Abstract： Objective To investigate inhibitory effect of targeting herpes simplex virus (HSV)-2 latency-asscciated transcript (LAT) open reading frame(ORF) on expression of LAT gene in activated latent HSV-2 infection. Methods The neural cell model of re-activated latent HSV-2 infection was established using the cell line SH-SY5Y. The morphology of SH-SY5Y cells with latent and re-activated HSV-2 infection was studied with phase-contrast microscopy, and PCR was used to verify the latent and re-activated HSV-2 infection. Three pairs of siRNA targeting HSV LAT mRNAwere designed and transfected into SH-SY5Y cells using LipofectamineTM 2000 reagent. The change in SH-SY5Y morphology under phase-contrast microscope was further studied. Moreover, the expression levels of HSV LAT mRNA before and at different time points after transfection were assayed by RT-PCR. Results Established latent HSV-2 infection in SH-SY5Y cells was completed in the presence of 60 μmol/L Aciclovir(ACV). Observational study showed that the longest latency of HSV-2 infection in SH-SY5Y cells was 14 days. After induced viral re-activation with heat stress at 43°C for 1.5 h, phase-contrast microscopy revealed a range of cytopathic effects in SH-SY5Y between 24 h and 72 h, with increasing cell degeneration and necrosis over time. PCR amplification of HSV-2 LAT genes and electrophoresis of PCR products confirmed the latent and re-activated infection of virus in the cells. There was a reduction in HSV LAT mRNA expression in the cells transfected with HSV LAT ORF-siRNA by 39％,51％ and 60％ at 24, 36 and 48 h, respectively. Conclusion Inhibition of LAT ORF may lead to down-regulated expression of HSV-2 LAT and glycoprotein G (gG) genes, which provides robust evidence for further studies on the role of LAT ORF in re-activation of latent HSV-2 infection.