Recovery of Infectious Human Parainfluenza Virus Type 3 from cDNA

AbstractInfectious HPIV3 was produced by the intracellular coexpression of four plasmid-borne cDNAs. These separately encoded a complete HPIV3 genome (negative-sense), the HPIV3 nucleocapsid protein N, the phosphoprotein P, and the polymerase protein L. The cDNA-encoded HPIV3 genome differed from the JS wildtype (wt) strain of HPIV3 used in its construction by seven point mutations: four of these are silent mutations in the HN or L gene coding regions that serve as markers of a cDNA-derived virus, two were introduced to create an amino acid substitution that ablates an epitope recognized by the HN-specific monoclonal neutralizing antibody 423/6, and the remaining point mutation results in an incidental amino acid substitution in the HN protein at amino acid position 263. The four plasmids were transfected into HEp-2 cell monolayers and their expression was driven by T7 RNA polymerase supplied by a vaccinia virus recombinant. The titer of virus present in the harvested transfection supernatant was low (