Search

We conducted an in-depth characterization of the Nipah virus (NiV) isolate previously obtained from a Pteropus lylei bat in Cambodia in 2003 (CSUR381). We performed full-genome sequencing and phylogenetic analyses and confirmed CSUR381 is part of the NiV-Malaysia genotype. In vitro studies revealed similar cell permissiveness and replication of CSUR381 (compared with 2 other NiV isolates) in both bat and human cell lines. Sequence alignments indicated conservation of the ephrin-B2 and ephrin-B3 receptor binding sites, the glycosylation site on the G attachment protein, as well as the editing site in phosphoprotein, suggesting production of nonstructural proteins V and W, known to counteract the host innate immunity. In the hamster animal model, CSUR381 induced lethal infections. Altogether, these data suggest that the Cambodia bat-derived NiV isolate has high pathogenic potential and, thus, provide insight for further studies and better risk assessment for future NiV outbreaks in Southeast Asia.

Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 1998. In pigs, infection resulted in a predominantly non-lethal respiratory disease; however, infection in humans resulted in over 100 deaths. Nipah virus has continued to re-emerge in Bangladesh and India, and person-to-person transmission appeared in the outbreak. Although a number of NiV vaccine studies have been reported, there are currently no vaccines or treatments licensed for human use. In this study, we have developed a recombinant measles virus (rMV) vaccine expressing NiV envelope glycoproteins (rMV-HL-G and rMV-Ed-G). Vaccinated hamsters were completely protected against NiV challenge, while the mortality of unvaccinated control hamsters was 90%. We trialed our vaccine in a non-human primate model, African green monkeys. Upon intraperitoneal infection with NiV, monkeys showed several clinical signs of disease including severe depression, reduced ability to move and decreased food ingestion and died at 7 days post infection (dpi). Intranasal and oral inoculation induced similar clinical illness in monkeys, evident around 9 dpi, and resulted in a moribund stage around 14 dpi. Two monkeys immunized subcutaneously with rMV-Ed-G showed no clinical illness prior to euthanasia after challenge with NiV. Viral RNA was not detected in any organ samples collected from vaccinated monkeys, and no pathological changes were found upon histopathological examination. From our findings, we propose that rMV-NiV-G is an appropriate NiV vaccine candidate for use in humans.

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.

Nipah virus (NiV) P gene encodes P protein and three accessory proteins (V, C and W). It has been reported that all four P gene products have IFN antagonist activity when the proteins were transiently expressed. However, the role of those accessory proteins in natural infection with NiV remains unknown. We generated recombinant NiVs lacking V, C or W protein, rNiV(V-), rNiV(C-), and rNiV(W-), respectively, to analyze the functions of these proteins in infected cells and the implications in in vivo pathogenicity. All the recombinants grew well in cell culture, although the maximum titers of rNiV(V-) and rNiV(C-) were lower than the other recombinants. The rNiV(V-), rNiV(C-) and rNiV(W-) suppressed the IFN response as well as the parental rNiV, thereby indicating that the lack of each accessory protein does not significantly affect the inhibition of IFN signaling in infected cells. In experimentally infected golden hamsters, rNiV(V-) and rNiV(C-) but not the rNiV(W-) virus showed a significant reduction in virulence. These results suggest that V and C proteins play key roles in NiV pathogenicity, and the roles are independent of their IFN-antagonist activity. This is the first report that identifies the molecular determinants of NiV in pathogenicity in vivo.

Hendra virus (HeV) and Nipah virus (NiV) are recently-emerged, closely related and highly pathogenic paramyxoviruses. We have analysed here the pathogenesis of the acute HeV infection using the new animal model, golden hamster (Mesocricetus auratus), which is highly susceptible to HeV infection. HeV-specific RNA and viral antigens were found in multiple organs and virus was isolated from different tissues. Dual pathogenic mechanism was observed: parenchymal infection in various organs, including the brain, with vasculitis and multinucleated syncytia in many blood vessels. Furthermore, monoclonal antibodies specific for the NiV fusion protein neutralized HeV in vitro and efficiently protected hamsters from HeV if given before infection. These results reveal the similarities between HeV and NiV pathogenesis, particularly in affecting both respiratory and neuronal system. They demonstrate that hamster presents a convenient novel animal model to study HeV infection, opening new perspectives to evaluate vaccine and therapeutic approaches against this emergent infectious disease.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A reverse transcription-PCR assay including a competitive internal control was developed on the basis of the most up-to-date genome information. Biotinylated amplification products were hybridized to DNA macroarrays on the surfaces of polymer supports, and hybridization events were visualized by incubation with a streptavidin-horseradish peroxidase conjugate and the formation of a visible substrate precipitate. Optimal assay conditions for the detection of as few as 6.3 genome copies per reaction were established. Eighteen geographically and historically diverse CCHF virus strains representing all clinically relevant isolates were detected. The feasibility of the assay for clinical diagnosis was validated with acute-phase patient samples from South Africa, Iran, and Pakistan. The assay provides a specific, sensitive, and rapid method for CCHF virus detection without requiring sophisticated equipment. It has usefulness for the clinical diagnosis and surveillance of CCHF infections under limited laboratory conditions in developing countries or in field situations.

Diagnostic systems for Lassa fever (LF), a viral hemorrhagic fever caused by Lassa virus (LASV), such as enzyme immunoassays for the detection of LASV antibodies and LASV antigens, were developed using the recombinant nucleoprotein (rNP) of LASV (LASV-rNP). The LASV-rNP was expressed in a recombinant baculovirus system. LASV-rNP was used as an antigen in the detection of LASV-antibodies and as an immunogen for the production of monoclonal antibodies. The LASV-rNP was also expressed in HeLa cells by transfection with the expression vector encoding cDNA of the LASV-NP gene. An immunoglobulin G enzyme-linked immunosorbent assay (ELISA) using LASV-rNP and an indirect immunofluorescence assay using LASV-rNP-expressing HeLa cells were confirmed to have high sensitivity and specificity in the detection of LASV-antibodies. A novel monoclonal antibody to LASV-rNP, monoclonal antibody 4A5, was established. A sandwich antigen capture (Ag-capture) ELISA using the monoclonal antibody and an anti-LASV-rNP rabbit serum as capture and detection antibodies, respectively, was then developed. Authentic LASV nucleoprotein in serum samples collected from hamsters experimentally infected with LASV was detected by the Ag-capture ELISA. The Ag-capture ELISA specifically detected LASV-rNP but not the rNPs of lymphocytic choriomeningitis virus or Junin virus. The sensitivity of the Ag-capture ELISA in detecting LASV antigens was comparable to that of reverse transcription-PCR in detecting LASV RNA. These LASV rNP-based diagnostics were confirmed to be useful in the diagnosis of LF even in institutes without a high containment laboratory, since the antigens can be prepared without manipulation of the infectious viruses.

We developed a real-time reverse transcription--PCR that detected 1,164 copies/mL of Crimean-Congo hemorrhagic fever virus per milliliter of serum at 95% probability (probit analysis) and was 100% concordant with nested PCR on 63 samples from 31 patients with confirmed infection. Infected patients who died appeared to have higher viral loads; low viral loads correlated with IgG detection.

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.

Crimean-Congo haemorrhagic fever (CCHF) is an arbovirus infection, which is transmitted through ticks or via blood and secretions. Until recently, human cases of CCHF were unknown in Turkey; however, several acute disease cases were reported in 2002. We report on the investigation of a cluster of suspected CCHF cases in the middle part of the Black Sea from May 2002 to October 2003. The medical charts that we reviewed were obtained from all local physicians and our field investigations. 'Suspected case' was defined with regard to time, place, and both clinical and laboratory characteristics. A total of 108 patients were defined as suspected case. Among them 36 patients were reached and blood samples taken for examination for CCHF by using ELISA and RT-PCR. According to the laboratory analysis, 80.6% (29/36) were acute cases and 8.3% (3/36) were past CCHF infections. The overall mortality rate was 5.6%. There was no nosocomial infection; however, there were 2 family clusters. Tick exposure was the most prevalent risk factor (74.2%). A multidisciplinary collaboration should be developed in order to understand the magnitude of the disease and also to keep it under control.

Specimens were obtained from the 3 Malagasy fruit bats, Pteropus rufus, Eidolon dupreanum, and Rousettus madagascariensis. Antibodies against Nipah, Hendra, and Tioman viruses were detected by immunoassay in 23 and by serum neutralization tests in 3 of 427 serum samples, which suggests that related viruses have circulated in Madagascar.

There have recently been large outbreaks of Marburg hemorrhagic fever (MHF) caused by Marburgvirus (MARV) in the Democratic Republic of Congo and Angola. The development of reliable diagnostic systems for MHF is urgently needed. An antigen-capture enzyme-linked immunosorbent assay (Ag-capture ELISA) using either of the two monoclonal antibodies (2A7 and 2H6) produced by immunizing mice with recombinant nucleoprotein of MARV was described (Journal of Medical Virology, 76, 111-118, 2005). In the present study, it was revealed that the Ag-capture ELISA specifically detected authentic MARV antigen and that the sensitivity of the Ag-capture ELISA was at a level similar to that of reverse-transcription polymerase chain reaction. These results suggest that the Ag-capture ELISA using the monoclonal antibodies, 2A7 and 2H6, is applicable to the diagnosis of MHF.

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.

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), which primarily targets human dendritic cells (DC) and macrophages (MP). Massive numbers of viral particles are released with no effect on the viability, activation or maturation of these cells. LV does not inhibit the activation of cells induced by sCD40L or LPS. We report here the consequences of exogenous activation of LV-infected human DC and MP for viral replication. The activation of cells with lipopolysaccharide or exogenous poly(I-C) and the transfection of cells with poly(I-C) strongly inhibited LV replication, at least partly by inducing type I interferon (IFN) synthesis. In contrast, cell stimulation with sCD40L did not induce type I IFN responses or inhibit LV release. Recombinant type I IFNs strongly inhibited LV replication in both cell types, whereas IFNgamma and IFNlambda did not. The modest type I IFN production observed in LV-infected MP, but not in DC, was involved in controlling LV replication in MP. These results provide an explanation for the slower replication of LV in MP than in DC, and suggest that type I IFNs are crucial in the control of LV.

Background: Foamy viruses are exogenous retroviruses that are highly endemic in non-human primates (NHPs). Recent studies, mainly performed in North America, indicated frequent simian foamy virus (SFV) infection in persons occupationally exposed to NHPs. This zoonotic infection was demonstrated mainly after bites by chimpanzees [Pan troglodytes (P. t.)] of the West African P. t. verus subspecies in primatology centers or zoos in the USA., Methods: We studied 32 chimpanzees from the Central African subspecies P. t. troglodytes and P. t. vellerosus, originating from Cameroon (29 cases) or Gabon (3 cases). We screened first plasma or sera of the animals with a Western blot detecting the SFVs Gag doublet proteins. Then, we performed two nested polymerase chain reactions (PCRs) amplifying a fragment of the integrase and LTR regions and, finally, we made phylogenetical analyses on the sequences obtained from the integrase PCR products., Results: By serological and/or molecular assays, we detected foamy viruses (FVs) infection in 14 chimpanzees. Sequence comparison and phylogenetic analyses of a 425 bp fragment of the integrase gene obtained for 10 of the 14 positive apes, demonstrated a wide diversity of new FVs strains that belong phylogenetically either to the P. t. troglodytes or P. t. vellerosus foamy viral clade., Conclusions: This study shows that chimpanzees living in these areas of Central Africa are infected by several specific foamy viruses. This raises, in such regions, the potential risk of a human retroviral infection of zoonotic origin linked to chimpanzees contacts, as already exemplified for STLV-1 and SIV infections.

We conducted a survey in Cambodia in 2000 on henipavirus infection among several bat species, including flying foxes, and persons exposed to these animals. Among 1,072 bat serum samples tested by enzyme-linked immunosorbent assay, antibodies reactive to Nipah virus (NiV) antigen were detected only in Pteropus lylei species; Cynopterus sphinx, Hipposideros larvatus, Scotophilus kuhlii, Chaerephon plicata, Taphozous melanopogon, and T. theobaldi species were negative. Seroneutralization applied on a subset of 156 serum samples confirmed these results. None of the 8 human serum samples was NiV seropositive with the seroneutralization test. One virus isolate exhibiting cytopathic effect with syncytia was obtained from 769 urine samples collected at roosts of P. lylei specimens. Partial molecular characterization of this isolate demonstrated that it was closely related to NiV. These results strengthen the hypothesis that flying foxes could be the natural host of NiV. Surveillance of human cases should be implemented.

A survey for the presence of simian foamy retroviruses (SFVs) was performed in 44 wild-caught apes and monkeys, including 27 gorillas, 11 mandrills and six drills, originating from south Cameroon or Gabon. Combined serological and/or nested-PCR assays indicated SFV infection among five Gorilla gorilla gorilla, seven Mandrillus sphinx and two Mandrillus leucophaeus. Sequences of a 425 bp fragment of the integrase gene were obtained for 11 animals. Phylogenetic studies indicated that strains from gorillas, mandrills and drills each formed a highly supported phylogenetic clade with, moreover, the existence of two different gorilla SFVs. This study demonstrates for the first time that these animals are naturally infected with specific SFVs. In the context of simian-to-human interspecies transmission, the results confirm that such viruses can also infect humans, as the SFVs identified in wild-caught animals were the same as those recently reported as infecting hunters living in the same geographical areas.

Lassa virus (LV) and Mopeia virus (MV) are closely related members of the Arenavirus genus, sharing 75% amino acid sequence identity. However, LV causes hemorrhagic fever in humans and nonhuman primates, whereas MV cannot induce disease. We have previously shown that antigen-presenting cells (APC)-macrophages (MP) and dendritic cells (DC)-sustain high replication rates of LV but are not activated, suggesting that they play a role in the immunosuppression observed in severe cases of Lassa fever. Here, we infected human APC with MV and analyzed the cellular responses induced. MV infection was productive in MP and even more so in DC. Apoptosis was not induced in either cell type. Moreover, unlike DC, MP were early and strongly activated in response to MV, as shown by the increased surface expression of CD86, CD80, CD54, CD40, and HLA-abc and by the production of mRNA encoding alpha interferon (IFN-alpha), IFN-beta, tumor necrosis factor alpha and interleukin-6. In addition, MV-infected MP produced less of the virus than DC, which was related to the fact that these cells secreted IFN-alpha. Thus, the strong activation of MP is probably a major event in the control of MV infection and may be involved in the induction of an adaptive immune response in infected hosts. These results may explain the difference in pathogenicity between LV and MV.

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.

Lassa fever is a hemorrhagic fever caused by Lassa virus (LV), an old-world Arenavirus. Little is known about the immune responses that occur during the disease, but protection seems to be linked to the induction of cellular responses specific for viral glycoproteins. Conversely, severe Lassa fever may be associated with immunosuppression. We studied the infection of human dendritic cells (DC) and macrophages (MP) by LV. Both these cell types are susceptible to LV infection. Viral nucleoprotein was detected in DC and MP, and high and moderate viral titers were obtained with culture supernatants of DC and MP, respectively. LV did not induce apoptosis in DC and MP. These cells were not activated by LV infection. No change was observed in the expression of surface molecules involved in activation, costimulation, adhesion, and Ag presentation following LV infection, or in the functional properties of DC. Inflammatory cytokine production was not detected at the mRNA or protein level after LV infection of DC and MP. Thus, MP, and particularly DC, are crucial targets for LV and are probably involved in the early replication of LV from the initial site of infection. The lack of activation and maturation of cells following infection may be associated with the immunosuppression observed in severe LV infection.

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.

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.

Some viral diseases are transmitted to human by arthropods (arboviroses), or by animals (zoonoses). Among more than 500 arboviruses and epizootic viruses that are classified into seven families, only a few are responsible for zoonoses or cause severe human diseases. Infected patients may show an acute disease associated with different symptoms, ranging from high fever to encephalitis, pulmonary distress, and haemorrhages. Some diseases show one or more of these symptoms and the factors responsible for severe outcomes, either linked to the virus, or to the host, or to the vector, remain poorly understood. Arboviroses and zoonoses are emerging or re-emerging diseases that need a multidisciplinary effort to control the propagation of the infectious agent and the pathogenesis in infected patients. Some viruses could be used for bioterrorism attacks. In virology, studies on the interactions of the viruses with their vectors and vertebrate hosts and on the pathophysiology of the infections will allow a better prevention of these diseases.

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.

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.

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.

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 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.

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.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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.

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.

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.

Although the WHO declared global smallpox eradication in 1980, the Orthopoxvirus remains a source of concern for several reasons. Firstly, stocks of the smallpox virus have been preserved for experimental use (at least officially in the USA and Russia) so that an escaped isolate could lead to reemergence and spread of the disease worldwide. Secondly discontinuation of smallpox vaccination programs has led to dwindling acquired immunity in the world population thus raising the risk of epidemic extension of several Orthopoxvirus zoonoses (e.g., monkeypox). Thirdly stocks of camelpox virus which is very similar to Smallpox virus and was intended for biological warfare were discovered during the Gulf War in 1991 and pose a potentially serious threat. Finally official stocks of Smallpox virus could be stolen and used by bioterrorists. Thus reemergence of Orthopoxvirus including smallpox, monkeypox, cowpox, and camelpox is a real danger and contingency planning is needed to define prophylactic and therapeutic strategies to prevent and/or stop an epidemics. Adverse effects from earlier smallpox vaccine (vaccinia) in healthy people or immunocompromised people (congenital or acquired as in HIV infected patients) are absolute contraindications to smallpox vaccination at this time. Further research is needed to develop new vaccines (e.g., attenuated isolates of vaccinia) and effective treatment. This is the only valid reasons for postponing planned destruction of remaining Smallpox virus stocks.

Human T-cell leukemia virus type 1 (HTLV-1) is a member of the Oncoretrovirinae family containing several viruses that have been associated with a low incidence of leukemia and sarcoma in mammals. Primates are susceptible to viruses of genera HTLV (humans) and STLV (other primates). The high degree of homology in genomic arrangement of HTLV and STLV is probably due to the existence of a common simian ancestor. Most infections are asymptomatic but a few cases exhibit blood diseases, e.g., T-lymphoma or T-cell leukemia, or neurologic disease, mainly, HTLV-1 associated myelopathy and tropical spastic paraparesia (HAM-TSP). The four major modes of viral transmission are vertical transmission from mother to child either in utero or, more commonly, during breastfeeding, sexual intercourse, blood transfusion, and intravenous drug use. Geographic distribution of HTLV-1 and its confinement to a few well-defined ecosystems have yet to be explained. Diagnosis is now easy and can reduce transmission by intravenous drugs use. Development of a vaccine seems possible given the low genetic variability of this virus.

A recent serological and molecular survey of a semifree-ranging colony of mandrills (Mandrillus sphinx) living in Gabon, central Africa, indicated that 6 of 102 animals, all males, were infected with simian T-cell lymphotropic virus type 1 (STLV-1). These animals naturally live in the same forest area as do human inhabitants (mostly Pygmies) who are infected by the recently described human T-cell lymphotropic virus type 1 (HTLV-1) subtype D. We therefore investigated whether these mandrills were infected with an STLV-1 related to HTLV-1 subtype D. Nucleotide and/or amino acid sequence analyses of complete or partial long terminal repeat (LTR), env, and rex regions showed that HTLV-1 subtype D-specific mutations were found in three of four STLV-1-infected mandrills, while the remaining monkey was infected by a different STLV-1 subtype. Phylogenetic studies conducted on the LTR as well as on the env gp21 region showed that these three new STLV-1 strains from mandrills fall in the same monophyletic clade, supported by high bootstrap values, as do the sequences of HTLV-1 subtype D. These data show, for the first time, the presence of the same subtype of primate T-cell lymphotropic virus type 1 in humans and wild-caught monkeys originating from the same geographical area. This strongly supports the hypothesis that mandrills are the natural reservoir of HTLV-1 subtype D, although the possibility that another monkey species living in the same area could be the original reservoir of both human and mandrill viruses cannot be excluded. Due to the quasi-identity of both human and monkey viruses, interspecies transmission episodes leading to such a clade may have occurred recently.

A highly divergent HIV-1 isolate, designated YBF 30, was obtained in 1995 from a 40-year-old Cameroonian woman with AIDS. Depending on the genes studied, phylogenetic analysis showed that YBF30 branched either with SIVcpz-gab or between SIVcpz-gab and HIV-1 group M. The structural genes and tat, vpr, and nef of YBF30 are approximately equidistant from those of HIV-1 group M and SIVcpz-gab. In contrast, vif and rev are closer to HIV-1 group M, and vpu is highly divergent. Using a YBF30 V3 loop peptide enzyme immunoassay, we screened 700 HIV-1-positive sera collected in Cameroon; three reacted strongly with the YBF30 peptides and one was confirmed as being related to YBF30 by genetic analysis of a pol fragment. YBF30 is as distinct from SIVcpz-gab as it is from HIV-1 group M and can thus be considered as the prototype strain of a new human immunodeficiency virus group.

Natural SIVmnd and STLVmnd infections of mandrills in a colony at the Centre International de Recherches Médicales de Franceville (CIRMF) in Gabon were investigated by genetic analysis to determine the extent of intracolony transmission. SIVmnd pol sequence analysis indicates that the six strains present in the colony belong to the SIVmnd lentivirus subgroup previously defined according to the only available prototype sequence (SIVmndGB1), which originated from the same colony. The intraanimal nucleotide diversity (1.1-3.1%) was similar in range to that reported in individuals infected by other HIV/SIVs. The interanimal diversity (0.5-4.3%) was not significantly different from that observed in each individual mandrill, indicating an epidemiological link among the SIVmnd isolates of distinct animals within the colony. Phylogenetic analysis of these isolates, together with seroepidemiological and behavior surveillance within the colony, indicates a predominant male-to-male transmission of SIVmnd that probably occurred during bouts of interanimal aggression. Moreover, our results suggest one case of vertical transmission of SIVmnd from a naturally infected founder female to one of her six offspring. The first genetic analysis of STLV isolates from mandrills is also reported here. Partial tax/rex sequences were used to evaluate the diversity between seven STLVmnd isolates and their phylogenetic relationships with other known strains of human and nonhuman primate T cell leukemia virus, types I and II (PTLV-I/II). They all belong to the PTLV-I subtype, but two genetically distinct STLVmnd groups were evidenced within the mandrill colony. The phylogenetic analyses of the STLVmnd isolates, together with seroepidemiological and behavior surveillance of the mandrills, indicate that intracolony transmissions of STLVmnd are also predominantly the result of male-to-male aggressive contacts.

Using the cluster-sampling method, the authors estimated the seroprevalence of 4 sexually transmitted diseases (STDs) among the sexually active general population in a city of 30,000 inhabitants in the east of Gabon. The seroprevalences were 2% for HIV-1, 13.8% for hepatitis B, 8.6% for Treponema pallidum and 59.6% for Chlamydia trachomatis. The seroprevalences of hepatitis B and chlamydia were stable over time and similar to those registered in other countries of central Africa. On the other hand, the seroprevalence of T. pallidum is notably low in comparison with these countries and seems to be decreasing. The seroprevalence of HIV-1 is also low but has doubled in 8 years in the city. Immigrant women from west Africa were a high-risk group for STDs but more generally, cohabiting was a risk factor for women.

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.

A seroprevalence survey was conducted for simian immunodeficiency virus (SIV) antibody in household pet monkeys in Gabon. Twenty-nine monkeys representing seven species were analyzed. By using human immunodeficiency virus type 2 (HIV-2)/SIVsm, SIVmnd, and SIVagm antigens, one red-capped mangabey (RCM) (Cercocebus torquatus torquatus) was identified as harboring SIV-cross-reactive antibodies. A virus isolate, termed SIVrcm, was subsequently established from this seropositive RCM by cocultivation of its peripheral blood mononuclear cells (PBMC) with PBMC from seronegative humans or RCMs. SIVrcm was also isolated by cocultivation of CD8-depleted RCM PBMC with Molt 4 clone 8 cells but not with CEMx174 cells. The lack of growth in CEMx174 cells distinguished this new SIV from all previously reported sooty mangabey-derived viruses (SIVsm), which grow well in this cell line. SIVrcm was also successfully transmitted (cell free) to human and rhesus PBMC as well as to Molt 4 clone 8 cells. To determine the evolutionary origins of this newly identified virus, subgenomic pol (475 bp) and gag (954 bp) gene fragments were amplified from infected cell culture DNA and sequenced. The position of SIVrcm relative to those of members of the other primate lentivirus lineages was then examined in evolutionary trees constructed from deduced protein sequences. This analysis revealed significantly discordant phylogenetic positions of SIVrcm in the two genomic regions. In trees derived from partial gag sequences, SIVrcm clustered independently from all other HIV and SIV strains, consistent with a new primate lentivirus lineage. However, in trees derived from pol sequences, SIVrcm grouped with the HIV-1/SIVcpz lineage. These findings suggest that the SIVrcm genome is mosaic and possibly is the result of a recombination event involving divergent lentiviruses in the distant past. Further analysis of this and other SIVrcm isolates may shed new light on the origin of HIV-1.

We investigated the early interactions between HIV-1, HIV-2, and simian immunodeficiency virus (SIV) envelope glycoproteins gp120(IIIB), gp105(ROD), and gp120(mac251), and human and macaque cells of the lymphocytic series. Our results demonstrate that the soluble viral glycoproteins induce a specific phospholipase A2 (PLA2) activation in lymphocytes through CD4. This PLA2 activation was induced after envelope glycoprotein-CD4 interaction and, because of its local membrane-destabilizing effect, may have important implications for preparing the lymphocyte membrane for fusion with the viral particle. However, this effect is not sufficient to accomplish fusion. These data indicate that the specific step of fusion may be downstream from PLA2 activation.