Development of a high-throughput rubella virus infectivity assay based on viral activation of caspases.

Rubella virus (RV, German measles) is a teratogenic agent that can lead to serious congenital defects after maternal infection during early pregnancy. Currently, the disease can be prevented effectively by available live attenuated vaccines. An important requisite for the manufacture and release of a safe and potent live virus vaccine is the measurement of the vaccine titer (potency), to ensure the correct dose and efficacy of the vaccine. One historical method for measuring potency is the endpoint dilution TCID(50) assay. Traditionally, RV TCID(50) titers are calculated after visual inspection of cells for presence of cytopathic effect (CPE). Such visual scoring is tedious and labor intensive. The development of a new TCID(50) readout method, based on a fluorescent molecular marker of RV-induced apoptosis, is described in this report. Further, in order to calculate TCID(50) potency a novel mathematical model was established to convert the numerical fluorescence measurements into categorical data. Finally, the assay parameters such as signal-to-noise ratio, robustness, variability and bias were optimized. This new readout method demonstrated strong concordance with the standard manual scoring of CPE, and therefore can provide a practical, objective and higher-throughput alternative to the traditional TCID(50) readout used for calculating titers of rubella virus.

Varicella-zoster virus infection induces autophagy in both cultured cells and human skin vesicles.

When grown in cultured cells, varicella-zoster virus (VZV) forms many aberrant light particles and produces low titers. Various studies have explored the reasons for such a phenotype and have pointed to impaired expression of specific late genes and at lysosomal targeting of egressing virions as possible causes. In the studies presented here, we report that the autophagic degradation pathway was induced at late time points after VZV infection of cultured cells, as documented by immunoblot analysis of the cellular proteins LC3B and p62/SQSTM1, along with electron microscopy analysis, which demonstrated the presence of both early autophagosomes and late autophagic compartments. Autophagy was induced in infected cells even in the presence of phosphonoacetic acid, an inhibitor of viral late gene expression, thus suggesting that accumulation of immediate-early and early viral gene products might be the major stimulus for its induction. We also showed that the autophagic response was not dependent on a specific cell substrate, virus strain, or type of inoculum. Finally, using immunofluorescence imaging, we demonstrated autophagosome-specific staining in human zoster vesicles but not in normal skin. Thus, our results document that this innate immune response pathway is a component of the VZV infectious cycle in both cultured cells and the human skin vesicle, the final site of virion formation in the infected human host.