Viral Exploration of Negative Acute Febrile Cases Observed during Chikungunya Outbreaks in Gabon.

Non-malarial febrile illness outbreaks were documented in 2007 and 2010 in Gabon. After investigation, these outbreaks were attributed to the chikungunya and dengue viruses (CHIKV and DENV). However, for more than half of the samples analyzed, the causative agent was not identified. Given the geographical and ecological position of Gabon, where there is a great animal and microbial diversity, the circulation of other emerging viruses was suspected in these samples lacking aetiology. A total of 436 undiagnosed samples, collected between 2007 and 2013, and originating from 14 urban, suburban, and rural Gabonese locations were selected. These samples were used for viral isolation on newborn mice and VERO cells. In samples with signs of viral replication, cell supernatants and brain suspensions were used to extract nucleic acids and perform real-time RT-PCR targeting specific arboviruses, i.e., CHIKV, DENV, yellow fever, Rift Valley fever, and West Nile and Zika viruses. Virus isolation was conclusive for 43 samples either on newborn mice or by cell culture. Virus identification by RT-PCR led to the identification of CHIKV in 37 isolates. A total of 18 complete genomes and 19 partial sequences containing the E2 and E1 genes of CHIKV were sequenced using next-generation sequencing technology or the Sanger method. Phylogenetic analysis of the complete genomes showed that all the sequences belong to the East Central South Africa lineage. Furthermore, we identified 2 distinct clusters. The first cluster was made up of sequences from the western part of Gabon, whereas the second cluster was made up of sequences from the southern regions, reflecting the way CHIKV spread across the country following its initial introduction in 2007. Similar results were obtained when analyzing the CHIKV genes of the E2 and E1 structural proteins. Moreover, study of the mutations found in the E2 and E1 structural proteins revealed the presence of several mutations that facilitate the adaptation to the Aedes albopictus mosquito, such as E2 I211T and E1 A226V, in all the Gabonese CHIKV strains. Finally, sequencing of 6 additional viral isolates failed to lead to any conclusive identification.

Origin of HTLV-1 in hunters of nonhuman primates in Central Africa.

Of 78 Gabonese individuals who had received bites from nonhuman primates (NHPs) while hunting, 7 were infected with human T lymphotropic virus (HTLV-1). Five had been bitten by gorillas and were infected with subtype B strains; however, a 12-year-old girl who was severely bitten by a Cercopithecus nictitans was infected with a subtype D strain that was closely related to the simian T lymphotropic virus (STLV-1) that infects this monkey species. Her mother was infected with a subtype B strain. These data confirm that hunters in Africa can be infected by HTLV-1 that is closely related to the strains circulating among local NHP game. Our findings strongly suggest that a severe bite represent a risk factor for STLV-1 acquisition.

Cross-species transmission of simian foamy virus to humans in rural Gabon, Central Africa.

In order to characterize simian foamy retroviruses (SFVs) in wild-born nonhuman primates (NHPs) in Gabon and to investigate cross-species transmission to humans, we obtained 497 NHP samples, composed of 286 blood and 211 tissue (bush meat) samples. Anti-SFV antibodies were found in 31 of 286 plasma samples (10.5%). The integrase gene sequence was found in 38/497 samples, including both blood and tissue samples, with novel SFVs in several Cercopithecus species. Of the 78 humans, mostly hunters, who had been bitten or scratched by NHPs, 19 were SFV seropositive, with 15 cases confirmed by PCR. All but one were infected with ape SFV. We thus found novel SFV strains in NHPs in Gabon and high cross-species transmission of SFVs from gorilla bites.

Recent introduction and rapid dissemination of Chikungunya virus and Dengue virus serotype 2 associated with human and mosquito coinfections in Gabon, central Africa.

BACKGROUND: Chikungunya virus (CHIKV) and Dengue virus serotype 2 (DENV-2) were recently introduced in central Africa, along with Aedes albopictus. Simultaneous outbreaks of CHIKV and DENV-2 have subsequently occurred, in Cameroon in 2006 and Gabon in 2007. METHODS: To study the spread of the 2 viruses, we conducted active surveillance of acute febrile syndromes throughout Gabon between 2007 and 2010. Diagnostic methods included quantitative real-time reverse-transcription polymerase chain reaction, and molecular characterization was based on partial envelope gene sequences. RESULTS: Between 2007 and 2010, 4287 acutely febrile patients were investigated for CHIKV and DENV-2 infections, of whom 1567 were CHIKV-positive, 376 DENV-2-positive, and 37 coinfected. We diagnosed 153 CHIKV and 11 DENV-2 cases in 2008, and 5 CHIKV and 9 DENV-2 cases in 2009. In 2010, CHIKV and DENV-2 caused a second large simultaneous outbreak. Among 2826 acutely febrile patients examined during this outbreak, 1112 were CHIKV-positive, 288 DENV-2-positive, and 28 coinfected. Mosquitoes were collected near the homes of coinfected patients, and 1 Aedes albopictus specimen was found to be positive for both CHIKV and DENV-2. CONCLUSIONS: These findings show the rapid dissemination of CHIKV and DENV-2 within a nonimmune population in a tropical African country, probably facilitated by the spread of Aedes albopictus. This has resulted in major simultaneous outbreaks with numerous coinfections in both human and mosquito.

Acute risk for hepatitis E virus infection among HIV-1-positive pregnant women in central Africa.

BACKGROUND: Hepatitis E virus (HEV), an enterically transmitted pathogen, is highly endemic in several African countries. Pregnant women are at particularly high risk for acute or severe hepatitis E. In Gabon, a central African country, the prevalence of antibodies to HEV among pregnant women is 14.1%. Recent studies have demonstrated unusual patterns of hepatitis E (chronic hepatitis, cirrhosis) among immunodeficient patients. FINDINGS: We investigated the prevalence of antibodies to HEV among pregnant women infected with HIV-1 or HTLV-1 in Gabon. Of 243 samples collected, 183 were positive for HIV-1 and 60 for HTLV-1; 16 women (6.6%) had IgG antibodies to HEV. The seroprevalence was higher among HIV-1-infected women (7.1%) than HTLV-1-infected women (5.0%). Moreover, the HIV-1 viral load was significantly increased (p ≤ 0.02) among women with past-HEV exposure (1.3E+05 vs 5.7E+04 copies per ml), whereas no difference was found in HTLV-1 proviral load (9.0E+01 vs 1.1E+03 copies per ml). CONCLUSIONS: These data provide evidence that HIV-1-infected women are at risk for acute or severe infection if they are exposed to HEV during pregnancy, with an increased viral load.

Prevalence, genetic diversity and antiretroviral drugs resistance-associated mutations among untreated HIV-1-infected pregnant women in Gabon, central Africa.

BACKGROUND: In Africa, the wide genetic diversity of HIV has resulted in emergence of new strains, rapid spread of this virus in sub-Saharan populations and therefore spread of the HIV epidemic throughout the continent. METHODS: To determine the prevalence of antibodies to HIV among a high-risk population in Gabon, 1098 and 2916 samples were collected from pregnant women in 2005 and 2008, respectively. HIV genotypes were evaluated in 107 HIV-1-positive samples to determine the circulating subtypes of strains and their resistance to antiretroviral drugs (ARVs). RESULTS: The seroprevalences were 6.3% in 2005 and 6.0% in 2008. The main subtype was recombinant CRF02_AG (46.7%), followed by the subtypes A (19.6%), G (10.3%), F (4.7%), H (1.9%) and D (0.9%) and the complex recombinants CRF06_cpx (1.9%) and CRF11_cpx (1.9%); 12.1% of subtypes could not be characterized. Analysis of ARVs resistance to the protease and reverse transcriptase coding regions showed mutations associated with extensive subtype polymorphism. In the present study, the HIV strains showed reduced susceptibility to ARVs (2.8%), particularly to protease inhibitors (1.9%) and nucleoside reverse transcriptase inhibitors (0.9%). CONCLUSIONS: The evolving genetic diversity of HIV calls for continuous monitoring of its molecular epidemiology in Gabon and in other central African countries.

Two distinct variants of simian foamy virus in naturally infected mandrills (Mandrillus sphinx) and cross-species transmission to humans.

BACKGROUND: Each of the pathogenic human retroviruses (HIV-1/2 and HTLV-1) has a nonhuman primate counterpart, and the presence of these retroviruses in humans results from interspecies transmission. The passage of another simian retrovirus, simian foamy virus (SFV), from apes or monkeys to humans has been reported. Mandrillus sphinx, a monkey species living in central Africa, is naturally infected with SFV. We evaluated the natural history of the virus in a free-ranging colony of mandrills and investigated possible transmission of mandrill SFV to humans. RESULTS: We studied 84 semi-free-ranging captive mandrills at the Primate Centre of the Centre International de Recherches Médicales de Franceville (Gabon) and 15 wild mandrills caught in various areas of the country. The presence of SFV was also evaluated in 20 people who worked closely with mandrills and other nonhuman primates. SFV infection was determined by specific serological (Western blot) and molecular (nested PCR of the integrase region in the polymerase gene) assays. Seropositivity for SFV was found in 70/84 (83%) captive and 9/15 (60%) wild-caught mandrills and in 2/20 (10%) humans. The 425-bp SFV integrase fragment was detected in peripheral blood DNA from 53 captive and 8 wild-caught mandrills and in two personnel. Sequence and phylogenetic studies demonstrated the presence of two distinct strains of mandrill SFV, one clade including SFVs from mandrills living in the northern part of Gabon and the second consisting of SFV from animals living in the south. One man who had been bitten 10 years earlier by a mandrill and another bitten 22 years earlier by a macaque were found to be SFV infected, both at the Primate Centre. The second man had a sequence close to SFVmac sequences. Comparative sequence analysis of the virus from the first man and from the mandrill showed nearly identical sequences, indicating genetic stability of SFV over time. CONCLUSION: Our results show a high prevalence of SFV infection in a semi-free-ranging colony of mandrills, with the presence of two different strains. We also showed transmission of SFV from a mandrill and a macaque to humans.

Prevalence and genetic diversity of hepatitis B and delta viruses in pregnant women in Gabon: molecular evidence that hepatitis delta virus clade 8 originates from and is endemic in central Africa.

Hepatitis B virus (HBV) surface antigen (HBsAg) was found in 9.2% of 1,186 pregnant women from Gabon, of whom 10.1% had the HBe antigen and 89.9% had anti-HBe antibodies. Antibodies to the hepatitis delta virus (HDV) were found in 15.6% of the HBsAg-positive women. The HBV strains were of the A3 and E genotypes. The HDV strains belonged to HDV clades 1 and 8. These results provide clear evidence that HDV clade 8 is indigenous to Africa.

New insights into prevalence, genetic diversity, and proviral load of human T-cell leukemia virus types 1 and 2 in pregnant women in Gabon in equatorial central Africa.

Human T-cell leukemia virus type 1 (HTLV-1) is highly endemic in areas of central Africa; mother-to-child transmission and sexual transmission are considered to be the predominant routes. To determine the prevalence and subtypes of HTLV-1/2 in pregnant women in Gabon, we conducted an epidemiological survey in the five main cities of the country. In 907 samples, the HTLV-1 seroprevalence was 2.1%, which is lower than that previously reported. Only one case of HTLV-2 infection was found. The HTLV-1 seroprevalence increased with age and differed between regions (P

Hepatitis E virus is highly prevalent among pregnant women in Gabon, central Africa, with different patterns between rural and urban areas.

Hepatitis E virus (HEV) is highly endemic in several African countries with high mortality rate among pregnant women. Nothing is known about the circulation of this virus in central Africa. We evaluated therefore the prevalence of anti-HEV IgG in samples collected from pregnant women living in the five main cities of Gabon, central Africa. We found that 14.1% (119/840) of pregnant women had anti-HEV IgG. The prevalence differed between regions and between age groups. In 391 newly collected samples from the region where the highest prevalence was found, a significant difference (p < 0.05) in seroprevalence was found between rural (6.4%) and urban (13.5%) areas. These data provide evidence of a high prevalence of HEV in Gabon, providing indirect evidence of past contact with this virus.

Revisiting human T-cell lymphotropic virus types 1 and 2 infections among rural population in Gabon, central Africa thirty years after the first analysis.

HTLV-1 infection is considered as highly endemic in central Africa. Thirty years ago, a first epidemiological study was performed in Gabon, central Africa, and revealed that the prevalence varied from 5.0 to 10.5%. To evaluate current distribution of HTLVs in Gabon, 4.381 samples were collected from rural population living in 220 villages distributed within the 9 provinces of country. HTLVs prevalence was determined using two ELISA tests and positive results were confirmed by Western Blot. The overall HTLV-1 seroprevalence was of 7.3% among the rural Gabonese population; with 5.4% for men and 9.0% for women. Prevalence of HTLV-1 differed by province, ranging from 2.3% to 12.5% into the rain forest. Being a woman older than 51 years represented a high risk for HTLV-1 acquisition. Hospitalization, operation/surgery, transfusion and medical abortion or fever, arthritis and abdominal pain are also significant risk factors. In addition, 0.1% of samples were found as HTLV-2 positive, while 12.0% had an indeterminate HTLV serological pattern. HTLV-3 and HTLV-4 were not found. Phylogenetic analysis was performed on 87 samples and demonstrated that HTLV-1 present in Gabon belongs mostly to subtype B, however the rare subtype D was also found. Altogether, our results demonstrate that almost thirty years after the first epidemiological study prevention of HTLVs infection is still an issue in Gabon.

Measuring terrestrial subsidies to aquatic food webs using stable isotopes of hydrogen.

Understanding river food webs requires distinguishing energy derived from primary production in the river itself (autochthonous) from that produced externally (allochthonous), yet there are no universally applicable and reliable techniques for doing so. We compared the natural abundance stable isotope ratios of hydrogen (deltaD) of allochthonous and autochthonous energy sources in four different aquatic ecosystems. We found that autochthonous organic matter is uniformly far more depleted in deuterium (lower deltaD values) than allochthonous: an average difference of approximately 100% per hundred. We also found that organisms at higher trophic levels, including both aquatic invertebrates and fish, have deltaD values intermediate between aquatic algae and terrestrial plants. The consistent differences between leaves and algae in deltaD among these four watersheds, along with the intermediate values in higher trophic levels, indicate that natural abundance hydrogen isotope signatures are a powerful tool for partitioning energy flow in aquatic ecosystems.

Hydrogen Isotopes as a Sentinel of Biological Invasion by the Japanese Beetle, Popillia japonica (Newman).

Invasive species alter ecosystems, threaten native and endangered species, and have negative economic impacts. Knowing where invading individuals are from and when they arrive to a new site can guide management. Here, we evaluated how well the stable hydrogen isotope composition (δ2H) records the recent origin and time since arrival of specimens of the invasive Japanese beetle (Popillia japonica Newman) captured near the Portland International Airport (Oregon, U.S.A.). The δ2H of Japanese beetle specimens collected from sites across the contiguous U.S.A. reflected the δ2H of local precipitation, a relationship similar to that documented for other organisms, and one confirming the utility of δ2H as a geographic fingerprint. Within weeks after experimental relocation to a new isotopic environment, the δ2H of beetles changed linearly with time, demonstrating the potential for δ2H to also mark the timing of arrival to a new location. We used a hierarchical Bayesian model to estimate the recent geographical origin and timing of arrival of each specimen based on its δ2H value. The geographic resolution was broad, with values consistent with multiple regions of origin in the eastern U.S.A., slightly favoring the southeastern U.S.A. as the more likely source. Beetles trapped from 2007-2010 had arrived 30 or more days prior to trapping, whereas the median time since arrival declined to 3-7 days for beetles trapped from 2012-2014. This reduction in the time between arrival and trapping at the Portland International Airport supports the efficacy of trapping and spraying to prevent establishment. More generally, our analysis shows how stable isotopes can serve as sentinels of biological invasions, verifying the efficacy of control measures, or, alternatively, indicating when those measures show signs of failure.

Longitudinal Analysis of Natural Killer Cells in Dengue Virus-Infected Patients in Comparison to Chikungunya and Chikungunya/Dengue Virus-Infected Patients.

BACKGROUND: Dengue virus (DENV) is the most prominent arbovirus worldwide, causing major epidemics in South-East Asia, South America and Africa. In 2010, a major DENV-2 outbreak occurred in Gabon with cases of patients co-infected with chikungunya virus (CHIKV). Although the innate immune response is thought to be of primordial importance in the development and outcome of arbovirus-associated pathologies, our knowledge of the role of natural killer (NK) cells during DENV-2 infection is in its infancy. METHODOLOGY: We performed the first extensive comparative longitudinal characterization of NK cells in patients infected by DENV-2, CHIKV or both viruses. Hierarchical clustering and principal component analyses were performed to discriminate between CHIKV and DENV-2 infected patients. PRINCIPAL FINDINGS: We observed that both activation and differentiation of NK cells are induced during the acute phase of infection by DENV-2 and CHIKV. Combinatorial analysis however, revealed that both arboviruses induced two different signatures of NK-cell responses, with CHIKV more associated with terminal differentiation, and DENV-2 with inhibitory KIRs. We show also that intracellular production of interferon-γ (IFN-γ) by NK cells is strongly stimulated in acute DENV-2 infection, compared to CHIKV. CONCLUSIONS/SIGNIFICANCE: Although specific differences were observed between CHIKV and DENV-2 infections, the significant remodeling of NK cell populations observed here suggests their potential roles in the control of both infections.

Short communication. Antiretroviral drug resistance among drug-naive HIV-1-infected individuals in Djibouti (Horn of Africa).

OBJECTIVE: To survey the frequency of genotypic antiretroviral resistance in drug-naive HIV-1-infected Djiboutians. METHODS: A national study was conducted in the general population of Djibouti in March 2002 to determine HIV-1 seroprevalence. Blood samples were collected anonymously and plasma samples scoring positive for HIV-1 antibodies were tested for viral load. Genotypic studies were performed with viral RNA from plasma using the consensus technique of the Agence Nationale de Recherche sur le SIDA (www.hivfrenchresistance.org). Mutations were identified using the International AIDS Society-USA resistance panel and resistant virus was defined according to the ANRS algorithm. RESULTS: A panel of 2423 individuals representing the general population of Djibouti was included. Antibodies were detected in 53 of 2423 samples tested. The HIV-1 seroprevalence in the general population was 2.2%. Genotype C was the most prevalent, and the other isolates were CRF_02 AG, or subtype A or D. Forty-seven of the 53 samples were tested for genotypic resistance, and mutations concerning all three classes of antiretrovirals were found. The most frequent were secondary mutations associated with protease inhibitors (PIs): M36I, R41K and K20I/R. A few strains displayed primary mutations (the non-nucleoside reverse transcriptase inhibitor [NNRTI]-associated mutations K101E, K103T, L100I and G190V; the PI-associated mutation N88D; and the NRTI-associated mutation K65R). The presence of these mutations may be due to the transmission of strains from treated patients. CONCLUSION: Substantial polymorphism and a few primary mutations are found in HIV-1 non-B subtype isolates from Djiboutian antiretroviral-drug-naive individuals. This needs to be taken into account to adapt antiretroviral regimens and prophylactic schedules locally.

Chikungunya, a paradigm of neglected tropical disease that emerged to be a new health global risk.

Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family that causes chronic and incapacitating arthralgia in human populations. Since its discovery in 1952, CHIKV was responsible for sporadic and infrequent outbreaks. However, since 2005, global Chikungunya outbreaks have occurred, inducing some fatalities and associated with severe and chronic morbidity. Chikungunya is thus considered as an important re-emerging public health problem in both tropical and temperate countries, where the distribution of the Aedes mosquito vectors continues to expand. This review highlights the most recent advances in our knowledge and understanding of the epidemiology, biology, treatment and vaccination strategies of CHIKV.

Zika virus in Gabon (Central Africa)--2007: a new threat from Aedes albopictus?

BACKGROUND: Chikungunya and dengue viruses emerged in Gabon in 2007, with large outbreaks primarily affecting the capital Libreville and several northern towns. Both viruses subsequently spread to the south-east of the country, with new outbreaks occurring in 2010. The mosquito species Aedes albopictus, that was known as a secondary vector for both viruses, recently invaded the country and was the primary vector involved in the Gabonese outbreaks. We conducted a retrospective study of human sera and mosquitoes collected in Gabon from 2007 to 2010, in order to identify other circulating arboviruses. METHODOLOGY/PRINCIPAL FINDINGS: Sample collections, including 4312 sera from patients presenting with painful febrile disease, and 4665 mosquitoes belonging to 9 species, split into 247 pools (including 137 pools of Aedes albopictus), were screened with molecular biology methods. Five human sera and two Aedes albopictus pools, all sampled in an urban setting during the 2007 outbreak, were positive for the flavivirus Zika (ZIKV). The ratio of Aedes albopictus pools positive for ZIKV was similar to that positive for dengue virus during the concomitant dengue outbreak suggesting similar mosquito infection rates and, presumably, underlying a human ZIKV outbreak. ZIKV sequences from the envelope and NS3 genes were amplified from a human serum sample. Phylogenetic analysis placed the Gabonese ZIKV at a basal position in the African lineage, pointing to ancestral genetic diversification and spread. CONCLUSIONS/SIGNIFICANCE: We provide the first direct evidence of human ZIKV infections in Gabon, and its first occurrence in the Asian tiger mosquito, Aedes albopictus. These data reveal an unusual natural life cycle for this virus, occurring in an urban environment, and potentially representing a new emerging threat due to this novel association with a highly invasive vector whose geographic range is still expanding across the globe.

First evidence of simultaneous circulation of three different dengue virus serotypes in Africa.

Gabon, in Central Africa, was affected for the first time in 2007 and then in 2010 by simultaneous outbreaks of chikungunya and Dengue serotype 2 (DENV-2) viruses. Through the national surveillance of dengue-like syndromes between 2007 and 2010, we observed continuous circulation of DENV-2 in a southward movement. This rapid spread of DENV-2 was associated with the emergence of DENV-1 in 2007 and DENV-3 in 2010. Interestingly, we detected six DENV-2 infected patients with hemorrhagic signs during the second outbreak in 2010. Although these cases do not meet all standard WHO criteria for severe Dengue with hemorrhage (formerly DHF), this is the first report of several dengue fever cases associated with hemorrhagic signs during a simultaneous circulation of different DENV serotypes in Africa. Together, these findings suggest that DENV is becoming more widely established on this continent and that DHF will likely become a serious public-health problem in the near future.

A chikungunya outbreak associated with the vector Aedes albopictus in remote villages of Gabon.

Chikungunya virus (CHIKV) recently caused major urban outbreaks in central African countries such as the Democratic Republic of Congo (DRC), Cameroon, and Gabon. In Gabon, the tiger mosquito Aedes albopictus was shown to be the main CHIKV vector during the 2007 outbreak. This invasive Asian species was first identified in Gabon in early 2007, and was thought to be restricted mainly to coastal provinces where urban epidemic CHIKV foci were recorded. Here we report a CHIKV outbreak in a small cluster of villages isolated in the deep forest of southern Gabon, in which A. albopictus was the main vector. This observation indicates concomitant geographic and ecological spread of CHIKV activity and A. albopictus in remote environments in central Africa, as well as an enhanced risk of propagation of epidemic arboviruses.

No clinical or biological difference between Chikungunya and Dengue Fever during the 2010 Gabonese outbreak.

Chikungunya (CHIKV) and Dengue (DENV) viruses, both arboviruses, have caused multiple outbreaks worldwide. Their clinical features are poorly described in Africa and there is no comparative study, although Chikungunya is considered as a dengue-like disease. We conducted a comparative study of clinical and biological data from CHIKV and DENV positive patients during the 2010 Gabonese outbreak. Patients consulting with general symptoms and having laboratory confirmation for CHIKV or DENV were included. Clinical and biological data were recorded. Statistical analyses were performed using Epi Info. A P value < 0.05 was considered significant. In all, 270 CHIKV+, 53 DENV+ and 20 co-infected patients were included in the study. Headaches, hemorrhage, leukopenia and lymphopenia were significantly (P respectively 0.01, 0.001, 0.02 and 0.001) more frequent in DENV+ patients than in CHIKV+. There was no additive effect of the two viruses.These clinical and hematological disorders are non specific and cannot assist for the differential diagnosis. These diseases are clinically indistinguishable, and need for laboratory confirmation.