Philippe Marianneau

Report of a fatal case of dengue infection with hepatitis: Demonstration of dengue antigens in hepatocytes and liver apoptosis

A fatal case of dengue (DEN) infection associated with a spleen rupture and with hepatitis is reported here. Microscopic studies showed numerous areas of spleen rupture with hematomas and revealed necrotic foci in liver samples obtained at autopsy. Although hepatitis was reported in several cases of DEN fever, the mechanism of liver injury remains poorly understood. In this case, immunohistochemistry showed that DEN viral antigens were mostly detected in hepatocytes surrounding the necrotic foci. By in situ detection of DNA fragmentation, apoptotic hepatocytes were found to be colocated with DEN virus-infected hepatocytes. These findings suggest that hepatocytes are the major sites of DEN virus replication in the liver and that DEN virus induces apoptosis of hepatocytes in vivo.

A Golden Hamster Model for Human Acute Nipah Virus Infection

A predominantly pig-to-human zoonotic infection caused by the novel Nipah virus emerged recently to cause severe morbidity and mortality in both animals and man. Human autopsy studies showed the pathogenesis to be related to systemic vasculitis that led to widespread thrombotic occlusion and microinfarction in most major organs especially in the central nervous system. There was also evidence of extravascular parenchymal infection, particularly near damaged vessels (Wong KT, Shieh WJ, Kumar S, Norain K, Abdullah W, Guarner J, Goldsmith CS, Chua KB, Lam SK, Tan CT, Goh KJ, Chong HT, Jusoh R, Rollin PE, Ksiazek TG, Zaki SR, Nipah Virus Pathology Working Group: Nipah virus infection: Pathology and pathogenesis of an emerging paramyxoviral zoonosis. Am J Pathol 2002, 161:2153-2167). We describe here a golden hamster (Mesocricetus auratus) model that appears to reproduce the pathology and pathogenesis of acute human Nipah infection. Hamsters infected by intranasal or intraperitoneal routes died within 9 to 29 days or 5 to 9 days, respectively. Pathological lesions were most severe and extensive in the hamster brain. Vasculitis, thrombosis, and more rarely, multinucleated endothelial syncytia, were found in blood vessels of multiple organs. Viral antigen and RNA were localized in both vascular and extravascular tissues including neurons, lung, kidney, and spleen, as demonstrated by immunohistochemistry and in situ hybridization, respectively. Paramyxoviral-type nucleocapsids were identified in neurons and in vessel walls. At the terminal stage of infection, virus and/or viral RNA could be recovered from most solid organs and urine, but not from serum. The golden hamster is proposed as a suitable model for further studies including pathogenesis studies, anti-viral drug testing, and vaccine development against acute Nipah infection.

Nipah Virus: Vaccination and Passive Protection Studies in a Hamster Model

ABSTRACT Nipah virus, a member of the paramyxovirus family, was first isolated and identified in 1999 when the virus crossed the species barrier from fruit bats to pigs and then infected humans, inducing an encephalitis with up to 40% mortality. At present there is no prophylaxis for Nipah virus. We investigated the possibility of vaccination and passive transfer of antibodies as interventions against this disease. We show that both of the Nipah virus glycoproteins (G and F) when expressed as vaccinia virus recombinants induced an immune response in hamsters which protected against a lethal challenge by Nipah virus. Similarly, passive transfer of antibody induced by either of the glycoproteins protected the animals. In both the active and passive immunization studies, however, the challenge virus was capable of hyperimmunizing the vaccinated animals, suggesting that although the virus replicates under these conditions, the immune system can eventually control the infection.

Early and Strong Immune Responses Are Associated with Control of Viral Replication and Recovery in Lassa Virus-Infected Cynomolgus Monkeys

ABSTRACT Lassa virus causes a hemorrhagic fever endemic in West Africa. The pathogenesis and the immune responses associated with the disease are poorly understood, and no vaccine is available. We followed virological, pathological, and immunological markers associated with fatal and nonfatal Lassa virus infection of cynomolgus monkeys. The clinical picture was characterized by fever, weight loss, depression, and acute respiratory syndrome. Transient thrombocytopenia and lymphopenia, lymphadenopathy, splenomegaly, infiltration of mononuclear cells, and alterations of the liver, lungs, and endothelia were observed. Survivors exhibited fewer lesions and a lower viral load than nonsurvivors. Although all animals developed strong humoral responses, antibodies appeared more rapidly in survivors and were directed against GP1, GP2, and NP. Type I interferons were detected early after infection in survivors but only during the terminal stages in fatalities. The mRNAs for CXCL10 (IP-10) and CXCL11 (I-TAC) were abundant in peripheral blood mononuclear cells and lymph nodes from infected animals, but plasma interleukin-6 was detected only in fatalities. In survivors, high activated-monocyte counts were followed by a rise in the total number of circulating monocytes. Activated T lymphocytes circulated in survivors, whereas T-cell activation was low and delayed in fatalities. In vitro stimulation with inactivated Lassa virus induced activation of T lymphocytes from all infected monkeys, but only lymphocytes from survivors proliferated. Thus, early and strong immune responses and control of viral replication were associated with recovery, whereas fatal infection was characterized by major alterations of the blood formula and, in organs, weak immune responses and uncontrolled viral replication.

Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs

We analysed the binding and infectivity of dengue virus serotype 1 (DEN-1) for the human hepatoma cell line HepG2 in comparison with the simian kidney cell line Vero. The higher susceptibility of Vero cells to DEN-1 correlated with greater binding affinity of DEN-1 to these cells. In contrast, the capacity of virus attachment was higher for HepG2 than for Vero cells. Profiles of DEN-1 binding at different pH were markedly different between the two cell types. A type-specific neutralizing monoclonal antibody reduced initial virus binding to both cell types similarly but complex- and group-specific neutralizing antibodies affected virus adhesion differently. Altogether, these results suggest the involvement of different receptors or receptors presented in a different environment on the cell surface in the two cell lines. The sensitivity to proteolytic enzymes and to ionic detergent of the binding sites on the two cell types was tested and results indicated that they may be multimeric proteins or protein complexes.

Differing Infection Patterns of Dengue and Yellow Fever Viruses in a Human Hepatoma Cell Line

Dengue (DEN) and yellow fever (YF) viruses are responsible for human diseases with symptoms ranging from mild fever to hepatitis and/or hemorrhages. Whereas DEN virus typically induces only limited foci of necrosis in the liver, YF virus infection is characterized by devastating lesions. In a human hepatoma cell line (HepG2), the kinetics of DEN and YF virus replication and release from the cells and the nature of host cell response to viral infection were compared. DEN virus infection was characterized by the early appearance of intracellular viral antigens, major ultrastructural cytopathic changes as early as 32 h after infection, extensive apoptotic cell death, and a low production of infectious particles. In contrast, YF virus grew exponentially to high titers and induced cytopathic changes only 72 h after infection. Differences between the infection processes of the two viruses observed in the hepatoma cell line may explain the different liver pathologies.

Apoptotic cell death in response to dengue virus infection: the pathogenesis of dengue haemorrhagic fever revisited.

BACKGROUNDnDengue virus infection may be asymptomatic or lead to undifferentiated febrile illness or dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS). The major clinical manifestations of DHF/DSS are high fever, haemorrhage, hepatomegaly and circulatory failure.nnnOBJECTIVESnThe relatively high level of viraemia only a few days after infection may reflect a large number of replication sites. However, the degree of cell injury in fatal cases of DHF/DSS is not sufficient to explain death and suggests metabolic disturbance rather than tissue destruction. This theory was investigated in this study.nnnRESULTSnWe demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo.nnnCONCLUSIONSnThe elimination of apoptotic bodies by phagocytic cells is a previously unsuspected pathway of dengue virus clearance from infected tissues. However, the mechanisms of host defence involving apoptosis and phagocytic cell activation may cause local tissue injury or transient homeostasis imbalance and may trigger further deleterious events.

Experimental infection of squirrel monkeys with nipah virus

We infected squirrel monkeys (Saimiri sciureus) with Nipah virus to determine the monkeys’ suitability for use as primate models in preclinical testing of preventive and therapeutic treatments. Infection of squirrel monkeys through intravenous injection was followed by high death rates associated with acute neurologic and respiratory illness and viral RNA and antigen production.

Measles Vaccine Expressing the Secreted Form of West Nile Virus Envelope Glycoprotein Induces Protective Immunity in Squirrel Monkeys, a New Model of West Nile Virus Infection

West Nile virus (WNV) is a mosquito-borne flavivirus that emerged in North America and caused numerous cases of human encephalitis, thus urging the development of a vaccine. We previously demonstrated the efficacy of a recombinant measles vaccine (MV) expressing the secreted form of the envelope glycoprotein from WNV to prevent WNV encephalitis in mice. In the present study, we investigated the capacity of this vaccine candidate to control WNV infection in a primate model. We first established experimental WNV infection of squirrel monkeys (Saimiri sciureus). A high titer of virus was detected in plasma on day 2 after infection, and viremia persisted for 5 days. A single immunization of recombinant MV-WNV vaccine elicited anti-WNV neutralizing antibodies that strongly reduced WNV viremia at challenge. This study demonstrates for the first time the capacity of a recombinant live attenuated measles vector to protect nonhuman primates from a heterologous infectious challenge.

Characterization of Lassa Virus Cell Entry and Neutralization with Lassa Virus Pseudoparticles

ABSTRACT The cell entry and humoral immune response of the human pathogen Lassa virus (LV), a biosafety level 4 (BSL4) Old World arenavirus, are not well characterized. LV pseudoparticles (LVpp) are a surrogate model system that has been used to decipher factors and routes involved in LV cell entry under BSL2 conditions. Here, we describe LVpp, which are highly infectious, with titers approaching those obtained with pseudoparticles displaying G protein of vesicular stomatitis virus and their the use for the characterization of LV cell entry and neutralization. Upon cell attachment, LVpp utilize endocytic vesicles for cell entry as described for many pH-dependent viruses. However, the fusion of the LV glycoproteins is activated at unusually low pH values, with optimal fusion occurring between pH 4.5 and 3, a pH range at which fusion characteristics of viral glycoproteins have so far remained largely unexplored. Consistent with a shifted pH optimum for fusion activation, we found wild-type LV and LVpp to display a remarkable resistance to exposure to low pH. Finally, LVpp allow the fast and quantifiable detection of neutralizing antibodies in human and animal sera and will thus facilitate the study of the humoral immune response in LV infections.