A HIGHLY RECOMBINOGENIC SYSTEM FOR THE RECOVERY OF INFECTIOUS SENDAI-PARAMYXOVIRUS FROM CDNA - GENERATION OF A NOVEL COPY-BACK NONDEFECTIVEINTERFERING VIRUS

We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucl eocapsid protein (NP), phosphoprotein and large proteins into cells in fected with a vaccinia virus which expresses T7 RNA polymerase, These cells were then injected into chicken eggs, in which SeV grows to very high titers, FL-3 was marked with a BglII site in the leader region a nd an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene , creating a new, out-of-frame, 5' proximal AUG, All the virus stocks generated eventually removed this impediment to NP expression, by eith er point mutation or recombination between FL-3 and pGEM-NP, The recov ery system was found to be highly recombinogenic, Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasm id containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new g enomic 3' end was found in the recombinant SeV in 12 out of 12 recover ies, Using this approach, a novel copy-back nondefective virus was gen erated which interferes with wild-type virus replication.