Isolation and identification of herpes simplex virus type 1
Objective To isolate and identify a strain of herpes simplex virus type 1(HSV-1)from blister fluid of patients with herpes labialis,and to compare its cytotoxicity,viral replication,and immune activation ability with HSV-1(F)in ovarian cancer cells.Methods A virus was isolated from the blister fluid of a patient with orolabial herpes,and was named HSV-1(YMM)after identification by gene sequencing and transmission electron microscopy.SKOV3,ES2,and ID8 cells were infected with HSV-1(YMM)and HSV-1(F)at multiplicity of infection(MOI)of 1 and 0.1,respectively.Crystal violet staining and CCK-8 assay were used to detect the cell viability at 48 h to evaluate the cytotoxicity of the virus.Vero cells were infected with HSV-1(YMM)and HSV-1(F)at MOI of 0.1,respectively.At 12,24,36,48,60 and 72 h,the virus titer was measured to evaluate the virus replication ability.SKOV3 cells were infected with HSV-1(YMM)and HSV-1(F)at MOI of 1,respectively.The relative expressions of interferon(IFN)-αand IFN-β mRNAs were detected by real-time fluorescence quantitative PCR at 24 h,and were compared.Results After gene sequencing and morphological identification,a spherical enveloped virus with a diameter of 100 to 125 nm was isolated,and its gene sequence completely matched to that of HSV-1(Sammple11-DOCK8)(MN401208.1).HSV-1(YMM)and HSV-1(F)at MOI of 1 had obvious toxic effects on SKOV3,ES2 and ID8 cells.The cytotoxic effects of HSV-1(YMM)and HSV-1(F)on SKOV3,ES2 and ID8 cells at MOI of 0.1 were significantly less than those at MOI of 1.There were no significant differences in the cytotoxicities of HSV-1(YMM)and HSV-1(F)on SKOV3,ES2 and ID8 cells at MOI of 1 or 0.1.The cell viabilities of SKOV3,ES2 and ID8 cells infected with HSV-1(YMM)and HSV-1(F)at MOI of 1 were lower than those at MOI of 0.1(P＜0.05).There were no significant differences in cell viabilities of SKOV3,ES2 and ID8 cells infected with HSV-1(YMM)and HSV-1(F)at MOI of 1 or 0.1(P＞0.05).At 12,24,36,48 and 60 h after the Vero cells were infected,there were no significant differences in the virus titers between HSV-1(YMM)and HSV-1(F)(P＞0.05).The viral titer of HSV-1(YMM)at 72 h[(7.4±4.6)X 107 PFUs/mL]was higher than that of HSV-1(F)[(2.3±0.5)X 107 PFUs/mL](t=2.857,P=0.046).The virus titers of HSV-1(YMM)and HSV-1(F)increased gradually with time from 12 to 48 h after infection.The relative expressions of IFN-α and IFN-β mRNAs were higher in SKOV3 cells infected with HSV-1(YMM)(11.9±7.8,24.1± 3.8)than those in SKOV3 cells infected with HSV-1(F)(4.1±2.5,12.2±2.2)(t=3.353,P=0.005;t=6.638,P=0.001).Conclusion HSV-1(YMM)isolated from blister fluid samples of patients with orolabial herpes has similar cytotoxicity as HSV-1(F),but it has stronger virus replication ability and stronger ability to induce the expression of type I interferon.
herpes simplex virus type 1cytotoxicityvirus replicationtype Ⅰ interferonovarian carcinoma cell
NETL
NSTL
万方数据
