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 recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that 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 CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight 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 in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.
Krishnamurthy
S
S
Virginia-Maryland Regional College of Veterinary Medicine, College Park, Maryland 20742, USA.
Huang
Z
Z
Samal
S K
SK
D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004274:DNA, Recombinant; D005822:Genetic Vectors; D016679:Genome, Viral; D009522:Newcastle disease virus / Q000235:genetics* / Q000472:pathogenicity; D016366:Open Reading Frames; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics; D014774:Virulence
D018076:DNA, Complementary; D004274:DNA, Recombinant; D015500:Chloramphenicol O-Acetyltransferase