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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
Inactivation of HSV-1 Using Methylene Blue and Visible Light: Correlation of TCID50 Reduction with Glycoprotein D RNA Expression by RT-qPCR
Leila Mousavi Dehaqani , Zohreh Sharifi *
Keywords: Virus Inactivation, Herpes Simplex Virus Infection, methylene blue, visible light, RT-PCR
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 References:
  1. Yao C-Y, Fu W-L. Biosensors for hepatitis B virus detection. World J Gastroenterol. 2014; 20(35): 12485-92. [DOI:10.3748/wjg.v20.i35.12485] [PMID] []
  2. Wang YF, Pang DW, Zhang ZL, Zheng HZ, Cao JP, Shen JT. Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J Med virol. 2003; 70(2): 205-11. [DOI:10.1002/jmv.10379] [PMID]
  3. Quint WG, Heijtink RA, Schirm J, Gerlich WH, Niesters HG. Reliability of methods for hepatitis B virus DNA detection. J Cli Microbiol. 1995; 33(1): 225-8. [DOI:10.1128/jcm.33.1.225-228.1995] [PMID] []
  4. Elikaei A, Hosseini SM, Sharifi Z, Latifi H, Nikbakht H, Mirshafiee H, et al. Methylene blue based device for pathogen reduction in human plasma. Iran J Ped Hematol Oncol. 2013; 3(3): 97-102.
  5. Ryan KJ, Ray CG. Medical microbiology: An introduction to infectious diseases: McGraw-Hill; Medical Publishing Division.2004.4th edition.
  6. Mettenleiter TC, Klupp BG, Granzow H. Herpesvirus assembly: a tale of two membranes. Curr Opin Microbiol. 2006; 9(4): 423-9. [DOI:10.1016/j.mib.2006.06.013] [PMID]
  7. McGeoch DJ, Rixon FJ, Davison AJ. Topics in herpesvirus genomics and evolution. Virus research. 2006; 117(1): 90-104. [DOI:10.1016/j.virusres.2006.01.002] [PMID]
  8. Clarke RW. Forces and Structures of the Herpes Simplex Virus (HSV) Entry Mechanism. ACS Infectious Diseases. 2015; 1(9): 403-15. [DOI:10.1021/acsinfecdis.5b00059] [PMID]
  9. Akhtar J, Shukla D. Viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry. FEBS J. 2009; 276(24): 7228-36. [DOI:10.1111/j.1742-4658.2009.07402.x] [PMID] []
  10. Gustafsson RK Engdahl E, Fogdell-Hahn A. Development and validation of aQ-PCR based TCID50 method for human herpesvirus 6. Virol J. 2012; 9: 311. [DOI:10.1186/1743-422X-9-311] [PMID] []
  11. Brandolini M, Taddei F , Marino MM , Grumiro L , Scalcione A , Turba ME, et al. Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Viruses. 2021 ; 13(6):1022. [DOI:10.3390/v13061022] [PMID] []
  12. Elikaei A, Sharifi Z, Hosseini SM, Latifi H, Musavi Hosseini MK. Inactivation of model viruses suspended in fresh frozen plasma using novel methylene blue based device. Iran J Microbiol. 2014; 6: 41-5.
  13. Reed LJ, Muench H. A Simple Method of Estimating Fifty Percent Endpoints. Am J Epidemiol. 1938: 27, 493-7. [DOI:10.1093/oxfordjournals.aje.a118408] []
  14. Lei C ,Yang J , Hu J, Sun X. On the Calculation of TCID50 for Quantitation of Virus Infectivity. Virol Sin. 2021: 36(1), 141- 4. [DOI:10.1007/s12250-020-00230-5] [PMID] []
  15. Zamanian M, Sharifi Z, Noormohammadi Z, Akbarzadeh T, Bineshian F. Antiviral effect of Artemisia aucheri aqueous extract on UL46 and US6 genes of HSV-1. Antivir Ther. 2021; 26(1-2): 43-8. [DOI:10.1177/13596535211039907] [PMID]
  16. Stelzer-Braid S, Walker GJ, Aggarwal A, Isaacs SR, Yeang M, Naing Z, et al. Virus isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for
  17. diagnostic and research purposes. Pathology .2020; 52: 760-3. [DOI:10.1016/j.pathol.2020.09.012] [PMID] []
  18. Lei C, Yang J, Hu J, Sun X. On the Calculation of TCID50 for Quantitation of Virus Infectivity. Virol Sin. 2021; 36: 141-4. [DOI:10.1007/s12250-020-00230-5] [PMID] []
  19. Amanat F, White KM, Miorin L, Strohmeier Sh, McMahon M, Meade P, et al. An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening. Curr Protoc Microbiol. 2020; 58(10): e108. [DOI:10.1002/cpmc.108] [PMID] []
  20. Logan M, Manalil J, Notte Ch, Kearse C, George S, Zeiser A, et al. A flow cytometric granularity assay for the quantification of infectious virus. Vaccine . 2019; 37(47): 7090- 9. [DOI:10.1016/j.vaccine.2019.02.059] [PMID]

Inactivation of HSV-1 Using Methylene Blue and
Visible Light: Correlation of TCID50 Reduction with
Glycoprotein D RNA Expression by RT-qPCR

Leila Mousavi Dehaqani1, Zahra Paz2, Zohreh Sharifi1       


1Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
2Blood Transfusion Resaerch Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran


Received: 2026/01/11
Accepted: 2026/04/04
 





     http://dx.doi.org/10.61186/bloodj.22.1.19
    



Citation:
Mousavi Dehaqani L, Paz Z, Sharifi Z. Inactivation of HSV-1 Using Methylene Blue and Visible Light: Correlation of TCID50 Reduction with Glycoprotein D RNA Expression by RT-qPCR. J Iran Blood Transfus. 2026: 23 (1): 12-21
    



Correspondence: Sharifi Z, Associate professor of Biological Products and Blood Safety Resaerch Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran.
Tel: (+9821) 82052152
E-mail:
1- Acridine Orange

1- Acridine Orange
1- Biological safety cabinet
1- Platelet Concentrate
2- Food and Drug Administration
3- Normal Skin Flora
4- Platelet Rich Plasma-Platelet Concentrate
5- Eosin-Methylene blue
6- Thioglycolate
z.sharifi@tmi.ac.ir  		 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
 
Copyright © 2025 Journal of Iranian Blood Transfusion, Published by Blood Transfusion Research Center.
This work is licensed under a Creative Common Attribution-Non Commercial 4.0 International license.






 
Type of Study: Research | Subject: Virology
References
1. Yao C-Y, Fu W-L. Biosensors for hepatitis B virus detection. World J Gastroenterol. 2014; 20(35): 12485-92. [DOI:10.3748/wjg.v20.i35.12485] [PMID] []
2. Wang YF, Pang DW, Zhang ZL, Zheng HZ, Cao JP, Shen JT. Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement. J Med virol. 2003; 70(2): 205-11. [DOI:10.1002/jmv.10379] [PMID]
3. Quint WG, Heijtink RA, Schirm J, Gerlich WH, Niesters HG. Reliability of methods for hepatitis B virus DNA detection. J Cli Microbiol. 1995; 33(1): 225-8. [DOI:10.1128/jcm.33.1.225-228.1995] [PMID] []
4. Elikaei A, Hosseini SM, Sharifi Z, Latifi H, Nikbakht H, Mirshafiee H, et al. Methylene blue based device for pathogen reduction in human plasma. Iran J Ped Hematol Oncol. 2013; 3(3): 97-102.
5. Ryan KJ, Ray CG. Medical microbiology: An introduction to infectious diseases: McGraw-Hill; Medical Publishing Division.2004.4th edition.
6. Mettenleiter TC, Klupp BG, Granzow H. Herpesvirus assembly: a tale of two membranes. Curr Opin Microbiol. 2006; 9(4): 423-9. [DOI:10.1016/j.mib.2006.06.013] [PMID]
7. McGeoch DJ, Rixon FJ, Davison AJ. Topics in herpesvirus genomics and evolution. Virus research. 2006; 117(1): 90-104. [DOI:10.1016/j.virusres.2006.01.002] [PMID]
8. Clarke RW. Forces and Structures of the Herpes Simplex Virus (HSV) Entry Mechanism. ACS Infectious Diseases. 2015; 1(9): 403-15. [DOI:10.1021/acsinfecdis.5b00059] [PMID]
9. Akhtar J, Shukla D. Viral entry mechanisms: cellular and viral mediators of herpes simplex virus entry. FEBS J. 2009; 276(24): 7228-36. [DOI:10.1111/j.1742-4658.2009.07402.x] [PMID] []
10. Gustafsson RK Engdahl E, Fogdell-Hahn A. Development and validation of aQ-PCR based TCID50 method for human herpesvirus 6. Virol J. 2012; 9: 311. [DOI:10.1186/1743-422X-9-311] [PMID] []
11. Brandolini M, Taddei F , Marino MM , Grumiro L , Scalcione A , Turba ME, et al. Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management. Viruses. 2021 ; 13(6):1022. [DOI:10.3390/v13061022] [PMID] []
12. Elikaei A, Sharifi Z, Hosseini SM, Latifi H, Musavi Hosseini MK. Inactivation of model viruses suspended in fresh frozen plasma using novel methylene blue based device. Iran J Microbiol. 2014; 6: 41-5.
13. Reed LJ, Muench H. A Simple Method of Estimating Fifty Percent Endpoints. Am J Epidemiol. 1938: 27, 493-7. [DOI:10.1093/oxfordjournals.aje.a118408] []
14. Lei C ,Yang J , Hu J, Sun X. On the Calculation of TCID50 for Quantitation of Virus Infectivity. Virol Sin. 2021: 36(1), 141- 4. [DOI:10.1007/s12250-020-00230-5] [PMID] []
15. Zamanian M, Sharifi Z, Noormohammadi Z, Akbarzadeh T, Bineshian F. Antiviral effect of Artemisia aucheri aqueous extract on UL46 and US6 genes of HSV-1. Antivir Ther. 2021; 26(1-2): 43-8. [DOI:10.1177/13596535211039907] [PMID]
16. Stelzer-Braid S, Walker GJ, Aggarwal A, Isaacs SR, Yeang M, Naing Z, et al. Virus isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for diagnostic and research purposes. Pathology .2020; 52: 760-3. [DOI:10.1016/j.pathol.2020.09.012] [PMID] []
17. Lei C, Yang J, Hu J, Sun X. On the Calculation of TCID50 for Quantitation of Virus Infectivity. Virol Sin. 2021; 36: 141-4. [DOI:10.1007/s12250-020-00230-5] [PMID] []
18. Amanat F, White KM, Miorin L, Strohmeier Sh, McMahon M, Meade P, et al. An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening. Curr Protoc Microbiol. 2020; 58(10): e108. [DOI:10.1002/cpmc.108] [PMID] []
19. Logan M, Manalil J, Notte Ch, Kearse C, George S, Zeiser A, et al. A flow cytometric granularity assay for the quantification of infectious virus. Vaccine . 2019; 37(47): 7090- 9. [DOI:10.1016/j.vaccine.2019.02.059] [PMID]
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