Neurovirulence Study Of I176R Mutation In The E-coding Region Of Japanese Encephalitis Virus

Japanese encephalitis is caused by Japanese encephalitis virus(JEV).This study aims at analysis JEV key mutations related to neurovirulence and identifying whether the E-I176 R mutation influence JEV neurovirulence.Firstly,six mutations related to neurovirulence were analysed.The whole sequence of JEV SCYA201201-1 and SCYA201201-0901 strain were previously sequencing.The neurovirulence of these two strains were tested.Based on the current knowledge of JEV,we analyzed and assumed that 6 amino acid substitutions(E-A72 T,E-I176 R,E-S251 Y,E273K;NS2B-V44A;NS4A-G168E)play main roles in the attenuation process.And then,JEV E-I176 R mutant virus was obtained using site-directed and reverse genetic system.The JEV E-I176 R mutant virus was identified using plaque formation test,PCR and sequencing experiments.in vitro characters of JEV E-I176 R mutant virus were investigated on Syrian baby hamster kidney cells,and there was no significant differences on both plaque formation and growth rate between JEV E-I176 R mutant virus and its control virus.At last,the neurovirulence of JEV E-I176 R mutant virus was investigated.84 of 3-week-old female mice were divided into 14 groups,and every virus titer was studied for three independent experiments.Intracranial challenge of mice with 200 and 2000 plaque-forming units(p.f.u.)of control virus caused 55.56% and 88.89% mortality,respectively,while challenge with 200 and 2000 p.f.u.of JEV E-I176 R mutant virus caused an 11.11% and 50% mortality,respectively.The results of standard quantitative RT-PCR showed that control virus could be detected in brain tissues at day 3 p.i.,peaking at day 5 p.i.,while JEV E-I176 R mutant virus levels remained stable and the viral titer was significantly lower than that of control.This study analysed 6 amino acid substitutions may be related to JEV neurovirulence,and confirmed E-176 was the key mutation of JEV neurovirulence.The mutation of E-I176 R could significant decrease the neurovirulence of JEV.This study provides critical information for understanding the molecular mechanism of JEV attenuation.