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.

The ORFs at the far left end of the genome, which include v-FLIP, v-OX2, DHFR, and v-Bcl-2, closely resembled both microbat and megabat proteins. A phylogenetic tree of v-OX2 and OX2 proteins from a variety of vertebrate species indicates that the viral homologue clusters with microbat sequences and that it is also near the megabat OX2 sequence (Fig. 6C) . Since a subset of these ORFs share identity to bat gene sequences, this serves as additional evidence that the natural host for BGHV8 is a Myotis species bat and that the microbat cells from which the cell line was derived naturally harbored the virus prior to establishment of the cell line. mRNA profile of BGHV8 in MVI-it cell line. We next utilized the RNA-seq data from the MVI-it cell line to profile the abundance of viral transcripts produced within the culture. Raw sequencing reads were mapped to the coding sequences (CDS) of the BGHV8 reference genome and are displayed in the Lightweight Genome Viewer (lwgv) (30) . A representative screen shot (Fig. 7A) captures read coverage at each of the 77 CDS. The full genome map is available at http://katahdin.mssm.edu/ravi/web/lwgv/ lwgv.cgi?annÏ­nbghv_3.ann. These coverage data, represented on a log 10 scale, indicate expression of various genes and highlight that the majority of the genome is transcribed within the MVI-it cell line. An RNA-seq analysis in CLC Genomics Workbench which determined the reads per kilobase of transcript per million mapped reads (RPKM) further indicates that a majority of the genes are transcribed (Fig. 7B) . The three most highly expressed genes were ORF17, ORF33, and ORF53 (all structural genes). A heat map imposed on the BGHV8 genome also illustrates the relative expression of each CDS (Fig. 7C) . It is important to note that these data represent a single time point within a culture of cells where infection was not synchronized, but these data provide the basis for future studies to fully identify transcription start and stop sites and specific promoters.