Article

A Novel Nobecovirus in an Epomophorus wahlbergi Bat from

Nairobi, Kenya
Meredith C. VanAcker 1,2, *,† , Koray Ergunay 3,4,5,6,† , Paul W. Webala 7 , Maureen Kamau 8 , Janerose Mutura 8 ,
Rashid Lebunge 8 , Griphin Ochieng Ochola 8 , Brian P. Bourke 3,4,5 , Emily G. McDermott 9 ,
Nicole L. Achee 10 , Le Jiang 11 , John P. Grieco 10 , Erick Keter 12 , Audrey Musanga 13 , Suzan Murray 2 ,
Jared A. Stabach 14 , Meggan E. Craft 15 , Eric M. Fèvre 16,17 , Yvonne-Marie Linton 3,4,5
and James Hassell 2,17,18

1 Department of Evolution, Ecology, and Organismal Biology, University of California,

Riverside, CA 92521, USA
2 Global Health Program, Smithsonian Institution, National Zoo and Conservation Biology Institute,
Washington, DC 20008, USA
3 Walter Reed Biosystematics Unit (WRBU), Smithsonian Institution, Museum Support Center,
Suitland, MD 20746, USA; [email protected] (K.E.)
4 One Health Branch, Walter Reed Army Institute of Research (WRAIR), Silver Spring, MD 20910, USA
5 Department of Entomology, Smithsonian Institution, National Museum of Natural History (NMNH),
Washington, DC 20560, USA
6 Virology Unit, Department of Medical Microbiology, Faculty of Medicine, Hacettepe University,
Ankara 06100, Turkey
7 Department of Forestry and Wildlife Management, Maasai Mara University, Narok 20500, Kenya;
[email protected]
8 Mpala Research Centre (MRC), Nanyuki 10400, Kenya; [email protected] (M.K.);
[email protected] (J.M.); [email protected] (R.L.)
9 Department of Entomology and Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA;
[email protected]
10 Department of Biological Sciences, Eck Institute for Global Health, University of Notre Dame,
Notre Dame, IN 46556, USA; [email protected] (N.L.A.)
11 Viral and Rickettsial Diseases Department, Infectious Diseases Directorate, Naval Medical Research
Center (NMRC), 503 Robert Grant Avenue, Silver Spring, MD 20910, USA
12 Department of Wildlife Management, University of Eldoret, Eldoret 30100, Kenya; [email protected]
13 College of Agriculture and Veterinary Sciences, University of Nairobi, Nairobi 00100, Kenya
14 Conservation Ecology Center, Smithsonian National Zoo and Conservation Biology Institute,
Academic Editors: Gábor Kemenesi,
Kornélia Kurucz and Andrew Pekosz Front Royal, VA 22630, USA; [email protected]
15 Department of Ecology, Evolution and Behavior, College of Biological Sciences, University of Minnesota,
Received: 3 February 2025
St. Paul, MN 55108, USA; [email protected]
Revised: 25 March 2025 16 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 3BX, UK;
Accepted: 8 April 2025 [email protected]
Published: 12 April 2025 17 International Livestock Research Institute (ILRI), Nairobi 00100, Kenya
18 Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT 06520, USA
Citation: VanAcker, M.C.; Ergunay,
* Correspondence: [email protected]
K.; Webala, P.W.; Kamau, M.; Mutura, † Shared first authorship.
J.; Lebunge, R.; Ochola, G.O.; Bourke,
B.P.; McDermott, E.G.; Achee, N.L.;
et al. A Novel Nobecovirus in an
Abstract: Most human emerging infectious diseases are zoonotic, originating in animal
Epomophorus wahlbergi Bat from hosts prior to spillover to humans. Prioritizing the surveillance of wildlife that overlaps
Nairobi, Kenya. Viruses 2025, 17, 557. with humans and human activities can increase the likelihood of detecting viruses with
https://doi.org/10.3390/v17040557 a high potential for human infection. Here, we obtained fecal swabs from two fruit bat
Copyright: © 2025 by the authors. species—Eidolon helvum (n = 6) and Epomophorus wahlbergi (n = 43) (family Pteropodidae)—
Licensee MDPI, Basel, Switzerland. in peridomestic habitats in Nairobi, Kenya, and used metagenome sequencing to detect
This article is an open access article microorganisms. A near-complete genome of a novel virus assigned taxonomically to the
distributed under the terms and
Coronaviridae family Betacoronavirus genus and Nobecovirus subclade was characterized from
conditions of the Creative Commons
E. wahlbergi. Phylogenetic analysis indicates this unique Nobecovirus clade shares a common
Attribution (CC BY) license
(https://creativecommons.org/
ancestor with Eidolon/Rousettus Nobecovirus subclades isolated from Madagascar, Kenya,
licenses/by/4.0/). and Cameroon. Recombination was detected across open reading frames, except the spike

Viruses 2025, 17, 557 https://doi.org/10.3390/v17040557

Viruses 2025, 17, 557 2 of 13

protein, in all BOOTSCAN analyses, indicating intra-host coinfection and genetic exchange
between genome regions. Although Nobecoviruses are currently bat-specific and are not
known to be zoonotic, the propensity of coronaviruses to undergo frequent recombination
events and the location of the virus alongside high human and livestock densities in one
of East Africa’s most rapidly developing cities justifies continued surveillance of animal
viruses in high-risk urban landscapes.

Keywords: East Africa; urban bat-borne coronavirus; peridomestic habitat; Betacoronavirus;

metagenomics; Nobecovirus; wildlife–livestock–human interface; Eidolon helvum

1. Introduction
Infectious diseases are a major threat to human, wildlife, and livestock health and are
a significant burden on global public health and economic systems. Efforts to elucidate the
environmental, epidemiological, and ecological factors that drive zoonotic viral emergence
specifically have increased our understanding of the detrimental and complex role of
land-use change in spillover [1,2] while exposing the expansive knowledge gap on host–
virus associations driven by unknown viral diversity [3]. A recent estimate posits that
10,000 viruses with zoonotic potential circulate in non-human mammals [3], the continued
discovery of which greatly improves our understanding of global viral emergence patterns.
Several studies have examined whether taxonomic animal groups have predictable
patterns in the number of zoonoses they host [4–6]. If certain host groups contribute
disproportionately to the zoonotic potential of virus species [4], efforts toward zoonotic
discovery and outbreak preparedness and response could be strategically prioritized to-
ward those groups. Bats (Mammalia: Chiroptera) specifically have been hypothesized
to be a “special” reservoir host based on immunological mechanisms that allow bats to
tolerate viral infection [7–9] and due to the links between bat-derived coronaviruses and
human epidemics, including severe acute respiratory syndrome (SARS-CoV), Middle East
respiratory syndrome-related coronavirus (MERS-CoV) [10], and the ongoing SARS-CoV-2
pandemic—whose ancestral genome relates to bat Sarbecoviruses [11,12]. Alternative
hypotheses have been proposed to predict which host species will likely harbor the next
human-infecting virus, including an animal order’s evolutionary divergence from hu-
mans [4]; the life history strategies of taxonomic groups, such as rodents, that facilitate
sympatry with humans [6]; and more recently, that the abundance of human-infecting
viruses are linked to the number of viruses maintained by each reservoir group, predicted
by a group’s species richness [5,13]. Although these hypotheses are still being debated,
they serve as a guide for key factors to consider when conducting viral surveillance of
wildlife reservoirs.
Urbanization is a key driver of zoonotic disease emergence, as anthropogenic distur-
bances from land conversion alter contact between humans, animals, and pathogens and
can result in pathogen spillover across novel interfaces. It is critical to monitor the viral
diversity circulating in urban reservoir hosts considering the role of urban development in
driving zoonotic emergence and because subsequent pathogen spread through human pop-
ulations in an urban landscape may occur at a higher frequency than in natural and rural
landscapes. Bats and rodents frequently share habitats with humans and human-dominated
landscapes, such as in cities, and represent the richest mammalian orders globally [14].
Further, the Afrotropical region (Africa south of the Sahara with Madagascar) hosts 31%
of mammal diversity across the tropics globally [15], with approximately 20% of all recog-
nized bat species occurring in continental Africa [16]. Over 100 species of bats are found in
Viruses 2025, 17, x FOR PEER REVIEW 3 of 15

Viruses 2025, 17, 557 3 of 13

bats are found in Kenya alone [17,18]. With diverse bat species occupying habitats that
overlap with [17,18].
Kenya alone rapid, often
Withunplanned
diverse baturbanization, Nairobi,
species occupying Kenya,that
habitats is an ideal location
overlap for
with rapid,
the surveillance
often unplannedof urban bats. Nairobi,
urbanization, Further, Kenya,
novel interactions and microbial
is an ideal location exchanges be-
for the surveillance of
tween wildlife reservoirs, livestock, and humans were previously documented
urban bats. Further, novel interactions and microbial exchanges between wildlife reservoirs, in Nairobi,
flagging
livestock,itand
as ahumans
potentiallywere high-risk
previouslylandscape for pathogen
documented in Nairobi,spillover [19].
flagging it Prior work has
as a potentially
identified a range of viruses in Kenyan bat species, including adenoviruses,
high-risk landscape for pathogen spillover [19]. Prior work has identified a range of viruses astroviruses,
caliciviruses,
in Kenyan bat coronaviruses,
species, including rhabdoviruses,
adenoviruses,rotaviruses,
astroviruses,paramyxoviruses, and filo-
caliciviruses, coronaviruses,
viruses [20–26].
rhabdoviruses, rotaviruses, paramyxoviruses, and filoviruses [20–26].
Members
Members of of the
the family
family Coronaviridae
Coronaviridae areare enveloped,
enveloped, positive-sense
positive-sense RNA RNA viruses
viruses that
that
infect four of the seven classes of vertebrates: mammals and birds
infect four of the seven classes of vertebrates: mammals and birds (Orthocoronaviruses), (Orthocoronaviruses),
amphibians
amphibians (Letoviruses),
(Letoviruses), and bony fish
and bony fish (Pitoviruses)
(Pitoviruses) [27].
[27]. InIn the
the subfamily
subfamilyOrthocoron-
Orthocoro-
navirinae,
avirinae, four genera are recognized, where Alphacoronavirus and Betacoronavirus are
four genera are recognized, where Alphacoronavirus and Betacoronavirus are
primarily
primarily associated
associated withwith bats
bats as
as hosts.
hosts. The
The Betacoronavirus
Betacoronavirus genus genus can
can be
be further
further broken
broken
down
down into
into subgenera:
subgenera: Sarbecovirus
Sarbecovirus (hosted
(hosted byby bats
bats inin the
the family
family Rhinolophidae),
Rhinolophidae), Merbe-
Merbe-
covirus
covirus (hosted
(hosted by by bats
bats inin the
the family
family Vespertilionidae), Nobecovirus
Nobecovirus (hosted
(hosted by by bats
bats in the
family
family Pteropodidae),
Pteropodidae), and and Hibecovirus
Hibecovirus (hosted
(hosted byby bats
bats inin family
family Hipposideridae)
Hipposideridae)[28–31].
[28–31].
Another subgenus, Embecovirus,
Embecovirus, is is primarily
primarilyassociated
associatedwith withrodents
rodentsandandbovids,
bovids,though
thougha
afew
fewbat
bathosts
hostshavehavebeen
beendescribed.
described.Here,Here,we weutilized
utilized metagenome
metagenome sequencing to ensure
unbiased
unbiased pathogen
pathogen screening
screening of of bat-derived
bat-derived samples
samples and and to to characterize
characterize thethe genome
genome of
the novel Betacoronavirus we detected
the novel Betacoronavirus we detected in in an urban fruit bat. This
This work provides integral
information
information on on the
the complex
complex epidemiology
epidemiology of of coronaviruses
coronaviruses in in wild
wild animal
animal populations,
populations,
especially those adapted
adapted to toanthropogenically
anthropogenicallydominated
dominatedlandscapes
landscapeswith with high densities
high densities of
of humans
humans and and livestock.
livestock.

Materials and
2. Materials and Methods
Methods
2.1. Study
2.1. Study Area
Area and
and Bat
Bat Sampling
Sampling
Between June
Between Juneand
andAugust
August2023,2023,bat
batsamples
sampleswerewere obtained
obtained from
from E. E. wahlbergi
wahlbergi andand
E.
E. helvum at eight households in Nairobi, Kenya (Figure 1). Individual households
helvum at eight households in Nairobi, Kenya (Figure 1). Individual households were se- were
selected
lected based
based onon suitablehabitats
suitable habitatspresent
presentfor
forfruit
fruitbat
batforaging
foraging(e.g.,
(e.g.,fruiting
fruiting trees),
trees), the
the
presence of flyways (e.g., in riverine vegetation), and recent eco-epidemiological
presence of flyways (e.g., in riverine vegetation), and recent eco-epidemiological data that data that
indicated the
indicated the presence
presence of E. wahlbergi
of E. wahlbergi and
and E.
E. helvum
helvum in
in the
the neighborhood
neighborhood [32].[32].

Figure
Figure 1.1. Regional
Regional map
map of
of East
East Africa
Africa with Nairobi County highlighted. WithinWithin Nairobi
Nairobi County
County
(right), we trapped
trappedforaging
foragingfruit
fruitbats
batsininwest
westand
andcentral
central Nairobi
Nairobi where
where suitable
suitable peridomestic
peridomestic hab-
habitats
werewere
itats found. BlueBlue
found. points indicate
points households
indicate where
households E. helvum
where E. helvum E. wahlbergi
and and werewere
E. wahlbergi trapped and
trapped
sampled, and green points represent households where only E. wahlbergi was trapped and sampled.
Viruses 2025, 17, 557 4 of 13

Bats were captured using two to three mist nets that were set up before sunset at
flyways within the residential property and checked every 15 min. Only E. wahlbergi and E.
helvum were retained for processing, while all other species were released at capture points.
One trap night was conducted at each site and took place between the hours of 17:00 and
24:00 or until 9 targeted bats were trapped and sampled. Bats were placed in separate clean
cloth bags to prevent cross-contamination and processed in order of capture. During bat
handling, individuals were identified with species using published keys [17]. We recorded
the body weight and forearm length of each bat and collected oropharyngeal and rectal
swabs, whole blood, and fecal and urine samples when available (IACUC #SI-22051 and
research permit WRTI-0247-11-22). All samples were directly stored in DNA/RNA Shield
(Zymo Research, Irvine, CA, USA) for sample preservation and transferred to a −80 ◦ C
freezer within 24–48 h from field collection. Bats were released following sample collection
from the processing area near the mist nets where individuals were trapped.

2.2. Sample Processing and Library Preparation

Swabs were individually processed for total nucleic acids, as described in detail in the
protocol in [33]. Briefly, the samples were lysed in Proteinase K and ATL Lysis Buffer with
Reagent DX (Qiagen, Valencia, CA, USA) with 0.1 mm zirconium oxide beads using a Bullet
Blender (Next Advance, Troy, NY, USA). The lysate was centrifuged, and the supernatant
was extracted using the IndiMag Pathogen Kit (Indical Bioscience, Leipzig, Germany)
with the KingFisher™ Flex Purification System (ThermoFisher Scientific, Waltham, MA,
USA). Nucleic acid concentrations were measured using a Qubit 4 Fluorometer following
manufacturer instructions.
Oxford Nanopore Sequencing (ONS) cDNA libraries were prepared using NEBNext
Ultra II RNA First Strand and Non-Directional RNA Second Strand Synthesis modules,
utilizing random primer mix (New England Biolabs, Ipswich, MA, USA), following DNase
treatment, as previously described [34]. Subsequently, double-stranded cDNA libraries
were generated using the NEBNext Ultra II End repair/dA-tailing Module and Quick
Ligation Modules (New England Biolabs), as well as a Ligation Sequencing Kit SQK-LSK109
(Oxford Nanopore Technologies, Oxford, UK). Cleanup and quantification were carried
out using Agencourt AMPure XP reagent (Beckman Coulter Biosciences, Indianapolis,
IN, USA) and a Qubit dsDNA HS Assay Kit (ThermoFisher Scientific). Samples were
individually barcoded with the Native Barcoding Expansion 96—EXP-NBD 196 (Oxford
Nanopore Technologies) and combined for sequencing. Sequencing libraries containing
24 barcoded pools were loaded on a single ONT R9.4.1 flow cell and run on a MinION
Mk1C device (Oxford Nanopore Technologies) for 48 h.

2.3. Data Processing and Phylogenetic Analysis

Base calling and demultiplexing were accomplished on the device with MinKNOW
operating software v21.11.7 (Oxford Nanopore Technologies) and Guppy v5.1.13 [35].
Raw reads were trimmed with Porechop to remove adapter sequences and then filtered
with NanoFilt to remove reads with q-scores ≤9 and read lengths ≤100 bp [35,36]. Bat
genomes were further removed using Minimap2 v2.24 and Samtools v1.9 [37,38]. The
processed data were aligned to the National Center for Biotechnology Information (NCBI)
non-redundant (NR) database using DIAMOND v2.0.14 and visualized using MEGAN6
(v6.25.9) [39,40]. The Flye assembler, developed for long single-molecule sequencing reads,
was used for de novo assemblies [41]. Sequences were handled using Geneious Prime
(v.2024.0.4) (Biomatters Ltd., Auckland, New Zealand). The BLASTn (2.13.0) and BLASTp
(2.13.0) algorithms were used for similarity searches in the NCBI database [42]. CLUSTALW
(2.0.11) was used for sequence alignment and pairwise comparisons [43]. Protein domain
Viruses 2025, 17, 557 5 of 13

and motif searches were performed using the NCBI conserved domain search tool and
MOTIF Search in the PFAM database [44,45].
Phylogenetic analysis was performed on sequences using IQ-TREE 2 and MEGA [46,47].
In IQ-TREE, the optimal evolutionary models and partitioning schemes were deter-
mined for amino acid sequence alignment using the automatic model selection tools
(-mMFP+MERGE). Amino acid models were restricted to those designed for viral se-
quences (-msub viral). A 70% majority-rule consensus tree was constructed by maximum
likelihood using 1000 replicates from the ultrafast bootstrap approximation approach (UF-
Boot) [48]. The UFBoot support values are more unbiased than normal bootstrap support,
and significant clade support is considered at ≥95% [48,49]. Standard bootstrap analysis
was carried out using MEGA v11.0.13 [47] for 500 replicates. The optimal analysis models
were selected using the built-in “Find Best DNA/protein-substitution model” tools. Maxi-
mum likelihood trees based on nucleotide sequences were constructed using the General
Time Reversible (GTR) model with a discrete Gamma distribution (+G) (ORF1a) and the
General Time Reversible (GTR) model with a discrete Gamma distribution and invariant
sites (+G+I) (ORF1b). Potential genetic exchange and recombination events were assessed
using the RDP, GENECONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN, and 3SEQ
tools in automated and manual analyses with default settings [50]. BOOTSCAN plots were
generated using SimPlot (version 3.5.1) [51].

3. Results
A total of 49 rectal swabs were processed from two fruit bat species, Wahlberg’s
epauletted fruit bats (E. wahlbergi, 43/49, 87.7%) and straw-colored fruit bats (E. helvum,
6/49, 12.2%). Viral contigs were generated by de novo assembly from a single E. wahlbergi
sample (1/49, 2.0%). Through the remapping of reads to assembled contigs and producing
alignments, a single virus contig was generated that revealed the identities of several
Nobecoviruses from Africa in BLAST queries.

3.1. Genome Annotation and Phylogenetic and Recombination Analyses

The virus contig (referred to as NRB24) comprised 21,973 nucleotides, covering 75–76%
of bat Nobecovirus genomes. Seven open reading frames (ORFs) were identified, includ-
ing partial ORF1a (1−7099) and complete ORF1b (7099–15,114), spike (S) glycoprotein
(15,086−18,910), ORF3 (18,911–19,609), envelope (E) protein (19,609−19,836), membrane
(M) glycoprotein (19,841−20,509), and nucleocapsid (N) protein (20,564−21,973, Figure S1).
As observed in coronaviruses, ORF1a and ORF1b partially overlap in NRB24, where ORF1b
is in the −1 reading frame relative to the ORF1a stop codon (7097−7099), enabling the
expression of ORF1b-encoded protein expression by cis-acting RNA elements that direct a
fraction of elongating ribosomes to slip (programmed ribosomal frameshifting) [52].
Pairwise comparisons of individual ORFs with Nobecoviruses from Africa revealed
identities of up to 77.6% and 91.9% in nucleotide and putative amino acid sequences,
respectively (Table S1). Maximum likelihood analysis based on complete putative ORF1a
and ORF1b nucleotide and amino acid sequences showed that NRB24 formed a separate
subclade, distinct from previously described Nobecovirus clades, with strong bootstrap
support (Figures 2 and S2) [53]. Similar tree topologies were observed in maximum like-
lihood trees constructed using S, ORF3, and N putative amino acid alignments as well
(Figure S3). In each tree, NRB24 remained distinct, sharing a common ancestor with the
African Nobecovirus clade, which includes all geographically related virus genomes. More-
over, a comparable tree topology was observed during the analysis of a shorter nucleotide
fragment encoding for virus RNA-dependent RNA polymerase (RdRp) (Figure 3), which
was documented to delineate major Nobecovirus clades [53]. However, the separate group-
 nucleotide fragment encoding for virus RNA-dependent RNA polymerase (RdRp) (Figure
3), which was documented to delineate major Nobecovirus clades [53]. However, the sep-
nucleotide
arate fragment
grouping encoding
of NRB24 wasfor
notvirus RNA-dependent
apparent RNA polymerase
in the maximum likelihood (RdRp) (Figure
trees based on
Viruses 2025, 17, 557 3), whichEwas
putative anddocumented toalignments,
M amino acid delineate major Nobecovirus
presumably clades
due to their [53]. However,
relatively thesizes
limited sep-
6 of 13
arate grouping
(Figure S3). of NRB24 was not apparent in the maximum likelihood trees based on
putative E and M amino acid alignments, presumably due to their relatively limited sizes
ing of NRB24
(Figure S3). was not apparent in the maximum likelihood trees based on putative E and
M aminoA acid alignments, presumably due to their relatively limited
B sizes (Figure S3).

A B

Figure 2. The maximum likelihood consensus tree of coronavirus ORF1a ((A): 2419 amino acids)
and ORF1b ((B): 2528 amino acids) sequences. The trees were constructed using 1000 replicates.
Figure 2. The maximum likelihood consensus tree of coronavirus ORF1a ((A): 2419 amino acids) and
Figure 2. achieving
Branches The maximum≥95% likelihood consensus
bootstrap support aretree of coronavirus
annotated with redORF1a ((A): 2419
dots. Viruses are amino acids)
indicated by
ORF1b ((B): 2528 amino acids) sequences. The trees were constructed using 1000 replicates. Branches
and ORF1b
GenBank ((B): 2528
accession, amino
name, andacids) sequences.
isolate The
identifier. trees and
NRB24 wererelated
constructed
virus using 1000
genomes arereplicates.
marked.
achieving ≥95% bootstrap support are annotated with red dots. Viruses are indicated by GenBank
Branches
SARS-CoV-2achieving
accession, name, ≥95%
Wuhan-Hu-1
and bootstrap
isolate support
(Sarbecovirus)
identifier. areand
and
NRB24 annotated
related with
Hipposideros red dots. Viruses
coronaviruses
virus genomes are indicated
are CD36/PDF0663
marked. by
(Hi-
SARS-CoV-2
GenBank
becovirus)accession,
Wuhan-Hu-1 name,
included asand
were(Sarbecovirus) isolate
outgroups
and identifier.
as needed.
Hipposideros NRB24 and related
coronaviruses virus genomes
CD36/PDF0663 are marked.
(Hibecovirus) were
SARS-CoV-2 Wuhan-Hu-1
included as outgroups (Sarbecovirus) and Hipposideros coronaviruses CD36/PDF0663 (Hi-
as needed.
becovirus) were included as outgroups as needed.

Figure 3. The maximum likelihood consensus tree of the coronavirus RNA-dependent RNA poly-
merase (RdRp) sequences (256 nucleotides). The trees were constructed using 1000 replicates.
Branches achieving ≥95% bootstrap support are annotated with red dots. Viruses are indicated
by GenBank accession, name, and isolate identifier. Previously described Nobecovirus clades [53] are
marked. SARS-CoV-2 Wuhan-Hu-1 (Sarbecovirus) and bat coronavirus Zhejiang2013 (Hibecovirus)
were included as outgroups.
 Branches achieving ≥95% bootstrap support are annotated with red dots. Viruses are indicated by
GenBank accession, name, and isolate identifier. Previously described Nobecovirus clades [53] are
Viruses 2025, 17, 557 marked. SARS-CoV-2 Wuhan-Hu-1 (Sarbecovirus) and bat coronavirus Zhejiang2013 (Hibecovirus)
7 of 13
were included as outgroups.

We carried
We carriedout
outfurther
furtherininsilico
silicoanalyses
analysesofofcomplete
completeORFORF sequences
sequences to to detect
detect poten-
potential
tial recombinations in Nobecoviruses from Africa, including NRB24. Probable
recombinations in Nobecoviruses from Africa, including NRB24. Probable recombinations recombina-
tions involving
involving multiple
multiple spots spots were detected
were detected using using all tools.
all tools. In theIn the BOOTSCAN
BOOTSCAN plot, re-
plot, recombi-
combination signals were observed to involve all ORFs but the S protein (Figure
nation signals were observed to involve all ORFs but the S protein (Figure 4), suggesting 4), sug-
gesting coinfections
coinfections and intra-host
and intra-host genetic exchange
genetic exchange driving genome
driving genome diversification.
diversification.

Figure 4. BOOTSCAN plot of African bat Nobecovirus genomes. The alignment encompasses
Figure 4. BOOTSCAN plot of African bat Nobecovirus genomes. The alignment encompasses 15096
15096 positions with the corresponding ORFs plotted on top. The plot was prepared within a sliding
positionsofwith
window 200 the
bp, corresponding ORFs
wide-centered on the plotted
positionon top. The
plotted, plot
with was
a 20 bpprepared
step size,within a sliding
for 1000 win-
replications
dow of 200on,
(GapStrip: bp,neighbor-joining,
wide-centered on the2.0),
T/t: position plotted,
with the with a 20 bp
bat coronavirus step size, for
CMR704-P12 1000 replications
genome (NC048212,
(GapStrip: on, neighbor-joining, T/t: 2.0), with the bat coronavirus CMR704-P12 genome
Eidolon helvum, Cameroon) as the query. Refer to Table 1 for a description of genomes in groups(NC048212,
Eidolon Kenya2024
labeled helvum, Cameroon)
(NRB24),as the query.Madagascar2018,
Kenya2006, Refer to Table 1 and
for aCameroon2013.
description of genomes in groups
labeled Kenya2024 (NRB24), Kenya2006, Madagascar2018, and Cameroon2013.
3.2. Analysis of Putative Viral Proteins
We examined the putative amino acid sequences of ORF1a (2365), ORF1b (2579),
S glycoprotein (1274), ORF3 (232), E protein (75), M glycoprotein (222), and N protein (469).
The ORF1a protein was partially identified with the virus non-structural protein (Nsp), and
3–10 conserved domains were identified (Table S2). Similarly, the complete ORF1b revealed
conserved motifs of the RdRp catalytic core, as well as Nsp13–16, with various functions
required for virus replication. The domains of the main coronavirus structural proteins, S,
E, M, and N were further observed in the NRB24 contig (Table 1).
In coronaviruses, the S protein is critical for host range and virulence, as it mediates
target cell attachment and the membrane fusion required for virus entry [27]. We identified
the S1-S2 subunits, the S1/S2 and furin cleavage sites, the fusion peptide region, and
associated motifs in NRB24 (Figure 5). Like other Nobecoviruses, NRB24 lacked the SARS-
CoV-2 receptor binding motif and shared S1/S2 and furin cleavage sites and fusion peptide
markers with Nobecoviruses.
Viruses 2025, 17, 557 8 of 13

Table 1. Comparative topology and conserved domains identified in complete regions in NRB24 and closely related virus genomes.

Epomophorus Eidolon Rousettus

Host Eidolon helvum Rousettus madagascariensis
wahlbergi helvum aegyptiacus
Location, Year Kenya, 2024 Kenya 2006 Kenya 2006 Cameroon, 2013 Madagascar, 2018
Isolate NRB24 KY24 KY06 CMR900 CMR66 CMR891-892 CMR705-P13 CMR704-P12 MIZ178 MIZ240
GenBank Accession PQ179293 HQ728482.1 HQ728483.1 MG693169.1 MG693170.1 MG693171.1 MG693172.1 NC_048212.1 OK067320.1 OK067321.1
Size 2579 2579 2588 2579 2588 2579 2579 2579 2579 2579
Catalytic core—RNA
1–823 1–823 1–823 1–823 1–823 1–823 1–823 1–823 1–823 1–823
polymerase (477363)
Nsp13—zinc-binding
824–918 824–918 824–918 824–918 824–918 824–918 824–918 824–918 824–918 824–918
domain (cl41714)
Nsp13—stalk (cl41715) 922–969 922–969 922–969 922–969 922–969 922–969 922–969 922–969 922–969 922–969
Nsp13—1B domain (cl41715) 973–1051 973–1051 973–1051 973–1051 973–1051 973–1051 973–1051 973–1051 973–1051 973–1051
ORF1b Nsp13—helicase domain (cl41748) 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413 1074–1413
Nsp14 (cl40464) 1428–1950 1428–1951 1428–1952 1428–1951 1428–1952 1428–1951 1428–1951 1428–1951 1428–1951 1428–1951
Nsp15—N terminal
1954–2014 1954–2014 1955–2015 1954–2014 1955–2015 1954–2014 1954–2014 1954–2014 1954–2014 1954–2014
domain (cl40469)
Nsp15—M domain (cl41717) 2018–2146 2018–2146 2019–2143 2018–2146 2019–2143 2018–2146 2018–2146 2018–2146 2018–2146 2018–2146
Nsp15—endoribonuclease domain (cl41718) 2144–2292 2144–2292 2141–2289 2144–2292 2141–2289 2144–2292 2144–2292 2144–2292 2144–2292 2144–2292
Nsp16—methyltransferase (461919, cl41719) 2297–2578 2323–2537 2297–2578 2323–2537 2297–2578 2323–2537 2323–2537 2323–2537 2297–2578 2323–2537
Size 1274 1264 1278 1273 1278 1271 1269 1269 1256 1265
S1—N terminal domain (cd21627) 30–323 33–318 36–327 33–326 36–327 33–325 31–322 31–322 34–312 42–321
S Receptor binding domain (cl09656) 367–521 362–516 371–521 370–528 371–521 369–523 366–524 366–524 356–511 365–519
S1/S2 cleavage + S2 fusion domain (cd22381) 538–1268 533–1258 538–1273 545–1267 538–1272 540–1265 541–1263 541–1263 528–1250 536–1259
ORF3 Size 232 238 220 238 220 238 239 239 238 238
Size 75 75 75 75 75 75 75 75 75 75
E Envelope protein (cl40474) - - - - - - 4–63 4–63 4–63 4–63
Size 222 221 222 221 223 221 221 221 221 221
M Matrix protein (cl40475) 11–222 5–222 7–222 8–221 7–223 5–221 10–221 10–221 8–221 8–221
Size 469 467 468 468 468 467 470 470 467 467
N Nucleocapsid protein (cl47612) 53–396 53–374 54–375 54–375 54–395 54–375 54–375 54–375 53–374 53–374
Locations are provided according to the individual viral proteins. NCBI accessions are provided in parentheses.
 fied the S1-S2 subunits, the S1/S2 and furin cleavage sites, the fusion peptide region, and
associated motifs in NRB24 (Figure 5). Like other Nobecoviruses, NRB24 lacked the SARS-
CoV-2 receptor binding motif and shared S1/S2 and furin cleavage sites and fusion pep-
Viruses 2025, 17, 557 tide markers with Nobecoviruses. 9 of 13

RBD s u pe r f a mi l y / d o ma i n
36 13 5
NC0 4 5 5 12 SARS- Co V2 - Wu h a n- Hu 1 RVQPTESI VRFPNI TNLCPFGEVFNATRFASVYAWNRKRI SNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVI RGDEVRQI APGQTGKI
HQ7 2 8 4 82 Ba t c o r o n a vi r u s KY2 4 RSQP- SAI VRLSSADGI CNVNYTHFLR- PPLPFYWRRFQI KSCKFDFNTLVSALPTYDLWCKGI SPERLGSMCFGAVTLDSMLI NTTHYNDLKSNVPDAF
HQ7 2 8 4 83 Ba t c o r o n a vi r u s KY0 6 RAQV- DGFVRVTQRGAYCRPPYNALLD- PPQPVVWRRFMLYDCAFDFSVVI DNLPTHQLQCYGI SPRRLASMCYSSVTI DVMRI NATHLNNLLNRVPDSF
MG6 9 3 1 69 Ba t c o r o n a vi r u s CMR9 0 RSSP- VDTVRI TNSAKACSVPYNLLET- PPLPFVWKRHSI SNCKFDFQALLSHLPSFQLRCYGI SPTKLSSMCFGTVSLDVMLVNVTHYNNLLNDVPDDF
MG6 9 3 1 70 Ba t c o r o n a vi r u s CMR6 6 RAQV- DGFVRVTQRGDYCQPPYADLLE- PPQPVVWRRFMLYNCAFDFSVVI DNLPTHQLQCYGI SPKRLASMCYSSVTI DVMRI NATHLNNLLNRVPDSF
MG6 9 3 1 71 Ba t c o r o n a vi r u s CMR8 9 1 RSQP- TTTVRLTSADGI CNVNYTHFVK- PPLPFYWRRFQI KSCKFDFNALVSALPTYDLWCTGI SPERLGSMCFGAVTLDSMLI NI THYNDFKANVPDAF
MG6 9 3 1 72 Ba t c o r o n a vi r u s CMR7 0 5 RSSP- TDNVRI TNSASSCSVPYSVLSR- PPLPFVWKRYAI SNCKFDFQALFSHLPTFQLRCFGI SPTKLATMCFGTVTLDI MLVNVTHYNNLLNDVPDDF
NC0 4 8 2 12 Ba t c o r o n a vi r u s CMR7 0 4 RSSP- TDNVRI TNSASSCSVPYSVLSR- PPLPFVWKRYAI SNCKFDFQALLSHLPTFQLRCFGI SPTKLATMCFGTVTLDI MLVNVTHYNNLLNDVPDDF
OK0 6 7 3 20 Ba t c o r o n a vi r u s MI Z1 7 RSQP- ASMVRLTSESEI CAVNYTTFLN- PPLPFYWRRYQI RGCKFDFNSLI TQLPTHDLWCKGI SPERLASMCFGAVTLDSMLI NVTHYSDLKANVPDPF
OK0 6 7 3 21 Ba t c o r o n a vi r u s MI Z2 4 RTQP- ASTVRLTSSDGLCNI NYTHFI E- PPLPFYWKRFQI KRCKFDFNSLVSALPTHDLWCHGI SPERLGSMCFGAVTLDSMLI NVTHYNDFKANVPDPF
NRB2 4 Ke n y a 2 0 2 4 RSQP- SAVVRLSSVASTCAVNYNLFKN- PPLPFWWRRNQI KGCKFDFHPQLQEFPTHDLWCKGI SPEKLGSMCYGSVTLDSMLI NVTHYSNFKANVPDPF

Re c e p t o r b i n d i n g mo t i f
13 6 235
NC0 4 5 5 12 SARS- Co V2 - Wu h a n- Hu 1 ADYNYKLPDDFTGCVI AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDI STEI YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELL
HQ7 2 8 4 82 Ba t c o r o n a vi r u s KY2 4 TRFNYKLPTNFYGCVHTYYLN- DSSFNYAVVND- - - PGWFQEI RPGGRPDNPGYLNTLAYKAASCNE- - - - - - - - - - - - - - - - - - - - KCFGMI VI SLTI A
HQ7 2 8 4 83 Ba t c o r o n a vi r u s KY0 6 SLYNYAVPDDFYGCLHAFYLN- - STTAYAVANQ- - - - - - - FRI NPGGRQSNSAYI ATVLNAAEYAKSY- - - - - - - - - - - - - - - - - - - FMYGLAVI TLKPA
MG6 9 3 1 69 Ba t c o r o n a vi r u s CMR9 0 SLYNYQLPRNFYGCLHSYYLPNSTTFNYI VASRSQSPSWVYNI SPGGRQPPSSFLNTLPGSAKPCQG- - - - - - - - - - - - - - - - - - - - SCLGLAVI SLSVA
MG6 9 3 1 70 Ba t c o r o n a vi r u s CMR6 6 SLYNYAVPDNFYGCLHAFYLN- - STTAYAVANR- - - - - - - FPI KPGGRQSNRDYI ATVLNAAHYTTFS- - - - - - - - - - - - - - - - - - - YI FGLAVI TLKPA
MG6 9 3 1 71 Ba t c o r o n a vi r u s CMR8 9 1 TRFNYKLPVNFYGCVHTYYLN- DTSFNYAVVNN- - - PSWSPEI KPGGRPNNAGYLSTI AYNAPSCNG- - - - - - - - - - - - - - - - - - - - KCLGMI VI SLTI A
MG6 9 3 1 72 Ba t c o r o n a vi r u s CMR7 0 5 SLYNYQLPRNFYGCLHSYYLPNDTAFSYTVASRI RYPSWVHSI TPGGRQPVGPFLDSLQSSSKPCTG- - - - - - - - - - - - - - - - - - - - SCLGLAVI SLSI A
NC0 4 8 2 12 Ba t c o r o n a vi r u s CMR7 0 4 SLYNYQLPRNFYGCLHSYYLPNDTAFSYTVASRI RYPSWVHSI TPGGRQPVGPFLDSLQSSSKPCTG- - - - - - - - - - - - - - - - - - - - SCLGLAVI SLSI A
OK0 6 7 3 20 Ba t c o r o n a vi r u s MI Z1 7 TRYNYKLPVDFYGCVHTYYLSNSTLFKYTVVNN- - - PHWFAEVVPGGRPNNDAFLATLEASATPCDG- - - - - - - - - - - - - - - - - - - - RCYGMVVI SLTI A
OK0 6 7 3 21 Ba t c o r o n a vi r u s MI Z2 4 TRFNYKLPTNFYGCVHTYYLN- DSSLKYVVVND- - - PHWFQEI RPGGRPDNSGYLATLVSNATSCNG- - - - - - - - - - - - - - - - - - - - KCLGMI VI SLTVA
NRB2 4 Ke n y a 2 0 2 4 TRFNYKLPNNFYGCVHTYYLN- GTSFKYDVAYK- - - NGYI SEI SPGGRPDNTLLLNTLAASARSCED- - - - - - - - - - - - - - - - - - - - KCYGMVVI SLTI P

23 6 258
NC0 4 5 5 12 SARS- Co V2 - Wu h a n- Hu 1 HAPATVCGPKKSTNLVKNKCVNF
HQ7 2 8 4 82 Ba t c o r o n a vi r u s KY2 4 SGNTMVCPVGNDTNVI SGVCVNY
HQ7 2 8 4 83 Ba t c o r o n a vi r u s KY0 6 SGNKLVCPI ANDTDI I TDRCVQY
MG6 9 3 1 69 Ba t c o r o n a vi r u s CMR9 0 SANKLVCPVGNDTDI VTGNCVNY
MG6 9 3 1 70 Ba t c o r o n a vi r u s CMR6 6 SGNKLVCPI ANDTDI I TERCVQY
MG6 9 3 1 71 Ba t c o r o n a vi r u s CMR8 9 1 SGNTMVCPI GNDTDVTQDVCVNY
MG6 9 3 1 72 Ba t c o r o n a vi r u s CMR7 0 5 SANKLVCPVGNDTDI VPDTCVNY
NC0 4 8 2 12 Ba t c o r o n a vi r u s CMR7 0 4 SANKLVCPVGNDTDI VPDTCVNY
OK0 6 7 3 20 Ba t c o r o n a vi r u s MI Z1 7 SGAAMVCPVGNDTSVVEGTCVNY
OK0 6 7 3 21 Ba t c o r o n a vi r u s MI Z2 4 SGNTMVCPI GNDTALLPDVCVNY
NRB2 4 Ke n y a 2 0 2 4 AGDTMVCPI GNDTTLI TDVCVN

S1 / S2 c l e a va g e r e g i on Fu s i o n p e p t i d e
38 9 426 51 3 55 0
NC0 4 5 5 12 SARS- Co V2 - Wu h a n- Hu 1 ASYQTQTNSPRRARSVASQSI I AYTMSLGAENSVAYSN DFGGFNFSQI LPDP- - - - SKPSKRSFI EDLLFNKVTLADAGF
HQ7 2 8 4 82 Ba t c o r o n a vi r u s KY2 4 LR- PTV- SARTLGGESMLELVLYDPLYD- - SLVPI TPV YTGDFNFTSLVGCLGTSCNQNSYRSALSDLLFNKVSVADPGF
HQ7 2 8 4 83 Ba t c o r o n a vi r u s KY0 6 LI NDTTVAVARAAGLPRLYLVNYDPLYDNNSATPMTPV FGGDYNFTGLMGCLGPNCGATTYRSALSDLLYDKVKI TDPGF
MG6 9 3 1 69 Ba t c o r o n a vi r u s CMR9 0 LQ- PSTI SRQGS- - - - TLLLVTYDPLSD- - TLTPI TPV YTGDFNFTSLVGCVGTDCDSKSYRSALSDLLFNKVSVADPGF
MG6 9 3 1 70 Ba t c o r o n a vi r u s CMR6 6 LI NDTTVAI AHAVGLPKLYLVNYDPLYDNNSATPI TPV FGGDYNFTGLMGCLGPNCGATTYRSALSDLLYDKVKI TDPGF
MG6 9 3 1 71 Ba t c o r o n a vi r u s CMR8 9 1 LR- PPQ- GARSMGDTSMLELVLYDPLYD- - SLVPI TPV YTGDFNFTSLVGCLGI SCNQNSYRSALSDLLFNKVSVADPGF
MG6 9 3 1 72 Ba t c o r o n a vi r u s CMR7 0 5 LQ- PPLI KGFEA- - - - TLSLVTYNPLAD- - SLTPI TPV YTGDFNFTSLVGCI GTDCDSKSHRSALSDLLFSKVSVADPGF
NC0 4 8 2 12 _ Ba t c o r o n a vi r u s CMR7 0 4 LQ- PSLI KGFEA- - - - TLSLVTYNPLAD- - SLTPI TPV YTGDFNFTSLVGCI GTDCDSKSHRSALSDLLFSKVSVADPGF
OK0 6 7 3 20 Ba t c o r o n a vi r u s MI Z1 7 LR- PPLTRSSAGG- - - LLELVTYDPLYE- - SLVPI TPV YTGDFNFTSLVGCLGTNCGANSYRSALSDLLFNKVSVADPGF
OK0 6 7 3 21 Ba t c o r o n a vi r u s MI Z2 4 LR- PSR- SRSSS- - SNLLELVLYDPLYD- - ALVPI TPV YTGDFNFTSLVGCLGTSCNQNSYRSAI SDLLFNKVSVADPGF
NRB2 4 Ke n y a 2 0 2 4 LR- PHNNTVRAMGASSMLELVRYDPLYG- - SVVPI TPV YTGDFNFTSLVGCLGTSCNTNSYRSSLSDLLFNKVSVADPGF
Fu r i n c l e a v a g e s i t e

Figure 5. Alignment of domains involved in receptor binding of coronavirus S (spike) protein. Amino
acid positions
Figure are provided
5. Alignment according
of domains to the in
involved SARS-CoV2 Wuhan-Hu-1
receptor binding strain. Conserved
of coronavirus residues
S (spike) are
protein.
highlighted (RBD domain, S1/S2 cleavage site, and fusion peptide: yellow; receptor
Amino acid positions are provided according to the SARS-CoV2 Wuhan-Hu-1 strain. Conserved binding motif
and furin cleavage site: cyan).
residues are highlighted (RBD domain, S1/S2 cleavage site, and fusion peptide: yellow; receptor
binding motif and furin cleavage site: cyan).
4. Discussion
Here, we report a partial Betacoronavirus genome representing a distinct Nobecovirus
subclade related to African Nobecoviruses, detected in a rectal swab from a Wahlberg’s
epauletted fruit bat (E. wahlbergi) from Nairobi, Kenya. Tentatively named NRB24, the par-
tial viral contig—covering >75% of Nobecovirus genomes—was generated by untargeted
metagenome screening. The contig encompassed viral structural proteins (S, E, M, and N),
as well as regions encoding for the core replication enzymes and main co-factors encoded
by ORF1a and ORF1b. In phylogenetic analyses, it emerged as a novel Nobecovirus sub-
clade, sharing a common ancestor with the Eidolon/Rousettus Nobecovirus subclade from
Africa. We detected several functional domains on the NRB24 putative structural and non-
structural proteins. Further analysis of the S protein revealed shared markers and cleavage
sites with Nobecoviruses. We did not detect RNA derived from NRB24 in oral swabs from
the same individual bat, suggesting probable tropism for gastrointestinal tissue.
Zoonotic capacity in particular subgenera, such as Sarbecoviruses (including SARS-
CoV and SARS-CoV-2) and Merbecoviruses (including MERS-CoV) has been documented
and caused spillover events with a significant health impact [28,29]. So far, no zoonotic
potential has been recognized in Nobecoviruses, which are described exclusively in fruit
bats of Pteropodidae [27,31]. Moreover, scarce information is available on Nobecovirus
cell receptors and target cells. Compared to other bat-associated Sarbecovirus and Mer-
becoviruses, Nobecoviruses have been observed to infect fewer bat host species, which
Viruses 2025, 17, 557 10 of 13

might indicate host specificity toward Rousettus and Eonycteris fruit bats (family Pteropo-
didae) [31,53]. Importantly, our findings describe another Pteropodidae species that can
harbor Nobecoviruses.
All Pteropodidae species are distributed in tropical and subtropical areas of the Old
World and have been documented to harbor zoonotic viruses, including the Ebola, Mar-
burg, Hendra, and Nipah viruses [31,54]. Despite a lack of direct evidence of Nobe-
coviruses, examples of virus spillover to humans and recombination events have been
documented [55,56], highlighting the potential of viruses hosted by Pteropodidae species.
In this study, we documented potential recombination events among various Nobecoviruses
of African origin, including the newly described NRB24, despite lacking the S protein,
which is crucial for the host range. As exemplified by SARS-CoV and MERS-CoV, re-
combinations facilitate direct bat-to-human spillover and cross-species emergence via
intermediary bridge hosts [28,29,57]. The potential of Nobecovirus to gain zoonotic po-
tential through recombinations should not be underestimated, particularly when fruit bat
hosts share habitats with potential intermediate hosts like livestock, as with our sites. It
is imperative to continue active surveillance of fruit bat species in the Afrotropics and
tropical and subtropical regions of Southeast Asia for emerging corona- and other viruses
and to better understand their diverse virome. Despite challenges in identifying adequate
bat habitats on private properties in a city environment, limiting our sample size, this
study provided novel viral sequences obtained in a non-invasive manner from urban bats,
contributing to our knowledge of Nobecoviruses and their bat host associations.

Supplementary Materials: The following supporting information can be downloaded at

https://www.mdpi.com/article/10.3390/v17040557/s1: Figure S1: NRB24 genome annotation of
seven open reading frames and genome coverage. Figure S2: Maximum likelihood tree of coron-
aviruses ORF1a (7472 nucleotides) and ORF1b (8033 nucleotides). Figure S3: Maximum likelihood
consensus tree of coronavirus S (3949 amino acids), E (75 amino acids), ORF3 (266 amino acids), M
(215 amino acids), and N (477 amino acids) sequences. Table S1: Pairwise comparison of nucleotide
and deduced amino acid sequences of NRB24 and related Nobecovirus ORFs. Table S2: Conserved
domains identified in NRB24 partial ORF1a putative amino acid sequence (n = 2365).

Author Contributions: Wildlife specimen collection and processing: M.C.V., P.W.W., E.K. and A.M.;
Laboratory analysis: M.K., J.M., R.L. and G.O.O.; Data processing and interpretation: K.E. and B.P.B.;
Manuscript drafting: K.E. and M.C.V.; Study design, M.C.V., J.H., E.M.F., J.A.S., M.E.C., Y.-M.L. and
S.M.; Administrative processing: Y.-M.L., E.G.M., N.L.A., L.J. and J.P.G. All authors have read and
agreed to the published version of the manuscript.

Funding: Sample collection was supported by the Smithsonian Institution’s George E. Burch Fel-
lowship funding. This work was supported by the US Army Medical Research and Development
Command under Contract Nos. W81XWH-21-C-0001, W81XWH-22-C-0093, and HT9425-23-C-0059.
The views, opinions, and/or findings contained in this report are those of the author(s) and should
not be construed as an official Department of the Army or Navy position, policy, or decision unless
so designated by other documentation. The opinions or assertions contained herein are the private
views of the authors and are not to be construed as official or as reflecting the true views of the
Departments of the Army, Navy, or Defense. Material contained within this publication has been
reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation
and/or publication. This manuscript was prepared while K.E. and B.P.B. held National Research
Council (NRC) Research Associateship Awards at the Walter Reed Biosystematics Unit through the
Walter Reed Army Institute of Research, Silver Spring, MD, USA. Field sites for this project were
established through the Urban Zoo project, supported by the UK Medical Research Council, the
Biotechnology and Biological Science Research Council (UK), the Economic and Social Research
Council (UK), and the Natural Environment Research Council (UK) through the Environmental
& Social Ecology of Human Infectious Diseases Initiative (ESEI), Grant Reference: G1100783/1.
Viruses 2025, 17, 557 11 of 13

This work also received support from the CGIAR One Health initiative “Protecting Human Health
Through a One Health Approach”, which was supported by contributors to the CGIAR Trust Fund
(https://www.cgiar.org/funders/, accessed on 1 February 2025).

Institutional Review Board Statement: The study protocol was approved by the National Zoological
Park Institutional Animal Care and Use Committee (NZP-IACUC #SI-22051) and by the Kenyan
Wildlife Research and Training Institute (WRTI-0247-11-22). All collection, sampling, and subsequent
experiments were performed in accordance with the approved protocols.

Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement: Raw sequence data generated in this study are available in the NCBI Se-
quence Read Archive (SRA) under project PRJNA1237873 and the assembled virus genome sequence
is available in GenBank under accession number PQ179293.

Acknowledgments: We want to deeply thank the household members who provided access to their
properties for bat sampling. This work would not have been possible without their generosity.

Conflicts of Interest: The authors declare no conflicts of interest.

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