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.

Genomics Workbench resulted in several contigs which contained ORFs organized in a manner consistent with a gammaherpesvirus genome. Due to the short length of the HiSeq reads, we were unable to completely assemble a single contig, suggesting that the HiSeq data were not sufficient to assemble through putative repeat regions (a hallmark feature of gammaherpesviruses). In an attempt to close these gaps, we took viral genomic DNA, performed sequence-independent single primer amplification (SISPA), and constructed Illumina sequencing libraries as previously described (23) . We next performed MiSeq analysis to obtain sequencing reads approximately 270 bp in length. Using the CLC genome finishing tool, these longer reads were used to join the contigs generated from the HiSeq data into a single sequence of 129,563 bases (Fig. 4) . To annotate the assembled BGHV8, we developed gammaherpesvirus-specific annotation software in the Viral Genome ORF Reader (VIGOR) software (24) with 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, following the gene and protein naming convention of the Equid herpesvirus 2 (EHV-2) reference genome (NC_001650.2). Every single gammaherpesvirus-specific gene and the core genes were identified in the annotation (Fig. 4 , yellow ORFs) with additional ORFs that did not match EHV-2 ORFs (Fig. 4 , blue ORFs). We were not able to fully resolve a repeat region around bp 122000, resulting in a disrupted reading frame for ORF73, which codes for the latency-associated nuclear antigen (LANA; Fig. 4 , gray ORF). Strikingly, the organization of the ORFs is consistent with EHV-2 and with other related gammaherpesviruses, and all features are present in the GenBank file KU220026.