Effect of non-structural protein amino acid site-directed mutation of dengue virus Ban18HK20 strain on virus proliferation and virulence / 中国生物制品学杂志

ABSTRACT

@#Objective To investigate the effect of non-structural protein amino acid mutations(NS1-G53D,NS3-E250V)on viral proliferation and virulence of Dengue virus(DENV)type 4 Ban18HK20 strain.Methods The mutant sites in Ban18-HK20 strain were determined by sequence alignment with an attenuated strain of DENV(PDK53). Point mutation plasmids pSPTM-Ban18HK20(G53D),pSPTM-Ban18HK20(E250V)and pSPTM-Ban18HK20(G53D+E250V)were constructed by using homologous recombination technique,and identified by enzymatic cleavage and sequencing. Viral RNA was obtained by in vitro transcription and then electrotransfected to Vero cells to rescue the point mutant virus. The biological characteristics of point mutant virus were identified by plaque assay,indirect immunofluorescence assay,virus genome sequencing,growth kinetics test,CCK-8 test and mouse neurotoxicity test.Results Enzyme digestion and sequencing confirmed that the point mutation plasmids were constructed correctly. The results of immunofluorescence assay and virus sequencing showed that the virus was rescued successfully. The point mutation virus showed smaller plaque than the wildtype virus. The virus titers of Ban18HK20(G53D),Ban18HK20(E250V)and Ban18HK20(G53D+E250V)reached the peak on the 4th day,which were 7. 67,7. 84 and 7. 78 LgPFU/mL,respectively. The titer of the wild-type virus reached the peak value 7. 68 LgPFU/mL on the 5th day. The proliferation activity of BHK21 cells infected with Ban18HK20(G53D)was significantly higher than those infected with the wild-type virus(P = 0. 003 6)and the proliferative activity of C6/36 cells infected with Ban18HK20(G53D)and Ban18HK20(G53D+E250V)was significantly higher than those infected with the wild-type virus(P < 0. 000 1). Both the point mutation virus and wild-type virus had no neurotoxicity to 4-week-old BALB/c mice. The neurotoxicity of point mutation viruses Ban18HK20(E250V)(LD_(50)=8. 51 PFU)and Ban18HK20(G53D+E250V)(LD_(50)= 0. 69 PFU)on 3-day-old BALB/c neonatal mice was lower than that of the wildtype virus(LD_(50)= 0. 69 PFU). The survival rate(60%)of nude mice injected with point mutation virus Ban18HK20(G53D +E250V)was higher than those injected with the wild-type virus(0)after intracerebral injection of the same amount of virus. The point mutation virus was genetically stable,and no recurrent mutation occurred in Vero cells after passage to the10th generation.Conclusion The non-structural protein NS1-G53D amino acid mutation weakens the cytotoxicity of Ban18HK20 strain to C6/36 and BHK21 cells,and the two-point combined mutation could weaken the neurotoxicity of Ban18HK20 strain in nude mice,which laid a foundation of the study of DENV virulence sites and the development of the vaccine.