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.

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.

Serology and Behavioral Perspectives on Ebola Virus Disease Among Bushmeat Vendors in Equateur, Democratic Republic of the Congo, After the 2018 Outbreak.

After the 2018 Ebola outbreak in Equateur Province, Democratic Republic of the Congo, we conducted behavioral interviews and collected samples from bushmeat vendors and primates in Mbandaka to test for evidence of Ebola virus exposure. Although participants indicated being aware of Ebola, they did not consider themselves at occupational risk for infection. We found antibodies against Zaire ebolavirus in one participant despite no reported history of disease or contact with infected individuals. Our data underline concerns of possible subclinical or undiagnosed Ebola virus infections and the importance and challenges of risk communication to populations who are occupationally exposed to bushmeat. Following the 2018 Ebola outbreak in Equateur Province, Democratic Republic of the Congo, bushmeat vendors interviewed in Mbandaka indicated being aware of Ebola, but did not consider themselves at occupational risk. Antibodies against Zaire ebolavirus were detected in one participant.

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.

Evidence of Low-Level Dengue Virus Circulation in the South Region of Cameroon in 2018.

Dengue fever is an understudied disease in many parts of Africa and little is known about its prevalence in Cameroon. We tested blood from 629 individuals from the South Region of Cameroon, collected over the course of one year, for flavivirus RNA using conventional broad range PCR. Flavivirus RNA corresponding to dengue virus (DENV) serotype 1 was identified in two individuals who were also diagnosed with malaria. This finding confirms previous reports that indicate the presence of low-level circulation of DENV in Cameroon and supports the concern that dengue fever may be underdiagnosed due to more prevalent diseases that have similar symptomology and insufficient diagnostic capacity.

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.

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.

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.

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.

African origin of the malaria parasite Plasmodium vivax.

Plasmodium vivax is the leading cause of human malaria in Asia and Latin America but is absent from most of central Africa due to the near fixation of a mutation that inhibits the expression of its receptor, the Duffy antigen, on human erythrocytes. The emergence of this protective allele is not understood because P. vivax is believed to have originated in Asia. Here we show, using a non-invasive approach, that wild chimpanzees and gorillas throughout central Africa are endemically infected with parasites that are closely related to human P. vivax. Sequence analyses reveal that ape parasites lack host specificity and are much more diverse than human parasites, which form a monophyletic lineage within the ape parasite radiation. These findings indicate that human P. vivax is of African origin and likely selected for the Duffy-negative mutation. All extant human P. vivax parasites are derived from a single ancestor that escaped out of Africa.

A gorilla reservoir for human T-lymphotropic virus type 4.

Of the seven known species of human retroviruses only one, human T-cell lymphotropic virus type 4 (HTLV-4), lacks a known animal reservoir. We report the largest screening for simian T-cell lymphotropic virus (STLV-4) to date in a wide range of captive and wild non-human primate (NHP) species from Cameroon. Among the 681 wild and 426 captive NHPs examined, we detected STLV-4 infection only among gorillas by using HTLV-4-specific quantitative polymerase chain reaction. The large number of samples analyzed, the diversity of NHP species examined, the geographic distribution of infected animals relative to the known HTLV-4 case, as well as detailed phylogenetic analyses on partial and full genomes, indicate that STLV-4 is endemic to gorillas, and that rather than being an ancient virus among humans, HTLV-4 emerged from a gorilla reservoir, likely through the hunting and butchering of wild gorillas. Our findings shed further light on the importance of gorillas as keystone reservoirs for the evolution and emergence of human infectious diseases and provide a clear course for preventing HTLV-4 emergence through management of human contact with wild gorillas, the development of improved assays for HTLV-4/STLV-4 detection and the ongoing monitoring of STLV-4 among gorillas and for HTLV-4 zoonosis among individuals exposed to gorilla populations.

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing.

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.

A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis.

In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.