A Novel Method to Titrate Herpes Simplex Virus-1 (HSV-1) Using Laser-Based Scanning of Near-Infrared Fluorophores Conjugated Antibodies

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2017 Jun 30

PMID 28659899

Citations 10

Authors

Marco Fabiani

Dolores Limongi

Anna Teresa Palamara

Giovanna De Chiara

Maria Elena Marcocci

Affiliations

Soon will be listed here.

Abstract

Among several strategies used for Herpes simplex virus (HSV) detection in biological specimens, standard plaque assay (SPA) remains the most reliable method to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may be particularly laborious for multiple sample analysis. Here we describe an innovative method to perform the titration of HSV type 1 (HSV-1) in different samples, using the "In-Cell Western" Assay (ICW) from LI-COR, a quantitative immunofluorescence assay that exploits laser-based scanning of near infrared (NIR). In particular, we employed NIR-immunodetection of viral proteins to monitor foci of HSV-1 infection in cell monolayers, and exploited an automated detection of their fluorescence intensity to evaluate virus titre. This innovative method produced similar and superimposable values compared to SPA, but it is faster and can be performed in 96 well plate, thus allowing to easily and quickly analyze and quantify many samples in parallel. These features make our method particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1 drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving data reproducibility. Overall, our method may represents a useful tool for both clinical and research purposes.

Citing Articles

Recurrent Herpes Simplex Virus Type 1 (HSV-1) Infection Modulates Neuronal Aging Marks in In Vitro and In Vivo Models.

Napoletani G, Protto V, Marcocci M, Nencioni L, Palamara A, De Chiara G Int J Mol Sci. 2021; 22(12).

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Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus.

Nguyen H, Sah N, Humphrey M, Rabkin S, Saha D J Vis Exp. 2021; (171).

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Experimental Data Based Machine Learning Classification Models with Predictive Ability to Select in Vitro Active Antiviral and Non-Toxic Essential Oils.

Sabatino M, Fabiani M, Bozovic M, Garzoli S, Antonini L, Marcocci M Molecules. 2020; 25(10).

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A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

Sarshar M, Scribano D, Tranquilli G, Di Pietro M, Filardo S, Zagaglia C BMC Microbiol. 2019; 19(1):252.

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Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays.

Masci A, Menesale E, Chen W, Co C, Lu X, Bergelson S Mol Ther Methods Clin Dev. 2019; 14:270-274.

PMID: 31489337 PMC: 6717064. DOI: 10.1016/j.omtm.2019.07.007.

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