Molecular studies on a human common cold virus, human rhinovirus nine.

The molecular cloning of the common cold virus, rhinovirus 9, was achieved using the hybrid (RNA:CDNA) cloning protocol first described by Wood and Lee (1976). Two experiments were required to clone the complete genome; the first was primed by poly dT17 while the second was primed by an oligonucleotide which annealed to a sequence in the P2 region of the genome. Plasmids containing cDNA representative of the rhinovirus 9 genome were identified by in situ hybridisation. Such plasmids were characterised by the restriction endonuclease mapping of their cDNA inserts. This analysis indicated that plasmids containing cDNA derived from the entire 7.1 Kb genome of rhinovirus 9 had been obtained. The cloned rhinovirus 9 cDNAs were sub-cloned into M13 sequencing vectors (Messing et al., 1977) either as restriction endonuclease fragments or as randomly sheared, end-repaired fragments. The nucleotide sequence of these derived sub-clones was determined by the chain termination protocol of Sanger and colleagues (1977). The subsequent analysis and interpretation of the complete nucleotide sequence of rhinovirus 9, and its derived amino acid sequence, was carried out using the computer programs of Staden (1980) and Devereux (1984).