Volume 23, Issue 1 (Spring 2026)
bloodj 2026, 23(1): 12-21 |
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Mousavi Dehaqani L, Sharifi Z. Inactivation of HSV-1 Using Methylene Blue and Visible Light: Correlation of TCID50 Reduction with Glycoprotein D RNA Expression by RT-qPCR. bloodj 2026; 23 (1) :12-21
URL: http://bloodjournal.ir/article-1-1609-en.html
URL: http://bloodjournal.ir/article-1-1609-en.html
Abstract: (203 Views)
A B S T R A C T
Background and Objectives
Despite Significant advances in blood safety, the risk of infectious agent transmission remains a challenge for blood transfusion centers. To enhance blood safety, methods for removing or inactivating viruses have been developed. This study focuses on the inactivation of the herpes simplex virus type 1 (HSV-1) using methylene blue and visible light in plasma and compares the efficacy of the qRT-PCR and TCID50 methods.
Materials and Methods
In a experimental study, vero cells were cultured in DMEM with 1% penicillin-streptomycin and 10% fetal calf serum at 37°C with 5% CO2. The virus thiter was determinded by infectivy these cells. Subsequently, the virus stock was added to plasma at 0.1 volume. The virus was added to five individual plasma units obtained from different blood donors. Five plasma samples containing virus were treated with 1 μM methylene blue and then irradiated for 10 min at 627 nm. Virus titration was performed in 96-well plates, and virus titer was measured before and after irradiation through 10-fold serial dilutions using the TCID50 method. The control group included plasma without virus, plasma containing virus irradiated without methylene blue, and plasma containing methylene blue without irradiation. After RNA extraction from the dilutions and cDNA construction, glycoprotein D expression was quantified by qRT-PCR. All experiments were repeated three times. Statistical analyses were performed using SPSS-26 and the Pearson correlation coefficient between the two tests was calculated.
Results
Before inactivation, the virus titer was determined by observing cytopathic effects in Vero cells as 107TCID50/mL. After inactivation with methylene blue and irradiation, the titer decreased to 102 TCID50/mL, representing a 5-log reduction. The mean threshold cycle (Ct) of the glycoprotein D gene in cell cultures containing non-inactivated HSV-1 (107TCID50/mL) was 22.6 (Ct = 22.6) whereas the mean Ct of the glycoprotein D gene in plasma samples after inactivation by methylene blue and visible light (102 TCID50/mL) was 28 (Ct = 28). This indicates a 15-fold decrease in glycoprotein D expression (p= 0.001).
Conclusions
A correlation was observed between the logarithmic values of TCID50 and Ct values obtained by qRT-PCR assay. Molecular methods based on glycoprotein D (gD mRNA) expression may be served as a usefull approach for detecting of active HSV infection.
Key words: Virus Inactivation, Herpes Simplex Virus Infection, methylene blue, visible light, RT-PCR
Background and Objectives
Despite Significant advances in blood safety, the risk of infectious agent transmission remains a challenge for blood transfusion centers. To enhance blood safety, methods for removing or inactivating viruses have been developed. This study focuses on the inactivation of the herpes simplex virus type 1 (HSV-1) using methylene blue and visible light in plasma and compares the efficacy of the qRT-PCR and TCID50 methods.
Materials and Methods
In a experimental study, vero cells were cultured in DMEM with 1% penicillin-streptomycin and 10% fetal calf serum at 37°C with 5% CO2. The virus thiter was determinded by infectivy these cells. Subsequently, the virus stock was added to plasma at 0.1 volume. The virus was added to five individual plasma units obtained from different blood donors. Five plasma samples containing virus were treated with 1 μM methylene blue and then irradiated for 10 min at 627 nm. Virus titration was performed in 96-well plates, and virus titer was measured before and after irradiation through 10-fold serial dilutions using the TCID50 method. The control group included plasma without virus, plasma containing virus irradiated without methylene blue, and plasma containing methylene blue without irradiation. After RNA extraction from the dilutions and cDNA construction, glycoprotein D expression was quantified by qRT-PCR. All experiments were repeated three times. Statistical analyses were performed using SPSS-26 and the Pearson correlation coefficient between the two tests was calculated.
Results
Before inactivation, the virus titer was determined by observing cytopathic effects in Vero cells as 107TCID50/mL. After inactivation with methylene blue and irradiation, the titer decreased to 102 TCID50/mL, representing a 5-log reduction. The mean threshold cycle (Ct) of the glycoprotein D gene in cell cultures containing non-inactivated HSV-1 (107TCID50/mL) was 22.6 (Ct = 22.6) whereas the mean Ct of the glycoprotein D gene in plasma samples after inactivation by methylene blue and visible light (102 TCID50/mL) was 28 (Ct = 28). This indicates a 15-fold decrease in glycoprotein D expression (p= 0.001).
Conclusions
A correlation was observed between the logarithmic values of TCID50 and Ct values obtained by qRT-PCR assay. Molecular methods based on glycoprotein D (gD mRNA) expression may be served as a usefull approach for detecting of active HSV infection.
Key words: Virus Inactivation, Herpes Simplex Virus Infection, methylene blue, visible light, RT-PCR
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