Isolation and Characterization of a Novel Gammaherpesvirus from a Microbat Cell Line

Shabman, Reed S.; Shrivastava, Susmita; Tsibane, Tshidi; Attie, Oliver; Jayaprakash, Anitha; Mire, Chad E.; Dilley, Kari E.; Puri, Vinita; Stockwell, Timothy B.; Geisbert, Thomas W.; Sachidanandam, Ravi; Basler, Christopher F.

BGHV8 genome construction and annotation. Extracted DNA from BGHV8-infected Vero cells was sheared (Covaris), and a library was generated for sequencing by Illumina HiSeq2500 to generate 100-bp reads. A second library from viral genomic DNA was generated by sequence-independent single primer amplification (SISPA) as previously described (23) and was sequenced on the Illumina MiSeq platform to generate trimmed sequencing reads approximately 270 bp in length. De novo assembly in CLC Genomics Workbench resulted in several contigs. Using the CLC genome finishing tool, longer MiSeq reads were used to join the contigs generated from the HiSeq data into a single sequence of approximately 130 kb. To annotate the assembled genome, we developed gammaherpesvirus-specific annotation software called Viral Genome ORF Reader (VIGOR) (24) , with very relaxed parameters on the percent similarity cutoff to accommodate the variation in herpesvirus genomes. VIGOR was used to predict genes, perform alignments, ensure the fidelity of open reading frames, and detect any potential sequencing or assembly errors. The annotation was manually verified and edited as required, according to the gene and protein naming convention of the Equid herpesvirus 2 (EHV-2) reference genome (NC_001650.2). We analyzed the assembled BGHV8 sequence in a tandem repeat finder program (http://tandem.bu.edu/trf/trf.html) which was previously used to identify repeat regions in a mouse gammaherpesvirus genome (25) .