The spread of dengue (DEN) worldwide combined with an increased severity of the DEN-associated clinical outcomes have made this mosquito-borne virus of great global public health importance. Progress in understanding DEN pathogenesis and in developing effective treatments has been hampered by the lack of a suitable small animal model. Most of the DEN clinical isolates and cell culture-passaged DEN virus strains reported so far require either host adaptation, inoculation with a high dose and/or intravenous administration to elicit a virulent phenotype in mice which results, at best, in a productive infection with no, few, or irrelevant disease manifestations, and with mice dying within few days at the peak of viremia. Here we describe a non-mouse-adapted DEN2 virus strain (D2Y98P) that is highly infectious in AG129 mice (lacking interferon-α/β and -γ receptors) upon intraperitoneal administration. Infection with a high dose of D2Y98P induced cytokine storm, massive organ damage, and severe vascular leakage, leading to haemorrhage and rapid death of the animals at the peak of viremia. In contrast, very interestingly and uniquely, infection with a low dose of D2Y98P led to asymptomatic viral dissemination and replication in relevant organs, followed by non-paralytic death of the animals few days after virus clearance, similar to the disease kinetic in humans. Spleen damage, liver dysfunction and increased vascular permeability, but no haemorrhage, were observed in moribund animals, suggesting intact vascular integrity, a cardinal feature in DEN shock syndrome. Infection with D2Y98P thus offers the opportunity to further decipher some of the aspects of dengue pathogenesis and provides a new platform for drug and vaccine testing.

The mechanism of action of a serotype-specific natural human antibody against dengue virus has been identified.

The sterically bulky carbene precursor 1,3-diisopropylbenzimidazolium bromide ( i Pr 2 -bimyH + Br -) (A) has been prepared by an improved method in 84% yield. Reaction of A with Pd(OAc) 2 and NaBr gave the dimeric Pd(II) benzimidazolin-2-ylidene complex [PdBr 2 ( i Pr 2 -bimy)] 2 (1), which can be easily cleaved by CH 3 CN, another equivalent of salt A, and triphenylphosphine to afford the novel benzannulated monocarbene complexes trans-[PdBr 2 (CH 3 CN)( i Pr 2 -bimy)] (2), ( i Pr 2 -bimyH)[PdBr 3 ( i Pr 2 -bimy)] (3), trans-[PdBr 2 ( i Pr 2 -bimy)(Ph 3 P)] (trans-4), and cis-[PdBr 2 ( i Pr 2 -bimy)(Ph 3 P)] (cis-4), respectively. All compounds have been fully characterized by multinuclei NMR spectroscopies and mass spectrometries (FAB, ESI). X-ray diffraction studies on single crystals of 1-3 and cis-4 revealed a square planar geometry and a fixed orientation of the N-isopropyl substituents with the C-H group pointing to the metal center to maximize C-H‚‚‚Pd interactions. The large downfield shift of the C-H protons in the 1 H NMR spectrum compared to the precursor A indicates that these C-H‚‚‚Pd interactions are retained in solution and better described as weak hydrogen bonds, rather than as agostic interactions. Furthermore, the molecular structures of especially complexes 2 and 3 clearly show a bending of the bromo ligands toward the carbene carbon atom in order to maximize intramolecular C carbene ‚‚‚Br interactions. The nature of these interactions can be attributed to a form of back-bonding to the formally vacant p-orbital of the C carbene atom with the electron density originating from the bromo ligands' lone pairs. A detailed study on the trans-cis isomerization of the mixed NHC-phosphine complexes 4 revealed that a cis arrangement in such complexes is thermodynamically favored. Furthermore, a preliminary catalytic study shows that complex 1 is highly active in the Suzuki-Miyaura coupling of aryl bromides and chlorides in pure water as environmentally benign solvent.

Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations.Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.

No abstract

Dengue (DEN) is a mosquito-borne viral disease that has become an increasing economic and health burden for the tropical and subtropical world. The lack of an appropriate animal model of DEN has greatly impeded the study of its pathogenesis and the development of vaccines/antivirals. We recently reported a DEN virus 2 (DENV-2) strain (D2Y98P) that lethally infects immunocompromised AG129 mice, resulting in organ damage or dysfunction and increased vascular permeability, hallmarks of severe DEN in patients (G. K. Tan et al., PLoS Negl. Trop. Dis. 4:e672, 2010). Here we report the identification of one critical virulence determinant of strain D2Y98P. By mutagenesis, we showed that a Phe-to-Leu alteration at amino acid position 52 in nonstructural protein NS4B completely abolished the pathogenicity of the D2Y98P virus, as evidenced by a lack of lethality and the absence of histological signs of disease, which correlated with reduced viral titers and intact vascular permeability. Conversely, a Leu-to-Phe alteration at position 52 of NS4B in nonvirulent DENV-2 strain TSV01 led to 80% lethality and increased viremia. The NS4B(Phe52) viruses displayed enhanced RNA synthesis in mammalian cells but not in mosquito cells. The increased viral RNA synthesis was independent of the ability of NS4B to interfere with the host type I interferon response. Overall, our results demonstrate that Phe at position 52 in NS4B confers virulence in mice on two independent DENV-2 strains through enhancement of viral RNA synthesis. In addition to providing further insights into the functional role of NS4B protein, our findings further support a direct relationship between viral loads and DEN pathogenesis in vivo, consistent with observations in DEN patients.Dengue (DEN) disease, caused by any one of the four serotypes of dengue virus (DENV), is a major global public health and economic burden. DENV affects 2.5 billion people worldwide, mostly in tropical and subtropical countries, leading to 50 to 100 million human infections each year (13). Clinical manifestations of DEN disease range from undifferentiated fever to DEN fever (DF) and DEN hemorrhagic fever (DHF), with plasma leakage that may lead to hypovolemic shock (DEN shock syndrome [DSS]). Therapeutic intervention is limited to fluid management for DEN patients who experience severe vascular leakage (41). Currently, no antiviral therapy or vaccine to fight DEN is available on the market (9, 31).DENV belongs to the genus Flavivirus within the family Flaviviridae. Besides DENV, many other flaviviruses are important human pathogens, including yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). DENV is a small enveloped virus with a positive single-strand RNA of approximately 10,700 nucleotides (18). The DENV genome encodes 10 proteins, comprising 3 structural proteins (capsid [C], premembrane [PrM], and envelope [E]) and 7 nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The structural proteins fo...

Reaction of the bromo-bridged dimeric monocarbene complex [PdBr 2 ( i Pr 2 -bimy)] 2 (1) with various bidentate N-heterocycles afforded novel linear dinuclear Pd(II) complexes {[PdBr 2 ( i Pr 2 -bimy)] 2 (µ-L)}-{µ-L ) 4,4′-bipyridine (2); µ-L ) 4,4′-bipyridylethane (3); µ-L ) 4,4′-bipyridylethylene (4)}. The mononuclear counterpart [PdBr 2 ( i Pr 2 -bimy)(pyridine)] ( 5) has also been synthesized by reaction of 1 with pyridine. All compounds have been fully characterized by multi-nuclei NMR spectroscopies, FAB mass spectrometry, and X-ray diffraction analyses. Molecular structures of 2-5 show a fixed orientation of the C-H protons in the N-isopropyl substituents toward the metal center, suggesting interesting C-H‚ ‚‚Pd preagostic interactions. These interactions are retained in solution as indicated by the large downfield shift of these C-H protons in the 1 H NMR spectrum. UV-vis and CV studies revealed that the dinuclear complexes 2-4 have electrochemical behavior similar to that of the mononuclear species 5.

Two stereoisomers of cyclobutane derivatives with pyridyl and carboxylic acid functionalities have been stereoselectively synthesized by a solid-state photochemical [2 + 2] cycloaddition reaction in quantitative yields. The head-to-head and head-to-tail parallel orientations of the monomers, required to obtain these two isomers, have been controlled by the anions present in the salts. The photoinert behavior of these salts in solution signifies the importance of the solid-state synthesis of these cyclobutane derivatives.