AN INFECTIOUS CLONE OF HUMAN PARAINFLUENZA VIRUS TYPE-3

A full length clone of the human parainfluenza virus type 3 (HPIV-3) g enome (called pHPIV-3) was constructed, and recombinant, infectious HP IV-3 was generated by transfecting pHPIV-3 and support plasmids encodi ng the HPIV-3 NP, P, and L proteins into HeLa cells infected with a va ccinia virus recombinant which expresses T7 RNA polymerase. T7 RNA pol ymerase promoters on the transfected plasmids direct the synthesis of transcripts encoding the NP, P, and L proteins and a full-length, posi tive-sense copy of the HPIV-3 genome, Generation of virus was dependen t on transfection of pHPIV-3 and the HPIV-3 P- and L-encoding plasmids . However, a plasmid encoding the NP protein was not required since NP was expressed from pHPIV-3. Recovered virus was neutralized by anti-H PIV-3 antisera and shown to contain specific base substitutions charac teristic of pHPIV-3. Recombination was shown to occur during recovery, as viruses with two distinct genotypes and phenotypes were isolated, The ability to produce infectious HPIV-3 engineered to contain specifi c alterations within the HPIV-3 genes and cis-acting elements expedite s the study of all aspects of the virus replication cycle. Additionall y, analysis of mutations may lead to the identification of attenuating genotypes, a key step in the development of a live virus vaccine.