Recovery of a virulent strain of Newcastle disease virus from cloned cDNA:Expression of a foreign gene results in growth retardation and attenuation

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombin ant NDV expressing an additional gene were generated entirely from cloned c DNAs. For this purpose, a full-length cDNA clone of the virus genome, repre sented in eight different subgenomic fragments, was assembled in a transcri ption plasmid between a T7 RNA polymerase promoter and a hepatitis delta vi rus ribozyme sequence, infectious NDV could be generated in the cells infec ted with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length pl asmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinan t virus was then amplified and recovered, either after inoculation of trans fection supernatant into the allantoic cavity of embryonated specific-patho gen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to tha t of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CA T open reading frame, flanked by NDV gene start and gene end sequence motif s, was inserted into the region between the HN and L genes of the full-leng th cDNA. This construct was then used in the generation of a recombinant ND V expressing CAT protein. The CAT gene was maintained stably for at least e ight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease i n yield. In addition, the virus showed an increase in mean death time for e ggs and a lower intracerebral pathogenicity index in day-old chicks, implic ating attenuation of the recombinant virus. Thus, introduction of an additi onal gene into the NDV genome represents a method to achieve growth retarda tion and attenuation. These results also indicate that NDV can be engineere d to express foreign protein stably and can be manipulated in the future fo r use as a vaccine vector. (C) 2000 Academic Press.