Closing the genomic gaps of rat cytomegalovirus ALL-03 in genome scaffold

Cytomegalovirus (CMV) has been known to cause acute, latent and persisting infections in animals and humans. Interestingly, Rat CMV ALL-03 strain (RCMV ALL-03) is unique among all previous RCMVs and Murine CMV strain in crossing the placenta naturally which can be used to study the CMV congenital infection. The partial genome sequence for this strain has been produced through Next Generation Sequencing technology using Illumina platform but the incompleteness limits the comprehensive study of genomics and proteomics. Thus, completing the genome sequence by closing all the gaps in draft genome and verify the position and function of all Open Reading Frame (ORF) can aid in future study for antiviral drug and vaccine development. The study was commenced by propagating RCMV ALL-03 extensively to a large amount in rat embryonic fibroblast (REF) cells. Infected cells exhibiting advance cytopathic effect were harvested and concentrated by 8% (w/v) PEG 6000. Concentrated viruses were then purified and proceeded with DNA extraction by conventional method. Four sets of forward and reverse primer for each gap were carefully designed using CLC Genomic workbench software. Four set of primers for each gap were used in Polymerase Chain Reaction (PCR) for gap verification purpose followed by single band of DNA in agarose gel sequenced and 8 templates of sequences were obtained for each gap. All these templates were compared and verified the original missing sequences hence inserting the actual missing sequences taken place in the previous partial sequence. Result shows that, false gap have been identified and all the misassemblies in genome sequence were corrected, thus producing a final completed genome sequence of RCMV ALL-03. From this study, the final number of base pairs of RCMV ALL-03 was 197,958 and has been arranged as single unique sequence flanked by 504 base pair terminal direct repeats. The overall content of G+C content was 46% and total 123 protein coding genes (CDS) were identified. Out of 123 CDS, 46 CDS (36.2%) were grouped into 8 functional classes such as capsid, glycoprotein, tegument, DNA replication, DNA packaging, nuclear egress, immune evasion and regulatory. Subsequently, all the positions of ORF were corrected and translation of protein has been verified. Next, phylogenetic analysis of RCMV ALL-03 based on conserved genes of herpesvirus revealed that this Malaysian isolate is closest to RCMV-English and RCMV-Berlin strains with 99% and 97% of homology were identified respectively. Moreover, from the tree, it is observed that the evolutionary relationship of RCMV ALL-03 with other strains of herpesviruses from all the three subfamilies. Interestingly, betaherpesvirus subfamily shown to be more closely related with gammaherpesviruses compare to alphaherpesviruses where beta- and gamma- share some of the functional ORF. Results presented have been submitted to genebank and the accession number was obtained. Further exploration of RCMV ALL-03 in future could provide more valuable information in understanding the pathogenesis of the virus as well as congenital infection which could serve as model to mimic the study of HCMV.

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