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.

Sialic acid recognition is a key determinant of influenza A virus tropism in murine trachea epithelial cell cultures.

Influenza A virus interacts with specific types of sialic acid during attachment and entry into susceptible cells. The precise amino acids in the hemagglutinin protein that control sialic acid binding specificity and affinity vary among antigenic subtypes. For H3 subtypes, amino acids 226 and 228 are critical for differentiating between alpha2,3- and alpha2,6-linked forms of sialic acid (SA). We demonstrate that position 190 of the HA from A/Udorn/307/72 (H3N2) plays an important role in the recognition of alpha2,3-SA, as changing the residue from a glutamic acid to an aspartic acid led to alteration of red blood cell hemagglutination and a complete loss of replication in differentiated, murine trachea epithelial cell cultures which express only alpha2,3-SA. This amino acid change had a minimal effect on virus replication in MDCK cells, suggesting subtle changes in receptor recognition by the H3 hemagglutinin can lead to significant alterations in cell and species tropism.

Using cryo-EM to measure the dipole potential of a lipid membrane.

The dipole potential of a lipid bilayer membrane accounts for its much larger permeability to anions than cations and affects the conformation and function of membrane proteins. The absolute value of the dipole potential has been very difficult to measure, although its value has been estimated to range from 200 to 1,000 mV from ion translocation rates, the surface potential of lipid monolayers, and molecular dynamics calculations. Here, a point charge probe method was used to investigate the dipole potentials of both ester and ether lipid membranes. The interactions between electrons and lipid molecules were recorded by phase-contrast imaging using cryo-EM. The magnitude and the profile of the dipole potential along the bilayer normal were obtained by subtracting the contribution of the atomic potential from the cryo-EM image intensity. The peak dipole potential was estimated to be 510 and 260 mV for diphytanoylphosphatidylcholine and diphytanylphosphatidylcholine, respectively.