INFECTIOUS RNA TRANSCRIBED FROM AN ENGINEERED FULL-LENGTH CDNA TEMPLATE OF THE GENOME OF A PESTIVIRUS

Infectious RNA was transcribed for the first time from a full-length c DNA template of the plus-strand RNA genome of a pestivirus, The genome of the C strain, which is a vaccine strain of classical swine fever v irus, was sequenced and used to synthesize the template. The cDNA sequ ence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids, Although there were mostly only small differences betwee n the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3' noncoding region of the C strain, Furthermor e, we showed that the sequences at the 5' and 3' termini of the C stra in are highly conserved among pestiviruses, We found that the infectiv ity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc- 133 depended on the correctness of the nucleotide sequence, The in vit ro transcripts of pPRKflc-133 were infectious, whereas those of pPRKfl c-113 were not, In fact, only 5 amino acids among the complete amino a cid sequence determined this difference in infectivity, However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiat ed from native C-strain virus, Therefore, we exchanged the region enco ding the antigenic N-terminal half of envelope protein E2 in pPRKflc-1 33 with the equivalent region of strain Brescia, The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc- 133 with monoclonal antibodies directed against envelope proteins E(rn s) and E2 of strain Brescia and the C strain, To be suitable for furth er vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus, In fact, we found that FLc -133, hybrid virus FLc-h6, and the C strain grew equally well, We conc luded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the re plication, virulence, cell and host tropism, and pathogenesis of class ical swine fever virus.