AMPLIFICATION OF THE FULL-LENGTH HEPATITIS-A VIRUS GENOME BY LONG REVERSE TRANSCRIPTION-PCR AND TRANSCRIPTION OF INFECTIOUS RNA DIRECTLY FROM THE AMPLICON

The genetic study of RNA viruses is greatly facilitated by the availab ility of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of ''long PCR'' to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to th e 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length ampli con of approximate to 7.5 kb. Further, since we engineered a T7 promot er in the sense primer, RNA could be transcribed directly from the amp licon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both th e HAV cDNA and the transcribed RNA, replicating HAV was detected by im munofluorescence microscopy and, following passage to other cell cultu res, by focus formation. The recovered virus displayed the cytopathic effect and targe plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcript ion-PCR procedure and demonstrates the potential of this method for pr oviding cDNAs of viral genomes and simplifying the construction of inf ectious clones.