Method for the growth of Herpes Simplex Virus (HSV-1 and HSV-2) in an oxygen-free environment

Abstract

Vaginal herpes infections occur under low oxygen conditions, including anoxia. Using an anaerobic (0% O2) cell system, the infection rate, early protein expression, and progeny viability were determined. Anaerobic HeLa 229 cells (0% O2; anaerobic DMEM; 24h) were infected with MOI 1 of HSV1/2, then incubated under aerobic (control) and anaerobic conditions. Infectivity (6h post-infection (pi)) was assessed by ONPG activity and enumerating X-gal-stained cells. HSV1/2 viral immediate-early entry, early entry, and late entry protein expression were determined by fluorescent antibody staining followed by HSV1/2 glycoprotein B mouse monoclonal antibody and DAPI. Statistical analysis was performed using t-test and data were considered significant when p ≤ 0.05 (GraphPad Prism). HSV1/2 infection under standard aerobic conditions was approximately twice that measured for anaerobically infected cells. In addition, the level of HSV-2 infectivity was significantly (p < 0.05) higher (~8-fold) than that of HSV-1, regardless of oxygen level. HSV entry stage protein expression (ICP8, ICP27, VP5) was detected for anaerobic HSV1/2. A similar pattern was observed for anaerobically grown progeny of HSV1/2, i.e., the number of progeny was higher when anaerobic progenies were grown under aerobic conditions (anaerobic to aerobic) as compared to those grown under anaerobic conditions (anaerobic to anaerobic). In addition, the progeny levels grown solely under anaerobic conditions trended higher for HSV-1 than HSV-2 progeny but were not significantly different. These findings show that HSV1/2 can infect and replicate in anaerobically growing cells and that oxygen is not essential for productive replication.