title: A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.
abstract: Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients' sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay. The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50of acyclovir was ∼0.28μg/ml, similar to the previously published IC50value. The IC50of novel antiviral candidates was between 1.6-3.1μg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32-1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests. Our direct qPCR method for the HSV-2 growth determination of antiviral IC50and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, 6720 Szeged, Hungary. Electronic address: virok.dezso.peter@med.u-szeged.hu.
authors.forename: Dezső P
authors.initials: DP
authors.lastname: Virók
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, 6720 Szeged, Hungary. Electronic address: ildiko.eszik@gmail.com.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Eszik
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, 6720 Szeged, Hungary. Electronic address: mosolygo.timea@med.u-szeged.hu.
authors.forename: Tímea
authors.initials: T
authors.lastname: Mosolygó
authors.affiliation: Research Program for Rational Drug Design in Dermatology and Rheumatology, Department of Dermatology, Paracelsus Medical University of Salzburg, Müllner Hauptstraße 48, A-5020 Salzburg, Austria; ProComCure Biotech, 5020 Salzburg, Austria. Electronic address: oender@procomcure.com.
authors.forename: Kamil
authors.initials: K
authors.lastname: Önder
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, 6720 Szeged, Hungary. Electronic address: endresz.valeria@med.u-szeged.hu.
authors.forename: Valéria
authors.initials: V
authors.lastname: Endrész
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, 6720 Szeged, Hungary. Electronic address: burian.katalin@med.u-szeged.hu.
authors.forename: Katalin
authors.initials: K
authors.lastname: Burián
mesh_terms: D000212:Acyclovir / Q000494:pharmacology; D057134:Antibodies, Neutralizing / Q000097:blood*; D000914:Antibodies, Viral / Q000097:blood*; D000998:Antiviral Agents / Q000494:pharmacology*; D016679:Genome, Viral; D006561:Herpes Simplex / Q000276:immunology; D018258:Herpesvirus 2, Human / Q000187:drug effects* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D020128:Inhibitory Concentration 50; D009500:Neutralization Tests; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000998:Antiviral Agents; D000212:Acyclovir
keywords: Antiviral; HSV; Neutralization; qPCR