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In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes. Dengue virus (DENV) is an enveloped, positive-strand RNA virus belonging to the family Flaviviridae, which also includes tick-borne encephalitis virus, yellow fever virus, and West Nile virus. Flavivirus virions contain three structural proteins: the C (capsid) protein, the M (membrane) protein, and the E (envelope) protein (12, 26). Multiple copies of the C protein associate with the viral RNA to form the nucleocapsid (26). The nucleocapsid is surrounded by a lipid bilayer in which the M and E glycoproteins are inserted. In the infected cell, the M protein is produced as a precursor protein called prM, which is believed to function as a chaperone during the folding

The type VI secretion system (T6SS) of Gram-negative bacteria has been involved in various processes, notably bacterial competition and eukaryotic cell subversion. Most Pseudomonas aeruginosa strains possess three T6SS gene clusters, but only the function of the first T6SS (H1-T6SS) has been clearly elucidated. It is involved in the secretion of three toxins (Tse1 to -3) that target bacterial competitors. In the case of the H2- and H3-T6SS, no clear function has been assigned, and only one effector has been associated with these systems. Yet the H2-T6SS was proposed to promote P. aeruginosa internalization in nonphagocytic epithelial cells. Although the H2-T6SS genetic organization is conserved across P. aeruginosa isolates, one feature is the presence of an additional transcriptional unit in the PA14 strain H2-T6SS cluster, which is divergent from the core H2-T6SS genes. A specific set of four genes encodes an Hcp protein (Hcp2), a VgrG protein (VgrG14), an Rhs element (PA14_43100 or RhsP2), and a protein with no homologies with previously characterized proteins (PA14_43090). In this study, we engineered a P. aeruginosa PA14 strain carrying an arabinose-inducible H2-T6SS on the chromosome. We showed that arabinose induction readily promotes assembly of the H2-T6SS, as seen by monitoring Hcp2 secretion. We further studied the secretion fate of VgrG14 and RhsP2, but these were not detectable in the extracellular medium. We finally investigated whether activation of the PA14 H2-T6SS gene cluster could influence phenotypic traits such as internalization in eukaryotic cells, and we reported noteworthy differences compared to strain PAO1, which may be accounted for by the described genetic differences.

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4(+) T cells, and CD8(+) T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected (YP T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of BI cells suggested that BL cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding, We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and Leads to the production of high levels of RV-specific intestinal IgA, In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA, Cotransfer of mixtures of purified B1 cells and Iii-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (BI-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA, To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4(+) T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4(+) T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4(+) T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4(+) T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.