The recombinant HVT vaccine strain vHVT19-NDV contains an expression cassette encoding the fusion (F) protein of Newcastle disease virus (NDV). The F coding sequence was derived from the Texas velogenic strain of NDV (genotype IV).

The expression cassette consists of the murine cytomegalovirus immediate-early (MCMV IE) promoter that directs expression of the NDV F gene, while the simian virus 40 (SV40) polyadenylation signal provides the transcription termination and polyadenylation sequence.

The expression cassette was inserted into the intergene 1 locus of the FC-126 strain of herpesvirus of turkeys (HVT; Marek's disease virus, serotype 3). This locus is situated between the HVT065 and HVT066 open reading frames, which are arranged in a tail-to-tail orientation. The insertion site is located between the putative polyadenylation signals of these two ORFs. The adjacent regions are therefore not considered to contain regulatory elements that would modulate expression of the inserted donor DNA.

The recombinant virus was generated by homologous recombination between the parental HVT FC-126 genome and the donor plasmid pEL198 following co-transfection of chicken embryo fibroblasts (CEFs). The donor plasmid was organized as left flanking arm – expression cassette-right flanking arm, allowing targeted integration of the expression cassette at the intergene 1 locus.

Note:

No DNA of bacterial origin from the donor plasmid was incorporated into the recombinant virus. No antibiotic-resistance or other resistance marker was used for selection. Recombinant virus expressing the inserted NDV F protein was selected using indirect immunofluorescence with an antibody recognizing the F protein.
Expression of the inserted F gene results in production of the NDV fusion protein in cells infected with vHVT19-NDV. The expressed protein serves as an antigen for induction of protective immunity against Newcastle disease, while the HVT vector retains its immunizing properties against Marek's disease.
The genetic and phenotypic stability of the recombinant virus was evaluated through five serial back-passages in chickens in a reversion-to-virulence study. Expression of the inserted NDV F gene was maintained in 100% of the viruses evaluated following the back-passages.