Bank vole immunoheterogeneity may limit Nephropatia Epidemica emergence in a French non-endemic region.

Ecoevolutionary processes affecting hosts, vectors and pathogens are important drivers of zoonotic disease emergence. In this study, we focused on nephropathia epidemica (NE), which is caused by Puumala hantavirus (PUUV) whose natural reservoir is the bank vole, Myodes glareolus. We questioned the possibility of NE emergence in a French region that is considered to be NE-free but that is adjacent to a NE-endemic region. We first confirmed the epidemiology of these two regions and we demonstrated the absence of spatial barriers that could have limited dispersal, and consequently, the spread of PUUV into the NE-free region. We next tested whether regional immunoheterogeneity could impact PUUV chances to circulate and persist in the NE-free region. We showed that bank voles from the NE-free region were sensitive to experimental PUUV infection. We observed high levels of immunoheterogeneity between individuals and also between regions. Antiviral gene expression (Tnf and Mx2) reached higher levels in bank voles from the NE-free region. During experimental infections, anti-PUUV antibody production was higher in bank voles from the NE-endemic region. These results indicated a lower susceptibility to PUUV for bank voles from this NE-free region, which might limit PUUV persistence and therefore, the risk of NE.

Crimean-Congo haemorrhagic fever.

Crimean-Congo haemorrhagic fever (CCHF) is one of the most widespread arboviroses in the world. It is present in Africa, south-east Europe, the Middle East and Asia. It is caused by a nairovirus (Bunyaviridae family) transmitted by several species of ticks. The geographical distribution of the disease coincides with the distribution of Hyalomma ticks. While infected livestock do not show signs of illness, humans are severely affected, with a high mortality rate. The most common symptoms are high fever, dizziness, headache, vomiting and haemorrhages. Pathogenesis studies in interferon-receptor-deficient mice indicated that the interferon response is crucial in controlling virus propagation and in protecting against the disease. Detection of the virus in biological material is currently performed by reverse-transcription polymerase chain reaction. Enzyme-linked immunosorbent assay and indirect immunofluorescence are used to detect the presence of CCHF virus-specific antibodies. In the 1970s, a formalin-inactivated vaccine prepared from suckling mouse brain was used in Eastern Europe and the former Soviet Union, but its efficacy remains to be proven. Treatment of patients with ribavirin is recommended by the World Health Organization, but it should be administered as early as possible. Although important progress has been made over the last few decades, many questions about the pathogenesis and epidemiology of the disease are still to be addressed and there is a need to develop efficient vaccines and antivirals.

Seroprevalence and incidence of Puumala orthohantavirus in its bank vole (Myodes glareolus) host population in northeastern France: Between-site and seasonal variability.

Given the difficulty of measuring pathogen transmission in wildlife, epidemiological studies frequently rely on cross-sectional seroprevalence. However, seropositivity indicates only exposure to a pathogen at an unknown time. By allowing to obtain repeated test results from individuals sampled multiple times over an extended period, longitudinal data help reduce this uncertainty. We used capture-mark-recapture data on bank vole (Myodes glareolus) individuals collected at four sites over ten years in northeastern France to investigate the impact of environmental variables on seroprevalence and incidence of Puumala orthohantavirus (PUUV). PUUV causes a chronic infection without apparent symptoms, that may however impair survival of its rodent host in the wild. Viral transmission between rodents may occur through direct contact or via the environment. Principal component analysis was used to deal with multicollinearity among environmental variables. Incidence and seroprevalence were investigated with either generalized estimating equations or Poisson regression models depending on the number of observations for each season. In spring, only the factor site was found to be significant for seroprevalence, while a principal component including meteorological conditions of the previous winter and the normalized difference vegetation index (NDVI) of both the previous winter and spring had a significant effect on incidence. In autumn, only the factor site was significant for incidence, while two principal components, including either the meteorological conditions of the autumn and previous spring or NDVI of the autumn significantly affected seroprevalence. We discuss these results in light of the particular demography of small mammals. We encourage other researchers to investigate the relationships between demographic parameters of wild host populations and the environment, by using both incidence and seroprevalence.

Environmental risk factors for haemorrhagic fever with renal syndrome in a French new epidemic area.

In France, haemorrhagic fever with renal syndrome (HFRS) is endemic along the Belgian border. However, this rodent-borne zoonosis caused by the Puumala virus has recently spread south to the Franche-Comté region. We investigated the space-time distribution of HFRS and evaluated the influence of environmental factors that drive the hantavirus reservoir abundance and/or the disease transmission in this area. A scan test clearly indicated space-time clustering, highlighting a single-year (2005) epidemic in the southern part of the region, preceded by a heat-wave 2 years earlier. A Bayesian regression approach showed an association between a variable reflecting biomass (normalized difference vegetation index) and HFRS incidence. The reasons why HFRS cases recently emerged remain largely unknown, and climate parameters alone do not reliably predict outbreaks. Concerted efforts that combine reservoir monitoring, surveillance, and investigation of human cases are warranted to better understand the epidemiological patterns of HFRS in this area.

[Crimean-Congo hemorrhagic fever: basics for general practitioners]. / La fièvre hémorragique de Crimée-Congo: l'essentiel pour le praticien.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease described in more than 30 countries in Europe, Asia and Africa. The causative agent is the Crimean-Congo hemorrhagic fever virus (CCHFV) that is a member of the genus Nairovirus of the family Bunyaviridae. CCHFV that is characterized by a high genetic variability is transmitted to humans by tick bites or contact with fluids from an infected individual or animal. The initial symptoms of CCHF are nonspecific and gradually progress to a hemorrhagic phase that can be lethal (case-fatality rate: 10 to 50%). Characteristic laboratory findings of CCHF are thrombocytopenia, elevated liver and muscle enzymes, and coagulation defects. The pathogenesis of CCHF remains unclear but might involve excessive pro-inflammatory cytokine production and dysfunction of the innate immune response. Diagnosis of CCHF is based mainly on isolation of the virus, identification of the viral genome by molecular techniques (RT-PCR), and serological detection of anti-CCHFV antibodies. There is currently no specific treatment for CCHFV infection and the efficacy of ribavirin is controversial. In absence of an effective vaccine, prevention is based mainly on vector control, protection measures, and information to increase the awareness of the population and of healthcare workers.

First insights into Puumala orthohantavirus circulation in a rodent population in Alsace, France.

In-depth knowledge on the mechanisms that maintain infection by a zoonotic pathogen in an animal reservoir is the key to predicting and preventing transmission to humans. The Puumala orthohantavirus (PUUV), the most prevalent orthohantavirus in Western Europe, causes a mild form of haemorrhagic fever with renal syndrome (HFRS) in humans. In France, this endemic illness affects the north-eastern part of the country. We conducted a 4-year capture-mark-recapture study in a bank vole population, combined with molecular analyses, to explore the epidemiological situation of PUUV in Alsace, a French region where human cases have occurred, but for which no studies have been conducted on this reservoir host. PUUV-infected bank voles were detected in the 2 years that showed high bank vole density with a prevalence of 4%. The individual PUUV sequences identified in this study were similar from year to year and similar to other French sequences. On a very small spatial scale, the distribution of seropositive bank voles was very heterogeneous in time and space. The short distances travelled on average by bank voles resulted in spatial clusters of seropositive rodents, which spread only very gradually throughout the year.

Spatial and Temporal Epidemiology of Nephropathia Epidemica Incidence and Hantavirus Seroprevalence in Rodent Hosts: Identification of the Main Environmental Factors in Europe.

In Europe, the increasing number of nephropathia epidemica (NE) infections in humans, caused by Puumala virus carried by bank voles (Myodes glareolus), has triggered studies of environmental factors driving these infections. NE infections have been shown to occur in specific geographical areas characterized by environmental factors that influence the distribution and dynamics of host populations and virus persistence in the soil. Here, we review the influence of environmental conditions (including climate factors, food availability and habitat conditions) with respect to incidence in humans and seroprevalence in rodents, considering both direct and indirect transmission pathways. For each type of environmental factor, results and discrepancies between studies are presented and examined in the light of biological hypotheses. Overall, food availability and temperature appear to be the main drivers of host seroprevalence and NE incidence, but data quality and statistical approaches varied greatly among studies. We highlight the issues that now need to be addressed and suggest improvements for study design in regard to the current knowledge on hantavirus epidemiology.

Identifying Areas Suitable for the Occurrence of Rift Valley Fever in North Africa: Implications for Surveillance.

Rift Valley fever (RVF) is a vector-borne zoonotic disease that has caused widespread outbreaks throughout Africa and the Arabian Peninsula, with serious consequences for livestock-based economies and public health. Although there have never been any reports of RVF in Morocco, Algeria, Tunisia and Libya, it is a priority disease in the Maghreb, due to the threat of introduction of the virus through transboundary livestock movements or infected mosquito vectors. However, the implementation of surveillance activities and early warning contingency plans requires better knowledge of the epidemiological situation. We conducted a multicriteria decision analysis, integrating host distribution with a combination of important ecological factors that drive mosquito abundance, to identify hotspots and suitable time periods for RVF enzootic circulation (i.e. stable transmission at a low to moderate level for an extended period of time) and an RVF epizootic event (i.e. a sudden occurrence of a large number of infected animals over a large geographic area) in the Maghreb. We also modelled vector species distribution using available information on vector presence and habitat preference. We found that the northern regions of the Maghreb were moderately suitable for RVF enzootics, but highly suitable for RVF epizootics. The vector species distribution model identified these regions as the most favourable mosquito habitats. Due to the low density of animal hosts and arid conditions, the desert region showed low RVF suitability, except in oases. However, the presence of competent vectors in putative unsuitable areas underlines the need for further assessments of mosquito habitat preference. This study produced monthly RVF suitability maps useful for animal health managers and veterinary services involved in designing risk-based surveillance programmes. The suitability maps can be further enhanced using existing country-specific sources of information and by incorporating knowledge - as it becomes available - on the epidemiology of the disease and distribution of vectors in the Maghreb.

Ultrastructural observations in hepatitis C virus-infected lymphoid cells.

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.

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.

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.

[Recent knowledge on the pathogenesis of yellow fever and questions for the future]. / Connaissances récentes sur la pathogénie de la fièvre jaune et questions pour le futur.

Yellow fever is an arthropod-borne disease with symptoms ranging from mild fever to acute hepatonephritis, hemorrhages and shock often fatal. The pathophysiology of severe yellow fever in humans and in monkeys susceptible to the virus is largely unknown. Yellow fever virus replicates in Küpffer cells and in hepatocytes in the liver. The degree of severity in yellow fever disease is linked to different factors related to virus virulence and to host susceptibility. A better knowledge of the complex interactions between the virus and the host is requested before initiating new actions in prophylaxy and therapy against yellow fever.

[Apoptotic cell death in response to dengue virus infection: what are the consequences of viral pathogenesis?]. / La mort cellulaire par apoptose en réponse à l'infection par le virus de la dengue: quelles conséquences dans la pathogénie virale?

Dengue is a human disease of viral etiology which may be fatal in its hemorragic form. It is widely spread in the tropical areas of the different continents and has been dramatically expanding over the past 30 years. Although an immunological disorder is thought to be involved in dengue physiological symptoms, the pathogenesis of dengue hemorragic fever has not yet been elucidated. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. Other factors, related to virus replication in specific host cells, could also contribute to the severity of the disease. Apoptotic cell death is one of the important consequences of dengue virus infection both in vitro and in vivo. Dengue replication triggers apoptotic signals in neurons and hepatocytes although the original effectors and kinetics differ. Implications of the ongoing apoptotic processes in viral pathogenesis will be further discussed.

Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean-Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East.

Several haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL-4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean-Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 10(5) -10(6) PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ~13-14% of global divergence with the tiled sequence, or stretches of ~20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism.

Induction of programmed cell death (apoptosis) by dengue virus in vitro and in vivo.

Dengue is a human disease which may be fatal in its hemorrhagic form. How dengue virus- and host-specified factors underlie virulence and pathogenesis is poorly understood. An immunological disorder is thought to be involved in dengue physiological symptoms. Whether the immune response is deleterious or beneficial to the host remains a matter of debate. In this review, we summarized developments in research on viral pathogenesis in the context of apoptosis triggered by dengue virus infection. Apoptosis, an active process of cell destruction, is one of the important consequences of dengue virus infection in vitro and in vivo. Dengue virus replication induces apoptosis in mouse neurons and human hepatocytes. The ability to activate this genetically programmed cell death pathway is dependent on both viral and cellular determinants.

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

BACKGROUND: Dengue 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. OBJECTIVES: The 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. RESULTS: We demonstrated that replication of dengue virus in infected cells induces stress leading to apoptotic cell death in vitro and in vivo. CONCLUSIONS: The 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.

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.

Recent advances in vaccines against viral haemorrhagic fevers.

Development of vaccines against viral haemorrhagic fevers is a public health priority. Recent advances in our knowledge of pathogenesis and of the immune responses elicited by these viruses emphasize the crucial role of the immune system in the control of infection, but also its probable involvement in pathogenesis. Several vaccine candidates against viral haemorrhagic fevers have been evaluated in animals during the past year. Together, these data suggest that a vaccine approach against viral haemorrhagic fevers is feasible, should induce well-balanced immune responses with cellular and humoral components, and should avoid the potential deleterious effects that are associated with such immune responses.

Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death.

The severe outcome of the dengue (DEN) virus infection known as DEN hemorrhagic fever-DEN shock syndrome (DHF-DSS) is, in some cases, accompanied by liver injury. Councilman bodies observed in liver biopsies of DHF-DSS cases may correspond to hepatocytes in apoptosis. We show here that infection of the hepatoma cell line HepG2 with DEN type 1 virus induced cell death typical of apoptosis late in the virus cycle. The transcription factor NF-kappaB was activated concomitantly with viral protein synthesis and thus before the appearance of apoptotic cells. Inhibition of apoptosis was observed when DEN virus-infected cells were treated with NF-kappaB decoys, indicating the involvement of this transcription factor in induction of cell death. Thus, infected hepatocytes appear to be subject to apoptosis in vitro, and this may be a key element in the pathophysiology of hepatic failure associated with DHF-DSS.