Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.Cytomegalovirus (CMV) is a major opportunistic pathogen in immunocompromised hosts. Long-term therapy with ganciclovir (GCV) and foscarnet (PFA) has been associated with development of clinical resistance and progression of disease (3,4,9,10). Standardized laboratory methods to rapidly and accurately determine the susceptibility of CMV isolates to antiviral drugs are needed. The inherent difficulties in working with CMV include the slow growth of the virus and the fact that CMV clinical isolates are strongly cell associated. Multiple passages in culture are typically required to generate sufficient extracellular virus for titration and testing. Standard susceptibility assays require the inhibition of viral replication in the presence of serial concentrations of drug. The slow growth of CMV has limited the usefulness of standard assays in patient management and in guiding clinical trials. Thus, the ultimate goal is to develop a rapid method that can be used directly on clinical samples to detect resistance mutations (1, 11). As a first step toward this goal, 12 laboratories in the CMV Resistance Working Group of the AIDS Clinical Trials Group (ACTG) have collaborated in formulating and testing a standardized plaque reduction assay to use as a "gold standard." In the first phase of this process, four characterized plaque-purified CMV strains (two laboratory strains and two clinical isolates) were distributed and tested in 12 laboratories using variations of a plaque reduction assay. In the second phase, a consensus assay was used to retest these strains, as well as six additional coded clinical isolates. The inhibition curves were calculated by computer modeling using a PROC NLIN program of SAS. The results are presented in this report. MATERIALS AND METHODS Cells and drugs.In phase 1, human diploid fibroblasts from a variety of sources, commercial and in-house, were used. In phase 2, MRHF (human foreskin) cell cultures (Biowhittaker, Walkersville, Md.) were used by all of the laboratories. GCV was provided by Syntex-Roche, and PFA was provided by Astra Pharmaceuticals. The drugs were prepared from common lots by the Viral Quality Assurance Laboratory of the ACTG at Rush-Presbyterian-St. Luke's Medical Center. GCV (4.5 mM) and PFA (20 mM) stocks were filter sterilized, shipped on dry i...
Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immune evasion proteins. For example, the MCV MC159 protein inhibits TNFR1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show here that, in HEK293T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 over-expression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IKK complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, co-immunoprecipitated with IKKγ. MC159 co-immunoprecipitated with IKKγ when using MEFs lacking either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ co-immunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159’s inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.
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