The attenuated Japanese encephalitis virus (JEV) strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E) protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439). Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.

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To explore the toxic effect of T-2 toxin on mouse Leydig cells and its underlying molecular mechanisms, we isolated Leydig cells from mature mice, set-up Leydig cells culture, treated cells with T-2 toxin, evaluated cell proliferation, detected the caspase-3 activity, mitochondrial activity and apoptosis rate, and measured the mRNA levels of Bcl-2, Bax, PARP and caspase-3. T-2 toxin inhibited cell proliferation at concentrations higher than 10 M or time more than 12 h, T-2 toxin also decreased Bcl-2 expression at the mRNA levels and mitochondrial activity at concentrations higher than 10 M. While, T-2 toxin increased the mRNA expressions of Bax and PARP at concentrations higher than 10 M and 10 M, respectively, triggered mitochondria-mediated apoptosis, activated downstream caspase-3, and then increased caspase-3 at the activity and mRNA levels at concentrations higher than 10 M. These data showed that T-2 toxin appears to activate specific intracellular death-related pathways leading to Bax-dependent caspase-3 activation and the induction of apoptosis in Leydig cells.