Insights into the Organization of the Poxvirus Multicomponent Entry-Fusion Complex from Proximity Analyses in Living Infected Cells.

Poxviruses are exceptional in having a complex entry-fusion complex (EFC) that is comprised of 11 conserved proteins embedded in the membrane of mature virions. However, the detailed architecture is unknown and only a few bimolecular protein interactions have been demonstrated by coimmunoprecipitation from detergent-treated lysates and by cross-linking. Here, we adapted the tripartite split green fluorescent protein (GFP) complementation system in order to analyze EFC protein contacts within living cells. This system employs a detector fragment called GFP1-9 comprised of nine GFP ß-strands. To achieve fluorescence, two additional 20-amino-acid fragments called GFP10 and GFP11 attached to interacting proteins are needed, providing the basis for identification of the latter. We constructed a novel recombinant vaccinia virus (VACV-GFP1-9) expressing GFP1-9 under a viral early/late promoter and plasmids with VACV late promoters regulating each of the EFC proteins with GFP10 or GFP11 attached to their ectodomains. GFP fluorescence was detected by confocal microscopy at sites of virion assembly in cells infected with VACV-GFP1-9 and cotransfected with plasmids expressing one EFC-GFP10 and one EFC-GFP11 interacting protein. Flow cytometry provided a quantitative way to determine the interaction of each EFC-GFP10 protein with every other EFC-GFP11 protein in the context of a normal infection in which all viral proteins are synthesized and assembled. Previous EFC protein interactions were confirmed, and new ones were discovered and corroborated by additional methods. Most remarkable was the finding that the small, hydrophobic O3 protein interacted with each of the other EFC proteins. IMPORTANCE Poxviruses are enveloped viruses with a DNA-containing core that enters cells following fusion of viral and host membranes. This essential step is a target for vaccines and therapeutics. The entry-fusion complex (EFC) of poxviruses is unusually complex and comprised of 11 conserved viral proteins. Determination of the structure of the EFC is a prerequisite for understanding the fusion mechanism. Here, we used a tripartite split green fluorescent protein assay to determine the proximity of individual EFC proteins in living cells. A network connecting components of the EFC was derived.

Microscopic differential cell counts in milk for the evaluation of inflammatory reactions in clinically healthy and subclinically infected bovine mammary glands.

Somatic cell count (SCC) is generally regarded as an indicator of udder health. A cut-off value of 100×10(3) cells/ml is currently used in Germany to differentiate between normal and abnormal secretion of quarters. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed analysis of the udder health status. The aim of this study was to differentiate somatic cells in foremilk samples of udder quarters classified as normal secreting by SCC <100×10(3) cells/ml. Twenty cows were selected and 72 normal secreting udder quarters were compared with a control group of six diseased quarters (SCC >100×10(3) cells/ml). In two severely diseased quarters of the control group (SCC of 967×10(3) cells/ml and 1824×10(3) cells/ml) Escherichia coli and Staphylococcus aureus were detected. DCC patterns of milk samples (n = 25) with very low SCC values of ≤6·25×10(3)cells/ml revealed high lymphocyte proportions of up to 92%. Milk cell populations in samples (n = 41) with SCC values of (>6·25 to ≤25)×10(3) cells/ml were also dominated by lymphocytes (mean value 47%), whereas DCC patterns of milk from udder quarters (n = 6) with SCC values (>25 to ≤100)×10(3)cells/ml changed. While in samples (n = 3) with SCC values of (27-33)×10(3) cells/ml macrophages were predominant (35-40%), three milk samples with (43-45)×10(3) cells/ml indicated already inflammatory reactions based on the predominance of polymorphonuclear leucocytes (PMN) (54-63%). In milk samples of diseased quarters PMN were categorically found as dominant cell population with proportions of ≥65%. Macrophages were the second predominant cell population in almost all samples tested in relationship to lymphocytes and PMN. To our knowledge, this is the first study evaluating cell populations in low SCC milk in detail. Udder quarters classified as normal secreting by SCC <100×10(3) cells/ml revealed already inflammatory processes based on DCC.