COMPLETION OF KUNJIN VIRUS-RNA SEQUENCE AND RECOVERY OF AN INFECTIOUSRNA TRANSCRIBED FROM STABLY CLONED FULL-LENGTH CDNA

Completion of the Kunjin virus (KUN) RNA sequence showed that it is th e longest flavivirus sequence reported (11,022 bases), commencing with a 5' noncoding region of 96 bases. The 3' noncoding sequence of 624 n ucleotides included a unique insertion sequence of 46 bases adjacent t o the stop codon, but otherwise it had Properties similar to those of RNAs of closely related flaviviruses. A full-length KUN cDNA clone whi ch could be stably propagated in Escherichia coli DH5 alpha was constr ucted; SP6 polymerase RNA transcripts from amplified cDNA were infecti ous when transfected into BHK-21 cells. A mutational change abolishing the BamHI restriction site at position 4049, leading to a conservativ e amino acid change of Arg-175 to Lys in the NS2A protein, was introdu ced into the cDNA during construction and was retained in the recovere d virus. Extra terminal nucleotides introduced during cloning of the c DNA were shown to be present in the in vitro RNA transcripts but absen t in the RNA of recovered virus. Although recovered virus differed fro m the parental KUN by a smaller plaque phenotype and delayed growth ra te in BHK-21 Cells and mice, it was very similar as assessed by severa l other criteria, such as peak titer during growth in cells, infectivi ty titer in cells and in mice, rate of adsorption and penetration in c ells, replication at 39 degrees C, and neurovirulence after intraperit oneal injection in mice. The KUN stably cloned cDNA will provide a use ful basis for future studies in defining and characterizing functional roles of all the gene products.