Analysis of the Genome of a Korean Isolate of the Pieris rapae Granulovirus Enabled by Its Separation from Total Host Genomic DNA by Pulse-Field Electrophoresis

Abstract

BackgroundMost traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. However, purification of virions may yield insufficient material for traditional sequencing. The electrophoretic method described here provides a strategy whereby the genomic DNA of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) could be recovered in sufficient amounts for sequencing by purifying it directly from total host DNA by pulse-field gel electrophoresis (PFGE).Methodology/Principal FindingsThe total genomic DNA of infected P. rapae was embedded in agarose plugs, treated with restriction nuclease and methylase, and then PFGE was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the purified viral DNA. The double-stranded circular genome of PiraGV-K was found to encode 120 open reading frames (ORFs), which covered 92% of the sequence. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (∼99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF 11), involved in the liquefaction of the host, were found in the genome.Conclusions/SignificanceThe recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the electrophoretic method. The method appears to be generally applicable to the analysis of genomes of large viruses.

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“…MEF DNA was prepared as described ( Aoidi et al, 2016 ). Purified DNA was digested with the Nhe I and Sbf I restriction endonucleases, fractionated by pulse field gel electrophoresis through 1% agarose gel, which was conducted in 0.5× TBE buffer (45 mM Tris-base, 1 mM EDTA, 45 mM boric acid) with a CHEF DR-II apparatus (Bio-Rad, Hercules, CA), with an initial switch time at 1 s and final switch time at 10 s for 20 h at 14°C and a voltage of 10 V/cm ( Herschleb et al, 2007 ; Jo et al, 2013 ). The gel was blotted onto N-Hybond membrane (GE Healthcare Life Sciences, Mississauga, ON, Canada), and hybridized to probe d described in …”