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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., Instituto de Biotecnologia y Biologia Molecular

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-AID/50 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. (authors)

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.

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

Two species of macaques, including two Macaca fascicularis from the Philippines, two M. fascicularis from Mauritius, and one Macaca mulatta, were experimentally infected with blood stages of Plasmodium coatneyi and followed during their clinical, parasitological, biological, and immunological evolution. Plasma cytokine (TNF-alpha, IL-1beta, IL-6, IFN-gamma) production peaked for all monkeys 11 days after inoculation, concomitantly with peaks of parasitemia. Only the M. fascicularis from the Philippines survived the infection. The main features, which discriminated nonfatal from fatal cases, were the observation in M. fascicularis from the Philippines of a mean CD4+/CD8+ ratio below I and of their ability, as revealed by mitogenic stimulation of whole blood, to produce increasing amounts of IFN-gamma as infection evolved. The contribution of environmental and genetic factors, which may differentiate the three groups of monkeys and therefore explain fatal or nonfatal evolution of the infection among them, is discussed.

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.

The development of control strategies for loiasis is of crucial importance in endemic areas and depends heavily on the accurate identification of occult-infected individuals. A polymerase chain reaction (PCR) and nested polymerase chain reaction (nested PCR) were developed and based on sequences of the repeat 3 region (15r3) of the gene encoding a Loa loa 15-kD protein. The assays was performed on 20 blood samples from occult-infected subjects and 30 from field-collected amicrofilaremic individuals. The size of the initial PCR product was 396 basepairs (bp). When this initial amplification using primers 15r3(1) and 15r3(2) was carried out for 30 cycles, the PCR products from three of the 20 occult-infected and five of the 30 amicrofilaremic individuals were visualized after electrophoresis by staining the gel with ethidium bromide. Subsequent Southern blotting and hybridization with the specific probe revealed hybridization in 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples but only after two days of exposure of the blot to the x-ray film. When the nested PCR was carried out (product size = 366 bp, primers 15r3(3) and 15r3(4)), 19 of 20 occult-infected and 23 of 30 amicrofilaremic samples that were positive by Southern hybridization of the initial PCR products were strongly positive by staining with ethidium bromide. Qualitative Southern blotting of the nested PCR products using the same probe previously described confirmed the ethidium bromide staining results after a very short exposure time of 4 hr. These results demonstrate that the nested PCR amplification product is specific and that its sensitivity in detecting occult loiasis is 95%. This approach has significant promise for the screening of large human populations for active loiasis without the requirement for blotting and hybridization of the PCR products.

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.

Accurate and specific diagnosis of human loiasis is of crucial importance in an endemic area where two-thirds of infected individuals are without circulating microfilariae (occult loiasis). By using the polymerase chain reaction (PCR) and specific primers to the repeat 3 region (15r3) of the gene coding for Loa loa 15-kDa polyprotein antigen, DNA was amplified from total blood lysate of occult-infected subjects. A 396-bp DNA fragment was specifically detected. We tested the specificity of this method by qualitative hybridization to PCR products using blood lysates of the following subjects: (1) from Gabon (80 individuals residing in L. loa endemic area where loiasis exists sympatrically with Mansonella perstans); (2) from Togo (12 individuals infected with Onchocerca volvulus and M. perstans); (3) from Tahiti (12 individuals infected with Wuchereria bancrofti); and (4) from Mali (12 individuals infected with O. volvulus and M. perstans). Samples from Gabon included 60 L. loa amicrofilaremics and 20 L. loa occult-infected subjects. Qualitative hybridization carried out at 50 degrees C on PCR products, using a 15r3-specific oligonucleotide probe, revealed hybridization with L. loa-infected samples from Gabon and four samples from Togo after 2 days exposure to the film. The positive samples from Togo were characterized by the use of nested PCR. Three nested PCR products have been sequenced. No differences were observed between the three sequences and they are 99.72% identical to L. loa 15r3. None of bancroftian-infected individuals from Tahiti, nor O. volvulus- and M. perstans-infected individuals from Mali reacted after 1 week's exposure (overexposure) to the film. This allows us to conclude first that our 15r3 PCR assay is specific for L. loa and secondly that L. loa infections occur in Togo. The sensitivity of this 15r3 PCR assay was further investigated with occult patients and field-collected amicrofilaremic samples. We found that 19 of the 20 occult-infected individuals were positive on Southern hybridization, whereas 35/60 amicrofilaremics were positive. These results have shown that the sensitivity of this assay in detecting unequivocal, parasitologically proven occult loiasis was 95%, while the specificity with regard to the sympatric M. perstans was 100%.

The International Center for Medical Research (CIRMF) is located in an area highly endemic for malaria in southeastern Gabon, where humans and apes (gorillas and chimpanzees) are living in the same geographic area. The presence of the CIRMF primate center housing apes (59 chimpanzees and nine gorillas in 1994) within the city of Franceville provided an opportunity to investigate the capability of cross-transmission of malaria species from humans to apes. The main vector of human malaria, Anopheles gambiae, was found in the primate center and in a nearby populated area of Franceville. Despite high malaria transmission in humans of both Plasmodium falciparum and P. malariae (mean of 43% cumulative prevalence in schoolchildren), none of the apes were found infected with plasmodia during a six-month investigation. However, low antibody levels against sporozoite and blood stages of both P. falciparum and P. malariae were detected in a few chimpanzees and gorillas. These results demonstrate that only rarely would apes be bitten in the field by mosquitoes infected with human malaria parasites. In the case of infection proven by serology, we did not find any evidence that blood-stage malaria parasites were able to the gametocyte stage. The absence of any established malaria transmission cycle within the primate colony of CIRMF indicates that apes cannot be considered as animal reservoirs for human malaria parasites in this environment.

Three outbreaks of Ebola hemorrhagic fever have recently occurred in Gabon. Virus has been isolated from clinical materials from all three outbreaks, and nucleotide sequence analysis of the glycoprotein gene of the isolates and virus present in clinical samples has been carried out. These data indicate that each of the three outbreaks should be considered an independent emergence of a different Ebola virus of the Zaire subtype. As in earlier Ebola virus outbreaks, no genetic variability was detected between virus samples taken during an individual outbreak.

Paired sera, saliva and cervicovaginal secretions from 17 HTLV-I-infected women (19-75 yr) were tested for total IgA and IgG, for IgA and IgG to the immunodominant region gp46/175-Pro-199, for serum IgG to the neutralizing domains gp46/ 190-Pro-199 and gp46/190-Ser-199, or for tax-rex proviral HTLV-DNA. Serum antibodies to gp46/ 175-Pro-199 were detected more frequently in the IgG (13/17) than in the IgA (5/17) isotypes. The majority (8/12) of anti-gp46/175-Pro-199-positive sera reacted also to gp46/190-Pro-199 or to gp46/ 190-Ser-199, demonstrating their neutralizing properties. In saliva, antibodies to gp46/175-Pro-199 were not generally detected. In cervicovaginal secretions, IgG to gp46/175-Pro-199, but not IgA, were detected in 6/15 (40%) patients. The mean specific activity of IgG to gp46/175-Pro-199 showed a trend to be higher in cervicovaginal secretions (218 +/- 109) than in sera (14 +/- 4). Furthermore, in all patients with cervicovaginal IgG to gp46/175-Pro-199, the cervicogaginal/serum ratio (19 +/- 6) of anti-gp46 IgG specific activities were markedly above 1. HTLV-DNA was detected in 4/17 salivas, and in 3/15 cervicovaginal secretions, all from patients demonstrating cervicovaginal synthesis of IgG to gp46/175-Pro-199. In conclusion, IgG to gp46/175-Pro-199 in cervicovaginal secretions, when present, appear to be produced primarily locally because of local HTLV-I excretion. Since anti-gp46/175-Pro-199 antibodies usually support reactivities to neutralizing domains, their presence could be relevant for limiting HTLV-I transmission via cervicovaginal secretions.

The reactivity of sera of 96 individuals infected with human T-cell leukemia virus type I (HTLV-I) was tested against various synthetic peptides corresponding to the gp46 immunodominant antigenic domains: residues 86-107, 175-199, and 239-261. The frequency of reactive sera was higher for 175-199 (93%) than for 239-261 (78%) or 86-107 (24%) with some variations in geographical regions and in diseases. The region 239-261 was extensively analyzed and five (linear or conformational) epitopes were found. The reactivity of sera toward functional or immunodominant domains may depend on the sequence of the infecting virus, and the role of three frequent substitutions (asparagine by tyrosine, proline by serine, and serine by proline or leucine at positions 93, 192, and 250 respectively) was established. Finally, the role of the genetic background of the host may condition the humoral immune response as individuals infected by HTLV-Is harboring the same predicted gp46 peptide sequence may recognize one, several, or all regions examined.

Reports of rare cases of suspected transmission of the human immunodeficiency virus (HIV) from mother to children by breast milk have been recently published. To study the factors that possibly limit HIV transmission through breast-feeding, milk samples obtamed from 15 healthy, seropositive mothers and 4 serobegative control subjects were studied for the presence of anti-HIV antibodies. All samples from seropositive women contained IgG antibody against envelope glycoproteins gpl6O and/or gpl20, and 11 of 15 samples contamed IgA antibodies against gpl60. IgA antibodies against other viral antigens were more rarely recovered, except against the internal proteins of the virus, p18 and p25. The finding of IgA antibodies to HIV-1 in breast milk establishes that the virus elicits a local immune response in heterosexual, seropositive women. The role of local antibodies in the postnatal transmission of HIV remains to be determined.

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.

Through PCR amplifications of the gene encoding the merozoite surface antigen 2, utilizing allele-specific 3D7 and FC27 probes, we have examined the prevalence of Plasmodium falciparum in children aged from 7 to 14 years living in a village located in the equatorial forest region of Central Africa (Gabon). Using this technique, 61% (100/163) of the blood samples were shown to be infected with P. falciparum with 24 alleles distinguished by size polymorphism and sequence type. The two main families (3D7 and FC27) and hybrid alleles were detected regardless of sex and hemoglobin phenotype. No age-related changes in prevalence of P. falciparum strains were observed; however, the prevalence of infection (42%) was significantly lower in individuals with the sickle-cell trait compared with their normal-hemoglobin counterparts (68%). Mixtures of genetically distinct parasite clones were present in 82% of children carrying the sickle-cell trait but in only 58% of normal-hemoglobin carriers. The significance of these observations regarding the design and interpretation of epidemiological investigations is discussed in the context of malaria transmission in the region studied.

Among the primates held at the CIRMF Primate Center, Gabon, no serological sign of SIV infection could be demonstrated in 68 cynomolgus monkeys, 60 chimpanzees, nine gorillas, and 12 sun-tailed monkeys, while seven of 102 mandrills and six of 24 vervets were infected with SIV. Six mandrills, seven vervets and ten cynomolgus monkeys exhibited a full HTLV type 1 Western blot profile. The sera of two gorillas and one chimpanzee presented with a positive but not typical HTLV Western blot profile. The sera of the gorillas lacked p24 antibodies, and the chimpanzee had a Western blot profile evocative of HTLV-II. All attempts to amplify viruses from these animals by PCR were unsuccessful. Two other chimpanzees and seven gorillas presented with indeterminate HTLV Western blot profiles. In the mandrill colony, only male animals were STLV seropositive and no sexual transmission to females was observed. SIV infection was also more frequent in male than female mandrills and sexual transmission appeared to be a rare event. No SRV infection was observed in macaques.

Nucleotide sequences of the central portion of gp120, including the third hypervariable (V3) loop, were obtained from lymphocytes cocultivated with SupT1 cells from 29 AIDS patients in Bangui, Central African Republic. These sequences displayed significantly greater diversity (average distance, 23%) than has been previously observed in isolates from comparably restricted geographical areas. Isolates belonging to four major subtypes of HIV-1 were found; the only subtype not represented was the North American/European subtype B. Unlike the situation in Zaire and Uganda, where subtypes A and D account equally for virtually all isolates of HIV-1, the predominant subtypes in the Central African Republic, accounting for two-thirds of the isolates, were subtypes A (10 isolates) and E (9 isolates). Subtype E represents a group of variants that have previously been found only in Thailand. Only one isolate belonging to subtype D was found. Also recovered were two isolates of subtype C, a subtype associated with southern African and Indian isolates but not previously detected in central Africa. These isolates, although clearly clustering with subtype C, formed a distinct subset, differing from one another by 8.8% and from the Indian and South African subtype C isolates by an average of 22.5%. High interpatient, intrasubtype variation was also seen among the CAR subtype A (average pairwise difference, 19.3%) and subtype E (10.9%) isolates. The diversity of V3 sequences in this set has implications for immunization protocols that rely on the recognition of V3. This study underscores the necessity of basing intervention strategies on knowledge of the particular sequences present in the target population or geographical area.

Semliki Forest (SF) virus was responsible for an outbreak of febrile illnesses in Bangui, Central African Republic (CAR), during October-December 1987. The virus was isolated at first from mosquitoes, mainly Aedes africanus, collected August-October in a gallery forest 100 km from Bangui. During October-December, 22 isolations of SF virus were made from serum samples collected from patients in Bangui presenting with fever, severe persistent headache, myalgia, arthralgia, and a convalescence marked by asthenia. During the same period, 8 SF virus isolates were obtained from mosquitoes collected in Bangui, mainly from Ae. aegypti. Europeans, particularly soldiers who had recently arrived from France, were affected. Antibodies to alphaviruses had been previously detected in a high proportion of resident human populations in CAR. We conclude that SF virus is a human pathogen.

A study conducted in 1996-97 at the Foundation Jeanne Ebori Hospital in Libreville Gabon identified an unexpectedly high prevalence of patients with HIV-2 and dual HIV-1/HIV-2 infection. During the 10-month study period 147 (14.3%) of 1029 serum samples tested were found to be HIV-positive by enzyme-linked immunosorbent assays. Use of synthetic peptides specific for the gp41 and the gp120 of HIV-1 and the gp36 of HIV-2 indicated that 141 (96%) of infected sera were exclusively HIV-1 4 were exclusively HIV-2 and 2 were positive for both HIV-1 and HIV-2. 2 of the 6 patients infected with HIV-2 or both HIV-1 and HIV-2 were immigrants from West Africa (Mali and Nigeria). 3 of the patients with HIV-2 infection were asymptomatic. The predominant HIV risk factor was heterosexual activity. These findings suggest the possibility of rare cases of HIV-1 and HIV-2 co-infections recombinant HIV-1 and HIV-2 strains and peculiar HIV-2 variants from Central Africa. More widespread entry of HIV-2 infection into Central Africa from Gabon could have major public health implications.

Sera of 381 adult people from 5 areas in Madagascar were tested by the indirect immunofluorescence method for antibodies against Congo-Crimean haemorrhagic fever and Rift Valley fever viruses (Bunyaviridae), Ebola (strains Zaire and Sudan) and Marburg viruses (Filoviridae), and Lassa virus (Arenaviridae). The highest prevalence rate was that of Ebola virus (4.5%). As no haemorrhagic syndrome has been found associated with this virus, the possible presence of a less pathogenic, antigenically related, strain is discussed. The prevalences of Congo-Crimean haemorrhagic fever and Rift Valley viruses were very low, despite previous viral isolations from potential vectors. No serum reacted against Lassa or Marburg antigens. The results are analysed in the light of the geographical and bioecological characteristics of Madagascar, which is a true 'microcontinent' very different from the African mainland.

To study the local immune response to human immunodeficiency virus type 1 (HIV-1) in women infected by or exposed to HIV-1 75 women were studied. 15 were IgG-seropositive but clinically asymptomatic 15 had acquired immune deficiency syndrome (AIDS) 15 were IgG-seronegative with seropositive husbands and 30 were healthy seronegative women who were selected as controls. Serum samples and vaginal secretions were tested for antibodies to HIV-1 IgG and IgA by Western blot analysis. Antibodies of the IgG and IgA classes were detected in serum samples and vaginal secretions from healthy seropositive women and from healthy seropositive women and from women with AIDS. Local IgG antibodies to all viral proteins were detected by Western blot tests. Genital IgA antibodies were mainly directed to the core proteins p18 and p25 the p68 reverse transcriptase and the gp160 and gp41 glycoproteins; IgA antibodies to the glycoprotein gp120 were rarely recovered. Antibodies of both the IgG and IgA classes in genital secretions were directed to all viral proteins including surface glycoproteins and could play a role in limiting the virus infectivity on normal mucosa. (authors)