Resurgence of Ebola virus in 2021 in Guinea suggests a new paradigm for outbreaks.

Seven years after the declaration of the first epidemic of Ebola virus disease in Guinea, the country faced a new outbreak-between 14 February and 19 June 2021-near the epicentre of the previous epidemic1,2. Here we use next-generation sequencing to generate complete or near-complete genomes of Zaire ebolavirus from samples obtained from 12 different patients. These genomes form a well-supported phylogenetic cluster with genomes from the previous outbreak, which indicates that the new outbreak was not the result of a new spillover event from an animal reservoir. The 2021 lineage shows considerably lower divergence than would be expected during sustained human-to-human transmission, which suggests a persistent infection with reduced replication or a period of latency. The resurgence of Zaire ebolavirus from humans five years after the end of the previous outbreak of Ebola virus disease reinforces the need for long-term medical and social care for patients who survive the disease, to reduce the risk of re-emergence and to prevent further stigmatization.

High seroreactivities to orthoebolaviruses in rural Cameroon: a case-control study on non-human primate bites and a cross-sectional survey in rural population.

BACKGROUND: Ebola (EBOV) and Sudan (SUDV) orthoebolaviruses are responsible for lethal haemorrhagic fever outbreaks in humans in Central and West Africa, and in apes that can be at the source of human outbreaks for EBOV. METHODS: To assess the risk of exposure to orthoebolaviruses through contact with non-human primates (NHP), we tested the presence of antibodies against different viral proteins with a microsphere-based multiplex immunoassay in a case-control study on bites from NHPs in forest areas from Cameroon (n=795), and in cross-sectional surveys from other rural populations (n=622) of the same country. RESULTS: Seroreactivities against at least two viral proteins were detected in 13% and 12% of the samples for EBOV and SUDV, respectively. Probability of seroreactivity was not associated with history of NHP bites, but was three times higher in Pygmies compared to Bantus. Although no neutralizing antibodies to EBOV and SUDV were detected in a selected series of highly reactive samples, avidity results indicate strong affinity to SUDV antigens. CONCLUSION: The detection of high level of seroreactivities against orthoebolaviruses in rural Cameroon where no outbreaks have been reported, raises the possibilities of silent circulation of orthoebolavirus, or of other not yet documented filoviruses, in these forested regions.

Co-Circulating Monkeypox and Swinepox Viruses, Democratic Republic of the Congo, 2022.

In September 2022, deaths of pigs manifesting pox-like lesions caused by swinepox virus were reported in Tshuapa Province, Democratic Republic of the Congo. Two human mpox cases were found concurrently in the surrounding community. Specific diagnostics and robust sequencing are needed to characterize multiple poxviruses and prevent potential poxvirus transmission.

Presumed Transmission of 2 Distinct Monkeypox Virus Variants from Central African Republic to Democratic Republic of the Congo.

We linked 4 mpox cases in South Ubangi, Democratic Republic of the Congo, to transboundary transmission from Central African Republic. Viral genome sequencing demonstrated that the monkeypox virus sequences belonged to distinct clusters of subclade Ia. This finding demonstrates the borderless nature of mpox and highlights the need for vigilant regional surveillance.

Use of strips of rapid diagnostic tests as a source of ribonucleic acid for genomic surveillance of viruses: an example of SARS-CoV-2.

BACKGROUND: This study aimed to demonstrate that the genomic material of SARS-CoV-2 can be isolated from strips of COVID-19 rapid diagnostic test cassettes. METHOD: It was a prospective cross-sectional study involving patients admitted to treatment centers and sampling sites in the city of Conakry, Guinea. A total of 121 patients were double sampled, and 9 more patients were tested only for RDT. PCR was conducted according to the protocol of the RunMei kit. Sequencing was performed by using the illumina COVIDSeq protocol. Nine COVID-19 RDTs without nasopharyngeal swabs were in addition tested. RESULT: Among the 130 COVID-19 RDTs, forty-seven were macroscopically positive, whereas seventy-two were positive according to PCR using RDT strip, while among the 121 VTM swabs, sixty-four were positive. Among eighty-three negative COVID-19 RDTs, twenty-seven were positive by PCR using RDT strip with a geometric mean Ct value of 32.49 cycles. Compared to those of PCR using VTM, the sensitivity and specificity of PCR using RDT strip were estimated to be 100% and 85.96%, respectively, with 93.39% test accuracy. Among the fifteen COVID-19 RDT extracts eligible for sequencing, eleven had sequences identical to those obtained via the standard method, with coverage between 75 and 99.6%. CONCLUSION: These results show that COVID-19 RDTs can be used as biological material for the genomic surveillance of SARS-CoV-2.

CD4 receptor diversity represents an ancient protection mechanism against primate lentiviruses.

Infection with human and simian immunodeficiency viruses (HIV/SIV) requires binding of the viral envelope glycoprotein (Env) to the host protein CD4 on the surface of immune cells. Although invariant in humans, the Env binding domain of the chimpanzee CD4 is highly polymorphic, with nine coding variants circulating in wild populations. Here, we show that within-species CD4 diversity is not unique to chimpanzees but found in many African primate species. Characterizing the outermost (D1) domain of the CD4 protein in over 500 monkeys and apes, we found polymorphic residues in 24 of 29 primate species, with as many as 11 different coding variants identified within a single species. D1 domain amino acid replacements affected SIV Env-mediated cell entry in a single-round infection assay, restricting infection in a strain- and allele-specific fashion. Several identical CD4 polymorphisms, including the addition of N-linked glycosylation sites, were found in primate species from different genera, providing striking examples of parallel evolution. Moreover, seven different guenons (Cercopithecus spp.) shared multiple distinct D1 domain variants, pointing to long-term trans-specific polymorphism. These data indicate that the HIV/SIV Env binding region of the primate CD4 protein is highly variable, both within and between species, and suggest that this diversity has been maintained by balancing selection for millions of years, at least in part to confer protection against primate lentiviruses. Although long-term SIV-infected species have evolved specific mechanisms to avoid disease progression, primate lentiviruses are intrinsically pathogenic and have left their mark on the host genome.

Interest of seroprevalence surveys for the epidemiological surveillance of the SARS-CoV-2 pandemic in African populations: Insights from the ARIACOV project in Benin.

BACKGROUND: Many SARS-CoV-2 seroprevalence surveys since the end of 2020 have disqualified the first misconception that Africa had been spared by the pandemic. Through the analysis of three SARS-CoV-2 seroprevalence surveys carried out in Benin as part of the ARIACOV project, we argue that the integration of epidemiological serosurveillance of the SARS-CoV-2 infection in the national surveillance packages would be of great use to refine the understanding of the COVID-19 pandemic in Africa. METHODS: We carried out three repeated cross-sectional surveys in Benin: two in Cotonou, the economic capital in March and May 2021, and one in Natitingou, a semi-rural city in the north of the country in August 2021. Total and weighted-by-age-group seroprevalences were estimated and the risk factors for SARS-CoV-2 infection were assessed by multivariate logistic regression. RESULTS: In Cotonou, a slight increase in overall age-standardised SARS-CoV-2 seroprevalence from 29.77% (95% CI: 23.12%-37.41%) at the first survey to 34.86% (95% CI: 31.57%-38.30%) at the second survey was observed. In Natitingou, the globally adjusted seroprevalence was 33.34% (95% CI: 27.75%-39.44%). A trend of high risk for SARS-CoV 2 seropositivity was observed in adults over 40 versus the young (less than 18 years old) during the first survey in Cotonou but no longer in the second survey. CONCLUSIONS: Our results show that, however, rapid organisation of preventive measures aimed at breaking the chains of transmission, they were ultimately unable to prevent a wide spread of the virus in the population. Routine serological surveillance on strategic sentinel sites and/or populations could constitute a cost-effective compromise to better anticipate the onset of new waves and define public health strategies.

Added Value of an Anti-Ebola Serology for the Management of Clinically Suspected Ebola Virus Disease Patients Discharged as Negative in an Epidemic Context.

BACKGROUND: Survivors from Ebola virus disease (EVD) may be at the origin of EVD resurgence. METHODS: Simultaneous reactivity to at least 2 Ebola virus or Zaire ebolavirus (EBOV) antigens was detected in 11 of 488 (2.3%; 95% confidence interval [CI], 1.1-4.0) suspected EVD patients who were discharged as negative after 2 consecutive negative tests during the 10th Ebola outbreak in the Democratic Republic of the Congo. RESULTS: After extrapolating the total number of individuals discharged as negative during the entire outbreak, we estimated a total of 1314 additional missed Ebola cases. CONCLUSIONS: These findings emphasize the usefulness of an EBOV serology analysis and the importance of extending epidemic surveillance to clinically suspected cases who were discharged as negative.

High Prevalence of Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (Anti-SARS-CoV-2) Antibodies After the First Wave of Coronavirus Disease 2019 (COVID-19) in Kinshasa, Democratic Republic of the Congo: Results of a Cross-sectional Household-Based Survey.

BACKGROUND: In October 2020, after the first wave of coronavirus disease 2019 (COVID-19), only 8290 confirmed cases were reported in Kinshasa, Democratic Republic of the Congo, but the real prevalence remains unknown. To guide public health policies, we aimed to describe the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) antibodies in the general population in Kinshasa. METHODS: We conducted a cross-sectional, household-based serosurvey between 22 October 2020 and 8 November 2020. Participants were interviewed at home and tested for antibodies against SARS-CoV-2 spike and nucleocapsid proteins in a Luminex-based assay. A positive serology was defined as a sample that reacted with both SARS-CoV-2 proteins (100% sensitivity, 99.7% specificity). The overall weighted, age-standardized prevalence was estimated and the infection-to-case ratio was calculated to determine the proportion of undiagnosed SARS-CoV-2 infections. RESULTS: A total of 1233 participants from 292 households were included (mean age, 32.4 years; 764 [61.2%] women). The overall weighted, age-standardized SARS-CoV-2 seroprevalence was 16.6% (95% CI: 14.0-19.5%). The estimated infection-to-case ratio was 292:1. Prevalence was higher among participants ≥40 years than among those <18 years (21.2% vs 14.9%, respectively; P < .05). It was also higher in participants who reported hospitalization than among those who did not (29.8% vs 16.0%, respectively; P < .05). However, differences were not significant in the multivariate model (P = .1). CONCLUSIONS: The prevalence of SARS-CoV-2 is much higher than the number of COVID-19 cases reported. These results justify the organization of a sequential series of serosurveys by public health authorities to adapt response measures to the dynamics of the pandemic.

Rapid Increase of Community SARS-CoV-2 Seroprevalence during Second Wave of COVID-19, Yaoundé, Cameroon.

We conducted 2 independent population-based SARS-CoV-2 serosurveys in Yaoundé, Cameroon, during January 27-February 6 and April 24-May 19, 2021. Overall age-standardized SARS-CoV-2 IgG seroprevalence increased from 18.6% in the first survey to 51.3% in the second (p<0.001). This finding illustrates high community transmission during the second wave of COVID-19.

CD4 receptor diversity in chimpanzees protects against SIV infection.

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.

Evolutionary history of human Plasmodium vivax revealed by genome-wide analyses of related ape parasites.

Wild-living African apes are endemically infected with parasites that are closely related to human Plasmodium vivax, a leading cause of malaria outside Africa. This finding suggests that the origin of P. vivax was in Africa, even though the parasite is now rare in humans there. To elucidate the emergence of human P. vivax and its relationship to the ape parasites, we analyzed genome sequence data of P. vivax strains infecting six chimpanzees and one gorilla from Cameroon, Gabon, and Côte d'Ivoire. We found that ape and human parasites share nearly identical core genomes, differing by only 2% of coding sequences. However, compared with the ape parasites, human strains of P. vivax exhibit about 10-fold less diversity and have a relative excess of nonsynonymous nucleotide polymorphisms, with site-frequency spectra suggesting they are subject to greatly relaxed purifying selection. These data suggest that human P. vivax has undergone an extreme bottleneck, followed by rapid population expansion. Investigating potential host-specificity determinants, we found that ape P. vivax parasites encode intact orthologs of three reticulocyte-binding protein genes (rbp2d, rbp2e, and rbp3), which are pseudogenes in all human P. vivax strains. However, binding studies of recombinant RBP2e and RBP3 proteins to human, chimpanzee, and gorilla erythrocytes revealed no evidence of host-specific barriers to red blood cell invasion. These data suggest that, from an ancient stock of P. vivax parasites capable of infecting both humans and apes, a severely bottlenecked lineage emerged out of Africa and underwent rapid population growth as it spread globally.

Role of Wildlife in Emergence of Ebola Virus in Kaigbono (Likati), Democratic Republic of the Congo, 2017.

After the 2017 Ebola virus (EBOV) outbreak in Likati, a district in northern Democratic Republic of the Congo, we sampled small mammals from the location where the primary case-patient presumably acquired the infection. None tested positive for EBOV RNA or antibodies against EBOV, highlighting the ongoing challenge in detecting animal reservoirs for EBOV.

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

Extensive Serological Survey of Multiple African Nonhuman Primate Species Reveals Low Prevalence of Immunoglobulin G Antibodies to 4 Ebola Virus Species.

Bats are considered a reservoir species for Ebola viruses, but nonhuman primates (NHPs) have represented a source of infection in several outbreaks in humans. Here we report serological screening of blood or fecal samples from monkeys (n = 2322) and apes (n = 2327). Thirty-six NHP species from Cameroon, Democratic Republic of the Congo, and Ivory Coast were tested with a sensitive and specific Luminex-based assay for immunoglobulin G antibodies to 4 Ebola virus species. Using the simultaneous presence of antibodies to nucleoproteins and glycoproteins to define positivity, we showed that specific Ebola virus antibodies are not widespread among NHPs. Only 1 mustached monkey (Cercopithecus cephus) from Cameroon was positive for Sudan ebolavirus. These observations support that NHPs are most likely intermediate hosts for Ebola viruses. With the increasing frequency of Ebola outbreaks, it is crucial to identify the animal reservoir and understand the ecology of Ebola viruses to inform disease control.

Rapid Confirmation of the Zaire Ebola Virus in the Outbreak of the Equateur Province in the Democratic Republic of Congo: Implications for Public Health Interventions.

Ten days after the declaration of the Ebola outbreak in the Democratic Republic of Congo, rapid identification of the species Zaire Ebola virus using partial gene amplification and nanopore sequencing backed up the use of the recombinant vesicular stomatitis virus-Zaire Ebola virus vaccine in the recommended ring vaccination strategy.

Primate lentiviruses use at least three alternative strategies to suppress NF-κB-mediated immune activation.

Primate lentiviruses have evolved sophisticated strategies to suppress the immune response of their host species. For example, HIV-2 and most simian immunodeficiency viruses (SIVs) use their accessory protein Nef to prevent T cell activation and antiviral gene expression by downmodulating the T cell receptor CD3. This Nef function was lost in HIV-1 and other vpu-encoding viruses suggesting that the acquisition of Vpu-mediated NF-κB inhibition reduced the selection pressure for inhibition of T cell activation by Nef. To obtain further insights into the modulation of NF-κB activity by primate lentiviral accessory factors, we analyzed 32 Vpr proteins from a large panel of divergent primate lentiviruses. We found that those of SIVcol and SIVolc infecting Colobinae monkeys showed the highest efficacy in suppressing NF-κB activation. Vpr-mediated inhibition of NF-κB resulted in decreased IFNß promoter activity and suppressed type I IFN induction in virally infected primary cells. Interestingly, SIVcol and SIVolc differ from all other primate lentiviruses investigated by the lack of both, a vpu gene and efficient Nef-mediated downmodulation of CD3. Thus, primate lentiviruses have evolved at least three alternative strategies to inhibit NF-κB-dependent immune activation. Functional analyses showed that the inhibitory activity of SIVolc and SIVcol Vprs is independent of DCAF1 and the induction of cell cycle arrest. While both Vprs target the IKK complex or a factor further downstream in the NF-κB signaling cascade, only SIVolc Vpr stabilizes IκBα and inhibits p65 phosphorylation. Notably, only de-novo synthesized but not virion-associated Vpr suppressed the activation of NF-κB, thus enabling NF-κB-dependent initiation of viral gene transcription during early stages of the replication cycle, while minimizing antiviral gene expression at later stages. Our findings highlight the key role of NF-κB in antiviral immunity and demonstrate that primate lentiviruses follow distinct evolutionary paths to modulate NF-κB-dependent expression of viral and antiviral genes.

[Tropical forests, changes in land uses and emerging infectious hazards]. / Forêts tropicales, changements d'usage des sols et risques infectieux émergents.

Tropical forests have the greatest biodiversity in macroorganisms on the planet, and they are also the richest in myriads of microorganisms for which so little is known today. Over the last 50 years, many of these microbial forms, that are naturally embedded into wildlife or the environment, e.g. soil, water, have revealed to be more or less dangerous pathogens for people exposed to these new natural threats, i.e. emerging infectious diseases. Here, we discuss about the extraordinary diversity of microorganisms that are present in tropical rainforests. We first present the main global distribution patterns for microbial forms at the interface between tropical wildlife and human, and second we provide an epidemiological picture on how microbial transmission from wild animals or the environment to people operates in tropical areas through four case-studies. We examine the animal hosts or environment, and transmission mechanisms involved in spillover of zoonotic or environmentally-persistent microbes, and identify land-use changes through deforestation for the development of agriculture, and contacts with wildlife notably through bush meat hunting as major drivers that facilitate mixing of diverse animal hosts and their microbial communities with human during practices. With an increase of deforestation in the tropics and more contacts between wildlife and people, new emerging disease events with high epidemic and pandemic potential will happen, that should guide new health policies and strategies at the global scale.

Survey of Ebola Viruses in Frugivorous and Insectivorous Bats in Guinea, Cameroon, and the Democratic Republic of the Congo, 2015-2017.

To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015-2017 from countries in Africa that have had previous Ebola outbreaks (Guinea, the Democratic Republic of the Congo) or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from >12 frugivorous and 27 insectivorous species; 2-37 (0.05%-0.92%) bats were seropositive for Zaire and 0-30 (0%-0.75%) bats for Sudan Ebola viruses. We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.

Assessment of the gorilla gut virome in association with natural simian immunodeficiency virus infection.

BACKGROUND: Simian immunodeficiency viruses (SIVs) of chimpanzees and gorillas from Central Africa crossed the species barrier at least four times giving rise to human immunodeficiency virus type 1 (HIV-1) groups M, N, O and P. The paradigm of non-pathogenic lentiviral infections has been challenged by observations of naturally infected chimpanzees with SIVcpz associated with a negative impact on their life span and reproduction, CD4+ T-lymphocyte loss and lymphoid tissue destruction. With the advent and dissemination of new generation sequencing technologies, novel promising markers of immune deficiency have been explored in human and nonhuman primate species, showing changes in the microbiome (dysbiosis) that might be associated with pathogenic conditions. The aim of the present study was to identify and compare enteric viromes of SIVgor-infected and uninfected gorillas using noninvasive sampling and ultradeep sequencing, and to assess the association of virome composition with potential SIVgor pathogenesis in their natural hosts. RESULTS: We analyzed both RNA and DNA virus libraries of 23 fecal samples from 11 SIVgor-infected (two samples from one animal) and 11 uninfected western lowland gorillas from Campo-Ma'an National Park (CP), in southwestern Cameroon. Three bacteriophage families (Siphoviridae, Myoviridae and Podoviridae) represented 67.5 and 68% of the total annotated reads in SIVgor-infected and uninfected individuals, respectively. Conversely, mammalian viral families, such as Herpesviridae and Reoviridae, previously associated with gut- and several mammalian diseases were significantly more abundant (p < 0.003) in the SIVgor-infected group. In the present study, we analyzed, for the first time, the enteric virome of gorillas and their association with SIVgor status. This also provided the first evidence of association of specific mammalian viral families and SIVgor in a putative dysbiosis context. CONCLUSIONS: Our results suggested that viromes might be potentially used as markers of lentiviral disease progression in wild gorilla populations. The diverse mammalian viral families, herein described in SIVgor-infected gorillas, may play a pivotal role in a disease progression still unclear in these animals but already well characterized in pathogenic lentiviral infections in other organisms. Larger sample sets should be further explored to reduce intrinsic sampling variation.