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J Virol Methods. 2015 Jun 1;217:79-86. doi: 10.1016/j.jviromet.2015.02.022. Epub 2015 Feb 28.

Generation of high-titre virus stocks using BrK.219, a B-cell line infected stably with recombinant Kaposi's sarcoma-associated herpesvirus.

Author information

1
Institute of Virology, Hannover Medical School, Carl-Neuberg-Str.1, Hannover D-30625, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany.
2
Department of Pediatric Hematology and Oncology, University Medical Centre Hamburg-Eppendorf, Hamburg D-20246, Germany.
3
Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.
4
Institute of Virology, Hannover Medical School, Carl-Neuberg-Str.1, Hannover D-30625, Germany; German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig, Germany. Electronic address: schulz.thomas@mh-hannover.de.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposi's sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 10(6) IU/ml in unconcentrated culture supernatants, representing a 10(3)-10(4)-fold improvement compared to conventional methods.

KEYWORDS:

BrK.219; KSHV; Kaposi's sarcoma; Kaposi's sarcoma-associated herpesvirus; high-titre virus stocks; human herpesvirus 8

PMID:
25736227
DOI:
10.1016/j.jviromet.2015.02.022
[Indexed for MEDLINE]

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