The genome of many plus-strand RNA viruses is infectious. In contrast, the genome of negative-stand or of double-stranded RNA viruses is not infectious. We are attempting to render a single gene segment of a negative strand RNA virus, influenza A virus, infectious by rescuing RNA copies of the gene prepared in vitro from cloned full-legnth DNA, in cells infected with a influenza A virus. To facilitate such a """"""""rescue"""""""", we chose a gene that bears a host range mutation that has a selective advantage. A full-legnth ds DNA copy of the influenza A WSN virus NA RNA was cloned. A complete cDNA copy of the gene was then expressed in an SV40 vector as a fully functional NA glycoprotein. Having established that the NA DNA was functional, we subcloned the DNA into a plasmid vector containing promoter sequences for phage RNA polymerases. (+) strand copies of NA DNA were generated in vitro. These transcripts necessarily contain short sequences at 5' and 3' ends copied from vector DNA. In the rescue experiment, primary AGMK cells were infected with a suitable recipient influenza A virus strain. After infection, cells were transfected with (+) strand RNA transcripts at hight multiplicity. After 16 to 18 hours, the medium and the AGMK cell monlayer were harvested and inoculated onto a MDBK cell monolayer. Growth in MDBK cells would indicate that """"""""rescue"""""""" of the WSN NA gene had occurred. Thus far, this approach has not succeeded. We are re-engineering the transcription vector to produce discrete NA copies of WSN NA DNA that lack terminal non-influenza sequences. We also plan to repeat the experiment in continuous monkey cell lines that persistently produce the influenza nucleoprotein, in one case, or the three influenza RNA polymerases, in another case. Possible such transformed cell lines may facilitate the rescue of transfected RNA.
