Viral entry defines the hepatitis E virus species barrier in murine hepatocytes

The Paslahepevirus balayani hepatitis E virus (HEV) and the distantly related Rocahepevirus ratti rat HEV pose a risk for zoonotic transmission to humans. However, the molecular determinants for HEV transmission between species remain unknown. Despite the broad host range including its ability to infect certain rodent species, infections of animals of the genus Mus within the subfamily Murinae are rarely documented. To dissect the molecular mechanisms underlying species barriers to HEV infection, this study aimed to investigate the virus replication cycle and immune-related determinants responsible for restricted HEV infection in murine hepatocytes. Murine hepatic cell lines supported moderate levels of zoonotic HEV replication and infectious virion production upon transfection of in vitro transcribed viral RNA. Notably, viral replication was not restricted by innate immune responses or presence of dominant restriction factors but was limited by absence of host-specific dependency factors. While successful attachment to murine hepatic cells was detected, mechanisms of viral entry differed between human and murine hepatocytes, correlating with murine cell lines and primary murine hepatocytes being refractory to HEV infection. In summary, the murine barrier to HEV infection is defined at the viral entry stage, specifically by a block post attachment and before viral replication is initiated. These findings shed new light on the fundamental role of viral entry mechanisms in defining HEV species tropism.