A Second Genome Sequence of an Enterovirus C99 Detected in a Healthy Chimpanzee.

We report the nearly complete genome sequence of an enterovirus 99 strain (Cpz-IJC08) detected in a healthy chimpanzee from the Tchimpounga Sanctuary in the Republic of Congo. According to the phylogeny, Cpz-IJC08 clustered with Cpz-IJC04, a previously identified chimpanzee enterovirus from the same sanctuary, isolated from an animal with signs of acute flaccid paralysis.

Genetic diversity and ecology of coronaviruses hosted by cave-dwelling bats in Gabon.

Little research on coronaviruses has been conducted on wild animals in Africa. Here, we screened a wide range of wild animals collected in six provinces and five caves of Gabon between 2009 and 2015. We collected a total of 1867 animal samples (cave-dwelling bats, rodents, non-human primates and other wild animals). We explored the diversity of CoVs and determined the factors driving the infection of CoVs in wild animals. Based on a nested reverse transcription-polymerase chain reaction, only bats, belonging to the Hipposideros gigas (4/156), Hipposideros cf. ruber (13/262) and Miniopterus inflatus (1/249) species, were found infected with CoVs. We identified alphacoronaviruses in H. gigas and H. cf. ruber and betacoronaviruses in H. gigas. All Alphacoronavirus sequences grouped with Human coronavirus 229E (HCoV-229E). Ecological analyses revealed that CoV infection was significantly found in July and October in H. gigas and in October and November in H. cf ruber. The prevalence in the Faucon cave was significantly higher. Our findings suggest that insectivorous bats harbor potentially zoonotic CoVs; highlight a probable seasonality of the infection in cave-dwelling bats from the North-East of Gabon and pointed to an association between the disturbance of the bats' habitat by human activities and CoV infection.

Identification of Dengue and Chikungunya Cases Among Suspected Cases of Yellow Fever in the Democratic Republic of the Congo.

For more than 95% of acute febrile jaundice cases identified through surveillance for yellow fever, a reemerging arthropod-borne viral disease, no etiological exploration is ever done. The aim of this study was to test for other arthropod-borne viruses that can induce the same symptoms in patients enrolled in the yellow fever surveillance in the Democratic Republic of the Congo (DRC). Of 652 patients included in the surveillance of yellow fever in DRC from January 2003 to January 2012, 453 patients that tested negative for yellow fever virus (YFV) immunoglobulin M (IgM) antibodies were selected for the study. Real-time polymerase chain reaction was performed for the detection of dengue, West Nile, Chikungunya, O'nyong-nyong, Rift Valley fever, Zika, and YFV. The average age of patients was 22.1 years. We reported 16 cases (3.5%; confidence interval [CI]: 0.8-5.2) of dengue (serotypes 1 and 2) and 2 cases (0.4%; CI: 0.0-1.0) of Chikungunya. Three patients were co-infected with the two serotypes of dengue virus. Three cases of dengue were found in early July 2010 from the city of Titule (Oriental province) during a laboratory-confirmed outbreak of yellow fever, suggesting simultaneous circulation of dengue and yellow fever viruses. This study showed that dengue and Chikungunya viruses are potential causes of acute febrile jaundice in the DRC and highlights the need to consider dengue and Chikungunya diagnosis in the integrated disease surveillance and response program in the DRC. A prospective study is necessary to establish the epidemiology of these diseases.

Diversity and role of cave-dwelling hematophagous insects in pathogen transmission in the Afrotropical region.

The progressive anthropization of caves for food resources or economic purposes increases human exposure to pathogens that naturally infect cave-dwelling animals. The presence of wild or domestic animals in the immediate surroundings of caves also may contribute to increasing the risk of emergence of such pathogens. Some zoonotic pathogens are transmitted through direct contact, but many others require arthropod vectors, such as blood-feeding insects. In Africa, hematophagous insects often play a key role in the epidemiology of many pathogens; however, their ecology in cave habitats remains poorly known. During the last decades, several investigations carried out in Afrotropical caves suggested the medical and veterinary importance particularly of insect taxa of the Diptera order. Therefore, the role of some of these insects as vectors of pathogens that infect cave-dwelling vertebrates has been studied. The present review summarizes these findings, brings insights into the diversity of cave-dwelling hematophagous Diptera and their involvement in pathogen transmission, and finally discusses new challenges and future research directions.

Serological Evidence for the Circulation of Rift Valley Fever Virus in Domestic Small Ruminants in Southern Gabon.

Rift Valley fever (RVF) is a zoonotic disease, which caused several epidemics in humans in many countries of Africa. Using an inhibition enzyme-linked immunosorbent assay (ELISA), real-time reverse transcription PCR, and nested one-step reverse transcription PCR, we conducted a cross-sectional study in populations of sheep and goats from the Mongo County in 2014 to determine the circulation of the Rift Valley fever virus (RVFV) in small ruminants from this area. From a total of 201 small ruminants (95 sheep and 106 goats), the overall IgG seroprevalence against the RVFV was 6.47% (13/201). No RVFV RNA was detected in the animal plasmas. Logistic regression analysis showed that age, species, sex, and locality were not the significant risk factors. The findings of this study highlight the risk of RVF for domestic ruminants bred in this region and for the human rural population living in contact with these animals and they emphasize the need to develop adequate control measures to limit this threat.

Exploring the diversity of blood-sucking Diptera in caves of Central Africa.

Caves house pathogenic microorganisms, some of which are transmitted by blood-sucking arthropods. In Africa, previous studies identified mosquitoes, sand flies and biting midges as the main potential vectors of cave-dwelling pathogens. However, to understand their involvement in pathogen spillover, it is crucial to characterize their diversity, community composition and dynamics. Using CDC light traps, we collected hematophagous Diptera in six caves of Gabon during one-shot or longitudinal sampling, and investigated their species diversity and dynamics in relation with external rainfall. Overall, we identified 68 species of mosquitoes, sand flies and biting midges, including 45 new records for Gabon. The dominant species were: Uranotaenia nigromaculata, Anopheles smithii s.l., Culex. rima group and Culex quasiguiarti for mosquitoes, Spelaeophlebotomus gigas and Spelaeomyia emilii for sand flies and the Culicoides trifasciellus group and Culicoides fulvithorax for biting midges. The survey revealed that species assemblages were cave-specific and included mainly troglophilous and trogloxenous species. Both diversity and abundance varied according to the cave and sampling time, and were significantly associated with rainfall. These associations were modulated by the cave specific environmental conditions. Moreover, the presence of trogloxenous and troglophilous species could be of high significance for pathogen transfers between cave and epigeous hosts, including humans.

High Prevalence and Diversity of Hepatitis Viruses in Suspected Cases of Yellow Fever in the Democratic Republic of Congo.

The majority of patients with acute febrile jaundice (>95%) identified through a yellow fever surveillance program in the Democratic Republic of Congo (DRC) test negative for antibodies against yellow fever virus. However, no etiological investigation has ever been carried out on these patients. Here, we tested for hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), and hepatitis E (HEV) viruses, all of which can cause acute febrile jaundice, in patients included in the yellow fever surveillance program in the DRC. On a total of 498 serum samples collected from suspected cases of yellow fever from January 2003 to January 2012, enzyme-linked immunosorbent assay (ELISA) techniques were used to screen for antibodies against HAV (IgM) and HEV (IgM) and for antigens and antibodies against HBV (HBsAg and anti-hepatitis B core protein [HBc] IgM, respectively), HCV, and HDV. Viral loads and genotypes were determined for HBV and HVD. Viral hepatitis serological markers were diagnosed in 218 (43.7%) patients. The seroprevalences were 16.7% for HAV, 24.6% for HBV, 2.3% for HCV, and 10.4% for HEV, and 26.1% of HBV-positive patients were also infected with HDV. Median viral loads were 4.19 × 105 IU/ml for HBV (range, 769 to 9.82 × 109 IU/ml) and 1.4 × 106 IU/ml for HDV (range, 3.1 × 102 to 2.9 × 108 IU/ml). Genotypes A, E, and D of HBV and genotype 1 of HDV were detected. These high hepatitis prevalence rates highlight the necessity to include screening for hepatitis viruses in the yellow fever surveillance program in the DRC.

Molecular identification of trypanosome species in trypanotolerant cattle from the south of Gabon.

The aim of this study was to provide information on trypanosome species infecting trypanotolerant cattle from southern Gabon. The study was conducted on 224 trypanotolerant cattle from three regions located in southern Gabon, using ITS1 primer-based PCR. Seventy-two (32%) N'dama cattle were found polymerase chain reaction (PCR) positive with trypanosomes. The overall prevalence of trypanosomosis was 57% (63/110), 4% (4/100), and 36% (5/14) in the Gala section of the Nyanga ranch, the Miyama ranch, and Ossiele, respectively. Trypanosoma congolense and Trypanosoma vivax were identified. In Gala section and Ossiele, T. congolense and T. vivax were found. In the Miyama ranch, only T. vivax was identified. Mixed infections were also found. The forest (9%) and savannah (63%) subgroups of T. congolense were identified. The presence of the two subgroups was detected in 16 out of 56 cattle (29%). T. congolense and T. vivax would appear to be the main agents responsible for bovine trypanosomosis in southern Gabon. Although trypanotolerant, N'dama cattle may serve as a reservoir, and this should be further studied. On the other hand, these trypanotolerant cattle can be reared in such tsetse infested areas, which gives them an advantage compared to other trypanosensitive breeds, and this shows that they represent a key factor in biodiversity which has to be promoted.

Bat flies (Diptera: Nycteribiidae and Streblidae) infesting cave-dwelling bats in Gabon: diversity, dynamics and potential role in Polychromophilus melanipherus transmission.

BACKGROUND: Evidence of haemosporidian infections in bats and bat flies has motivated a growing interest in characterizing their transmission cycles. In Gabon (Central Africa), many caves house massive colonies of bats that are known hosts of Polychromophilus Dionisi parasites, presumably transmitted by blood-sucking bat flies. However, the role of bat flies in bat malaria transmission remains under-documented. METHODS: An entomological survey was carried out in four caves in Gabon to investigate bat fly diversity, infestation rates and host preferences and to determine their role in Polychromophilus parasite transmission. Bat flies were sampled for 2-4 consecutive nights each month from February to April 2011 (Faucon and Zadie caves) and from May 2012 to April 2013 (Kessipoughou and Djibilong caves). Bat flies isolated from the fur of each captured bat were morphologically identified and screened for infection by haemosporidian parasites using primers targeting the mitochondrial cytochrome b gene. RESULTS: Among the 1,154 bats captured and identified as Miniopterus inflatus Thomas (n = 354), Hipposideros caffer Sundevall complex (n = 285), Hipposideros gigas Wagner (n = 317), Rousettus aegyptiacus Geoffroy (n = 157, and Coleura afra Peters (n = 41), 439 (38.0 %) were infested by bat flies. The 1,063 bat flies recovered from bats belonged to five taxa: Nycteribia schmidlii scotti Falcoz, Eucampsipoda africana Theodor, Penicillidia fulvida Bigot, Brachytarsina allaudi Falcoz and Raymondia huberi Frauenfeld group. The mean infestation rate varied significantly according to the bat species (ANOVA, F (4,75) = 13.15, P < 0.001) and a strong association effect between bat fly species and host bat species was observed. Polychromophilus melanipherus Dionisi was mainly detected in N. s. scotti and P. fulvida and less frequently in E. africana, R. huberi group and B. allaudi bat flies. These results suggest that N. s. scotti and P. fulvida could potentially be involved in P. melanipherus transmission among cave-dwelling bats. Sequence analysis revealed eight haplotypes of P. melanipherus. CONCLUSIONS: This work represents the first documented record of the cave-dwelling bat fly fauna in Gabon and significantly contributes to our understanding of bat fly host-feeding behavior and their respective roles in Polychromophilus transmission.

[The Emergence of Ebola virus in humans: a long process not yet fully understood]. / L'Émergence du virus EBOLA chez l'homme: un long processus pas totalement élucidé.

Since 1976 Ebola virus regularly has caused small deadly outbreaks in Central Africa, usually controlled in a few months. For the first time, an Ebola epidemic of exceptional magnitude dramatically engulfed several countries in West Africa since December 2013. Major failures of implementing measures to prevent human-to-human transmissions are the main cause of this large-scale Ebola outbreak. After about one-week incubation period, the Ebola virus disease is characterized by a sudden onset of high fever leading to multiple hemorrhages and to widespread organ failure. Several bat species constitute the main reservoirs of Ebola viruses. Human contamination would occur either directly from bats, widely consumed by the local populations, or through animal species susceptible to Ebola infection, such as chimpanzees and gorillas. Alongside this "natural cycle", an "epidemic cycle" involving domestic animals living in villages such as dogs or pigs, is seriously suggested. Thus, according to the diversity of concerned animals and their clinical infectionform, modalities of human contamination can be multiple and are still largely unknown. In this context, all efforts that could be made to unravel the mystery of the Ebola virus emergence in humans and clarify modalities of the virus transmission, would allow for predicting or for anticipating the future occurrence of epidemics. This review aims to provide an exhaustive inventory of the Ebola ecology to highlight events governing the virus transmission to humans that still remain unsolved.

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.

Cutaneous manifestations of filovirus infections.

Ebolavirus and Marburgvirus, two filoviruses belonging to the Filoviridae family, are among the most virulent pathogens for humans and non-human primates, causing outbreaks of fulminant hemorrhagic fever (HF) in Central African countries with case fatality rates of up to 90%. Fruit bats are the likely reservoir, and human infection occurs through contact with bats or infected large-animal carcasses or by person-to-person contact (through body fluids, medical care, and burial practices). Schematically, clinical manifestations occur in three successive phases and include general, gastrointestinal, and mucocutaneous disorders. Death usually results from hemorrhagic complications. Cutaneous manifestations rarely make a major contribution to disease severity but can assist with the diagnosis. Rash, the main cutaneous disorder, is nonspecific and cannot guide the differential diagnosis. Immunohistochemical examination of skin biopsy or necropsy specimens can confirm the diagnosis.

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.

Clinical forms of chikungunya in Gabon, 2010.

BACKGROUND: Chikungunya virus (CHIKV) has caused multiple outbreaks in tropical and temperate areas worldwide, but the clinical and biological features of this disease are poorly described, particularly in Africa. We report a prospective study of clinical and biological features during an outbreak that occurred in Franceville, Gabon in 2010. METHODOLOGY/PRINCIPAL FINDINGS: We collected, in suspect cases (individuals presenting with at least one of the following symptoms or signs: fever, arthralgias, myalgias, headaches, rash, fatigue, nausea, vomiting, diarrhea, bleeding, or jaundice), blood samples, demographic and clinical characteristics and outcome. Hematological and biochemical tests, blood smears for malaria parasites and quantitative PCR for CHIKV then dengue virus were performed. CHIKV+ patients with concomitant malaria and/or dengue were excluded from the study. From May to July 2010, data on 270 laboratory-confirmed CHIK patients were recorded. Fever and arthralgias were reported by respectively 85% and 90% of patients, while myalgias, rash and hemorrhage were noted in 73%, 42% and 2% of patients. The patients were grouped into 4 clinical categories depending on the existence of fever and/or joint pain. On this basis, mixed forms accounted for 78.5% of cases, arthralgic forms 12.6%, febrile forms 6.7% and unusual forms (without fever and arthralgias) 2.2%. No cases of organ failure or death were reported. Elevated liver enzyme and creatinine levels, anemia and lymphocytopenia were the predominant biological abnormalities, and lymphocytopenia was more severe in patients with high viral loads (p = 0.01). CONCLUSIONS/SIGNIFICANCE: During CHIK epidemics, some patients may not have classical symptoms. The existence of unusual forms and the absence of severe forms of CHIK call for surveillance to detect any change in pathogenicity.

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.

Epidemiology of concomitant infection due to Loa loa and Mansonella perstans in Gabon.

BACKGROUND: The filarial parasites Loa loa and Mansonnella perstans are endemic in the central and western African forest block. Loa loa is pathogenic and represents a major obstacle to the control of co-endemic filariae because its treatment can cause fatal complications such as encephalitis. METHODOLOGY/PRINCIPAL FINDINGS: 4392 individuals aged over 15 years were studied both by direct examination and a concentration technique. The overall prevalence rates were 22.4% for Loa loa microfilaremia, 10.2% for M. perstans microfilaremia, and 3.2% for mixed infection. The prevalence of both filariae was higher in the forest ecosystem than in savannah and lakeland (p<0.0001). The intensity of microfilariae (mf) was also higher in the forest ecosystem for both parasites. The prevalence and intensity of microfilaria were both influenced by age and gender. Correlations were found between the prevalence and intensity of Loa loa microfilariae (r = 0.215 p = 0.036), and between the prevalence of Loa loa and the prevalence of individuals with microfilaria >8000 mf/ml (r = 0.624; p<0.0001) and microfilariae >30 000 mf/ml (r = 0.319, p = 0.002). In contrast, the prevalence of pruritis and Calabar swellings correlated negatively with the prevalence of Loa loa microfilaria (r = -0.219, p = 0.032; r = -0.220; p = 0.031, respectively). Pruritis, Calabar swellings and eye worm were not associated with L. loa mf intensity (r = -0.144, p = 0.162; r-0.061, p = 0.558; and r = 0.051, p = 0.624, respectively), or with the prevalence or intensity of M. perstans microfilariae. CONCLUSIONS/SIGNIFICANCE: This map of the distribution of filariae in Gabon should prove helpful for control programs. Our findings confirm the spatial uniformity of the relationship between parasitological indices. Clinical manifestations point to a relationship between filariae and allergy.

Detection of Ebola virus in oral fluid specimens during outbreaks of Ebola virus hemorrhagic fever in the Republic of Congo.

BACKGROUND: Patients who have refused to provide blood samples has meant that there have been significant delays in confirming outbreaks of Ebola virus hemorrhagic fever (EVHF). During the 2 EVHF outbreaks in the Republic of Congo in 2003, we assessed the use of oral fluid specimens versus serum samples for laboratory confirmation of cases of EVHF. METHODS: Serum and oral fluid specimens were obtained from 24 patients with suspected Ebola and 10 healthy control subjects. Specimens were analyzed for immunoglobulin G antibodies by enzyme-linked immunosorbent assay (ELISA) and for Ebola virus by antigen detection ELISA and reverse-transcriptase polymerase chain reaction (RT-PCR). Oral fluid specimens were collected with a commercially available collection device. RESULTS: We failed to detect antibodies against Ebola in the oral fluid specimens obtained from patients whose serum samples were seropositive. All patients with positive serum RT-PCR results also had positive results for their oral fluid specimens. CONCLUSIONS: This study demonstrates the usefulness of oral fluid samples for the investigation of Ebola outbreaks, but further development in antibodies and antigen detection in oral fluid specimens is needed before these samples are used for filovirus surveillance activities in Africa.

The natural history of Ebola virus in Africa.

Several countries spanning the equatorial forest regions of Africa have had outbreaks of Ebola hemorrhagic fever over the last three decades. This article is an overview of the many published investigations of how Ebola virus circulates in its natural environment, focusing on the viral reservoir, susceptible animal species, environmental conditions favoring inter-species transmission, and how the infection is transmitted to humans. Major breakthroughs have been made in recent years but many outstanding questions must be dealt with if we are to prevent human outbreaks by interfering with the viral life cycle.