[Background] Ni-CE (CE) strain is an attenuated rabies virus after 100 passages of a laboratory strain, a virulent strain Nishigahara (Ni), in chicken embryo fibroblast cells. Ni strain dose not induce cytopathic effect (CPE). In contrast, CE strain induces CPE. Furthermore, it is reported that Ni mutants replaced an amino acid substitution at position 95 in the matrix protein from CE strain (Ni(95CEM)) induces CPE, while CE mutants replaced the same position from Ni strain (CE(95NiM)) does not induce CPE. It is suggested that this amino acid substitution at position 95 participates in the induction of cell death (Mita et al ., Virus Res., 2008). However, this phenomenon is yet to be fully understood. Here, to understand the mechanism of this phenomenon, we performed chronologic analysis of the number of viable cells infected with Ni, CE, Ni(95CEM) and CE(95NiM), and we also analyzed the induction of first apoptosis and last apoptosis chronologically. [Materials and methods] Human neuroblastoma SK-N-SH cells were infected with Ni, CE, Ni(95CEM), CE(95NiM) at the multiplicity of infection (MOI) of 3. According to Cell Titer-Glo 2.0 Assay kit, chronological luminescence was used to analyze the intracellular ATP as the signal of viable cells. Furthermore, using Real Time-Glo Annexin V Apoptosis and Necrosis Assay kit, luminescence was used to analyze the exposure of phosphatidylserine (PS) as the signal of first apoptosis, and fluorescence was used to analyze the degree of cell membrane disruption as the signal of last apoptosis. [Results and discussion] CE and Ni(95CEM) infected-cells showed lower ATP signal than Ni and CE(95NiM) from 48 hours post-infection (h pi) to 72 h pi. It was indicated that CE and Ni(95CEM) infected-cells induced stronger cell death than Ni and CE(95NiM). About PS exposure, every infected-cells showed a high growth of luminescence from 18 h pi to 36 h pi. After that, CE and Ni(95CEM) showed lower luminescence than Ni and CE(95CEM). This result indicated that the luminescent probe did not bind PS because of the cell membrane disruption induced by CE and Ni(95CEM). While CE and Ni(95CEM) infected-cells showed higher fluorescence than Ni and CE(95NiM). It indicated that CE and Ni(95CEM) induced cell membrane disruption and cell death through PS exposure, while Ni and CE(95NiM) did not induce cell membrane disruption despite PS exposure. In conclusion, these results suggested that amino acid substitution at position 95 in matrix protein participates in cell membrane disruption in the period of last apoptosis.
