Establishing cell model of herpes simplex virus type 2 latent infection and reactivation in SH-SY5Y cells

Objective: To establish cell model of herpes simplex virus type 2 latent infection and reactivation in SHSY5Y cells. Methods: Effects of ACV on SH-SY5Y cells were observed after cells were treated with ACV concentrations of 20, 40, 60, 80, 100, 120, or 140 μmol/L, and also cells morphology was observed under phase contrast microscope after HSV-2 was inoculated into SH-SY5Y cells using MOI of 0.1, 1, 10 and 100. The optimum condition of temperature and heating duration were approached using temperature of 41°C, 42°C, 43°C, 44°C, 45°C and heating duration of 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h to induce HSV-2 reactivation. The optimum concentration of Forskolin was also decided using 25, 50, 75, 100, 125 μmoL/L to reactivate the virus from latency. The viral gG gene and the LAT gene were used to verify HSV-2 latent infection and reactivation in cells by PCR and sequencing. The gG gene was quantitative detected by real-time PCR on the optimum state of latent infection and reactivation. The morphological changes were observed when the virus was reactivated from latency. Results: 60 μmoL/L ACV was the optimum concentration to establish latent state in SH-SY5Y cells. Virus titers between 1~10 MOI were the suitable infective dose of HSV-2 to establish cell model from latency to reactivation. In our research, the time of virus latency in SH-SY5Y cells could reach up to 14 d. Heat stress of 43°C, 1.5 h or Forskolin of 75 μmoL/L was the optimum condition to induce virus reactivation. Individual cells showed pathological changes after reactivation for 24 h and expanded after 48 h, as some cells fused into multinuclear giants with chromosome near the edge of the nuclear envelopes or broken nuclei. Viral LAT gene had expressed in SH-SY5Y cells latently infected with HSV-2 for 4, 6, or 8 d, while the gG gene had not expressed at all. Both LAT and gG genes expressed at 12, 24, and 36 h after heator chemical-induced reactivation. The gG gene expression after reactivation was 9.68 times higher than that in the latent period after normalizing to GAPDH expression. Conclusions: The cell model system of HSV-2 latent infection and reactivation in SH-SY5Y cells was established. The research provided usefulness for study on HSV-2 of latency, reactivation and pathogenic mechanism.