Evidence for circulation of Rift Valley fever virus in wildlife and domestic animals in a forest environment in Gabon, Central Africa.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by the Rift Valley fever virus (RVFV) that can infect domestic and wild animals. Although the RVFV transmission cycle has been well documented across Africa in savanna ecosystems, little is known about its transmission in tropical rainforest settings, particularly in Central Africa. We therefore conducted a survey in northeastern Gabon to assess RVFV circulation among wild and domestic animals. Among 163 wildlife samples tested using RVFV-specific RT-qPCR, four ruminants belonging to subfamily Cephalophinae were detected positive. The phylogenetic analysis revealed that the four RVFV sequences clustered together with a virus isolated in Namibia within the well-structured Egyptian clade. A cross-sectional survey conducted on sheep, goats and dogs living in villages within the same area determined the IgG RVFV-specific antibody prevalence using cELISA. Out of the 306 small ruminants tested (214 goats, 92 sheep), an overall antibody prevalence of 15.4% (95% CI [11.5-19.9]) was observed with a higher rate in goats than in sheep (20.1% versus 3.3%). RVFV-specific antibodies were detected in a single dog out of the 26 tested. Neither age, sex of domestic animals nor season was found to be significant risk factors of RVFV occurrence. Our findings highlight sylvatic circulation of RVFV for the first time in Gabon. These results stress the need to develop adequate surveillance plan measures to better control the public health threat of RVFV.

A Sanger approach based on overlapping fragments to screen SARS-CoV-2 variants.

Since the beginning of the COVID-19 pandemic, the SARS-CoV-2 virus has undergone various genetic mutations which have led to the emergence of variants. The World Health Organization (WHO) defines Variants of Concern (VOCs) and Variants of Interest (VOIs) according to several criteria. These include significant changes in the transmissibility and pathogenicity of the virus characterized by mutations in the spike gene coding the spike glycoprotein. In this study, we designed ten Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) assays in order to identify mutations of SARS-CoV-2 in overlapping fragments. Each assay contained mutations on the fragments sequenced by a Sanger method. The genomic analysis of the fragments allowed to identify the variant according to the position of the mutations. The assembly of the 10 fragments refined the analysis, highlighting all the mutations present in the S gene. Finally, a comparison of methods using a Next-Generation Sequencing (NGS) approches for samples enabled the method to be validated. By this method we have highlighted a characteristic mutation of the lineage B of SARS-CoV-2. We showed the circulation of SARS-CoV-2 belonging to lineage A and B in the beginning of the pandemic in Gabon. We have identified the Alpha, Delta and Omicron variants. This method would allow laboratories with limited financial means or without NGS instrument to obtain sequences of the S gene. This method wase very effective to highlight the circulation of variants, in particular VOCs and VOIs, in this developing country, Gabon, during the COVID-19 pandemic.

Burden of hepatitis B virus infection in pregnant women attending antenatal clinics in the southern Gabon.

Objectives: Hepatitis B virus (HBV) infection remains a public health threat in middle- and low-income countries, where mother-to-child transmission plays an important role. The aim of this study was to assess the burden of this infection among pregnant women in southern Gabon and the risk of vertical transmission. Methods: The study was a prospective investigation conducted from April 2021 to January 2022. Study participants were pregnant women aged 18 and over attending antenatal clinics in Franceville. Blood samples were collected to test for HBV surface antigen, anti-hepatitis B core, hepatitis B e antigen, and anti-hepatitis B e markers and to assess HBV infection. Results: We recruited 901 women with a median age of 26 years (interquartile range: 21-32). Overall prevalence of infection was 3.9% (confidence interval: 2.7-5.4%). 418/901 or 46.4% were anti-hepatitis B core positive. Among HBV surface antigen-positive women, 1/35 were hepatitis B e antigen-positive with a viral load >200,000 IU/ml. Over 64% of participants had no information about HBV infection, and none knew that the virus could be transmitted from mother to child. Conclusions: This study reveals a low HBV prevalence in pregnant women in Gabon and a low risk of vertical transmission of the virus. However, the rate of exposure of the population to the virus remains high and calls for improving actions and interventions for potential elimination goals.

Molecular Identification of Enteric Viruses in Domestic Animals in Northeastern Gabon, Central Africa.

Astroviruses (AstVs), enteroviruses (EVs), and caliciviruses (CaVs) infect several vertebrate taxa. Transmitted through the fecal-oral route, these enteric viruses are highly resistant and can survive in the environment, thereby increasing their zoonotic potential. Here, we screened for AstVs, EVs, and CaVs to investigate the role of domestic animals in the emergence of zoonoses, because they are situated at the human/wildlife interface, particularly in rural forested areas in Central Africa. Rectal swabs were obtained from 123 goats, 41 sheep, and 76 dogs in 10 villages located in northeastern Gabon. Extracted RNA reverse-transcribed into cDNA was used to detect AstVs, EVs, and CaVs by amplification of the RNA-dependent RNA polymerase (RdRp), or capsid protein (VP1) gene using PCR. A total of 23 samples tested positive, including 17 goats for AstVs, 2 goats, 2 sheep, 1 dog for EVs, and 1 dog for CaVs. Phylogenetic analyses revealed that AstV RdRp sequences clustered with sheep-, goat-, or bovine-related AstVs. In addition, one goat and two sheep VP1 sequences clustered with caprine/ovine-related Evs within the Enterovirus G species, and the CaV was a canine vesivirus. However, human-pathogenic Evs, EV-B80 and EV-C99, were detected in goats and dogs, raising questions on the maintenance of viruses able to infect humans.

Epidemiology of first cases of SARS-CoV-2 infection, from March to April 2020, in Gabon.

Background After the first cases of coronaviruses disease 2019 (COVID-19) in China in January 2020, we conducted an epidemiological surveillance of COVID-19 in Gabon. Methods We led molecular investigations on nasopharyngeal and oropharyngeal samples from the 1161 first suspected cases of COVID-19. We diagnosed the first case of COVID-19 on March, 12 2020. Results Among those suspected cases, 83 were confirmed cases. There was no significant difference in prevalence of SARS-CoV-2 between age groups (p=0.14). 73% were asymptomatic. The viral loads were significantly higher in the nasopharyngeal samples than in the oropharyngeal samples (p=0.03). There was no significant difference in viral loads between age groups (p=0.9895) and no correlation between clinical symptoms and viral loads (p=0.06042). A phylogenetic analysis performed with five sequences of the spike S gene showed that two sequences had the D614G mutation. Conclusion In conclusion, this study provides the first molecular data from Gabon concerning the COVID-19 pandemic. The data showed that most of the infected people were asymptomatic. The viral load was higher in the nasopharyngeal samples. The S gene analyzed suggested both introduction of the D614 and G614 variant in Gabon.

Screening and Whole Genome Sequencing of SARS-CoV-2 Circulating During the First Three Waves of the COVID-19 Pandemic in Libreville and the Haut-Ogooué Province in Gabon.

Since the onset of the COVID-19 pandemic, the SARS-CoV-2 viral dynamics in Africa have been less documented than on other continents. In Gabon, a Central African country, a total number of 37,511 cases of COVID-19 and 281 deaths have been reported as of December 8, 2021. After the first COVID-19 case was reported on March 12, 2020, in the capital Libreville, the country experienced two successive waves. The first one, occurred in March 2020 to August 2020, and the second one in January 2021 to May 2021. The third wave began in September 2021 and ended in November 2021. In order to reduce the data gap regarding the dynamics of SARS-CoV-2 in Central Africa, we performed a retrospective genotyping study using 1,006 samples collected from COVID-19 patients in Gabon from 2020 to 2021. Using SARS-CoV-2 variant screening by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and whole genome sequencing (WGS), we genotyped 809 SARS-CoV-2 samples through qRT-PCR and identified to generated 291 new genomes. It allowed us to describe specific mutations and changes in the SARS-CoV-2 variants in Gabon. The qRT-PCR screening of 809 positive samples from March 2020 to September 2021 showed that 119 SARS-CoV-2 samples (14.7%) were classified as VOC Alpha (Pangolin lineage B.1.1.7), one (0.1%) was a VOC Beta (B.1.351), and 198 (24.5 %) were VOC Delta (B.1.617.2), while 491 samples (60.7%) remained negative for the variants sought. The B1.1 variant was predominant during the first wave while the VOC Alpha dominated the second wave. The B1.617.2 Delta variant is currently the dominant variant of the third wave. Similarly, the analysis of the 291 genome sequences indicated that the dominant variant during the first wave was lineage B.1.1, while the dominant variants of the second wave were lineages B.1.1.7 (50.6%) and B.1.1.318 (36.4%). The third wave started with the circulation of the Delta variant (B.1.617). Finally, we compared these results to the SARS-CoV-2 sequences reported in other African, European, American and Asian countries. Sequences of Gabonese SARS-CoV-2 strains presented the highest similarities with those of France, Belgium and neighboring countries of Central Africa, as well as West Africa.

Molecular analyses of human rabies virus associated with encephalitis in two children in Gabon.

Rabies is a zoonotic neurological life-threatening neglected tropical disease present worldwide, and Gabon is listed as an endemic country. However, despite strong clinical suspicion in humans and molecular confirmation of rabies virus (RABV) infections in dogs for several decades, no molecularly confirmed human case in Gabon has ever been reported. In this study, we describe two cases of human rabies and provide the first molecular diagnostic report on suspected human rabies cases in Gabon. Our results showed that the RABVs isolated from the patients are closely related to other RABV strains belonging to the African 1A subclade in the Cosmopolitan lineage isolated more than 20 years ago from Gabonese dog brains, suggesting that only this species circulates in the country. Because both patients had a history of dog bites a few weeks before symptom onset and the main causative agent of human rabies worldwide is dog-associated RABV, we conclude that dogs are likely to be the source of human infection in this study.

Biochemical and hematological factors associated with COVID-19 severity among Gabonese patients: A retrospective cohort study.

The COVID-19 disease presents a large range of clinical manifestations and includes asymptomatic, mild, and severe cases. The level of severity is related to parameters associated with immunity, genetics, and biochemistry. Africa shows one of the lowest COVID-19 fatality rates but very few data on the biochemical markers of COVID-19 in patients and the factors associated with disease severity are available for the continent. In Gabon, the COVID-19 fatality rate is only 0.63% but almost no data on biomarkers in COVID-19 patients have been published. Both the number of COVID-19 cases and the mortality rate reported in Africa in general, and in Gabon in particular, are lower than in non-African countries. As such, understanding the factors associated with disease severity in Gabonese patients is a crucial step to better understand the disease in the African context and prepare for future COVID-19 waves and other epidemics of emerging diseases. Here, we compared biochemical and hematological markers among 753 Gabonese COVID-19 patients with asymptomatic (184/753), mild/moderate (420/753), and severe/critical (149/753) forms of the disease using an Analysis of Variance (ANOVA) or a Kruskal-Wallis (KW) test. We modeled these parameters together with comorbidities, age, and sex to predict factors associated with disease severity by using a "binomial generalized linear model" utilizing the "package" stats of R software version 4.0.2. Our results showed that almost all the biochemical and hematological parameters (except creatinine, phosphorus, D-dimers, platelets, and monocytes) varied according to disease severity. However, age and the dysfunction of organs like the kidney, liver, and lung together with the decrease of electrolytes (chloride, potassium, and sodium) are the best predictors of disease severity in Gabonese patients.

First investigation of pathogenic bacteria, protozoa and viruses in rodents and shrews in context of forest-savannah-urban areas interface in the city of Franceville (Gabon).

Rodents are reservoirs of numerous zoonotic diseases caused by bacteria, protozoans, or viruses. In Gabon, the circulation and maintenance of rodent-borne zoonotic infectious agents are poorly studied and are often limited to one type of pathogen. Among the three existing studies on this topic, two are focused on a zoonotic virus, and the third is focused on rodent Plasmodium. In this study, we searched for a wide range of bacteria, protozoa and viruses in different organs of rodents from the town of Franceville in Gabon. Samples from one hundred and ninety-eight (198) small mammals captured, including two invasive rodent species, five native rodent species and 19 shrews belonging to the Soricidae family, were screened. The investigated pathogens were bacteria from the Rickettsiaceae and Anaplasmataceae families, Mycoplasma spp., Bartonella spp., Borrelia spp., Orientia spp., Occidentia spp., Leptospira spp., Streptobacillus moniliformis, Coxiella burnetii, and Yersinia pestis; parasites from class Kinetoplastida spp. (Leishmania spp., Trypanosoma spp.), Piroplasmidae spp., and Toxoplasma gondii; and viruses from Paramyxoviridae, Hantaviridae, Flaviviridae and Mammarenavirus spp. We identified the following pathogenic bacteria: Anaplasma spp. (8.1%; 16/198), Bartonella spp. (6.6%; 13/198), Coxiella spp. (5.1%; 10/198) and Leptospira spp. (3.5%; 7/198); and protozoans: Piroplasma sp. (1%; 2/198), Toxoplasma gondii (0.5%; 1/198), and Trypanosoma sp. (7%; 14/198). None of the targeted viral genes were detected. These pathogens were found in Gabonese rodents, mainly Lophuromys sp., Lemniscomys striatus and Praomys sp. We also identified new genotypes: Candidatus Bartonella gabonensis and Uncultured Anaplasma spp. This study shows that rodents in Gabon harbor some human pathogenic bacteria and protozoans. It is necessary to determine whether the identified microorganisms are capable of undergoing zoonotic transmission from rodents to humans and if they may be responsible for human cases of febrile disease of unknown etiology in Gabon.

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.

Comparison of apoptosis in human primary pulmonary endothelial cells and a brain microvascular endothelial cell line co-cultured with Plasmodium falciparum field isolates.

BACKGROUND: Plasmodium falciparum infection can progress unpredictably to severe forms including respiratory distress and cerebral malaria. The mechanisms underlying the variable natural course of malaria remain elusive. METHODS: The cerebral microvascular endothelial cells-D3 and lung endothelial cells both from human were cultured separately and challenged with P. falciparum field isolates taken directly from malaria patients or 3D7 strain (in vitro maintained culture). The capacity of these P. falciparum isolates to induce endothelial cell apoptosis via cytoadherence or not was then assessed. RESULTS: Overall, 27 P. falciparum isolates were collected from patients with uncomplicated malaria (n = 25) or severe malaria (n = 2). About half the isolates (n = 17) were able to bind brain endothelial cells (12 isolates, 44%) or lung endothelial cells (17 isolates, 63%) or both (12 isolates, 44%). Sixteen (59%) of the 27 isolates were apoptogenic for brain and/or lung endothelial cells. The apoptosis stimulus could be cytoadherence, direct cell-cell contact without cytoadherence, or diffusible soluble factors. While some of the apoptogenic isolates used two stimuli (direct contact with or without cytoadherence, plus soluble factors) to induce apoptosis, others used only one. Among the 16 apoptogenic isolates, eight specifically targeted brain endothelial cells, one lung endothelial cells, and seven both. CONCLUSION: These results indicate that the brain microvascular cell line was more susceptible to apoptosis triggered by P. falciparum than the primary pulmonary endothelial cells and may have relevance to host-parasite interaction.

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.

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.

Molecular characterization of Orf virus in goats in Gabon, Central Africa.

BACKGROUND: Orf or contagious ecthyma is a zoonotic viral infection with a potential serious health threat for the small ruminants industry as well as humans. It is currently emerging in new territories. RESULTS: Eight suspected clinical cases of pustular dermatitis in goats occurred in the rural area of Tebe, in south-eastern Gabon, in January 2013. The orf virus (ORFV) was detected by high-throughput sequencing on sera, buccal swabs and scab pool samples. It was confirmed in six out of eight sick goats by using specific PCR targeting the major envelope protein (B2L) and the orf virus interferon resistance (VIR) genes. Phylogenetic analysis revealed that the Gabonese strain and South Korean strains evolved from a common ancestor, suggesting an Asian origin of the ORFV' Gabonese strain. CONCLUSIONS: This study provides the molecular detection of the ORFV strain involved in the cases of pustular dermatitis in goats and highlights its circulation in Gabon.

Evidence of lymphocytic choriomeningitis virus (LCMV) in domestic mice in Gabon: risk of emergence of LCMV encephalitis in Central Africa.

Lymphocytic choriomeningitis virus (LCMV) can cause acute fatal disease on all continents but was never detected in Africa. We report the first detection of LCMV RNA in a common European house mouse (Mus musculus domesticus) in Africa. Phylogenetic analyses show a close relationship with North American strains. These findings suggest that there is a risk of the appearance of LCMV acute encephalitis cases. This is a perfect example of virus dissemination by its natural host that may have dramatic public health consequences.

P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC) activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF), PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.

Identification of an unclassified paramyxovirus in Coleura afra: a potential case of host specificity.

Bats are known to harbor multiple paramyxoviruses. Despite the creation of two new genera, Aquaparamyxovirus and Ferlavirus, to accommodate this increasing diversity, several recently isolated or characterized viruses remain unclassified beyond the subfamily level. In the present study, among 985 bats belonging to 6 species sampled in the Belinga caves of Gabon, RNA of an unclassified paramyxovirus (Belinga bat virus, BelPV) was discovered in 14 African sheath-tailed bats (Coleura afra), one of which exhibited several hemorrhagic lesions at necropsy, and viral sequence was obtained in two animals. Phylogenetically, BelPV is related to J virus and Beilong virus (BeiPV), two other unclassified paramyxoviruses isolated from rodents. In the diseased BelPV-infected C. afra individual, high viral load was detected in the heart, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by J virus. BelPV was not detected in other tested bat species sharing the same roosting sites and living in very close proximity with C. afra in the two caves sampled, suggesting that this virus may be host-specific for C. afra. The mode of transmission of this paramyxovirus in bat populations remains to be discovered.

Atorvastatin prevents Plasmodium falciparum cytoadherence and endothelial damage.

BACKGROUND: The adhesion of Plasmodium falciparum parasitized red blood cell (PRBC) to human endothelial cells (EC) induces inflammatory processes, coagulation cascades, oxidative stress and apoptosis. These pathological processes are suspected to be responsible for the blood-brain-barrier and other organs' endothelial dysfunctions observed in fatal cases of malaria. Atorvastatin, a drug that belongs to the lowering cholesterol molecule family of statins, has been shown to ameliorate endothelial functions and is widely used in patients with cardiovascular disorders. METHODS: The effect of this compound on PRBC induced endothelial impairments was assessed using endothelial co-culture models. RESULTS: Atorvastatin pre-treatment of EC was found to reduce the expression of adhesion molecules and P. falciparum cytoadherence, to protect cells against PRBC-induced apoptosis and to enhance endothelial monolayer integrity during co-incubation with parasites. CONCLUSIONS: These results might suggest a potential interest use of atorvastatin as a protective treatment to interfere with the pathophysiological cascades leading to severe malaria.