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.
Highlights
Baculoviruses represent a diverse group of viruses with covalently closed, double-stranded, circular, supercoiled genomes, with sizes varying from 80 to 180 kb, encoding between 90 and 180 genes
Most genome sequencing projects rely on the wholegenome shotgun (WGS) method, which uses the Sanger technique to sequence genomic libraries over conventionally mapped clones using bacterial artificial chromosome (BAC), cosmid or fosmid libraries [24,25,26]
This has helped the focus on BAC-based physical map construction and its integration with high-density genetic maps that have benefited from next-generation sequencing (NGS) platforms and highthroughput array platforms [29,30]
Summary
Baculoviruses represent a diverse group of viruses with covalently closed, double-stranded, circular, supercoiled genomes, with sizes varying from 80 to 180 kb, encoding between 90 and 180 genes. The larger genomes of the Group II alphabaculoviruses can be attributed to a combination of repeated genes that are not found in the smaller genomes. This is in contrast to the betabaculoviruses genomes, varying from 101 kb in the case of Plutella xylostella granulovirus (PlxyGV) [4] to 178 kb in Xestia c-nigrum granulovirus (XecnGV) [5]. Despite the large difference in gene content in betabaculovirus genomes, as reflected in this range of sizes, their genomes are surprisingly collinear, compared with alphabaculoviruses, which show a high degree of variation [6,7]. Most traditional genome sequencing projects involving viruses include the culture and purification of the virus particles. 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 pulsefield gel electrophoresis (PFGE)
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