRNA was developed to express the cloning atypical rotaviruses of unknown sequence directly from very small quantities of human fecal specimens. The human stool sample is an extremely hostile environment from which to extract undegraded RNA, but the successful cloning of all 11 genome segments of group C rotavirus RNA suggests that this method may be generally applicable to other dsRNA viruses from a variety of biological sources. The procedure can be divided into the following four sections: (1) purification and analysis of dsRNA from fecal samples; (2) radiolabeling and purification of primer 1; (3) ligation of primer 1 to dsRNA; and (4) cDNA synthesis and amplification by PCR.