Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.

Dengue virus (DENV 1-4) represents a major emerging arthropod-borne pathogen. All four DENV serotypes are prevalent in the (sub) tropical regions of the world and infect 50-100 million individuals annually. Whereas the majority of DENV infections proceed asymptomatically or result in self-limited dengue fever, an increasing number of patients present more severe manifestations, such as dengue hemorrhagic fever and dengue shock syndrome. In this review we will give an overview of the infectious life cycle of DENV and will discuss the viral and host factors that are important in controlling DENV infection.

Cells infected with dengue virus release a high proportion of immature prM-containing virions. In accordance, substantial levels of prM antibodies are found in sera of infected humans. Furthermore, it has been recently described that the rates of prM antibody responses are significantly higher in patients with secondary infection compared to those with primary infection. This suggests that immature dengue virus may play a role in disease pathogenesis. Interestingly, however, numerous functional studies have revealed that immature particles lack the ability to infect cells. In this report, we show that fully immature dengue particles become highly infectious upon interaction with prM antibodies. We demonstrate that prM antibodies facilitate efficient binding and cell entry of immature particles into Fc-receptor-expressing cells. In addition, enzymatic activity of furin is critical to render the internalized immature virus infectious. Together, these data suggest that during a secondary infection or primary infection of infants born to dengue-immune mothers, immature particles have the potential to be highly infectious and hence may contribute to the development of severe disease.

SUMMARY We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O’nyong’nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

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 and assembly of the E protein (2, 27). The E glycoproteins are assembled as homodimers on the surface of mature virions and mediate the infectious entry of flaviviruses into cells (14, 21). The crystal structure of the major external part of the E glycoprotein has been solved and reveals that the protein contains three distinct domains: domain I is the structurally central domain, domain II is the dimerization domain and contains the fusion peptide, and domain III has an immunoglobulin-like fold and mediates receptor binding (1,6,7,21,39).The initial step in the viral life cycle of DENV is attachment of the virus to a cellular receptor. DENV has been proposed to bind to the glycosaminoglycan heparan sulfate, ...

Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent advances in the understanding of flavivirus cell entry with specific emphasis on the recent study of Zaitseva and coworkers, indicating that anionic lipids might play a crucial role in the fusion process of dengue virus [1].

Prior to the release of flavivirus particles from infected cells, the viral surface protein prM is cleaved to M by the cellular enzyme furin. For dengue virus (DENV), this maturation process appears to be very inefficient since a high proportion of progeny virions contain uncleaved prM. Furthermore, it has been reported that prM-containing DENV particles are infectious. These observations contradict the general assumption that prM processing is required to render virus particles infectious. Therefore, in this study, we reinvestigated the infectious properties of immature DENV virions. DENV particles were produced in furin-deficient LoVo cells. We observed that DENV-infected LoVo cells secrete high numbers of prM-containing particles. Subsequent analysis of the infectious titre revealed that immature particles lack the ability to infect cells, the infectious unit to particle ratio being 10 000-fold reduced compared with that of wild-type virus. Our results indicate that cleavage of prM to M is required for DENV infectivity.Dengue virus (DENV) is a mosquito-transmitted pathogen, which together with other arthropod-borne viruses like yellow fever virus, West Nile virus and tick-borne encephalitis virus (TBEV) are members of the genus Flavivirus. Flaviviruses are small, enveloped, icosahedral viruses with a single copy of a positive-strand RNA genome. The viral envelope anchors two transmembrane proteins: the small (8 kDa) membrane protein M and the major (51-60 kDa) envelope glycoprotein E (Lindenbach & Rice, 2001). The E glycoproteins are organized in 90 homodimers, which lie flat on the viral surface (Kuhn et al., 2002;Kuhn & Rossmann, 2005).Assembly of virus particles is initiated in the endoplasmic reticulum by formation of an immature virion. In immature particles, the E protein forms a heterodimeric association with prM, the precursor protein of M. Each particle contains 60 trimers of E-prM heterodimers and thus differs structurally from a mature virion (Kuhn et al., 2002;Lorenz et al., 2002;Zhang et al., 2003bZhang et al., , 2004. Maturation of flavivirus particles occurs during transport through the exocytic pathway. The viral envelope proteins are believed to undergo conformational changes triggered by the low pH in the lumen of the trans-Golgi network (TGN). Shortly before or during the final release of the virions, prM is cleaved by the host cell endoprotease furin into virion-associated M and a soluble peptide (Mackenzie & Westaway, 2001;Wengler & Wengler, 1989).The biological significance of the maturation process has been investigated in considerable detail, particularly for TBEV. Characterization of the infectious properties of prM-containing TBEV virions revealed that these particles cannot undergo the structural rearrangements required for membrane fusion (Guirakhoo et al., 1991;Heinz et al., 1994a;Stadler et al., 1997). Consequently, immature particles are considered to be non-infectious (Elshuber et al., 2003;Elshuber & Mandl, 2005; Heinz et al., 1994b;Stadler et al., 1997). These results led to ...