Development of a green fluorescent protein reporter cell line to reduce biohazards associated with detection of infectious Cercopithecine herpesvirus 1 (monkey B virus) in clinical specimens

Abstract

Detection of infectious viruses in clinical samples typically relies on daily examination of inoculated cell cultures for appearance of virus-induced cytopathic effect (CPE), with subsequent immunologic or genetic analysis to identify the specific virus producing the CPE. Performing virus isolation on samples suspected of containing Cercopithecine herpesvirus 1 (monkey B virus [BV]) is dangerous due to the extreme neuropathogenicity of this virus in humans, and minimally requires biosafety level 3 (BSL-3) facilities. To provide a safer method of detecting infectious BV in clinical samples, the eucaryotic green fluorescent protein (GFP) coding sequence was flanked with BV sequences containing transcriptional control elements. This construct was placed into a stealth vector and transfected into Vero cells, then stable transformed cell lines were selected. These cells express GFP only when infected with BV or other related primate herpesviruses. Expression of GFP allows detection of infectious BV in cultures sooner and more reliably than does standard microscopic observation for CPE. The ability to detect BV by GFP expression eliminates the need for further testing to identify the virus as an alpha-herpesvirus following development of CPE, thus allowing cell cultures to be sealed at inoculation. Although not entirely specific for BV, this cell line will make detection of infectious BV in samples collected from macaques safer to perform.