Rescue of Newcastle Disease Virus from Cloned cDNA: Evidence that Cleavability of the Fusion Protein Is a Major Determinant for Virulence

TLDR

A full-length cDNA clone of the NDV vaccine strain LaSota was assembled from overlapping fragments and inserted into a plasmid downstream of a T7 promoter and a hepatitis delta virus ribozyme. Transfection of this plasmid into fowlpoxvirus‑infected cells expressing T7 RNA polymerase produced antigenomic NDV RNA, which was replicated and transcribed by NP, P, and L proteins from cotransfected plasmids; the resulting RNA was inoculated into embryonated eggs to recover virus, and targeted nucleotide changes were introduced to alter the fusion protein cleavage site. The unmodified clone yielded a nonvirulent virus (ICPI = 0.00), whereas the clone with a consensus cleavage site produced a highly virulent strain (ICPI = 1.28) and showed markedly increased F0 cleavage, confirming that F0 protease cleavage is a major virulence determinant.

Abstract

ABSTRACT A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR↓L) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR↓F). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.