Marie-Thérèse Drouet

Introduction of West Nile virus in the Middle East by Migrating White Storks

West Nile virus (WNV) was isolated in a flock of 1,200 migrating white storks that landed in Eilat, a town in southern Israel, on August 26, 1998. Strong, hot westerly winds had forced the storks to fly under considerable physical stress before reaching the agricultural land surrounding the town. Most of the flock were fledglings, <1 year old, which had hatched in Europe. Thirteen dead or dying storks were collected 2 days after arrival and submitted to the laboratory for examination. Four WNV isolates were obtained from their brains. Out of 11 storks tested six days after arrival, three had WNV-neutralizing antibodies. Comparative analysis of full-length genomic sequences of a stork isolate and a 1999 flamingo isolate from the USA showed 28 nucleotide (nt) (0.25%) and 10 amino acid (0.3%) changes. Sequence analysis of the envelope gene of the stork isolate showed almost complete identity with isolates from Israeli domestic geese in 1998 and 1999 and from a nonmigrating, white-eyed gull in 1999. Since these storks were migrating southwards for the first time and had not flown over Israel, we assume that they had become infected with WNV at some point along their route of migration in Europe.

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.

Liver histopathology and biological correlates in five cases of fatal dengue fever in Vietnamese children.

Abstract. We studied five fatal cases of dengue haemorrhagic fever (DHF), confirmed using the reverse transcriptase-polymerase chain reaction (RT-PCR) method, in Vietnamese children. The liver seems to be a target for dengue virus, so postmortem examinations were performed to investigate elementary lesions, local recruitment of inflammatory cells and whether the virus was present in target cells of the liver. We detected severe, diffuse hepatitis with midzonal necrosis and steatosis in two patients, focal areas of necrosis in two patients, and normal histology in one patient. Dengue virus antigen was detected using immunohistochemistry in hepatocytes from necrotic areas in four cases. There was no recruitment of polymorphonuclear cells, and no lymphocytes were detected in the liver lesions of patients who died from DHF. Lymphocytic infiltration occurred in only one hepatitis B virus-positive patient, with no signs of chronic hepatitis. Kupffer cells had mostly been destroyed in cases with focal or severe necrosis. TUNEL tests were positive in necrotic areas, with positive cells forming clusters, suggesting that an apoptotic mechanism was involved. Thus, we suggest that the hepatocyte and Kupffer cells may be target cells supporting virus replication and that the councilman body is an apoptotic cell, as in the pathogenesis of yellow fever.

The Secreted Form of Dengue Virus Nonstructural Protein NS1 Is Endocytosed by Hepatocytes and Accumulates in Late Endosomes: Implications for Viral Infectivity

ABSTRACT The flavivirus nonstructural protein NS1 is expressed as three discrete species in infected mammalian cells: an intracellular, membrane-associated form essential for viral replication, a cell surface-associated form that may be involved in signal transduction, and a secreted form (sNS1), the biological properties of which remain elusive. To determine the distribution of the dengue virus (DEN) sNS1 protein in vivo, we have analyzed by immunohistological means the tissue tropism of purified DEN sNS1 injected intravenously into adult mice. The sNS1 protein was found predominantly associated with the liver, where hepatocytes appeared to represent a major target cell. We further showed that sNS1 could be efficiently endocytosed by human Huh7 and HepG2 hepatocytes in vitro. After its internalization, the protein was detected intracellularly for at least 48 h without being substantially degraded. Colocalization studies of sNS1 with markers of the endolysosomal compartments revealed that the protein was specifically targeted to lysobisphosphatidic acid-rich structures reminiscent of late endosomes, as confirmed by electron microscopy. Intracellular accumulation of sNS1 in Huh7 cells enhanced the fluid phase uptake of rhodamine-labeled dextran. Furthermore, preincubation of Huh7 cells with sNS1 increased dengue virus production after infection with the homologous strain of DEN-1 virus. Our results demonstrate that the accumulation of DEN sNS1 in the late endosomal compartment of hepatocytes potentializes subsequent dengue virus infection in vitro, raising the possibility that sNS1 may contribute to viral propagation in vivo.

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.

Variations in Biological Features of West Nile Viruses

Abstract: Pathological findings in humans, horses, and birds with West Nile (WN) encephalitis show neuronal degeneration and necrosis in the central nervous system (CNS), with diffuse inflammation. The mechanisms of WN viral penetration of the CNS and pathophysiology of the encephalitis remain largely unknown. Since 1996, several epizootics involving hundreds of humans, horses, and thousands of wild and domestic bird cases of encephalitis and mortality have been reported in Europe, North Africa, the Middle East, Russia, and the USA (see specific chapters in this issue). However, biological and molecular markers of virus virulence should be characterized to assess whether novel strains with increased virulence are responsible for this recent proliferation of outbreaks.

Molecular detection and characterization of yellow fever virus in blood and liver specimens of a non-vaccinated fatal human case

A yellow fever virus of a South American genotype was identified in the liver and blood samples of a non‐vaccinated European patient after his return from Brazil. ELISA tests were negative for IgG and positive for IgM against yellow fever. Yellow fever proteins in the formalin‐fixed and paraffin‐embedded liver biopsy were detected by immunohistochemical procedures. Viral RNA extracted from the liver tissue was also detected using an RT‐semi‐nested PCR procedure and molecular hybridization. Alignment of the sequence obtained from a gene fragment amplified by RT‐semi‐nested PCR directly from a blood sample with those of African and South American yellow fever virus strains identified a Brazilian topotype as being responsible for the disease. RT‐semi‐nested PCR may be used advantageously for clinical specimens for rapid and specific diagnosis, and with archival biopsy material for retrospective studies. J. Med. Virol. 53:212–217, 1997.

Interaction of dengue virus with human kupffer cells in vitro

Kupffer cells (KG). or liver macrophdges~, play a plvolal role In tht! pathogenesis of viral infection. Accordingly, they have the initial opportunity to clear the bulk of the virus from the circulation and depending on the type and efficiency of their interaction with the virus as well as its nature, several outcomes ranging from fecovery to persistent hepatitis, fulminant liver failure and death are possible. Dengue virus, like yellow fever virus, is a member of the flavivirus genus in the far

Detection of dengue virus RNA by reverse transcription-polymerase chain reaction in the liver and lymphoid organs but not in the brain in fatal human infection.

ly Raviviridae. Both agents are responsible for a large diversity of infections especiaily hemorrhagk fever. The role of the KG and the nature of their interactions with dengue virus in the course of the disease are not well know. In this study, we have infected primary cultures of human Kcs with dengue virus. The penetration of the virus into the cells and its cytopatbic effect have been examined by electron microscopy.We examined the production of infectious p&rticles. the synthesis of intracellular viral antigens and the induction of the cell death by apoptosis and followed the production of several cytokines and nit& oxide by infected Kcs. Our results show that viral particles are taken up by Kcs mainly by phagocytosis and, less frequently, by receptor mediated endoc)itosis in clathrin-coated pits. Several pictures suggested a process of fusion. No infectious particles were released from Kcs.Viral proteins were synthesized in abouth 10% of the cells, 24 hours after~infection. The dattern of the immunofluorescence was either speckled or concentrated in huge patches. Apoptotic cells could be detected by TUNEL assay and electron microscopy and these were phagucytosed and degraded by neibhourinl! Kcs. Infected Kcs svnthetized TNF-CX, IFN-a and NO as earlv as 6 hours &er infection, and k-6 after 24hours. Our results show that Kcs are not permissive for denguc virus. In ;t low number of cells an abortive infection resulting in apoptotic cell death was observed. Dengue virus induces Kcs activation. Viral particles as well as viral antigen-containing cells were rapidly taken up and degraded. LECHAIRE Jean-Pierre, FREBOURG Ghislaine and GAILL Frampise.