Clinical Manifestations of an Outbreak of Monkeypox Virus in Captive Chimpanzees in Cameroon, 2016.

Monkeypox virus (MPXV) is a reemerging virus of global concern. An outbreak of clade I MPXV affected 20 captive chimpanzees in Cameroon in 2016. We describe the epidemiology, virology, phylogenetics, and clinical progression of this outbreak. Clinical signs included exanthema, facial swelling, perilaryngeal swelling, and eschar. Mpox can be lethal in captive chimpanzees, with death likely resulting from respiratory complications. We advise avoiding anesthesia in animals with respiratory signs to reduce the likelihood of death. This outbreak presented a risk to animal care staff. There is a need for increased awareness and a One Health approach to preparation for outbreaks in wildlife rescue centers in primate range states where MPXV occurs. Control measures should include quarantining affected animals, limiting human contacts, surveillance of humans and animals, use of personal protective equipment, and regular decontamination of enclosures.

Coronavirus surveillance of wildlife in the Lao People's Democratic Republic detects viral RNA in rodents.

Coronaviruses can become zoonotic, as in the case of COVID-19, and hunting, sale, and consumption of wild animals in Southeast Asia increases the risk for such incidents. We sampled and tested rodents (851) and other mammals and found betacoronavirus RNA in 12 rodents. The sequences belong to two separate genetic clusters and are closely related to those of known rodent coronaviruses detected in the region and distantly related to those of human coronaviruses OC43 and HKU1. Considering the close human-wildlife contact with many species in and beyond the region, a better understanding of virus diversity is urgently needed for the mitigation of future risks.

DNA of diverse adenoviruses detected in Cameroonian rodent and shrew species.

Rodent adenoviruses are important models for human disease. In contrast to the over 70 adenovirus types isolated from humans, few rodent adenoviruses are known, despite the vast diversity of rodent species. PCR and Sanger sequencing were used to investigate adenovirus diversity in wild rodents and shrews in Cameroon. Adenovirus DNA was detected in 13.8% of animals (n = 218). All detected sequences differ from known adenovirus types by more than 10% at the amino acid level, thus indicating up to 14 novel adenovirus species. These results highlight the diversity of rodent adenoviruses, their phylogeny, and opportunities for studying alternative adenovirus rodent models.

Antimicrobial resistance from a one health perspective in Cameroon: a systematic review and meta-analysis.

BACKGROUND: Antimicrobial resistance (AMR) is widely acknowledged as a global health problem, yet in many parts of the world its magnitude is not well elucidated. A baseline assessment of the AMR prevalence is a priority for implementation of laboratory-based AMR surveillance This review, focused on a One health approach, aimed at describing the current status of AMR in Cameroon. METHODS: PubMed, Google Scholar and African Journals Online databases were searched for articles published in English and French in accordance with the PRISMA guidelines. Retrieval and screening of article was done using a structured search string with strict inclusion/exclusion criteria. Free-text and grey literature were obtained by contacting the authors directly. The pooled prevalence and 95% confidence intervals were calculated for each pathogen-antibiotic pairs using random-effects models. RESULT: Amongst 97 full-text articles reviewed, 66 met the eligibility criteria. The studies originated from the Centre (24; 36.4%), South-West (16; 24.2%), West (13; 19.7%), Littoral (9; 13.6%) and other (4; 6.1%) regions of Cameroon. These studies reported AMR in human (45; 68.2%), animals (9; 13.6%) and the environment (12; 18.2%). Overall, 19 species of bacteria were tested against 48 antibiotics. These organisms were resistant to all classes of antibiotics and showed high levels of multidrug resistance. Escherichia coli, Klebsiella pneumoniae and Staphylococcus spp were reported in 23, 19 and 18 of the human studies and revealed multidrug resistance (MDR) rates of 47.1% [95% CI (37.3-57.2%)], 51.0% [95% CI (42.0-59.9)] and 45.2% [95% CI (38.0-54.7)], respectively. Salmonella spp was reported in 6 of the animal studies and showed a MDR rate of 46.2% [95% CI (39.2-53.5%)] while Staphylococcus spp in 8 of environment studies showed MDR rate of 67.1% [95% CI (55.2-77.2%)]. CONCLUSION: This review shows that resistance to commonly prescribed antibiotics in Cameroon is high. The findings emphasize the urgent need to address gaps in the standardization of AMR diagnostics, reporting and use of available information to optimize treatment guidelines for the arsenal of antibiotics. Effective AMR surveillance through continued data sharing, large-scale collaboration, and coordination of all stakeholders is essential to understand and manage the AMR national burden.

Phyloepidemiological Analysis Reveals that Viral Divergence Led to the Paucity of Simian Immunodeficiency Virus SIVmus/gsn/mon Infections in Wild Populations.

Human immunodeficiency virus type 1 (HIV-1) is the result of cross-species transmission of simian immunodeficiency virus from chimpanzees (SIVcpz). SIVcpz is a chimeric virus which shares common ancestors with viruses infecting red-capped mangabeys and a subset of guenon species. The epidemiology of SIV infection in hominoids is characterized by low prevalences and an uneven geographic distribution. Surveys in Cameroon indicated that two closely related members of the guenon species subset, mustached guenons and greater spot-nosed guenons, infected with SIVmus and SIVgsn, respectively, also have low rates of SIV infections in their populations. Compared to that for other monkeys, including red-capped mangabeys and closely related guenon species, such an epidemiology is unusual. By intensifying sampling of geographically distinct populations of mustached and greater spot-nosed guenons in Gabon and including large sample sets of mona guenons from Cameroon, we add strong support to the hypothesis that the paucity of SIV infections in wild populations is a general feature of this monophyletic group of viruses. Furthermore, comparative phylogenetic analysis reveals that this phenotype is a feature of this group of viruses infecting phylogenetically disparate hosts, suggesting that this epidemiological phenotype results from infection with these HIV-1-related viruses rather than from a common host factor. Thus, these HIV-1-related viruses, i.e., SIVcpz and the guenon viruses which share an ancestor with part of the SIVcpz genome, have an epidemiology distinct from that found for SIVs in other African primate species.IMPORTANCE Stable virus-host relationships are established over multiple generations. The prevalence of viral infections in any given host is determined by various factors. Stable virus-host relationships of viruses that are able to cause persistent infections and exist with high incidences of infection are generally characterized by a lack of morbidity prior to host reproduction. Such is the case for cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections of humans. SIV infections of most African primate species also satisfy these criteria, with these infections found at a high prevalence and with rare cases of clinical disease. In contrast, SIVcpz, the ancestor of HIV-1, has a different epidemiology, and it has been reported that infected animals suffer from an AIDS-like disease in the wild. Here we conclusively demonstrate that viruses which are closely related to SIVcpz and infect a subset of guenon monkeys show an epidemiology resembling that of SIVcpz.

High Herpesvirus Diversity in Wild Rodent and Shrew Species in Central Africa.

OBJECTIVE: Herpesviruses belong to a diverse order of large DNA viruses that can cause diseases in humans and animals. With the goal of gathering information about the distribution and diversity of herpesviruses in wild rodent and shrew species in central Africa, animals in Cameroon and the Democratic Republic of the Congo were sampled and tested by PCR for the presence of herpesvirus DNA. METHODS: A broad range PCRs targeting either the Polymerase or the terminase gene were used for virus detection. Amplified products from PCR were sequenced and isolates analysed for phylogenetic placement. RESULTS: Overall, samples of 1,004 animals of various rodent and shrew species were tested and 24 were found to be positive for herpesvirus DNA. Six of these samples contained strains of known viruses, while the other positive samples revealed DNA sequences putatively belonging to 11 previously undescribed herpesviruses. The new isolates are beta- and gammaherpesviruses and the shrew isolates appear to form a separate cluster within the Betaherpesvirinae subfamily. CONCLUSION: The diversity of viruses detected is higher than in similar studies in Europe and Asia. The high diversity of rodent and shrew species occurring in central Africa may be the reason for a higher diversity in herpesviruses in this area.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

Highly pathogenic avian influenza A/H5N1 Clade 2.3.2.1c virus in poultry in Cameroon, 2016-2017.

In May 2016, highly pathogenic avian influenza virus of the subtype A/H5N1 was detected in Cameroon in an industrial poultry farm at Mvog-Betsi, Yaoundé (Centre region), with a recorded sudden increase of deaths among chickens, and an overall mortality rate of 75%. The virus spread further and caused new outbreaks in some parts of the country. In total, 21 outbreaks were confirmed from May 2016 to March 2017 (six in the Centre, six in the West, eight in the South and one in the Adamaoua regions). This resulted in an estimated total loss of 138,252 birds (44,451 deaths due to infection and 93,801 stamped out). Only domestic birds (chickens, ducks and geese) were affected in farms as well as in poultry markets. The outbreaks occurred in three waves, the first from May to June 2016, the second in September 2016 and the last wave in March 2017. The topology of the phylogeny based on the haemagglutinin gene segment indicated that the causative H5N1 viruses fall within the genetic clade 2.3.2.1c, within the same group as the A/H5N1 viruses collected in Niger in 2015 and 2016. More importantly, the gene constellation of four representative viruses showed evidence of H5N1/H9N2 intra-clade reassortment. Additional epidemiological and genetic data from affected countries in West Africa are needed to better trace the origin, spread and evolution of A/H5N1 in Cameroon. RESEARCH HIGHLIGHTS HPAI A/H5N1 was detected in May 2016 in domestic chickens in Yaoundé-Cameroon. Twenty-one outbreaks in total were confirmed from May 2016 to March 2017. The causative H5N1 viruses fall within the genetic clade 2.3.2.1c. The viral gene constellation showed evidence of H5N1/H9N2 intra-clade reassortment.

Female sex workers' empowerment strategies amid HIV-related socioeconomic vulnerabilities in Cameroon.

Research has consistently demonstrated that female sex workers use a variety of empowerment strategies to protect one another and their families. This study examines the strategies Cameroonian sex workers employ to do so. In-depth interviews and focus-group discussions were conducted with 100 sex workers. Coded texts were analysed for recurring themes. Sex workers reported being concerned with physical violence and sexual assault and demands from authorities for bribes to avoid fines and/or imprisonment. Women described strategies such as 'looking out for' each other when faced with security threats. Many reported staying in sex work to provide for their children through education and other circumstances to allow them to lead a better life. Sex worker mothers reported not using condoms when clients offered higher pay, or with intimate partners, even when they understood the risk of HIV transmission to themselves. Concern for their children's quality of life took precedence over HIV-related risks, even when sex workers were the children's primary carers. A sex worker empowerment programme with a focus on family-oriented services could offer an effective and novel approach to increasing coverage of HIV prevention, treatment and care in Cameroon.

Gender-based violence against female sex workers in Cameroon: prevalence and associations with sexual HIV risk and access to health services and justice.

BACKGROUND/OBJECTIVES: Female sex workers (FSWs) are at risk for HIV and physical and sexual gender-based violence (GBV). We describe the prevalence of lifetime GBV and its associations with HIV risk behaviour, access to health services and barriers in accessing justice among FSWs in Cameroon. METHODS: FSWs (n=1817) were recruited for a cross-sectional study through snowball sampling in seven cities in Cameroon. We examined associations of lifetime GBV with key outcomes via adjusted logistic regression models. RESULTS: Overall, 60% (1098/1817) had experienced physical or sexual violence in their lifetime. GBV was associated with inconsistent condom use with clients (adjusted OR (AOR) 1.49, 95% CI 1.18 to 1.87), being offered more money for condomless sex (AOR 2.09, 95% CI 1.56 to 2.79), having had a condom slip or break (AOR 1.53, 95% CI 1.25 to 1.87) and difficulty suggesting condoms with non-paying partners (AOR 1.47, 95% CI 1.16 to 1.87). Violence was also associated with fear of health services (AOR 2.25, 95% CI 1.61 to 3.16) and mistreatment in a health centre (AOR 1.66, 95% CI 1.01 to 2.73). Access to justice was constrained for FSWs with a GBV history, specifically feeling that police did not protect them (AOR 1.41, 95% CI 1.12 to 1.78). DISCUSSION: Among FSWs in Cameroon, violence is prevalent and undermines HIV prevention and access to healthcare and justice. Violence is highly relevant to FSWs' ability to successfully negotiate condom use and engage in healthcare. In this setting of criminalised sex work, an integrated, multisectoral GBV-HIV strategy that attends to structural risk is needed to enhance safety, HIV prevention and access to care and justice.

Reorganization and expansion of the nidoviral family Arteriviridae.

The family Arteriviridae presently includes a single genus Arterivirus. This genus includes four species as the taxonomic homes for equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), porcine respiratory and reproductive syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV), respectively. A revision of this classification is urgently needed to accommodate the recent description of eleven highly divergent simian arteriviruses in diverse African nonhuman primates, one novel arterivirus in an African forest giant pouched rat, and a novel arterivirus in common brushtails in New Zealand. In addition, the current arterivirus nomenclature is not in accordance with the most recent version of the International Code of Virus Classification and Nomenclature. Here we outline an updated, amended, and improved arterivirus taxonomy based on current data. Taxon-specific sequence cut-offs are established relying on a newly established open reading frame 1b phylogeny and pairwise sequence comparison (PASC) of coding-complete arterivirus genomes. As a result, the current genus Arterivirus is replaced by five genera: Equartevirus (for EAV), Rodartevirus (LDV + PRRSV), Simartevirus (SHFV + simian arteriviruses), Nesartevirus (for the arterivirus from forest giant pouched rats), and Dipartevirus (common brushtail arterivirus). The current species Porcine reproductive and respiratory syndrome virus is divided into two species to accommodate the clear divergence of the European and American "types" of PRRSV, both of which now receive virus status. The current species Simian hemorrhagic fever virus is divided into nine species to accommodate the twelve known simian arteriviruses. Non-Latinized binomial species names are introduced to replace all current species names to clearly differentiate them from virus names, which remain largely unchanged.

Discovery of prosimian and afrotherian foamy viruses and potential cross species transmissions amidst stable and ancient mammalian co-evolution.

BACKGROUND: Foamy viruses (FVs) are a unique subfamily of retroviruses that are widely distributed in mammals. Owing to the availability of sequences from diverse mammals coupled with their pattern of codivergence with their hosts, FVs have one of the best-understood viral evolutionary histories ever documented, estimated to have an ancient origin. Nonetheless, our knowledge of some parts of FV evolution, notably that of prosimian and afrotherian FVs, is far from complete due to the lack of sequence data. RESULTS: Here, we report the complete genome of the first extant prosimian FV (PSFV) isolated from a lorisiforme galago (PSFVgal), and a novel partial endogenous viral element with high sequence similarity to FVs, present in the afrotherian Cape golden mole genome (ChrEFV). We also further characterize a previously discovered endogenous PSFV present in the aye-aye genome (PSFVaye). Using phylogenetic methods and available FV sequence data, we show a deep divergence and stable co-evolution of FVs in eutherian mammals over 100 million years. Nonetheless, we found that the evolutionary histories of bat, aye-aye, and New World monkey FVs conflict with the evolutionary histories of their hosts. By combining sequence analysis and biogeographical knowledge, we propose explanations for these mismatches in FV-host evolutionary history. CONCLUSION: Our discovery of ChrEFV has expanded the FV host range to cover the whole eutherian clade, and our evolutionary analyses suggest a stable mammalian FV-host co-speciation pattern which extends as deep as the exafroplacentalian basal diversification. Nonetheless, two possible cases of host switching were observed. One was among New World monkey FVs, and the other involves PSFVaye and a bat FV which may involve cross-species transmission at the level of mammalian orders. Our results highlight the value of integrating multiple sources of information to elucidate the evolutionary history of viruses, including continental and geographical histories, ancestral host locations, in addition to the natural history of host and virus.

A hybrid phylogenetic-phylogenomic approach for species tree estimation in African Agama lizards with applications to biogeography, character evolution, and diversification.

Africa is renowned for its biodiversity and endemicity, yet little is known about the factors shaping them across the continent. African Agama lizards (45 species) have a pan-continental distribution, making them an ideal model for investigating biogeography. Many species have evolved conspicuous sexually dimorphic traits, including extravagant breeding coloration in adult males, large adult male body sizes, and variability in social systems among colorful versus drab species. We present a comprehensive time-calibrated species tree for Agama, and their close relatives, using a hybrid phylogenetic-phylogenomic approach that combines traditional Sanger sequence data from five loci for 57 species (146 samples) with anchored phylogenomic data from 215 nuclear genes for 23 species. The Sanger data are analyzed using coalescent-based species tree inference using (*)BEAST, and the resulting posterior distribution of species trees is attenuated using the phylogenomic tree as a backbone constraint. The result is a time-calibrated species tree for Agama that includes 95% of all species, multiple samples for most species, strong support for the major clades, and strong support for most of the initial divergence events. Diversification within Agama began approximately 23 million years ago (Ma), and separate radiations in Southern, East, West, and Northern Africa have been diversifying for >10Myr. A suite of traits (morphological, coloration, and sociality) are tightly correlated and show a strong signal of high morphological disparity within clades, whereby the subsequent evolution of convergent phenotypes has accompanied diversification into new biogeographic areas.

Correlates of prior HIV testing among men who have sex with men in Cameroon: a cross-sectional analysis.

BACKGROUND: Regular HIV testing is vital for timely linkage to the HIV care continuum and ensuring the success of behavioral and biomedical interventions to prevent HIV acquisition. Men who have sex with men (MSM) are a key population for HIV prevention, treatment, and care efforts globally. This study measures the factors associated with prior HIV testing among MSM in Cameroon. METHODS: In 2011, 272 and 239 MSM aged ≥ 18 were recruited from Douala and Yaoundé respectively using respondent-driven sampling (RDS) for a cross-sectional surveillance study. Participants completed a structured socio-behavioral survey and were offered HIV and syphilis testing and counseling. RESULTS: The majority of men self-reported ever testing for HIV (81.6%; 413/506) and receiving their last HIV test result (95.4%; 394/413). Testing in the last 12 months was more prevalent in Douala (63.3%; 169/267) compared to Yaoundé (55.9%; 132/236). Median frequency of testing was every 18 months in Douala and every two years in Yaoundé. In multivariate RDS-weighted analysis, correlates of ever testing for HIV in Douala were: having higher than secondary education compared to having secondary education or less (aOR = 3.51, 95% CI: 1.32-9.34), ever accessing a community-based HIV service for MSM (aOR = 3.37, 95% CI: 1.57-7.24) and having ≥4 male oral or anal sexual partners in the past 12 months (aOR = 2.49, 1.08-5.74). In Yaoundé, having higher than secondary education (aOR = 7.96, 95% CI: 1.31-48.41) was associated with ever testing for HIV. CONCLUSIONS: Supporting regular HIV testing and linkage to care is important in a context of high HIV prevalence and limited use of condoms and condom-compatible lubricants. Building the capacity of MSM organizations and mainstream health services to deliver affordable, integrated, confidential, and MSM-sensitive HIV testing may assist in effectively engaging more MSM in the HIV treatment cascade. Giving specific attention to MSM who are younger, of lower socioeconomic status and less connected to community-based MSM organizations may increase HIV testing uptake. Given the levels of HIV testing and high HIV prevalence among MSM in Cameroon, optimizing the safe and effective provision and uptake of antiretroviral-based prevention and treatment approaches is paramount in changing the trajectory of the HIV epidemic among these men and within their sexual networks.

Genomic analyses reveal poaching hotspots and illegal trade in pangolins from Africa to Asia.

The white-bellied pangolin (Phataginus tricuspis) is the world's most trafficked mammal and is at risk of extinction. Reducing the illegal wildlife trade requires an understanding of its origins. Using a genomic approach for tracing confiscations and analyzing 111 samples collected from known geographic localities in Africa and 643 seized scales from Asia between 2012 and 2018, we found that poaching pressures shifted over time from West to Central Africa. Recently, Cameroon's southern border has emerged as a site of intense poaching. Using data from seizures representing nearly 1 million African pangolins, we identified Nigeria as one important hub for trafficking, where scales are amassed and transshipped to markets in Asia. This origin-to-destination approach offers new opportunities to disrupt the illegal wildlife trade and to guide anti-trafficking measures.

High seroprevalence of enterovirus infections in apes and old world monkeys.

To estimate population exposure of apes and Old World monkeys in Africa to enteroviruses (EVs), we conducted a seroepidemiologic study of serotype-specific neutralizing antibodies against 3 EV types. Detection of species A, B, and D EVs infecting wild chimpanzees demonstrates their potential widespread circulation in primates.

Novel simian foamy virus infections from multiple monkey species in women from the Democratic Republic of Congo.

BACKGROUND: Zoonotic transmission of simian retroviruses in Central Africa is ongoing and can result in pandemic human infection. While simian foamy virus (SFV) infection was reported in primate hunters in Cameroon and Gabon, little is known about the distribution of SFV in Africa and whether human-to-human transmission and disease occur. We screened 3,334 plasmas from persons living in rural villages in central Democratic Republic of Congo (DRC) using SFV-specific EIA and Western blot (WB) tests. PCR amplification of SFV polymerase sequences from DNA extracted from buffy coats was used to measure proviral loads. Phylogenetic analysis was used to define the NHP species origin of SFV. Participants completed questionnaires to capture NHP exposure information. RESULTS: Sixteen (0.5%) samples were WB-positive; 12 of 16 were from women (75%, 95% confidence limits 47.6%, 92.7%). Sequence analysis detected SFV in three women originating from Angolan colobus or red-tailed monkeys; both monkeys are hunted frequently in DRC. NHP exposure varied and infected women lived in distant villages suggesting a wide and potentially diverse distribution of SFV infections across DRC. Plasmas from 22 contacts of 8 WB-positive participants were all WB negative suggesting no secondary viral transmission. Proviral loads in the three women ranged from 14 - 1,755 copies/105 cells. CONCLUSIONS: Our study documents SFV infection in rural DRC for the first time and identifies infections with novel SFV variants from Colobus and red-tailed monkeys. Unlike previous studies, women were not at lower risk for SFV infection in our population, providing opportunities for spread of SFV both horizontally and vertically. However, limited testing of close contacts of WB-positive persons did not identify human-to-human transmission. Combined with the broad behavioral risk and distribution of NHPs across DRC, our results suggest that SFV infection may have a wider geographic distribution within DRC. These results also reinforce the potential for an increased SFV prevalence throughout the forested regions of Africa where humans and simians co-exist. Our finding of endemic foci of SFV infection in DRC will facilitate longitudinal studies to determine the potential for person-to-person transmissibility and pathogenicity of these zoonotic retroviral infections.

The origin of malignant malaria.

Plasmodium falciparum, the causative agent of malignant malaria, is among the most severe human infectious diseases. The closest known relative of P. falciparum is a chimpanzee parasite, Plasmodium reichenowi, of which one single isolate was previously known. The co-speciation hypothesis suggests that both parasites evolved separately from a common ancestor over the last 5-7 million years, in parallel with the divergence of their hosts, the hominin and chimpanzee lineages. Genetic analysis of eight new isolates of P. reichenowi, from wild and wild-born captive chimpanzees in Cameroon and Côte d'Ivoire, shows that P. reichenowi is a geographically widespread and genetically diverse chimpanzee parasite. The genetic lineage comprising the totality of global P. falciparum is fully included within the much broader genetic diversity of P. reichenowi. This finding is inconsistent with the co-speciation hypothesis. Phylogenetic analysis indicates that all extant P. falciparum populations originated from P. reichenowi, likely by a single host transfer, which may have occurred as early as 2-3 million years ago, or as recently as 10,000 years ago. The evolutionary history of this relationship may be explained by two critical genetic mutations. First, inactivation of the CMAH gene in the human lineage rendered human ancestors unable to generate the sialic acid Neu5Gc from its precursor Neu5Ac, and likely made humans resistant to P. reichenowi. More recently, mutations in the dominant invasion receptor EBA 175 in the P. falciparum lineage provided the parasite with preference for the overabundant Neu5Ac precursor, accounting for its extreme human pathogenicity.

Wildlife in Cameroon harbor diverse coronaviruses, including many closely related to human coronavirus 229E.

Zoonotic spillover of animal viruses into human populations is a continuous and increasing public health risk. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the global impact of emergence. Considering the history and diversity of coronaviruses (CoVs), especially in bats, SARS-CoV-2 will likely not be the last to spillover from animals into human populations. We sampled and tested wildlife in the Central African country Cameroon to determine which CoVs are circulating and how they relate to previously detected human and animal CoVs. We collected animal and ecological data at sampling locations and used family-level consensus PCR combined with amplicon sequencing for virus detection. Between 2003 and 2018, samples were collected from 6,580 animals of several different orders. CoV RNA was detected in 175 bats, a civet, and a shrew. The CoV RNAs detected in the bats represented 17 different genetic clusters, coinciding with alpha (n = 8) and beta (n = 9) CoVs. Sequences resembling human CoV-229E (HCoV-229E) were found in 40 Hipposideridae bats. Phylogenetic analyses place the human-derived HCoV-229E isolates closest to those from camels in terms of the S and N genes but closest to isolates from bats for the envelope, membrane, and RNA-dependent RNA polymerase genes. The CoV RNA positivity rate in bats varied significantly (P < 0.001) between the wet (8.2 per cent) and dry seasons (4.5 per cent). Most sampled species accordingly had a wet season high and dry season low, while for some the opposite was found. Eight of the suspected CoV species of which we detected RNA appear to be entirely novel CoV species, which suggests that CoV diversity in African wildlife is still rather poorly understood. The detection of multiple different variants of HCoV-229E-like viruses supports the bat reservoir hypothesis for this virus, with the phylogenetic results casting some doubt on camels as an intermediate host. The findings also support the previously proposed influence of ecological factors on CoV circulation, indicating a high level of underlying complexity to the viral ecology. These results indicate the importance of investing in surveillance activities among wild animals to detect all potential threats as well as sentinel surveillance among exposed humans to determine emerging threats.

Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats.

Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel alpha- and betacoronaviruses in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.