Evidence for circulation of Rift Valley fever virus in wildlife and domestic animals in a forest environment in Gabon, Central Africa.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by the Rift Valley fever virus (RVFV) that can infect domestic and wild animals. Although the RVFV transmission cycle has been well documented across Africa in savanna ecosystems, little is known about its transmission in tropical rainforest settings, particularly in Central Africa. We therefore conducted a survey in northeastern Gabon to assess RVFV circulation among wild and domestic animals. Among 163 wildlife samples tested using RVFV-specific RT-qPCR, four ruminants belonging to subfamily Cephalophinae were detected positive. The phylogenetic analysis revealed that the four RVFV sequences clustered together with a virus isolated in Namibia within the well-structured Egyptian clade. A cross-sectional survey conducted on sheep, goats and dogs living in villages within the same area determined the IgG RVFV-specific antibody prevalence using cELISA. Out of the 306 small ruminants tested (214 goats, 92 sheep), an overall antibody prevalence of 15.4% (95% CI [11.5-19.9]) was observed with a higher rate in goats than in sheep (20.1% versus 3.3%). RVFV-specific antibodies were detected in a single dog out of the 26 tested. Neither age, sex of domestic animals nor season was found to be significant risk factors of RVFV occurrence. Our findings highlight sylvatic circulation of RVFV for the first time in Gabon. These results stress the need to develop adequate surveillance plan measures to better control the public health threat of RVFV.

Evidence of Transmission and Circulation of Deltacron XD Recombinant Severe Acute Respiratory Syndrome Coronavirus 2 in Northwest France.

In February 2022, samples collected in northwest France showed discordant molecular results. After virological and epidemiological investigations, 17 cases of Deltacron XD recombinant severe acute respiratory syndrome coronavirus 2 were confirmed by sequencing or suspected due to epidemiological links, showing evidence of an extended transmission event and circulation of this form, with low clinical severity.

Detection of SARS-CoV-2 in two cats during the second wave of the COVID-19 pandemic in France.

Although there are several reports in the literature of SARS-CoV-2 infection in cats, few SARS-CoV-2 sequences from infected cats have been published. In this study, SARS-CoV-2 infection was evaluated in two cats by clinical observation, molecular biology (qPCR and NGS), and serology (microsphere immunoassay and seroneutralization). Following the observation of symptomatic SARS-CoV-2 infection in two cats, infection status was confirmed by RT-qPCR and, in one cat, serological analysis for antibodies against N-protein and S-protein, as well as neutralizing antibodies. Comparative analysis of five SARS-CoV-2 sequence fragments obtained from one of the cats showed that this infection was not with one of the three recently emerged variants of SARS-CoV-2. This study provides additional information on the clinical, molecular, and serological aspects of SARS-CoV-2 infection in cats.

Report of One-Year Prospective Surveillance of SARS-CoV-2 in Dogs and Cats in France with Various Exposure Risks: Confirmation of a Low Prevalence of Shedding, Detection and Complete Sequencing of an Alpha Variant in a Cat.

Despite the probable zoonotic origin of SARS-CoV-2, only limited research efforts have been made to understand the role of companion animals in SARS-CoV-2 epidemiology. According to recent serological prevalence studies, human-to-companion animal transmission is quite frequent, which led us to consider that the risk of SARS-CoV-2 transmission from animal to human, albeit negligible in the present context, may have been underestimated. In this study, we provide the results of a prospective survey that was conducted to evaluate the SARS-CoV-2 isolation rate by qRT-PCR in dogs and cats with different exposure risks and clinical statuses. From April 2020 to April 2021, we analyzed 367 samples and investigated the presence of SARS-CoV-2 RNA using qRT-PCR. Only four animals tested positive, all of them being cats. Three cats were asymptomatic and one presented a coryza-like syndrome. We describe in detail the infection in two cats and the associated clinical characteristics. Importantly, we obtained SARS-CoV-2 genomes from one infected animal and characterized them as Alpha variants. This represents the first identification of the SARS-CoV-2 Alpha variant in an infected animal in France.

Nectar-feeding bats and birds show parallel molecular adaptations in sugar metabolism enzymes.

In most vertebrates, the demand for glucose as the primary substrate for cellular respiration is met by the breakdown of complex carbohydrates, or energy is obtained by protein and lipid catabolism. In contrast, a few bat and bird species have convergently evolved to subsist on nectar, a sugar-rich mixture of glucose, fructose, and sucrose.1-4 How these nectar-feeders have adapted to cope with life-long high sugar intake while avoiding the onset of metabolic syndrome and diabetes5-7 is not understood. We analyzed gene sequences obtained from 127 taxa, including 22 nectar-feeding bat and bird genera that collectively encompass four independent origins of nectarivory. We show these divergent taxa have undergone pervasive molecular adaptation in sugar catabolism pathways, including parallel selection in key glycolytic and fructolytic enzymes. We also uncover convergent amino acid substitutions in the otherwise evolutionarily conserved aldolase B (ALDOB), which catalyzes rate-limiting steps in fructolysis and glycolysis, and the mitochondrial gatekeeper pyruvate dehydrogenase (PDH), which links glycolysis and the tricarboxylic acid cycle. Metabolomic profile and enzyme functional assays are consistent with increased respiratory flux in nectar-feeding bats and help explain how these taxa can both sustain hovering flight and efficiently clear simple sugars. Taken together, our results indicate that nectar-feeding bats and birds have undergone metabolic adaptations that have enabled them to exploit a unique energy-rich dietary niche among vertebrates.

Interrogating Phylogenetic Discordance Resolves Deep Splits in the Rapid Radiation of Old World Fruit Bats (Chiroptera: Pteropodidae).

The family Pteropodidae (Old World fruit bats) comprises $>$200 species distributed across the Old World tropics and subtropics. Most pteropodids feed on fruit, suggesting an early origin of frugivory, although several lineages have shifted to nectar-based diets. Pteropodids are of exceptional conservation concern with $>$50% of species considered threatened, yet the systematics of this group has long been debated, with uncertainty surrounding early splits attributed to an ancient rapid diversification. Resolving the relationships among the main pteropodid lineages is essential if we are to fully understand their evolutionary distinctiveness, and the extent to which these bats have transitioned to nectar-feeding. Here we generated orthologous sequences for $>$1400 nuclear protein-coding genes (2.8 million base pairs) across 114 species from 43 genera of Old World fruit bats (57% and 96% of extant species- and genus-level diversity, respectively), and combined phylogenomic inference with filtering by information content to resolve systematic relationships among the major lineages. Concatenation and coalescent-based methods recovered three distinct backbone topologies that were not able to be reconciled by filtering via phylogenetic information content. Concordance analysis and gene genealogy interrogation show that one topology is consistently the best supported, and that observed phylogenetic conflicts arise from both gene tree error and deep incomplete lineage sorting. In addition to resolving long-standing inconsistencies in the reported relationships among major lineages, we show that Old World fruit bats have likely undergone at least seven independent dietary transitions from frugivory to nectarivory. Finally, we use this phylogeny to identify and describe one new genus. [Chiroptera; coalescence; concordance; incomplete lineage sorting; nectar feeder; species tree; target enrichment.].

Comparative phylogeography of African fruit bats (Chiroptera, Pteropodidae) provide new insights into the outbreak of Ebola virus disease in West Africa, 2014-2016.

Both Ebolavirus and Marburgvirus were detected in several fruit bat species of the family Pteropodidae, suggesting that this taxon plays a key role in the life cycle of filoviruses. After four decades of Zaire Ebolavirus (ZEBOV) outbreaks in Central Africa, the virus was detected for the first time in West Africa in 2014. To better understand the role of fruit bats as potential reservoirs and circulating hosts between Central and West Africa, we examine here the phylogeny and comparative phylogeography of Pteropodidae. Our phylogenetic results confirm the existence of four independent lineages of African fruit bats: the genera Eidolon and Rousettus, and the tribes Epomophorini and Scotonycterini, and indicate that the three species suspected to represent ZEBOV reservoir hosts (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) belong to an African clade that diversified rapidly around 8-7 Mya. To test for phylogeographic structure and for recent gene flow from Central to West Africa, we analysed the nucleotide variation of 675 cytochrome b gene (Cytb) sequences, representing eight fruit bat species collected in 48 geographic localities. Within Epomophorina, our mitochondrial data do not support the monophyly of two genera (Epomops and Epomophorus) and four species (Epomophorus gambianus, Epomops franqueti, Epomops buettikoferi, and Micropteropus pusillus). In Epomops, however, we found two geographic haplogroups corresponding to the Congo Basin and Upper Guinea forests, respectively. By contrast, we found no genetic differentiation between Central and West African populations for all species known to make seasonal movements, Eidolon helvum, E. gambianus, H. monstrosus, M. pusillus, Nanonycteris veldkampii, and Rousettus aegyptiacus. Our results suggest that only three fruit bat species were able to disperse directly ZEBOV from the Congo Basin to Upper Guinea: E. helvum, H. monstrosus, and R. aegyptiacus.

Development and characterization of 10 microsatellite markers in the Cape horseshoe bat, Rhinolophus capensis (Chiroptera, Rhinolophidae) and cross-amplification in southern African Rhinolophus species.

BACKGROUND: The Cape horseshoe bat, Rhinolophus capensis, is endemic to the Cape region of South Africa. Coalescent analysis of mitochondrial DNA sequence data suggests extensive historical gene flow between populations despite strong geographic variation of their echolocation call phenotype. Nevertheless the fine-scale genetic structure and evolutionary ecology of R. capensis remains poorly understood. Here we describe the development of 10 novel polymorphic microsatellite loci to investigate of the dispersal ecology of R. capensis and to facilitate taxonomic studies of Rhinolophus species in southern Africa. FINDINGS: We report 10 microsatellite primer pairs that consistently amplify scorable and polymorphic loci across 12 African rhinolophid species. Initial analysis of two populations of R. capensis from South Africa revealed moderate to high levels of allelic variation with 4-14 alleles per locus and observed heterozygosities of 0.450-0.900. No evidence of linkage disequilibrium was observed and eight of the loci showed no departure from Hardy-Weinberg equilibrium. Cross-species utility of these markers revealed consistently amplifiable polymorphic loci in eleven additional rhinolophid species. CONCLUSIONS: The cross-amplification success of the microsatellites developed here provides a cost-effective set of population genetic marker for the study of rhinolophid evolutionary ecology and conservation in southern Africa.

The comparative phylogeography of fruit bats of the tribe Scotonycterini (Chiroptera, Pteropodidae) reveals cryptic species diversity related to African Pleistocene forest refugia.

The hypothesis of Pleistocene forest refugia was tested using comparative phylogeography of Scotonycterini, a fruit bat tribe endemic to Africa containing four species: Scotonycteris zenkeri, Casinycteris argynnis, C. campomaanensis, and C. ophiodon. Patterns of genetic structure were assessed using 105 Scotonycterini (including material from three holotypes) collected at 37 localities, and DNA sequences from the mitochondrial cytochrome b gene (1140 nt) and 12 nuclear introns (9641 nt). Phylogenetic trees and molecular dating were inferred by Bayesian methods. Multilocus analyses were performed using supermatrix, SuperTRI, and *BEAST approaches. Mitochondrial analyses reveal strong phylogeographical structure in Scotonycteris, with four divergent haplogroups (4.9-8.7%), from Upper Guinea, Cameroon, western Equatorial Africa, and eastern Democratic Republic of the Congo (DRC). In C. argynnis, we identify two mtDNA haplogroups corresponding to western and eastern Equatorial Africa (1.4-2.1%). In C. ophiodon, the mtDNA haplotypes from Cameroon and Ivory Coast differ by only 1.3%. Nuclear analyses confirm the validity of the recently described C. campomaanensis and indicate that western and eastern populations of C. argynnis are not fully isolated. All mtDNA clusters detected in Scotonycteris are found to be monophyletic based on the nuclear dataset, except in eastern DRC. In the nuclear tree, the clade from western Equatorial Africa is closely related to individuals from eastern DRC, whereas in the mitochondrial tree it appears to be the sister-group of the Cameroon clade. Migrate-n analyses support gene flow from western Equatorial Africa to eastern DRC. Molecular dating indicates that Pleistocene forest refugia have played an important role in shaping the evolution of Scotonycterini, with two phases of allopatric speciation at approximately 2.7 and 1.6 Mya, resulting from isolation in three main forest areas corresponding to Upper Guinea, Cameroon, and Equatorial Africa. Two cryptic species and two subspecies are described herein in the genus Scotonycteris. Female philopatry and male biased dispersal are supported for the smallest taxa, i.e., the three species of Scotonycteris and C. argynnis. The Congo, Ntem, and Sanaga rivers are identified as biogeographic barriers to the dispersal of Scotonycteris during interglacial periods. A greater capacity for long-distance dispersal is inferred for the largest species, C. ophiodon.

Molecular systematics and phylogeography of the tribe Myonycterini (Mammalia, Pteropodidae) inferred from mitochondrial and nuclear markers.

The tribe Myonycterini comprises five fruit bat species of the family Pteropodidae, which are endemic to tropical Africa. Previous studies have produced conflicting results about their interspecific relationships. Here, we performed a comparative phylogeographic analysis based on 148 complete cytochrome b gene sequences from the three species distributed in West Africa and Central Africa (Myonycteris torquata, Lissonycteris angolensis and Megaloglossus woermanni). In addition, we investigated phylogenetic relationships within the tribe Myonycterini, using a matrix including 29 terminal taxa and 7235 nucleotide characters, corresponding to an alignment of two mitochondrial genes and seven nuclear introns. Our phylogenetic analyses confirmed that the genus Megaloglossus belongs to the tribe Myonycterini. Further, the genus Rousettus is paraphyletic, with R. lanosus, sometimes placed in the genus Stenonycteris, being the sister-group of the tribes Myonycterini and Epomophorini. Our phylogeographic results showed that populations of Myonycteris torquata and Megaloglossus woermanni from the Upper Guinea Forest are highly divergent from those of the Congo Basin Forest. Based on our molecular data, we recommended several taxonomic changes. First, Stenonycteris should be recognized as a separate genus from Rousettus and composed of S. lanosus. This genus should be elevated to a new tribe, Stenonycterini, within the subfamily Epomophorinae. This result shows that the evolution of lingual echolocation was more complicated than previously accepted. Second, the genus Lissonycteris is synonymised with Myonycteris. Third, the populations from West Africa formerly included in Myonycteris torquata and Megaloglossus woermanni are now placed in two distinct species, respectively, Myonycteris leptodon and Megaloglossus azagnyi sp. nov. Our molecular dating estimates show that the three phases of taxonomic diversification detected within the tribe Myonycterini can be related to three distinct decreases in tree cover vegetation, at 6.5-6, 2.7-2.5, and 1.8-1.6Ma. Our results suggest that the high nucleotide distance between Ebolavirus Côte d'Ivoire and Ebolavirus Zaire can be correlated with the Plio/Pleistocene divergence between their putative reservoir host species, i.e., Myonycteris leptodon and Myonycteris torquata, respectively.

DNA barcoding of African fruit bats (Mammalia, Pteropodidae). The mitochondrial genome does not provide a reliable discrimination between Epomophorus gambianus and Micropteropus pusillus.

Sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene have been shown to be useful for species identification in various groups of animals. However, the DNA barcoding approach has never been tested on African fruit bats of the family Pteropodidae (Mammalia, Chiroptera). In this study, the COI gene was sequenced from 120 bats collected in the Central African Republic and belonging to either Epomophorus gambianus or Micropteropus pusillus, two species easily diagnosed on the basis of morphological characters, such as body size, skull shape and palatal ridges. Two additional molecular markers were used for comparisons: the complete mitochondrial cytochrome b gene and the intron 7 of the nuclear ß-fibrinogen (FGB) gene. Our results reveal an unexpected discordance between mitochondrial and nuclear genes. The nuclear FGB signal agrees with our morphological identifications, as the three alleles detected for E. gambianus are divergent from the fourteen alleles found for M. pusillus. By contrast, this taxonomic distinction is not recovered with the analyses of mitochondrial genes, which support rather a polyphyletic pattern for both species. The conflict between molecular markers is explained by multiple mtDNA introgression events from M. pusillus into E. gambianus or, alternatively, by incomplete lineage sorting of mtDNA haplotypes associated with positive selection on FGB alleles of M. pusillus. Our work shows the failure of DNA barcoding to discriminate between two morphologically distinct fruit bat species and highlights the importance of using both mitochondrial and nuclear markers for taxonomic identification.