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.

Predicting the potential for zoonotic transmission and host associations for novel viruses.

Host-virus associations have co-evolved under ecological and evolutionary selection pressures that shape cross-species transmission and spillover to humans. Observed virus-host associations provide relevant context for newly discovered wildlife viruses to assess knowledge gaps in host-range and estimate pathways for potential human infection. Using models to predict virus-host networks, we predicted the likelihood of humans as hosts for 513 newly discovered viruses detected by large-scale wildlife surveillance at high-risk animal-human interfaces in Africa, Asia, and Latin America. Predictions indicated that novel coronaviruses are likely to infect a greater number of host species than viruses from other families. Our models further characterize novel viruses through prioritization scores and directly inform surveillance targets to identify host ranges for newly discovered viruses.

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.

Enterovirus Sequence Data Obtained from Primate Samples in Central Africa Suggest a High Prevalence of Enteroviruses.

Enteroviruses infect humans and animals and can cause disease, and some may be transmitted across species barriers. We tested Central African wildlife and found Enterovirus RNA in primates (17) and rodents (2). Some sequences were very similar, while others were dissimilar to known species, highlighting the underexplored enterovirus diversity in wildlife.

Coronavirus surveillance in wildlife from two Congo basin countries detects RNA of multiple species circulating in bats and rodents.

Coronaviruses play an important role as pathogens of humans and animals, and the emergence of epidemics like SARS, MERS and COVID-19 is closely linked to zoonotic transmission events primarily from wild animals. Bats have been found to be an important source of coronaviruses with some of them having the potential to infect humans, with other animals serving as intermediate or alternate hosts or reservoirs. Host diversity may be an important contributor to viral diversity and thus the potential for zoonotic events. To date, limited research has been done in Africa on this topic, in particular in the Congo Basin despite frequent contact between humans and wildlife in this region. We sampled and, using consensus coronavirus PCR-primers, tested 3,561 wild animals for coronavirus RNA. The focus was on bats (38%), rodents (38%), and primates (23%) that posed an elevated risk for contact with people, and we found coronavirus RNA in 121 animals, of which all but two were bats. Depending on the taxonomic family, bats were significantly more likely to be coronavirus RNA-positive when sampled either in the wet (Pteropodidae and Rhinolophidae) or dry season (Hipposideridae, Miniopteridae, Molossidae, and Vespertilionidae). The detected RNA sequences correspond to 15 alpha- and 6 betacoronaviruses, with some of them being very similar (>95% nucleotide identities) to known coronaviruses and others being more unique and potentially representing novel viruses. In seven of the bats, we detected RNA most closely related to sequences of the human common cold coronaviruses 229E or NL63 (>80% nucleotide identities). The findings highlight the potential for coronavirus spillover, especially in regions with a high diversity of bats and close human contact, and reinforces the need for ongoing surveillance.

Evaluation of bat adenoviruses suggests co-evolution and host roosting behaviour as drivers for diversity.

Adenoviruses (AdVs) are diverse pathogens of humans and animals, with several dozen bat AdVs already identified. Considering that over 100 human AdVs are known, and the huge diversity of bat species, many bat AdVs likely remain undiscovered. To learn more about AdV prevalence, diversity and evolution, we sampled and tested bats in Cameroon using several PCR assays for viral and host DNA. AdV DNA was detected in 14 % of the 671 sampled animals belonging to 37 different bat species. There was a correlation between species roosting in larger groups and AdV DNA detection. The detected AdV DNA belonged to between 28 and 44 different, mostly previously unknown, mastadenovirus species. The novel isolates are phylogenetically diverse and while some cluster with known viruses, others appear to form divergent new clusters. The phylogenetic tree of novel and previously known bat AdVs does not mirror that of the various host species, but does contain structures consistent with a degree of virus-host co-evolution. Given that closely related isolates were found in different host species, it seems likely that at least some bat AdVs have jumped species barriers, probably in the more recent past; however, the tree is also consistent with such events having taken place throughout bat AdV evolution. AdV diversity was highest in bat species roosting in large groups. The study significantly increased the diversity of AdVs known to be harboured by bats, and suggests that host behaviours, such as roosting size, may be what limits some AdVs to one species rather than an inability of AdVs to infect other related hosts.

Bridging the gap: Using reservoir ecology and human serosurveys to estimate Lassa virus spillover in West Africa.

Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections.

Human Interactions with Bat Populations in Bombali, Sierra Leone.

Human contact with bats has been epidemiologically linked to several of the most recent Ebola outbreaks, including the 2014 West Africa epidemic and the 2007 Luebo, Democratic Republic of the Congo, outbreak. While fruit bats remain the likely primary reservoir for Ebola virus (Zaire ebolavirus), recent wildlife surveillance efforts have identified a new species of ebolavirus (Bombali ebolavirus) in microchiropteran insect-eating bats in West and East Africa. Given the role of bats as potential Ebola reservoirs and sources of spillover into human populations, it is critically important to understand the circumstances and behaviors that bring human populations into close contact with bats. This study explores two sites in Bombali, Sierra Leone, where human populations have had close contact with microchiropteran bats via household infestations and fruit bats by hunting practices. Through interviews and focus groups, we identify the knowledge, beliefs, perceptions, and behaviors that may potentially protect or expose individuals to zoonotic spillover through direct and indirect contact with bats. We also describe how this research was used to develop a risk reduction and outreach tool for living safely with bats.

Assessing Climate Change Impact on Ecosystems and Infectious Disease: Important Roles for Genomic Sequencing and a One Health Perspective.

Changes in the Earth's climate and weather continue to impact the planet's ecosystems, including the interface of infectious disease agents with their hosts and vectors. Environmental disasters, natural and human-made activities raise risk factors that indirectly facilitate infectious disease outbreaks. Subsequently, changes in habitat, displaced populations, and environmental stresses that affect the survival of species are amplified over time. The recurrence and spread of vector-borne (e.g., mosquito, tick, aphid) human, animal, and plant pathogens to new geographic locations are also influenced by climate change. The distribution and range of humans, agricultural animals and plants, wildlife and native plants, as well as vectors, parasites, and microbes that cause neglected diseases of the tropics as well as other global regions are also impacted. In addition, genomic sequencing can now be applied to detect signatures of infectious pathogens as they move into new regions. Molecular detection assays complement metagenomic sequencing to help us understand the microbial community found within the microbiomes of hosts and vectors, and help us uncover mechanistic relationships between climate variability and pathogen transmission. Our understanding of, and responses to, such complex dynamics and their impacts can be enhanced through effective, multi-sectoral One Health engagement coupled with applications of both traditional and novel technologies. Concerted efforts are needed to further harness and leverage technology that can identify and track these impacts of climate changes in order to mitigate and adapt to their effects.

Implementing One Health approaches to confront emerging and re-emerging zoonotic disease threats: lessons from PREDICT.

Recurring outbreaks of emerging and re-emerging zoonoses, such as Ebola virus disease, avian influenza, and Nipah virus, serve as a reminder that the health of humans, animals, and the environment are interconnected and that early response to emerging zoonotic pathogens requires a coordinated, interdisciplinary, cross-sectoral approach. As our world becomes increasingly connected, emerging diseases pose a greater threat, requiring coordination at local, regional, and global levels. One Health is a multisectoral, transdisciplinary, and collaborative approach promoted to more effectively address these complex health threats. Despite strong advocacy for One Health, challenges for practical implementation remain. Here we discuss the value of the One Health approach for addressing global health challenges. We also share strategies applied to achieve successful outcomes through the USAID Emerging Pandemic Threats Program PREDICT project, which serve as useful case studies for implementing One Health approaches. Lastly, we explore methods for promoting more formal One Health implementation to capitalize on the added value of shared knowledge and leveraged resources.

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.

Achieving Health Security and Threat Reduction through Sharing Sequence Data.

With the rapid development and broad applications of next-generation sequencing platforms and bioinformatic analytical tools, genomics has become a popular area for biosurveillance and international scientific collaboration. Governments from countries including the United States (US), Canada, Germany, and the United Kingdom have leveraged these advancements to support international cooperative programs that aim to reduce biological threats and build scientific capacity worldwide. A recent conference panel addressed the impacts of the enhancement of genomic sequencing capabilities through three major US bioengagement programs on international scientific engagement and biosecurity risk reduction. The panel contrasted the risks and benefits of supporting the enhancement of genomic sequencing capabilities through international scientific engagement to achieve biological threat reduction and global health security. The lower costs and new bioinformatic tools available have led to the greater application of sequencing to biosurveillance. Strengthening sequencing capabilities globally for the diagnosis and detection of infectious diseases through mutual collaborations has a high return on investment for increasing global health security. International collaborations based on genomics and shared sequence data can build and leverage scientific networks and improve the timeliness and accuracy of disease surveillance reporting needed to identify and mitigate infectious disease outbreaks and comply with international norms. Further efforts to promote scientific transparency within international collaboration will improve trust, reduce threats, and promote global health security.

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.

Author Correction: The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses.

In the version of this Article originally published, the bat species for 12 individuals were incorrectly identified in Supplementary Table 1 and 2. After resequencing the MT-CytB and MT-CO1 segments and reviewing the data, the authors have corrected the errors for these 12 animals. In the amended version of the Supplementary Information, Supplementary Tables 1 and 2 have been replaced to include the corrected host species information. None of the 12 bats affected were positive for the Bombali virus, and the conclusions of the study are therefore unchanged.

The discovery of Bombali virus adds further support for bats as hosts of ebolaviruses.

Here we describe the complete genome of a new ebolavirus, Bombali virus (BOMV) detected in free-tailed bats in Sierra Leone (little free-tailed (Chaerephon pumilus) and Angolan free-tailed (Mops condylurus)). The bats were found roosting inside houses, indicating the potential for human transmission. We show that the viral glycoprotein can mediate entry into human cells. However, further studies are required to investigate whether exposure has actually occurred or if BOMV is pathogenic in humans.

Correction: Serologic and behavioral risk survey of workers with wildlife contact in China.

[This corrects the article DOI: 10.1371/journal.pone.0194647.].

Serologic and behavioral risk survey of workers with wildlife contact in China.

We report on a study conducted in Guangdong Province, China, to characterize behaviors and perceptions associated with transmission of pathogens with pandemic potential in highly exposed human populations at the animal-human interface. A risk factor/exposure survey was administered to individuals with high levels of exposure to wildlife. Serological testing was performed to evaluate prior infection with several wildlife viral pathogens. Follow up serology was performed on a subset of the cohort as well as close contacts of individuals. 1,312 individuals were enrolled in the study. Contact with a wide range of wildlife species was reported in both occupational and occasional contexts. The overall proportion of individuals seropositive to any of the tested wildlife pathogens was approximately 4.0%. However, persons employed as butchers demonstrated a seropositivity of 9.0% to at least one pathogen of interest. By contrast, individuals working as hunters had lower rates of seropositivity. Among the study population, a number of other behaviors showed correlation with seropositivity, including contact with particular wildlife species such as field rats. These results demonstrate the need to further explore zoonotic risks of particular activities regarding wildlife contact, and to better understand risks of persons working as butchers with wildlife species.

DNA indicative of human bocaviruses detected in non-human primates in the Democratic Republic of the Congo.

Bocaparvoviruses are members of the family Parvovirinae and human bocaviruses have been found to be associated with respiratory and gastrointestinal disease. There are four known human bocaviruses, as well as several distinct ones in great apes. The goal of the presented study was to detect other non-human primate (NHP) bocaviruses in NHP species in the Democratic Republic of the Congo using conventional broad-range PCR. We found bocavirus DNA in blood and tissues samples in 6 out of 620 NHPs, and all isolates showed very high identity (>97 %) with human bocaviruses 2 or 3. These findings suggest cross-species transmission of bocaviruses between humans and NHPs.

Promoting Scientific Transparency to Facilitate the Safe and Open International Exchange of Biological Materials and Electronic Data.

Scientific communication, collaboration and progress are enhanced through the exchange of data, materials and ideas. Recent advances in technology, commercial proprietary discovery and current local and global events (e.g., emerging human, animal and plant disease outbreaks) have increased the demand, and shortened optimal timelines for material and data exchange, both domestically and internationally. Specific circumstances in each case, such as the type of material being transferred (i.e., select agent, disease-causing agent and assessed biosafety risk level) and current events, dictate the level of agreements and requirements. Recent lessons learned from emerging disease issues and emergencies have demonstrated that human engagement and increased science diplomacy are needed to reinforce and sustain biosafety and biosecurity practices and processes, for better scientific transparency. A reasonable and accepted framework of guidance for open sharing of data and materials is needed that can be applied on multiple cooperative levels, including global and national. Although numerous agreement variations already exist for the exchange of materials and data, regulations to guide the development of both the language and implementation of such agreements are limited. Without such regulations, scientific exchange is often restricted, limiting opportunities for international capacity building, collaboration and cooperation. In this article, we present and discuss several international case histories that illustrate the complex nature of scientific exchange. Recommendations are made for a dual bottom-up and top-down approach that includes all stakeholders from beginning negotiation stages to emphasize trust and cooperation. The broader aim of this approach is to increase international scientific transparency and trust in a safe and open manner, supporting increased global one health security.