Reverse Genetics Assembly of Newcastle Disease Virus Genome Template Using Asis-Sal-Pac BioBrick Strategy

The BioBrick construction as an approach in synthetic biology provides the ability to assemble various gene fragments. To date, different BioBrick strategies have been exploited for assembly and cloning of a variety of gene fragments. We present a new BioBrick strategy, here referred as Asis-Sal-Pac BioBrick, which we used for the assembly of NDV as a candidate for single-stranded non-segmented, negative-sense RNA genome viruses.

In the present study, we isolated three NDVs from clinical samples which were classified into the VIId genotype based on their pathogenicity and phylogenetic analyses. Then, SalI, AsisI, and PacI enzymes were used to design and develop a novel BioBrick strategy, which enabled us to assemble the NDV genome, adopting the "rule of six". In this method, in each assembly step, the restriction sites in the newly formed destination plasmid are reproduced, which will be used for the next insertion. In this study using two overlapping PCRs, the cleavage site of the F gene was also modified from ¹¹²RRQKRF¹¹⁷to ¹¹²GRQGRL¹¹⁷ in order to generate the attenuated recombinant NDV. Finally, in order to construct the recombinant NDV viruses, the plasmids harboring the assembled full-length genome of the NDV and the helper plasmids were co-transfected into T7-BHK cells. The rescue of the recombinant NDVwas confirmed by RT-PCR and HA tests.

These findings suggest that the combination of reverse genetic technology and BioBrick assembly have the potential to be applied for the development of novel vaccine candidates. This promising strategy provides an effective and reliable approach to make genotype-matched vaccines against specific NDV strains or any other virus.