Heavy water (D2O) could enhance thermosta-bility of some viruses. However, the underlying mechanism is not clear. Here we report the development of a matrix-aided gas-isotope-ratio mass spectrometry method that allows direct determination of deuterium/hydrogen (D/H) ratio in D2O-treated Japanese encephalitis virus (JEV), hepatitis A virus (HAV) and RNA from D2O-treated HAV. The D/H ratio was expressed as δDSMOW.. Our experiments showed that δDSMOW values increased significantly in D2O-treated viral samples compared to normal controls, and increment in δDSMOW of D2O, treated viral samples was in a fine linear relationship with increment in amount of samples loaded in BSA matrix. Our experiments also indicated that increased δDSMOW of D2O-treated virus correlated well with its enhanced thermostability. The results suggested that hydrogen-deuterium exchange occurred in viral particles and its RNA structure as a result of D2O-treatment. Furthermore, such exchange could cause changes in viral phenotype, However, the underlying mechanism is not clear. Here we report the development of a matrix-aided gas-isotope-ratio mass spectrometry method that allows direct determination of deuterium / hydrogen ( The D / H ratio was expressed as δDSMOW .. Our experiments showed that δDSMOW values ​​increased significantly in (D) / H) ratio in D2O-treated Japanese encephalitis virus (JEV), hepatitis A virus (HAV) and RNA from D2O- treated HAV. D2O-treated viral samples compared to normal controls, and increment in δDSMOW of D2O, treated viral samples was in a fine linear relationship with increment in amount of samples loaded in BSA matrix. Our experiments also indicated that increased δDSMOW of D2O-treated virus correlated well with its enhanced thermostability. The results suggested that hydrogen-deuterium exchange occurred in viral particles and its RNA structure as a result of D2O-treatment. Furthermore, such exchange could cause changes in viral phenotype,