Aa. Khromykh et Eg. Westaway, COMPLETION OF KUNJIN VIRUS-RNA SEQUENCE AND RECOVERY OF AN INFECTIOUSRNA TRANSCRIBED FROM STABLY CLONED FULL-LENGTH CDNA, Journal of virology, 68(7), 1994, pp. 4580-4588
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.