Use of a BJAB-derived cell line for isolation of human herpesvirus 8

Abstract

Establishment of latently infected cell lines from primary effusion lymphomas (PEL) presently is the most efficient system for the propagation of clinical strains of human herpesvirus 8 (HHV-8) in culture. Here we describe a new approach to culture productively replicating HHV-8 from patient samples. A BJAB-derived B-cell line, BBF, was found to retain HHV-8 longer, to support the latent and lytic replication programs, and to produce transmissible virus. Supernatants from n-butyrate-treated peripheral blood mononuclear cells of 24 HHV-8-seropositive renal transplant recipients were used to infect BBF cells, and replicating virus was detected in cultures from 11 patients. Moreover, BBF cells infected with saliva strains showed a highly productive profile regardless of the initial viral load, which confirms that infectious HHV-8 can be present in saliva and also suggests that saliva strains may exhibit a high tropism for B lymphocytes. In conclusion, we established an in vitro system that efficiently detects HHV-8 in samples with low viral loads and that produces infectious progeny. BBF cells can be used to propagate HHV-8 from different biological samples as well as to clarify important issues related to virus-cell interactions in a context distinct from endothelial and PEL-derived cell lines.

FIG. 1.

Nested PCR analysis of BJAB, BBF, HEK, 293, and RD4 cell lines in long-term infection experiments with increasing MOIs. ORF26-specific primers were used to detect HHV-8 sequences in the DNA extracted from virus-exposed cell lines at 2, 5, 12, and 15 days p.i. BBF cells were found to retain HHV-8 sequences longer, since a specific signal was amplified at 5 days p.i. by using an MOI of 0.25 and at 15 days p.i. by using an MOI of 2.5. The asterisks below the 233-bp band indicate that the signal was also present in the first-round PCR.

FIG. 2.

Viral gene expression in HHV-8-infected BBF cells. RT-nested PCR analyses were performed on RNA extracted from HHV-8-infected BBF cells after 1, 2, 3, 5, 7, and 9 days p.i. A 205-bp ORF50-specific transcript was amplified after 1 and 2 days p.i., and a 243-bp ORFK8.1-specific transcript was amplified after 2 and 3 days p.i. An ORF73-specific band (214 bp) was detected throughout, together with a longer product amplified from viral DNA. In fact, primers were designed to amplify a spliced product and, therefore, to discriminate any amplification product deriving from genomic DNA. Lanes: M, 1-kb DNA ladder marker (Invitrogen); 1, water control; 2, nested water control; 3 to 8, RNA extracted from BBF cells after 1, 2, 3, 5, 7, and 9 days p.i., respectively; 9, noninduced (for the ORF73-specific transcript) or induced (for the ORF50- and ORFK8.1-specific transcripts) CRO-AP/3 RNA; 10, CRO-AP/3 DNA.

FIG. 3.

Expression of HHV-8 latent and lytic proteins in infected BBF cells. (A) Phase-contrast photograph of HHV-8-infected BBF cells (B, C, and D) Presence of the punctate nuclear pattern typical of the LANA protein in some BBF cells after 2 and 3 days p.i. (Alexa 488 label, confocal images). (E) Uninfected BBF cells stained with anti-LANA monoclonal antibody (Alexa 488 label). (F and G) Double-staining IFA with anti-vIL-6 polyclonal antibody (green; Alexa 488 label) and anti-PF8 monoclonal antibody (red; Alexa 594 label). (H) Merged image of vIL-6 (green) and PF8 (red) signals demonstrating that, in infected noninduced BBF cells, almost all cells that expressed vIL-6 coexpressed PF8, thus suggesting a tendency to spontaneously progress toward the early lytic phase. Magnifications, ×40.

FIG. 4.

Transmission of replication-competent virus from HHV-8-infected BBF cells. Uninfected BBF cells were exposed to supernatants from noninduced 6-day-infected BBF cells and analyzed for the presence of HHV-8 transcripts by RT-nested PCR after 2 and 3 days p.i. A 205-bp ORF50-specific transcript was amplified after 3 days p.i., in addition to a 1,162-bp product amplified from viral DNA and detected after 2 and 3 days p.i., suggesting that infected BBF cells release transmissible and replication-competent viral progeny. Lanes: M, 1-kb DNA ladder marker; 1, water control; 2, nested water control; 3 and 4, RNA extracted from reinfected BBF cells after 2 and 3 days p.i., respectively; 5, induced CRO-AP/3 RNA.

FIG. 5.

Kinetics of HHV-8 replication of saliva strains in BBF cells. A highly productive infection was observed in BBF cells infected with 100 μl of cell-free saliva samples from eight KS patients; virus production was measured in culture supernatants by real-time PCR and is reported as GEs per ml of supernatant. The kinetics of virus replication suggest that BBF cells can spontaneously support the lytic phase of HHV-8 infectious particles present in saliva.