A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos

Abstract

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

FIG. 1

Recombinant NDV constructs. A schematic representation of the NDV gene order in the negative-strand genomic RNA is shown. Sequences around the editing site (positions 2274 to 2300) are presented in a positive sense. The modifications resulting in interruption of the A stretch in NDV-P1, deletions of six nucleotides of the conserved editing site in NDV-Δ6, and the creation of a stop codon in the trans-V frame of NDV-Vstop (Vstop) are shown in boxes. +G indicates the position for insertion of nontemplated G residue(s).

FIG. 2

Low-level V protein expression in NDV-P1-infected cells. BSR-T7/5 cells were infected with rNDV or NDV-P1 at a multiplicity of infection of approximately 0.01. Eighteen hours after infection, cells were processed for indirect immunofluorescence after incubation with MAbs specific for NP protein (top) or for F protein (bottom) or anti-V peptide serum (middle). Although the levels of NP and F protein expressions in cells infected with both viruses were indistinguishable, the level of V protein expression was considerably lower in cells infected with NDV-P1 than in those infected with rNDV.

FIG. 3

NP and V proteins of sucrose-purified recombinant viruses. Virions in the allantoic fluid of infected embryonated eggs were purified by centrifugation through 20% sucrose. The volumes loaded for NDV-P1 were 4.5-fold greater than those for rNDV in order to normalize for NP protein content. Samples were loaded in duplicate, and blots were incubated with anti-NP MAb (lanes 1 through 3) or with anti-V peptide serum (lanes 4 through 6). AF, allantoic fluid from noninfected embryonated eggs; P1, NDV-P1.

FIG. 4

P-gene mRNA editing in NDV-P1-infected cells. mRNA sequences in the regions of the editing site with the unedited P ORF or with insertion of one G residue (+G) coding for V ORF are shown. NDV-P1 (P1) edits its P-gene mRNA in spite of the interruption of the five A residues by A-to-G substitution (∗).

FIG. 5

Pathogenicity of rNDV and NDV-P1 in SPF chicken embryos. Eleven-day-old embryonated SPF chicken eggs were inoculated with the parent rNDV (passage 3) or the mutant NDV-P1 (passage 5) and incubated for 7 days or until the embryos had died. NDV-P1 caused no embryo mortality for 7 days at all indicated doses (0.2 ml/egg), whereas rNDV was lethal at a dose as low as 1 EID₅₀/ml (approximately 10% mortality). Embryos inoculated with rNDV started to die as early as 3 days postinoculation at higher doses.