Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

Abstract

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication

“…Homologous recombination was performed in Saccharomyces cerevisiae (strain RFY 206, Mata his3 Δ 200 leu2-3 lys2 Δ 201 ura3-52 trp1 Δ ::hisG ) [ 27 ] transformed by lithium acetate (LiAc) [ 28 ]. Colonies were screened in Yeast Nitrogen Base (YNB) without tryptophan [ 29 ] and cloning was confirmed by PCR, using the oligonucleotides CHIKV-3′UTR-F and pBSC-SpeI-3′CHIKV-R ( Table 1 ). Finally, Escherichia coli (strain DH10B) (Invitrogen, Waltham, MA, USA) was transformed with the positive clones [ 29 ] and plasmid DNA was extracted (QIAGEN Plasmid Midi Kit, QIAGEN, Germantown, MD, USA) and used as described below.…”

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

“… 16 These properties make this approach easier, less laborious, and more efficient than traditional cloning strategies. 26 Along with other applications using viruses, 24 this methodology has successfully been used to overcome the problems of instability of some flavivirus recombinant cDNA genomes in E. coli . 20 , 27 A prerequisite for homologous recombination in yeast is the presence of nucleotide sequence homology (15–50 bp) at the ends of the DNA fragments and the vector to be recombined, necessary to recruit the yeast recombination machinery.…”

“…The three PCR products corresponding to the whole IBSP4ncp ORF plus the Bam HI-digested vector pBSC-NADL-HDR were introduced simultaneously into the yeast S. cerevisiae strain RFY206 using the lithium acetate method. 24 After transformation, yeast cells were inoculated in YNB solid medium (Yeast Nitrogen Base – Sigma–Aldrich) without tryptophan and maintained at 30 °C for up to three days. Five recombinant yeast colonies were chosen, picked and amplified in YNB medium.…”

Infectious bursal disease virus in poultry: current status and future prospects

“…Different reverse genetic systems for IBDV have been described, which can be used for better IBDV characterization and for IBDV vaccine development. 108 , 109 A modified IBDV that contained an authentic 3′ RNA sequence generated by cis -acting hepatitis delta virus ribozyme was less pathogenic to the BF compared with a cell line-adapted variant E IBDV strain, but induced higher Ab responses as early as day 7 post infection. 110 The risk of reversion to virulence of the genetically modified viruses may exist.…”