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

Quantitation of bat gammaherpesvirus 8-infected cells and release in supernatant from multiple cell lines. (A) Quantitative PCR (qPCR) to measure the amount of viral DNA released into the supernatant of seven representative cell lines at days 1, 3, and 5 postinfection. (B) Similar to panel A, but mRNA from infected cells was analyzed by reverse transcription followed by qPCR. (C) Representative plaque assay of BGHV8 on Vero cells. (D) Representative 50% tissue culture infectious dose (TCID₅₀) data from BGHV8 on Vero cells.

Electron microscopy of the MVI-it cell line identifies viral particles that appear to be BGHV8. (A) Representative EM image from the MVI-it culture. The black and red boxes highlight enlarged regions directly below the image above. Both areas identify budding and fully budded herpesvirus virions. (B) Representative EM image from the MVI-it culture. The blue and green boxes highlight enlarged regions directly below the image above. Both areas highlight intracellular herpesvirus particles, and the blue box captures virions within an inclusion body. Courtesy of Vsevolod Popov and Krystle Agans, reproduced with permission.

Assembly of the novel bat gammaherpesvirus genome highlights ORFs related to EHV-2 and accessory ORFs related to other gammaherpesviruses. To assemble a single contig of 129,563 bp, contigs assembled with 100-bp reads from Illumina HiSeq were joined with 270-bp reads from Illumina MiSeq data. The genome was annotated using the Viral Genome ORF Reader (VIGOR). Open reading frames are highlighted as follows: yellow, ORFs with homology to EHV-2; blue, ORFs with homology to other herpesvirus proteins; black, repeat regions; gray, putative LANA gene with an internal unresolved repeat region. ncRNA, noncoding RNA.

gB protein phylogenic tree highlights similarities with other bat herpesviruses and EHV-2. The tree displays multiple gammaherpesvirus protein sequences, and human herpesvirus 1 represents the outgroup. BGHV8 gB is an 839-amino-acid protein. An alignment was generated for a 263-amino-acid region of gB from positions 498 to 760 of BGHV8. All sequences were trimmed to include only the corresponding region, and an alignment was derived with an LG substitution model and 100 bootstraps with a scale of 0.4. The inset on the right side displays an enlarged image highlighted corresponding to the gray box in the left image to display the sequences most closely related to BGHV8. Red denotes BGHV8, and blue labels denote gB from viruses obtained from bat species. The most closely related sequence from this analysis comes from a gB sequence of Myotis ricketti herpesvirus 2 (AFM85235.1 ). Triangles represent multiple sequences which were collapsed to enhance presentation. To see the tree with no collapsed sequences and corresponding accession numbers, see Fig. S1 in the supplemental material.

BGHV8 v-OX2 clusters with gammaherpesviruses and Myotis sequences. (A) Representative alignment of BGHV8 OX2 with KSHV v-OX2 and Myotis davidii OX2. Dark red indicates identical residues, pink indicates conservation between two of the three alignments, and blue indicates less conserved regions. (B) Tree of representative viral OX2 ORFs demonstrates the relationship of BGHV8 v-OX2 to other gammaherpesviruses. Bold black denotes the penguinpox virus v-OX2 outgroup, black indicates gammaherpesvirus v-OX2, and pink denotes betaherpesvirus v-OX2. The red label highlights the location of BGHV8. (C) Tree of representative mammalian OX2 ORFs demonstrates the relatedness of BGHV8 OX2 to those from bat species. Blue labels denote microbat sequences, and green labels denote megabat sequences. The red label highlights the location of BGHV8.