Effect Of Japanese Encephalitis Virus NS1’ Protein On Virulence In Mouse

Objective:To studythe expression of Japanese encephalitis virus NS1’protein and its effect on neurovirulenceand neuroinvasiveness in mouse.Methods:Taking Japanese encephalitis virus vaccine strain SA14-14-2 and Japanese encephalitis wild virus strain SA14 as the templates,the A66G and G66A site directed mutation of NS2A gene was carried out by overlapping extension PCR and molecular cloning technology.After enzyme digestion and sequencing identification,a large number of site directed mutation plasmids were extracted,the plasmid was linearized with Xho Ⅰand transcribed in vitro to obtain viral RNA.The RNA was introduced into BHK21 cells by electro-transfection to rescue the mutant virus SA14-14-2(A66G)and SA14(G66A).The viruses were identified by plaque test,Indirectimmunofluorescence,Western blot and virus sequencing.Growth curves of the mutant virus were plotted,and exploring the effect of the mutant virus on BHK21 cells.The neurovirulence and neuroinvasiveness in mouse brain of mutant viruses were detected by animal experiments,so as to judge the effect of the 66th base in NS2A on the expression of NS1’ proteinand the effect of NS1’ protein on the neurovirulence and neuroinvasiveness of virus in mouse brain.Results:After enzyme digestion and sequencing identification,it was confirmed that the infectious clones of SA14-14-2(A66G)and SA14(G66A)were successfully constructed.The successful rescue of mutant viruses SA14-14-2(A66G)and SA14(G66A)was confirmed by plaque experiment,virus sequencing and Indirectimmunofluorescence experiment.Western blot showed that Japanese encephalitis vaccine strains SA 14-14-2 and SA14(G66A)did not express NS1’ protein,but Japanese encephalitis wild strains SA14 and SA14-14-2(A66G)expressed NS1’ protein.The growth curve showed that there was no significant difference in the growth characteristics between the mutant strain and the parent strain.Cell proliferation activity test showed that the expression of NS1’ protein inhibited the proliferation of BHK21 cells,and there was significant difference(P<0.01).The plaque experiment found that SA14(G66A)formed smaller plaque than SA14,and SA14-14-2(A66G)formed larger plaque than SA14-14-2.The LD50 of the vaccine strains SA14-14-2 and SA14-14-2(A66G)in 1-week-old mice brain were 297.9 PFU(0.02 ml)and 143.4 PFU(0.02 ml),and the LD50 of the wild strains SA14 and SA14(G66A)in 3-week-old mice brain were 0.8 PFU(0.03 ml)and 2.3 PFU(0.03 ml).The LD50 of vaccine strains SA14-14-2 and SA14-14-2(A66G)on neuroinvasiveness of 2-week-old mice were>5.65×106 PFU(0.5 ml)and>1.46×106 PFU(0.5 ml).The LD50 of wild strains SA14 and SA14(G66A)on neuroinvasiveness of 3-week-old mice were 1.3×103 PFU(0.5 ml)and 1.2×104 PFU(0.5 ml).Conclusion:The base at position 66 of Japanese encephalitis virus NS2A is the key site affecting the expression of NS1’ protein.The expression of NS1’ protein can improve the neurovirulence and neuroinvasiveness of the virus in mouse,and the effect on the neuroinvasiveness is more obvious.