{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T22:00:53Z","timestamp":1777672853449,"version":"3.51.4"},"reference-count":34,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2023,12,20]],"date-time":"2023-12-20T00:00:00Z","timestamp":1703030400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Japan Agency for Medical Research and Development (AMED)","award":["JP233fa627001"],"award-info":[{"award-number":["JP233fa627001"]}]},{"name":"Japan Agency for Medical Research and Development (AMED)","award":["JP22fk0108123"],"award-info":[{"award-number":["JP22fk0108123"]}]},{"name":"Japan Agency for Medical Research and Development (AMED)","award":["23wm0225033h0001"],"award-info":[{"award-number":["23wm0225033h0001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Flavivirus infections, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), present significant global public health challenges. For successful vaccine design, the assessment of neutralizing antibody activity requires reliable and robust methodologies for determining antibody titers. Although the plaque reduction neutralization test (PRNT) is commonly acknowledged as the gold standard, it has limitations in terms of time and cost, and its usage may be limited in resource-limited settings. To address these challenges, we introduced the micro-neutralization test (MNT) as a simplified alternative to the PRNT. The MNT employs a 96-well plate format, conducts microscale neutralization assays, and assesses cell viability by dissolving cells to create a uniform color solution, which is measured with a spectrometer. In this study, we evaluated the utility of the MNT by contrasting the end-point titers of the MNT and PRNT using 4 monoclonal antibodies, 15 non-human primate serum samples, and 2 therapeutic drug candidates across flaviviruses. The results demonstrated a strong correlation between the MNT and PRNT titers, affirming the robustness and reproducibility of the MNT for evaluating control measures against flaviviruses. This research contributes valuable insights toward the development of a cost-effective antibody titer testing approach that is particularly suitable for resource-limited settings.<\/jats:p>","DOI":"10.3390\/pathogens13010008","type":"journal-article","created":{"date-parts":[[2023,12,20]],"date-time":"2023-12-20T11:24:33Z","timestamp":1703071473000},"page":"8","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Utility of an In-Vitro Micro-Neutralizing Test in Comparison to a Plaque Reduction Neutralization Test for Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Serology and Drug Screening"],"prefix":"10.3390","volume":"13","author":[{"given":"Kazumi","family":"Haga","sequence":"first","affiliation":[{"name":"Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhenying (Nancy)","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biology, Emory College of Art and Science, Emory University, Atlanta, GA 30322, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7804-966X","authenticated-orcid":false,"given":"Misao","family":"Himeno","sequence":"additional","affiliation":[{"name":"Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ryuichi","family":"Majima","sequence":"additional","affiliation":[{"name":"Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8103-6659","authenticated-orcid":false,"given":"Meng Ling","family":"Moi","sequence":"additional","affiliation":[{"name":"Department of Developmental Medical Sciences, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1038\/s41564-020-0714-0","article-title":"The Continued Threat of Emerging Flaviviruses","volume":"5","author":"Pierson","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1038\/nature12060","article-title":"The Global Distribution and Burden of Dengue","volume":"496","author":"Bhatt","year":"2013","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1007\/s13365-014-0285-z","article-title":"Flaviviruses, an Expanding Threat in Public Health: Focus on Dengue, West Nile, and Japanese Encephalitis Virus","volume":"20","author":"Daep","year":"2014","journal-title":"J. Neurovirol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1038\/s41586-018-0446-y","article-title":"The Emergence of Zika Virus and Its New Clinical Syndromes","volume":"560","author":"Pierson","year":"2018","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4703","DOI":"10.3390\/v6114703","article-title":"Flavivirus-Mosquito Interactions","volume":"6","author":"Huang","year":"2014","journal-title":"Viruses"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Islam, M.T., Quispe, C., Herrera-Bravo, J., Sarkar, C., Sharma, R., Garg, N., Fredes, L.I., Martorell, M., Alshehri, M.M., and Sharifi-Rad, J. (2021). Production, Transmission, Pathogenesis, and Control of Dengue Virus: A Literature-Based Undivided Perspective. BioMed Res. Int., 2021.","DOI":"10.1155\/2021\/4224816"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Mulvey, P., Duong, V., Boyer, S., Burgess, G., Williams, D.T., Dussart, P., and Horwood, P.F. (2021). The Ecology and Evolution of Japanese Encephalitis Virus. Pathogens, 10.","DOI":"10.3390\/pathogens10121534"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.coviro.2016.11.007","article-title":"Flavivirus Transmission Focusing on Zika","volume":"22","author":"Vasilakis","year":"2017","journal-title":"Curr. Opin. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"S98","DOI":"10.1038\/nm1144","article-title":"Emerging Flaviviruses: The Spread and Resurgence of Japanese Encephalitis, West Nile and Dengue Viruses","volume":"10","author":"Mackenzie","year":"2004","journal-title":"Nat. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3","DOI":"10.4103\/0974-777X.176140","article-title":"The Emergence of Zika Virus as a Global Health Security Threat: A Review and a Consensus Statement of the INDUSEM Joint Working Group (JWG)","volume":"8","author":"Sikka","year":"2016","journal-title":"J. Glob. Infect. Dis."},{"key":"ref_11","unstructured":"Michael, B.N., Axel, K., John, E., Chusak, P., Jose, L.S.M., Renu, D.D., Raman, V., and Olaf, H. (2009). Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control: New Edition, World Health Organization. WHO Guidelines Approved by the Guidelines Review Committee."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"471","DOI":"10.3201\/eid1103.040285","article-title":"Japanese Encephalitis Virus in Meningitis Patients, Japan","volume":"11","author":"Kuwayama","year":"2005","journal-title":"Emerg. Infect. Dis."},{"key":"ref_13","first-page":"95","article-title":"Zika Virus and the Never-Ending Story of Emerging Pathogens and Transfusion Medicine","volume":"14","author":"Marano","year":"2016","journal-title":"Blood Transfus."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1038\/nrd.2017.33","article-title":"Broad-Spectrum Agents for Flaviviral Infections: Dengue, Zika and Beyond","volume":"16","author":"Boldescu","year":"2017","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_15","first-page":"285","article-title":"A Plaque Reduction Test for Dengue Virus Neutralizing Antibodies","volume":"99","author":"Russell","year":"1967","journal-title":"J. Immunol. Baltim. Md. 1950"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/S0065-3527(03)61001-8","article-title":"Serodiagnosis of Flaviviral Infections and Vaccinations in Humans","volume":"61","author":"Kuno","year":"2003","journal-title":"Adv. Virus Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.ebiom.2017.03.006","article-title":"A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients","volume":"17","author":"Shan","year":"2017","journal-title":"EBioMedicine"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.jviromet.2009.09.018","article-title":"Development of an Antibody-Dependent Enhancement Assay for Dengue Virus Using Stable BHK-21 Cell Lines Expressing Fc GammaRIIA","volume":"163","author":"Moi","year":"2010","journal-title":"J. Virol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.antiviral.2018.12.016","article-title":"Dengue Drug Discovery: Progress, Challenges and Outlook","volume":"163","author":"Lim","year":"2019","journal-title":"Antivir. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1021\/acs.jmedchem.9b00775","article-title":"Drugs for the Treatment of Zika Virus Infection","volume":"63","author":"Bernatchez","year":"2020","journal-title":"J. Med. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2391","DOI":"10.1002\/cmdc.202000464","article-title":"Broad-Spectrum Flavivirus Inhibitors: A Medicinal Chemistry Point of View","volume":"15","author":"Felicetti","year":"2020","journal-title":"ChemMedChem"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Samrat, S.K., Xu, J., Li, Z., Zhou, J., and Li, H. (2022). Antiviral Agents against Flavivirus Protease: Prospect and Future Direction. Pathogens, 11.","DOI":"10.20944\/preprints202201.0468.v1"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/S0166-3542(02)00185-7","article-title":"Interferon, Ribavirin, 6-Azauridine and Glycyrrhizin: Antiviral Compounds Active against Pathogenic Flaviviruses","volume":"58","author":"Crance","year":"2003","journal-title":"Antivir. Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/j.chom.2008.08.004","article-title":"Structural Insights into the Mechanisms of Antibody-Mediated Neutralization of Flavivirus Infection: Implications for Vaccine Development","volume":"4","author":"Pierson","year":"2008","journal-title":"Cell Host Microbe"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"728","DOI":"10.1073\/pnas.1522136113","article-title":"Neutralizing Antibody Titers against Dengue Virus Correlate with Protection from Symptomatic Infection in a Longitudinal Cohort","volume":"113","author":"Katzelnick","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5925","DOI":"10.1016\/j.vaccine.2011.06.062","article-title":"Correlation of Protection against Japanese Encephalitis Virus and JE Vaccine (IXIARO\u00ae) Induced Neutralizing Antibody Titers","volume":"29","author":"Klade","year":"2011","journal-title":"Vaccine"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"eaaw9066","DOI":"10.1126\/scitranslmed.aaw9066","article-title":"Distinct Neutralizing Antibody Correlates of Protection among Related Zika Virus Vaccines Identify a Role for Antibody Quality","volume":"12","author":"Maciejewski","year":"2020","journal-title":"Sci. Transl. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/0022-1759(83)90303-4","article-title":"Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays","volume":"65","author":"Mosmann","year":"1983","journal-title":"J. Immunol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/0022-1759(89)90397-9","article-title":"Re-Examination and Further Development of a Precise and Rapid Dye Method for Measuring Cell Growth\/Cell Kill","volume":"119","author":"Hansen","year":"1989","journal-title":"J. Immunol. Methods"},{"key":"ref_30","first-page":"50820","article-title":"Assays for the Identification of Novel Antivirals against Bluetongue Virus","volume":"80","author":"Gu","year":"2013","journal-title":"J. Vis. Exp. JoVE"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"825","DOI":"10.4269\/ajtmh.2009.08-0625","article-title":"Dengue Plaque Reduction Neutralization Test (PRNT) in Primary and Secondary Dengue Virus Infections: How Alterations in Assay Conditions Impact Performance","volume":"81","author":"Thomas","year":"2009","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3114","DOI":"10.1038\/s41596-021-00536-y","article-title":"Quantification of SARS-CoV-2 Neutralizing Antibody by Wild-Type Plaque Reduction Neutralization, Microneutralization and Pseudotyped Virus Neutralization Assays","volume":"16","author":"Bewley","year":"2021","journal-title":"Nat. Protoc."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hu, Y., Ma, C., and Wang, J. (2022). Cytopathic Effect Assay and Plaque Assay to Evaluate in Vitro Activity of Antiviral Compounds Against Human Coronaviruses 229E, OC43, and NL63. Bio Protoc., 12.","DOI":"10.21769\/BioProtoc.4314"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Amanat, F., White, K.M., Miorin, L., Strohmeier, S., McMahon, M., Meade, P., Liu, W., Albrecht, R.A., Simon, V., and Martinez-Sobrido, L. (2020). An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening. Curr. Protoc. Microbiol., 58.","DOI":"10.1002\/cpmc.108"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/1\/8\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:42:21Z","timestamp":1760132541000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/13\/1\/8"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,12,20]]},"references-count":34,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2024,1]]}},"alternative-id":["pathogens13010008"],"URL":"https:\/\/doi.org\/10.3390\/pathogens13010008","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,12,20]]}}}