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.

RNA sequencing and analysis of the MVI-it cell line. Cell line CRL-6012 was obtained from the American Type Culture Collection (ATCC) and passaged until cells reached a confluent monolayer in a 75-centimeter flask. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. RNA was isolated with Trizol (Invitrogen), and mRNA was subjected to next-generation sequencing on the Genome Analyzer II (42-bp single-read data). De novo assembly of raw read data generated 5,818 contigs, labeled as BatA1 to BatA5818. Nucleotide sequences from each contig were translated into protein space, and protein sequences were subjected to BLAST search against both the human and nonredundant protein databases, uncovering contigs with homology to herpesvirus protein sequences. For RNA-seq analysis, sequencing reads were mapped against each BGHV8 coding sequence (CDS) and in CLC Genomics Workbench (version 8.5) using the CLC RNA-seq tool. Total reads and the RPKM for each CDS were determined.