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

Abstract

Background 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. Results 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. Conclusions 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.

Nucleotide sequence characterization, amino acid variations and 3D structural analysis of HN protein of the NDV VIId genotype

“…However, there were conflicts in some of the results of these studies (Jin et al., 2017 ; Huang et al., 2004 ; Khattar et al., 2009 ). The primary targets of a host's immune response to ND are HN and F proteins, so these two proteins have been considered the main objectives for developing several ND vaccines (Yan et al., 2020 ; Tavassoli et al., 2019 ; Izquierdo‐Lara et al., 2019 ).…”

Rapid construction of infectious clones for distinct Newcastle disease virus genotypes

“…Until now, there are three major strategies used to assemble the NDV full-length cDNA into transcription vectors. The first conventional method is sequential cloning based on naturally occurring RE sites in the genome or artificially introduced RE sites ( 14 , 15 , 27 ). Several cDNA fragments spanning the entire genome of NDV are RT-PCR-amplified and sequentially joined using the RE sites.…”

“…Since the first recombinant NDV was rescued by using the reverse genetics technique in 1999 ( 11 ), many different NDV strains have been successfully rescued ( 12 – 15 ). In all these cases, cloning the full-length NDV genome has remained the most challenging and laborious of all the steps ( 16 ).…”

Rescue of an enterotropic Newcastle disease virus strain ZM10 from cloned cDNA and stable expressing an inserted foreign gene

“…Moreover, the NDV genome has to meet particular requirements for successful rescue such as the rule-of-six [25,26] and generation of the precise 3′ and 5′ ends [24,26]. To get the precise integral NDV genome, the traditional method is dividing the NDV genome into a set of short fragments from 6 to 11 [10,14,[27][28][29][30][31], and it takes months to complete for the multiple DNA fragments cloning [23]. In this study, the genome of NDV was divided into three fragments 6, 6 and 3 kb.…”