Determination of antiviral efficacy against lymphotropic herpesviruses utilizing flow cytometry
ABSTRACT Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV-6), and human herpesvirus type 8 (HHV-8) comprise a group of lymphotropic herpesviruses which are responsible for a wide range of diseases, including lymphoproliferative disorders and tumors. We have developed several flow cytometric assay (FACS) systems to evaluate antiviral efficacy against EBV, HHV-6 and HHV-8. Assays using either EBV or HHV-8, members of the gammaherpesvirus subfamily, have shown that while EBV responds well to acyclovir (ACV), HHV-8 was most sensitive to cidofovir (CDV). Since HHV-6 strains are divided into two sub-groups, A and B, we evaluated antiviral efficacy for strains from each group. The group A strain, HHV-6(GS), was inhibited by foscarnet (PFA), CDV and ganciclovir (GCV) in both Sup-T1 and HSB-2 cell lines. HHV-6(Z-29), a representative group B virus, was inhibited by GCV and CDV but not by PFA. Our findings indicate that flow cytometry can be utilized to efficiently evaluate new antiviral agents against lymphotropic herpesviruses and that the results are comparable to those obtained by other methods such as immunofluorescence.
- SourceAvailable from: Tsutomu Nakashima
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- "ACV inhibits virus-associated DNA polymerase, suppressing virus replication. Although some reports have indicated that ACV is effective against EBV in vitro , , clear efficacy in actual clinical practice has not been established , . Using the tonsillar infection model, we showed that ACV exerts a dose-dependent antiviral action. "
ABSTRACT: Epstein-Barr virus (EBV) may cause a variety of virus-associated diseases, but no antiviral agents have yet been developed against this virus. Animal models are thus indispensable for the pathological analysis of EBV-related infections and the elucidation of therapeutic methods. To establish a model system for the study of EBV infection, we tested the ability of B95-8 virus and recombinant EBV expressing enhanced green fluorescent protein (EGFP) to replicate in human lymphoid tissue. Human tonsil tissues that had been surgically removed during routine tonsillectomy were sectioned into small blocks and placed on top of collagen sponge gels in culture medium at the air-interface, then a cell-free viral suspension was directly applied to the top of each tissue block. Increasing levels of EBV DNA in culture medium were observed after 12-15 days through 24 days post-infection in tissue models infected with B95-8 and EGFP-EBV. Expression levels of eight EBV-associated genes in cells collected from culture medium were increased during culture. EBV-encoded small RNA-positive cells were detected in the interfollicular areas in paraffin-embedded sections. Flow cytometric analyses revealed that most EGFP(+) cells were CD3(-) CD56(-) CD19(+) HLA-DR(+), and represented both naïve (immunoglobulin D(+)) and memory (CD27(+)) B cells. Moreover, EBV replication in this model was suppressed by acyclovir treatment in a dose-dependent manner. These data suggest that this model has potential for use in the pathological analysis of local tissues at the time of primary infection, as well as for screening novel antiviral agents.PLoS ONE 10/2011; 6(10):e25490. DOI:10.1371/journal.pone.0025490 · 3.23 Impact Factor
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- "Only limited data have been published regarding the efficacy of antiviral therapy on HHV-6 [Long et al., 2003]. In a study on the effectiveness of ganciclovir against HHV-6, there was growing evidence that ganciclovir inhibits HHV-6 replication in the salivary glands [Ljungman et al., 2007]. "
ABSTRACT: Viral infections remain important causes of morbidity and mortality in hematopoietic cell transplant recipients. More recent developments in preparative regimens and graft manipulations, as well as the control of well-recognized post-transplant infections by the introduction of prophylaxis and preemptive strategies, have influenced the timing and the epidemiology of infections. As new pathogens, such as human metapneumovirus (HMPV), human bocavirus, human coronaviruses HCoV-NL63 and HCoV-HKU1, human herpesviruses HHV-6 and HHV-7, and polyomaviruses, have emerged, it is fundamental to determine the significance of the newly discovered viruses and their role in the transplantation field. This article summarizes recent data on epidemiology and laboratory diagnosis of new pathogens, as well as clinical features and management of the associated infectious complications. J. Med. Virol. 82:528-538, 2010. (c) 2010 Wiley-Liss, Inc.Journal of Medical Virology 03/2010; 82(3):528-38. DOI:10.1002/jmv.21696 · 2.35 Impact Factor
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- "Since Epstein-Barr virus (EBV) replicates only in lymphocytes, plaque reduction assays were not possible and other assays have been developed. These included incorporation of radioactivity in purified viral DNA (Lin et al., 1984), dot blot assays with radioactive DNA probes (Bacon & Boyd, 1995), ELISA based assays (Bacon & Boyd, 1995,Williams et al., 2003), flow cytometry assays using intact cells (Long et al., 2003), slot blot assays with purified DNA and non radioactive DNA probes (Meerbach et al., 2000), and "
ABSTRACT: There is a need for additional therapies for Epstein-Barr virus (EBV) infections, but the routine screening of large numbers of potential inhibitors has been difficult due to the laborious nature of traditional assays. A new rapid assay was developed to evaluate compounds for antiviral activity against this virus that is both rapid and robust. Test compounds are added to cultures of Akata cells in 96-well plates that have been induced to undergo a lytic infection. Viral DNA produced during the infection is transferred to a membrane and quantified using a non-radioactive DNA hybridization assay. This assay was validated using a set of compounds with known activity against EBV and results compared favorably to an established real-time PCR assay. Subsequent experience with this assay has confirmed that it offers improved efficiency and robustness compared to other assays used routinely to evaluate candidate compounds for antiviral activity against EBV.Journal of Virological Methods 10/2007; 144(1-2):86-90. DOI:10.1016/j.jviromet.2007.04.001 · 1.78 Impact Factor