Kunjin virus replicon-based vaccines expressing Ebola virus glycoprotein GP protect the guinea pig against lethal Ebola virus infection.

Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)-derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor-truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection.

[Filovirus]. / Les filovirus.

Since forty years Marburg and Ebola viruses emerge frequently in Africa and are responsible of viral hemorragic fever outbreaks with high mortality rate. Despite intensive research programs, these viruses remain mysterious: the reservoir is not clearly defined, and the mechanisms leading to their high pathogenicity are poorly understood; a defective or inadapted immune response seems to be the main factor. No specific treatment nor vaccine are available for humans. But encouraging results have been obtained in the treatment of filovirus infections in non human primate model with different products, as recombinant nematode anticoagulant protein, anti sens phosphorodiamidate morpholino oligomers or small interfering RNA.As vaccines, recombinantVSV expressing the GP of filovirus or adenovirus expressing the GP and NP of filovirus are very promising in macaque models.

Poly(I)-poly(C12U) but not ribavirin prevents death in a hamster model of Nipah virus infection.

Clinical nonrandomized trials demonstrate some efficacy for ribavirin in the treatment of patients with severe Nipah virus-induced encephalitis. We report here that EICAR, the 5-ethynyl analogue of ribavirin, and the OMP-decarboxylase inhibitors 6-aza-uridine and pyrazofurin have strong antiviral activity against Nipah virus replication in vitro. Ribavirin and 6-aza-uridine were tested further in hamsters infected with a lethal dose of Nipah virus. The activity of these small-molecule inhibitors was compared with that of the interferon inducer poly(I)-poly(C(12)U). Both ribavirin and 6-aza-uridine were able to delay but not prevent Nipah virus-induced mortality. Poly(I)-poly(C(12)U), at 3 mg/kg of body weight daily from the day of infection to 10 days postinfection, prevented mortality in 5 of 6 infected animals.

[Epidemics of Ebola haemorrhagic fever in Gabon (1994-2002). Epidemiologic aspects and considerations on control measures]. / Les epidémies de fièvre hémorragique due au virus Ebola au Gabon (1994 - 2002): aspects epidémiologiques et réflexions sur les mesures de contrôle.

Based on the description of the four Ebola haemorrhagic fever epidemics (EHF) occurred in Gabon between 1994 and 2002, the authors are considering the cultural and psycho-sociological aspects accounting for the difficulty to implement control measures. On the whole, the result of these raging epidemics came up to 207 cases and 150 dead (lethality: 72%). Analysing precisely the aspects of the third epidemic and pointing up the possible factors explaining its spreading far beyond its epicentre, the authors bring about the limits of measures not always understood by local populations. The discussion will deal with the possibilities of a better surveillance, a quick management of intervention means including a regional permanent pre-alert and taking into account the issue raised by the possible Ebola virus endemic.

Nipah virus: vaccination and passive protection studies in a hamster model.

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.

[Ebola: a virus endemic to central Africa?]. / Ebola: un virus endemique en Afrique centrale?

From October 2001 to March 2002, an outbreak of Ebola haemorrhagic fever occurred in the North-Eastern Gabon (63 cases) and neighbouring Congo (57 cases). It was the fourth epidemic in North Eastern Gabon since 1994. Meanwhile this outbreak differed from the previous epidemics: at least five different emerging sources of the virus in the human population were observed from the local fauna resulting in fears of an endemic Ebola virus in the area. The control of the outbreak was uneasy because of the unfriendly attitude of the local population related to the restrictive measures for the isolation of suspected patients and the epidemiological surveillance. Such rejection process emphasizes the need of a continuous increasing public awareness.

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.

A novel gamma 2-herpesvirus of the Rhadinovirus 2 lineage in chimpanzees.

Old World monkeys and, recently, African great apes have been shown, by serology and polymerase chain reaction (PCR), to harbor different gamma2-herpesviruses closely related to Kaposi's sarcoma-associated Herpesvirus (KSHV). Although the presence of two distinct lineages of KSHV-like rhadinoviruses, RV1 and RV2, has been revealed in Old World primates (including African green monkeys, macaques, and, recently, mandrills), viruses belonging to the RV2 genogroup have not yet been identified from great apes. Indeed, the three yet known gamma2-herpesviruses in chimpanzees (PanRHV1a/PtRV1, PanRHV1b) and gorillas (GorRHV1) belong to the RV1 group. To investigate the putative existence of a new RV2 Rhadinovirus in chimpanzees and gorillas we have used the degenerate consensus primer PCR strategy for the Herpesviral DNA polymerase gene on 40 wild-caught animals. This study led to the discovery, in common chimpanzees, of a novel gamma2-herpesvirus belonging to the RV2 genogroup, termed Pan Rhadino-herpesvirus 2 (PanRHV2). Use of specific primers and internal oligonucleotide probes demonstrated the presence of this novel gamma2-herpesvirus in three wild-caught animals. Comparison of a 1092-bp fragment of the DNA polymerase obtained from these three animals of the Pan troglodytes troglodytes subspecies, one from Gabon and the two others from Cameroon, revealed <1% of nucleotide divergence. The geographic colocalization as well as the phylogenetic "relationship" of the human and simian gamma2-herpesviruses support the model according to which herpesviruses have diversified from a common ancestor in a manner mediating cospeciation of herpesviruses with their host species. By demonstrating the existence of two distinct Rhadinovirus lineages in common chimpanzees, our finding indicates the possible existence of a novel human gamma2-herpesvirus belonging to the RV2 genogroup.

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.

Simian homologues of human gamma-2 and betaherpesviruses in mandrill and drill monkeys.

Recent serological and molecular surveys of different primate species allowed the characterization of several Kaposi's sarcoma-associated herpesvirus (KSHV) homologues in macaques, African green monkeys, chimpanzees, and gorillas. Identification of these new primate rhadinoviruses revealed the existence of two distinct genogroups, called RV1 and RV2. Using a degenerate consensus primer PCR method for the herpesvirus DNA polymerase gene, the presence of KSHV homologues has been investigated in two semi-free-ranging colonies of eight drill (Mandrillus leucophaeus), five mandrill (Mandrillus sphinx), and two hybrid (Mandrillus leucophaeus-Mandrillus sphinx) monkeys, living in Cameroon and Gabon, Central Africa. This search revealed the existence of not only two distinct KSHV homologues, each one belonging to one of the two rhadinovirus genogroups, but also of two new betaherpesvirus sequences, one being close to cytomegaloviruses and the other being related to human herpesviruses 6 and 7 (HHV-6 and -7). The latter viruses are the first simian HHV-6 and -7 homologues identified to date. These data show that mandrill and drill monkeys are the hosts of at least four novel distinct herpesviruses. Moreover, mandrills, like macaques and African green monkeys, harbor also two distinct gamma-2 herpesviruses, thus strongly suggesting that a second gamma-2 herpesvirus, belonging to the RV2 genogroup, may exist in humans.

Protection of cynomolgus macaque against cervicovaginal transmission of SIVmac251 by the spermicide benzalkonium chloride.

We evaluated the potential effectiveness of a spermicide cationic surfactant, benzalkonium chloride (BZK), to prevent the transmission of simian immunodeficiency virus (SIV) after intravaginal inoculation in 12 cynomolgus macaques. The inoculation procedure involved deposition of 6.7 ivag-AID50 of cell-free SIVmac251 into the receiving vagina, four times over a 2-week period, at the end of the luteal phase of the menstrual cycle. Six randomly selected females received vaginally foam containing BZK (7.37%, wt/wt) before each inoculation (BZK group), whereas the remaining were not pretreated (control group). In controls, 5 animals presented persistent SIV infection and 1 had a transient viremia. The number of uninfected animals was higher in the BZK group (6 of 6) than in controls (0 of 6). These findings demonstrate that BZK placed in the vaginal receptacle prior to SIV inoculation provides a significant protection in vivo. The wide spectrum of antimicrobial activities of BZK (including HIV) in addition to its efficiency to block the transmucosal passage of SIV in the macaque model qualifies this drug as an attractive topical microbicide to prevent sexually transmitted infections in humans.

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.

Mutations in CCR5-coding sequences are not associated with SIV carrier status in African nonhuman primates.

African monkeys can be naturally infected with SIV but do not progress to AIDS. Since mutations in the human CCR5 gene have been shown to influence susceptibility to HIV infection and disease progression, we have now investigated whether mutations in CCR5-coding sequences in African nonhuman primates can explain species-specific differences in susceptibility to lentiviral infection. The animals studied comprise chronically infected monkeys corresponding to four natural hosts of SIV (Cercopithecus aethiops, Cercopithecus pygerythrus, Cercopithecus sabaeus, and Cercopithecus tantalus), noninfected animals from three species that are known to be susceptible to SIV infection (Cercopithecus patas, Cercopithecus Ihoesti, and Pan troglodytes), and monkeys of six species that do not carry SIV in the wild (Cercocebus galeritus, Cercocebus aterrimus, Cercopithecus ascanius, Cercopithecus nictitans, Cercopithecus neglectus, and Cercopithecus cephus). We observed a high degree of genetic divergence among the species. The rate of accumulation of amino acid mutations was, however, not higher in SIV carriers than in other nonhuman primates. No homozygous premature stop codons, deletions, or frameshift mutations were detected. In at least two animals, one infected AGM (Cercopithecus tantalus) and one noninfected monkey (Cercocebus aterrimus), the CCR5 alleles identified encode functional proteins, as they were identical in terms of amino acid sequence to that of functional CCR5 reported in the literature. We found no other consistent differences in the genetic variability of CCR5-coding sequences between the nonhuman primates that are carriers of SIV and those that are not.

Leptospirosis and Ebola virus infection in five gold-panning villages in northeastern Gabon.

An exhaustive epidemiologic and serologic survey was carried out in five gold-panning villages situated in northeastern Gabon to estimate the degree of exposure of to leptospirosis and Ebola virus. The seroprevalence was 15.7% for leptospirosis and 10.2% for Ebola virus. Sixty years after the last seroepidemiologic survey of leptospirosis in Gabon, this study demonstrates the persistence of this infection among the endemic population and the need to consider it as a potential cause of hemorrhagic fever in Gabon. There was no significant statistical correlation between the serologic status of populations exposed to both infectious agents, indicating the lack of common risk factors for these diseases.

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.