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.

Multiple primate cell lines are permissive for BGHV8 transcription and replication. In an initial attempt to propagate BGHV8, we transferred supernatant from the MVI-it cell line to Vero cells. Within 18 h after supernatant transfer, cytopathic effect (CPE) was observed in the culture (Fig. 1A to D) . Moreover, syncytia were observed upon nucleus staining following infection ( Fig. 1E to G), a previously described feature of herpesvirus infection in vitro, including infection by equine gammaherpesvirus 2 (21) . To confirm that the CPE was due to the BGHV8 identified by deep sequencing, we designed quantitative PCR (qPCR) primers based on the mRNA contigs from our RNA-seq data set (see Materials and Methods). To detect viral replication and the release of progeny virions, we isolated DNA from supernatants of cells infected with BGHV8 and measured DNA copy numbers by qPCR at 1, 3, and 5 days postinfection ( Fig. 2A) . In parallel, total RNA from each cell line was harvested and oligo(dT) primers were used to reverse transcribe mRNA from both mock-and BGHV8-infected cell lines (Fig. 2B) . For both samples, signal was measured with primer pairs specific for a BGHV8 capsid gene (related to EHV ORF25). Consistent with previous CPE results, BGHV8 was able to both transcribe mRNA and generate DNA copies in select cell lines ( Fig. 2A and B). Specifically, we detected BGHV8 genome amplification and mRNA expression in Vero, A549, and Huh7 cells, cell lines derived from African green monkey kidney, human lung, and human liver, respectively.