The mechanisms of each subset of immune cells contributing to the pathogenesis of viral hepatitis remain incompletely understood. In this study, we examined the role of liver CD4(-) CD8(-) (double negative, DN) T cells during murine hepatitis virus strain 3 (MHV-3)-induced hepatitis in C3H/HeJ mice. We demonstrate that predominant population of DN T cells in the liver of healthy or MHV-3-infected mice express TCR gamma delta(+). The proportion of TCR gamma delta(+) DN T cells in liver CD3(+) T cells was markedly increased after MHV-3 infection. Adoptive transfer of TCR gamma delta(+). DNT cells led to dramatically decreased survival in MHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALT and AST levels. It was found that these cells were hyperactivated after MHV-3 infection with a production of TNF-alpha, IFN-gamma, IL-2 and IL-17A. Highly activated liver TCR gamma delta(+) DN T cells were cytotoxic to MHV-3-infected hepatocytes in vitro and this effect did not require cell-cell contact. Moreover, the cytotoxic effect of liver TCR gamma delta(+) DN T cells against hepatocytes involves TNF-alpha pathway, but not IL-17A or IFN-gamma. These results indicate that liver TCR gamma delta(+). DN T cells play a critical role in the liver injury in MHV-3-induced hepatitis, via a TNF-alpha dependent pathway. (C) 2012 Elsevier Ltd. All rights reserved.