A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies.

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 IC50 of acyclovir was ∼0.28µg/ml, similar to the previously published IC50 value. The IC50 of 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 IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells.

Chlamydiae are obligate intracellular bacteria developing in an intracytoplasmic niche, the inclusion. Chlamydia growth measurement by inclusion counting is a key task in the development of novel antichlamydial antibiotics and in vaccine studies. Most of the current counting methods rely on the immunofluorescent staining of the inclusions and either manual or automatic microscopy detection and enumeration. The manual method is highly labor intensive, while the automatic methods are either medium-throughput or require automatic microscopy. The sensitive and specific PCR technology could be an effective method for growth related chlamydial DNA detection; however the currently described PCR approaches have a major limitation, the requirement of purification of DNA or RNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of Chlamydia trachomatis DNA directly from the infected HeLa cells. With our method we were able to detect the bacterial growth in a 4 log scale (multiplicity of infection (MOI): 64 to 0.0039), with high correlation between the biological and technical replicates. As a further proof of the method, we applied the direct qPCR for antibiotic minimum inhibitory concentration (MIC) measurements. The measured MICs of moxifloxacin, tetracycline, clarithromycin and compound PCC00213 were 0.031 µg/ml, 0.031 µg/ml, 0.0039 µg/ml and 6.2 µg/ml respectively, identical or close to the already published MIC values. Our direct qPCR method for chlamydial growth and antibiotic MIC determination is less time-consuming, more objective and more sensitive than the currently applied manual or automatic fluorescent microscopy- based methods.

Possible contribution of epigenetic changes in the development of schizophrenia-like behavior in vasopressin-deficient Brattleboro rats.

Schizophrenia-like symptoms were detected in vasopressin-deficient (di/di) Brattleboro rats, and it was also suggested that schizophrenia might have an epigenetic component. We aimed to clarify if epigenetic changes contribute to schizophrenia-like behavior of this strain. Behavioral (locomotion by telemetry, cognition by novel object recognition, social recognition and social avoidance test, attention by pre-pulse inhibition) and epigenetic differences were compared between wild type and di/di animals. DNA methyltransferase1 (DNMT1), DNMT3a, as well as COMT, GAD, VGLUT1, 5HT2A, BDNF mRNA levels in prefrontal brain region and hippocampus were studied by qRT-PCR. Histone3 (H3) and H4 acetylation (Ac) were studied by western-blot followed by region specific examination of H3 lysine9 (K9) acetylation by immunohistochemistry. Impaired cognitive, social and attention behavior of di/di rats confirmed schizophrenia-like symptoms in our local colony. The pan-AcH3 immunoreactivity was lower in prefrontal region and elevated in the hippocampus of di/di animals. We found lower immunopositive cell number in the dorsal peduncular prefrontal cortex and the ventral lateral septum and increased AcH3K9 immunoreactivity in CA1 region of di/di animals. There were no major significant alterations in the studied mRNA levels. We confirmed that Brattleboro rat is a good preclinical model of schizophrenia. Its schizophrenia-like behavioral alteration was accompanied by changes in H3 acetylation in the prefrontal region and hippocampus. This may contribute to disturbances of many schizophrenia-related substances leading to development of schizophrenia-like symptoms. Our studies confirmed that not a single gene, rather fine changes in an array of molecules are responsible for the majority of schizophrenia cases.