Characterization and phylogenetic analysis of new bat astroviruses detected in Gabon, Central Africa.

Astroviruses are emerging RNA viruses that cause enteropathogenic infections in humans and in other mammals. The identification of astroviruses in a wide range of animals highlights the zoonotic importance of these viruses. Bats can harbor many different viruses, among which some are highly pathogenic for humans (for instance, Nipah, Ebola and SARS coronavirus), and also several astroviruses. As some RNA viruses can be directly transmitted from bats to humans, it is crucial to collect data about their frequency, genetic diversity and phylogenetic characterization. In this study, we report the molecular identification of 44 new astroviruses (with a detection rate of 4.5%) in 962 apparently healthy bats that belong to five different species and that were captured in different caves in North-East Gabon, Central Africa. Our results show that bat astroviruses form a group that is genetically distinct from astroviruses infecting other mammals. Moreover, these astroviruses showed an important genetic diversity and low host restriction in bat species.

Ebola and Marburg haemorrhagic fever.

Ebolaviruses and Marburgviruses (family Filoviridae) are among the most virulent pathogens for humans and great apes causing severe haemorrhagic fever and death within a matter of days. This group of viruses is characterized by a linear, non-segmented, single-stranded RNA genome of negative polarity. The overall burden of filovirus infections is minimal and negligible compared to the devastation caused by malnutrition and other infectious diseases prevalent in Africa such as malaria, dengue or tuberculosis. In this paper, we review the knowledge gained on the eco/epidemiology, the pathogenesis and the disease control measures for Marburg and Ebola viruses developed over the last 15 years. The overall progress is promising given the little attention that these pathogen have achieved in the past; however, more is to come over the next decade given the more recent interest in these pathogens as potential public and animal health concerns. Licensing of therapeutic and prophylactic options may be achievable over the next 5-10 years.

Ebola and Marburg haemorrhagic fever viruses: major scientific advances, but a relatively minor public health threat for Africa.

Ebola and Marburg viruses are the only members of the Filoviridae family (order Mononegavirales), a group of viruses characterized by a linear, non-segmented, single-strand negative RNA genome. They are among the most virulent pathogens for humans and great apes, causing acute haemorrhagic fever and death within a matter of days. Since their discovery 50 years ago, filoviruses have caused only a few outbreaks, with 2317 clinical cases and 1671 confirmed deaths, which is negligible compared with the devastation caused by malnutrition and other infectious diseases prevalent in Africa (malaria, cholera, AIDS, dengue, tuberculosis …). Yet considerable human and financial resourses have been devoted to research on these viruses during the past two decades, partly because of their potential use as bioweapons. As a result, our understanding of the ecology, host interactions, and control of these viruses has improved considerably.

Spatial and temporal patterns of Zaire ebolavirus antibody prevalence in the possible reservoir bat species.

To characterize the distribution of Zaire ebolavirus (ZEBOV) infection within the 3 bat species (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) that are possible reservoirs, we collected 1390 bats during 2003-2006 in Gabon and the Republic of the Congo. Detection of ZEBOV immunoglobulin G (IgG) in 40 specimens supports the role of these bat species as the ZEBOV reservoirs. ZEBOV IgG prevalence rates (5%) were homogeneous across epidemic and nonepidemic regions during outbreaks, indicating that infected bats may well be present in nonepidemic regions of central Africa. ZEBOV IgG prevalence decreased, significantly, to 1% after the outbreaks, suggesting that the percentage of IgG-positive bats is associated with virus transmission to other animal species and outbreak appearance. The large number of ZEBOV IgG-positive adult bats and pregnant H. monstrosus females suggests virus transmission within bat populations through fighting and sexual contact. Our study, thus, helps to describe Ebola virus circulation in bats and offers some insight into the appearance of outbreaks.

Ebola virus circulation in Africa: a balance between clinical expression and epidemiological silence.

Nearly thirty years after the first epidemics, Ebola virus (EBOV) remains hardly described, its transmission unclear and its reservoir elusive. Soon after the Ebola fever outbreak and virus discovery in 1976 and in order to investigate the distribution of EBOV in Central Africa, several countries including a range of ecological zones were investigated in the early 1980s, using extensive survey: Central African Republic (CAR), Cameroon, Chad, Congo, Gabon and Equatorial Guinea. Since 1992, ELISA antibody test along with a RT-PCR have been used to detect specific virus antibodies and characterize viral RNA. The widely separated geographic locations of outbreaks have suggested that the reservoir and the transmission cycle of EBOV are probably closely associated with the rain forest ecosystem, what is supported by the distribution of antibodies. The fact that outbreaks seldom occur suggests the presence of a rare or ecologically isolated animal reservoir having few contacts with humans and non-human primates. However various serological investigations showed a high prevalence in humans without any pathology reported. This suggests a circulation of both pathogenic and non-pathogenic strains as well as more frequent contacts with man than expected, and could partially explain fifteen years of Ebola fever silence between the emergence and re-emergence of Ebola virus in the Congolese basin. Nowadays, largely enlightened by the study of recent epizootic and epidemic manifestations of EBOV in Gabon and neighboring countries, EBOV natural history starts to be understood as for the fundamentals of epizootic in non-human primates and chains of transmission.

[Clinical management of patients and deceased during the Ebola outbreak from October to December 2003 in Republic of Congo]. / Prise en charge des malades et des défunts lors de l'epidémie de fièvre hémorragique due au virus Ebola d'octobre a décembre 2003 au Congo.

Outbreaks of Ebola virus hemorrhagic fever (EVHF) have been reported since 2001 in the Cuvette Ouest department, a forested area located in the Western North of Congo. At the end of October 2003 a new alarm came from this department which was quickly confirmed as being an epidemic of EVHF. The outbreak response was organized by the ministry of health with the assistance of an international team under the aegis of WHO. The case management of suspect cases was done in an isolation ward set up at the hospital; when patients refused to go to the ward for care they were isolated in their house according to a protocol "transmission risks reduction at home". Safe burials were performed by specialized teams which respected the major aspects of the funeral to allow the process of mourning of the families. An active surveillance system was set up in order to organize the detection of new cases and the follow-up of their contacts. A case definition was adopted. From October 11 to December 2, 2003, 35 cases including 29 deaths were reported, 16 cases were laboratory confirmed. The first four cases had been exposed to monkey meat (Cercopithecus nictitans). The epidemic spread was due to family transmission. The population interpretation of the disease, in particular questions around wizards and evil-minded persons, is a factor which must be taken into account by the medical teams during communication meetings for behavioral change of the populations. The case management of patient in isolation wards to prevent the transmission of the virus in the community remains the most effective means to dam up Ebola virus hemorrhagic fever outbreaks. The good perception by the community of the safe funerary procedures is an important aspect in the establishment of confidence relations with the local population.

[Multiple Ebola virus haemorrhagic fever outbreaks in Gabon, from October 2001 to April 2002]. / Plusieurs epidémies de fièvre hémorragique due au virus Ebola au Gabon, d'octobre 2001 a avril 2002.

Outbreaks of Ebola virus haemorrhagic fever have been reported from 1994 to 1996 in the province of Ogooué Ivindo, a forest zone situated in the Northeast of Gabon. Each time, the great primates had been identified as the initial source of human infection. End of November 2001 a new alert came from this province, rapidly confirmed as a EVHV outbreak. The response was given by the Ministry of Health with the help of an international team under the aegis of WHO. An active monitoring system was implemented in the three districts hit by the epidemic (Zadié, Ivindo and Mpassa) to organize the detection of cases and their follow-up. A case definition has been set up, the suspected cases were isolated at hospital, at home or in lazarets and serological tests were performed. These tests consisted of the detection of antigen or specific IgG and the RT-PCR. A classification of cases was made according to the results of biological tests, clinical and epidemiological data. The contact subjects were kept watch over for 21 days. 65 cases were recorded among which 53 deaths. The first human case, a hunter died on the 28th of October 2001. The epidemic spreads over through family transmission and nosocomial contamination. Four distinct primary foci have been identified together with an isolated case situated in the South East of Gabon, 580 km away from the epicenter. Deaths happened within a delay of 6 days. The last death has been recorded on the 22nd of March 2002 and the end of the outbreak was declared on the 6th of May 2002. The epidemic spreads over the Gabon just next. Unexplained deaths of animals had been mentionned in the nearby forests as soon as August 2001: great primates and cephalophus. Samples taken from their carcasses confirmed a concomitant animal epidemic.

A serological survey of Ebola virus infection in central African nonhuman primates.

We used an ELISA to determine the prevalence of IgG antibodies specific for the Zaire subtype of Ebola virus in 790 nonhuman primates, belonging to 20 species, studied between 1985 and 2000 in Cameroon, Gabon, and the Republic of Congo. The seroprevalence rate of Ebola antibody in wild-born chimpanzees was 12.9%, indicating that (1) Ebola virus circulates in the forests of a large region of central Africa, including countries such as Cameroon, where no human cases of Ebola infections have been reported; (2) Ebola virus was present in the area before recent outbreaks in humans; (3) chimpanzees are continuously in contact with the virus; and (4) nonlethal Ebola infection can occur in chimpanzees. These results, together with the unexpected detection of Ebola-specific IgG in other species (5 drills, 1 baboon, 1 mandrill, and 1 Cercopithecus), may help to narrow the search for the reservoir of Ebola virus. They also suggest that future Ebola outbreaks may occur anywhere in the central African forest region.

Inflammatory responses in Ebola virus-infected patients.

Ebola virus subtype Zaire (Ebo-Z) induces acute haemorrhagic fever and a 60-80% mortality rate in humans. Inflammatory responses were monitored in victims and survivors of Ebo-Z haemorrhagic fever during two recent outbreaks in Gabon. Survivors were characterized by a transient release in plasma of interleukin-1beta (IL-1beta), IL-6, tumour necrosis factor-alpha (TNFalpha), macrophage inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta early in the disease, followed by circulation of IL-1 receptor antagonist (IL-1RA) and soluble receptors for TNFalpha (sTNF-R) and IL-6 (sIL-6R) towards the end of the symptomatic phase and after recovery. Fatal infection was associated with moderate levels of TNFalpha and IL-6, and high levels of IL-10, IL-1RA and sTNF-R, in the days before death, while IL-1beta was not detected and MIP-1alpha and MIP-1beta concentrations were similar to those of endemic controls. Simultaneous massive activation of monocytes/macrophages, the main target of Ebo-Z, was suggested in fatal infection by elevated neopterin levels. Thus, presence of IL-1beta and of elevated concentrations of IL-6 in plasma during the symptomatic phase can be used as markers of non-fatal infection, while release of IL-10 and of high levels of neopterin and IL-1RA in plasma as soon as a few days after the disease onset is indicative of a fatal outcome. In conclusion, recovery from Ebo-Z infection is associated with early and well-regulated inflammatory responses, which may be crucial in controlling viral replication and inducing specific immunity. In contrast, defective inflammatory responses and massive monocyte/macrophage activation were associated with fatal outcome.

Early immune responses accompanying human asymptomatic Ebola infections.

In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFN gamma, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V beta repertoire usage showed that TCR-V beta 12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus.

Human asymptomatic Ebola infection and strong inflammatory response.

BACKGROUND: Ebola virus is one of the most virulent pathogens, killing a very high proportion of patients within 5-7 days. Two outbreaks of fulminating haemorrhagic fever occurred in northern Gabon in 1996, with a 70% case-fatality rate. During both outbreaks we identified some individuals in direct contact with sick patients who never developed symptoms. We aimed to determine whether these individuals were indeed infected with Ebola virus, and how they maintained asymptomatic status. METHODS: Blood was collected from 24 close contacts of symptomatic patients. These asymptomatic individuals were sampled 2, 3, or 4 times during a 1-month period after the first exposure to symptomatic patients. Serum samples were analysed for the presence of Ebola antigens, virus-specific IgM and IgG (by ELISA and western blot), and different cytokines and chemokines. RNA was extracted from peripheral blood mononuclear cells, and reverse transcriptase-PCR assays were done to amplify RNA of Ebola virus. PCR products were then sequenced. FINDINGS: 11 of 24 asymptomatic individuals developed both IgM and IgG responses to Ebola antigens, indicating viral infection. Western-blot analysis showed that IgG responses were directed to nucleoprotein and viral protein of 40 kDa. The glycoprotein and viral protein of 24 kDa genes showed no nucleotide differences between symptomatic and asymptomatic individuals. Asymptomatic individuals had a strong inflammatory response characterised by high circulating concentrations of cytokines and chemokines. INTERPRETATION: This study showed that asymptomatic, replicative Ebola infection can and does occur in human beings. The lack of genetic differences between symptomatic and asymptomatic individuals suggest that asymptomatic Ebola infection did not result from viral mutations. Elucidation of the factors related to the genesis of the strong inflammatory response occurring early during the infectious process in these asymptomatic individuals could increase our understanding of the disease.

Diagnosis of Ebola haemorrhagic fever by RT-PCR in an epidemic setting.

This study reports the first field evaluation of a new diagnostic technique for Ebola virus disease with sensitivity and specificity. Ebola virus causes rare but fulminating outbreaks in Equatorial Africa. Rapid differentiation from other infections is critical for timely implementation of public health measures. Patients usually die before developing antibodies, necessitating rapid virus detection. A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed, implemented and evaluated at Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon, to detect Ebola viral RNA in peripheral blood mononuclear cells (PBMC). Twenty-six laboratory-confirmed patients during and 5 after the acute phase of Ebola haemorrhagic fever, 15 healthy controls and 20 febrile patients not infected with Ebola virus were studied. RT-PCR results were compared with ELISA antigen capture, and Ebola specific IgM and IgG antibody detection. Ebola virus RNA was amplified from 26/26 specimens from the acute phase, 3/5 during recovery, 0/20 febrile patients and 1/15 negative controls. Sensitivity of RT-PCR in identifying acute infection and early convalescence compared with antigen or IgM detection was 100% and 91% respectively, and specificity compared with antigen detection and IgM assay combined was 97%. Antigen capture detected only 83% of those identified by PCR, and IgM only 67%. Ebola virus RNA was detected in all 13 fatalities, only 5 of whom had IgM and none IgG. RT-PCR detected Ebola RNA in PBMC one to three weeks after disappearance of symptoms when antigen was undetectable. RT-PCR was the most sensitive method and able to detect virus from early acute disease throughout early recovery.

Apoptosis in fatal Ebola infection. Does the virus toll the bell for immune system?

In fatal Ebola virus hemorrhagic fever massive intravascular apoptosis develops rapidly following infection and progressing relentlessly until death. While data suggest that T lymphocytes are mainly deleted by apoptosis in PBMC of human fatal cases, experimental Ebola infection in animal models have shown some evidence of destruction of lymphocytes in spleen and lymph nodes probably involving both T and B cells. Nevertheless, we are able to conclude from the accumulated evidence that early interactions between Ebola virus and the immune system, probably via macrophages, main targets for viral replication, lead to massive destruction of immune cells in fatal cases.

Sequence conservation of repeat 3 region of the gene coding for the 15 kDa polyprotein within human and simian Loa loa.

The human and simian strains of Loa loa microfilariae are morphologically identical even though their periodicities vary. When using primate models (Mandrillus sphinx) of human loaisis for vaccination trials, the absence of any ongoing simian L. loa infection must be demonstrated. Nested primers derived from a human strain of L. loa (targeted on the repeat 3 region of the gene encoding the 15 kDa polyprotein; 15r3) amplified at 366 bp sequence from simian L. loa genomic DNA and blood lysates from mandrills infected with simian L. loa. This nested-PCR assay has been tested on 12 amicrofilaremic (AMF) mandrills (without filarial microfilariae) and was positive in four mandrills. The nested-PCR product derived from simian L. loa genomic DNA and from three of four AMF mandrills has been sequenced. No difference was observed between the four sequences, which, in addition, were 99.18% identical to the 15r3 of human L. loa. Therefore, the 15r3 sequence is conserved within human and simian L. loa. These results suggest that the four PCR-positive mandrills without circulating microfilariae had occult simian L. loa infections. The study demonstrates the ability of a nested-PCR assay to identify animals naturally infected with simian L. loa.

Defective humoral responses and extensive intravascular apoptosis are associated with fatal outcome in Ebola virus-infected patients.

Ebola virus is very pathogenic in humans. It induces an acute hemorrhagic fever that leads to death in about 70% of patients. We compared the immune responses of patients who died from Ebola virus disease with those who survived during two large outbreaks in 1996 in Gabon. In survivors, early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells, which was indicated by the upregulation of FasL, perforin, CD28 and gamma interferon mRNA in peripheral blood mononuclear cells. In contrast, fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM. Early activation of T cells, indicated by mRNA patterns in peripheral blood mononuclear cells and considerable release of gamma interferon in plasma, was followed in the days preceding death by the disappearance of T cell-related mRNA (including CD3 and CD8). DNA fragmentation in blood leukocytes and release of 41/7 nuclear matrix protein in plasma indicated that massive intravascular apoptosis proceeded relentlessly during the last 5 days of life. Thus, events very early in Ebola virus infection determine the control of viral replication and recovery or catastrophic illness and death.

Ebola hemorrhagic fever outbreaks in Gabon, 1994-1997: epidemiologic and health control issues.

From the end of 1994 to the beginning of 1995, 49 patients with hemorrhagic symptoms were hospitalized in the Makokou General Hospital in northeastern Gabon. Yellow fever (YF) virus was first diagnosed in serum by use of polymerase chain reaction followed by blotting, and a vaccination campaign was immediately instituted. The epidemic, known as the fall 1994 epidemic, ended 6 weeks later. However, some aspects of this epidemic were atypical of YF infection, so a retrospective check for other etiologic agents was undertaken. Ebola (EBO) virus was found to be present concomitantly with YF virus in the epidemic. Two other epidemics (spring and fall 1996) occurred in the same province. GP and L genes of EBO virus isolates from all three epidemics were partially sequenced, which showed a difference of <0.1% in the base pairs. Sequencing also showed that all isolates were very similar to subtype Zaire EBO virus isolates from the Democratic Republic of the Congo.

[Ebola virus: what the practitioner needs to know]. / Virus Ebola: l'essentiel pour le praticien.

The Ebola virus is an RNA virus of Filoviridae family. The earliest documented fatal epidemic of Ebola hemorrhagic occurred in 1976. There are four genetically different subtypes of Ebola virus. The virus remains in the blood for several weeks, can maintain its infectivity for several weeks at 20 degrees C outside the body, and survives for several weeks in corpses. Isolation of Ebola virus requires level 4 laboratory security conditions. Specimens are obtained by culturing mammal cells. Identification is achieved using reference serums. Serologic diagnosis is made using mainly ELISA technique for immunocapture of IgM or EBO Ag. The natural reservoir for Ebola virus is unknown. One possibility is that each isolated strain has a different reservoir. In recorded outbreaks, the index case has often had a history of contact with non-human primates. However since these animals are also highly sensitive to the virus, they cannot be considered as reservoirs but only as intermediate hosts. Transmission requires close contact such as occurs in association with health care, local customs, or funeral rites. In humans, infection causes hemorrhagic fever that progresses to diarrhea within 5 to 10 days. Recovery is observed in only 25% of cases. During outbreaks containment depends on implementation of simple precautions including isolation of suspected cases, appropriate protective clothing, disinfection with hypochlorite solutions, and proper waste disposal.