Rousettus Aegyptiacus Research Articles

Generating bat primary and immortalised cell-lines from wing biopsies.

Bats are becoming recognised as new model species to study naturally evolved mammalian extended healthspan and disease tolerance. However, this research is limited by the lack of bat specific cellular resources. Here we describe an optimised protocol to develop both primary and immortalised fibroblast cell-lines from wing biopsy punches from the Egyptian fruit bat, Rousettus aegyptiacus. We show that the immortalised cell lines and primary cells show similar characteristics in their proliferative capacity and response to oxidative stress. They also exhibited a similar response in their NF-κB immune response to TLR agonists including SARS-CoV2. As wing punches can be acquired non-lethally, these methods can be used to develop primary and immortalised cells, from potentially any bat species, including those of conservation concern that cannot be sacrificed. This can expand the scope of bat species that can be studied in the future, and the development of key cellular resources required to functionally validate the regulators of bats' unique longevity.

Reproductive cycle of the Egyptian Fruit Bat, Rousettus aegyptiacus (Chiroptera: Petropodidae) in northern Egypt

The reproductive patterns of both sexes of the Egyptian Fruit Bat, Rousettus aegyptiacus, were studied in a population inhabiting a roost within Giza Governorate, northern Egypt. Bat specimens were collected over a period of 13 months extending from June 2017 to June 2018, inclusive. The reproductive cycle of males consisted of four consecutive phases, namely the active phase in autumn and the early winter months (October to January), the regressing phase in late winter and early spring months (February to April), the regressed phase in late spring and summer (May to August), and the recrudescence phase in early autumn (September). No active sperms were observed in smears of the cauda epididymis during the regressed phase (June–August) and males were considered to be sexually inactive during this phase. Varying percentages of reproductively active females (pregnant, lactating, pregnant and lactating) were collected in all months, with peaks in summer (June–July) and late autumn and early winter (November–January).

Increased Susceptibility of Rousettus aegyptiacus Bats to Respiratory SARS-CoV-2 Challenge Despite Its Distinct Tropism for Gut Epithelia in Bats

Increasing evidence suggests bats are the ancestral hosts of the majority of coronaviruses. In general, coronaviruses primarily target the gastrointestinal system, while some strains, especially Betacoronaviruses with the most relevant representatives SARS-CoV, MERS-CoV, and SARS-CoV-2, also cause severe respiratory disease in humans and other mammals. We previously reported the susceptibility of Rousettus aegyptiacus (Egyptian fruit bats) to intranasal SARS-CoV-2 infection. Here, we compared their permissiveness to an oral infection versus respiratory challenge (intranasal or orotracheal) by assessing virus shedding, host immune responses, tissue-specific pathology, and physiological parameters. While respiratory challenge with a moderate infection dose of 1 × 104 TCID50 caused a systemic infection with oral and nasal shedding of replication-competent virus, the oral challenge only induced nasal shedding of low levels of viral RNA. Even after a challenge with a higher infection dose of 1 × 106 TCID50, no replication-competent virus was detectable in any of the samples of the orally challenged bats. We postulate that SARS-CoV-2 is inactivated by HCl and digested by pepsin in the stomach of R. aegyptiacus, thereby decreasing the efficiency of an oral infection. Therefore, fecal shedding of RNA seems to depend on systemic dissemination upon respiratory infection. These findings may influence our general understanding of the pathophysiology of coronavirus infections in bats.

Structural Analysis of Virus Regulatory N6-Methyladenosine (m6A) Machinery of the Black Flying Fox (Pteropus alecto) and the Egyptian Fruit Bat (Rousettus aegyptiacus) Shows Evolutionary Conservation Amongst Mammals

Background: N6-methyladenosine (m6A) is an abundant RNA epitranscriptomic modification in eukaryotes. The m6A machinery includes cellular writer, eraser and reader proteins that regulate m6A. Pteropus alecto (P. alecto) (the Australian black flying fox) and Rousettus aegyptiacus (R. aegyptiacus) (the Egyptian fruit bat) are bats associated with several viral zoonoses yet neglected in the field of m6A epigenetics studies. Objectives: This study utilises various bioinformatics and in silico tools to genetically identify, characterise and annotate the m6A machinery in P. alecto and R. aegyptiacus. Methods: A range of bioinformatic tools were deployed to comprehensively characterise all known m6A-associated proteins of P. alecto and R. aegyptiacus. Results: Phylogenetically, the m6A fat mass and obesity-associated protein (FTO) eraser placed the order Chiroptera (an order including all bat species) in a separate clade. Additionally, it showed the lowest identity matrices in P. alecto and R. aegyptiacus when compared to other mammals (74.1% and 72.8%) and Homo sapiens (84.0% and 76.1%), respectively. When compared to humans, genetic loci-based analysis of P. alecto and R. aegyptiacus showed syntenic conservation in multiple flanking genes of 8 out the 10 m6A-associated genes. Furthermore, amino acid alignment and protein tertiary structure of the two bats’ m6A machinery demonstrated conservation in the writers but not in erasers and readers, compared to humans. Conclusions: These studies provide foundational annotation and genetic characterisation of m6A machinery in two important species of bats which can be exploited to study bat–virus interactions at the interface of epitranscriptomics.

Why do bats fly into cave doors? Inattentional blindness in echolocating animals.

Echolocating bats can navigate complex 3D environments by integrating prior knowledge of spatial layouts and real-time sensory cues. This study demonstrates that inattentional blindness to sensory information undermines successful navigation in Egyptian fruit bats, Rousettus aegyptiacus , a species that has access to vision and echolocation to traverse natural environments. Bats flew over repeated trials to a perch at a fixed location in the light, allowing them to navigate using both vision and echolocation. The experiment was then repeated in the dark to exclude the bat's use of vision. The perch was subsequently displaced by either 15 or 30 cm in one of six different directions (up, down, left, right, front, back). Echolocation behavior was recorded using a 25-channel microphone array, while flight paths were tracked using 13 motion capture cameras. The directional aim of echolocation clicks served as a metric for the bat's spatial attention to locations in their environment. In the light, bats modified their flight paths to successfully land on a perch that was moved 15 cm but surprisingly, often failed to land on it when displaced by 30 cm. In the dark, bats often failed to land on the perch after it was moved by only 15 cm. Landing failures suggest that learned spatial priors invoked inattentional blindness to changes in the environment, which interfered with successful navigation. In both the light and dark, when bats failed to land on the perch at its new location, they directed their attention toward the original perch position. Performance differences in the light and dark suggest that the bat's attentional spotlight may be narrower when it relies on echolocation than vision. To our knowledge, these findings provide the first evidence of inattentional blindness in a flying echolocating animal, demonstrating that spatial priors can dominate sensory processing during navigation.

Thermomechanical properties of bat and human red blood cells—Implications for hibernation

Hibernation is a widespread and highly efficient mechanism to save energy in mammals. However, one major challenge of hibernation is maintaining blood circulation at low body temperatures, which strongly depends on the viscoelastic properties of red blood cells (RBCs). Here, we examined at physiologically relevant timescales the thermomechanical properties of hundreds of thousands of individual RBCs from the hibernating common noctule bat (Nyctalus noctula), the nonhibernating Egyptian fruit bat (Rousettus aegyptiacus), and humans (Homo sapiens). We exposed RBCs to temperatures encountered during normothermia and hibernation and found a significant increase in elasticity and viscosity with decreasing temperatures. Our data demonstrate that temperature adjustment of RBCs is mainly driven by membrane properties and not the cytosol while viscous dissipation in the membrane of both bat species exceeds the one in humans by a factor of 15. Finally, our results show that RBCs from both bat species reveal a transition to a more viscous-like state when temperature decreases. This process on a minute timescale has an effect size that is comparable with fluctuations in RBC viscoelasticity over the course of the year, implying that environmental factors, such as diets, have a lower impact on the capability of RBCs to respond to different temperatures than general physical properties of the cell membrane. In summary, our findings suggest membrane viscoelasticity as a promising target for identifying mechanisms that could be manipulated to ensure blood circulation at low body temperatures in humans, which may be one first step toward safe synthetic torpor in medicine and space flight.

Elevated vitamin D levels in diurnally-active female fruit bats

Animal species have evolved to enhance their survival by focusing their temporal activity on specific parts of the diurnal-nocturnal cycle. Various factors, including inter-specific competition and anti-predator behavior, as well as anthropogenic effects like light pollution, have prompted some species to expand or shift their temporal niches. Our study focuses on the temporal niche shift of the Egyptian fruit bat (Rousettus aegyptiacus) to diurnal activity in Israel.Through an extensive citizen-science study, we assessed the distribution of these bats’ diurnal activity across Israel. We also documented the sex and age of bats from a colony known for its diurnal activity and collected blood samples from them for metabolic analysis.Our findings indicate that the shift toward daytime activity predominantly takes place in urban settings and is mostly exhibited by females. We found a significant physiological effect of this temporal shift, namely: diurnal bats’ vitamin D levels were significantly higher, and their parathyroid hormone (PTH) levels were significantly lower than those of nocturnal bats.We suggest that the reproductive metabolic demands of female bats might be a key factor driving this shift to diurnal activity. We hypothesize that the increase in vitamin D, derived from sunlight hours, might play a crucial role in regulating calcium homeostasis, thus contributing to the bats’ physiological needs during the reproduction season.

Spatial memory obviates following behaviour in an information centre of wild fruit bats.

According to the information centre hypothesis (ICH), colonial species use social information in roosts to locate ephemeral resources. Validating the ICH necessitates showing that uninformed individuals follow informed ones to the new resource. However, following behaviour may not be essential when individuals have a good memory of the resources' locations. For instance, Egyptian fruit bats forage on spatially predictable trees, but some bear fruit at unpredictable times. These circumstances suggest an alternative ICH pathway in which bats learn when fruits emerge from social cues in the roost but then use spatial memory to locate them without following conspecifics. Here, using an unique field manipulation and high-frequency tracking data, we test for this alternative pathway: we introduced bats smeared with the fruit odour of the unpredictably fruiting Ficus sycomorus trees to the roost, when they bore no fruits, and then tracked the movement of conspecifics exposed to the manipulated social cue. As predicted, bats visited the F. sycomorus trees with significantly higher probabilities than during routine foraging trips (of >200 bats). Our results show how the integration of spatial memory and social cues leads to efficient resource tracking and highlight the value of using large movement datasets and field experiments in behavioural ecology. This article is part of the theme issue 'The spatial-social interface: a theoretical and empirical integration'.

Human infection with IsrRAPXV, a novel zoonotic bat-derived poxvirus.

Bats are recognized as the natural reservoir of several zoonotic viruses that pose a threat to public health worldwide. In our recent reports we describe the identification of a novel poxvirus, IsrRAPXV, in Egyptian fruit bats. This poxvirus is associated with high morbidity and mortality in bats. Herein, we describe the identification of poxvirus in a female patient hospitalized with systemic symptoms and severe painful skin lesions on her hands. We performed qPCR, whole genome sequencing and phylogenetic analysis to identify and characterize this poxvirus as the etiologic agent. The patient interacted with wounded and sick bats as a volunteer in a bat shelter run by the Israel bat sanctuary organization. Samples collected from the patient's skin lesions were positive for the presence of IsrRAPXV by PCR. Additionally, phylogenetic analysis showed that this virus is identical to IsrRAPXV originally described by us as the causative agent of skin lesions in fruit bats. Our finding suggest that IsrRAPXV is zoonotic and therefore veterinarians and volunteers working in bats shelter should meticulously follow the guidelines of working with bats and use required personal protective equipment.

Rousettus aegyptiacus Fruit Bats Do Not Support Productive Replication of Cedar Virus upon Experimental Challenge.

Cedar henipavirus (CedV), which was isolated from the urine of pteropodid bats in Australia, belongs to the genus Henipavirus in the family of Paramyxoviridae. It is closely related to the Hendra virus (HeV) and Nipah virus (NiV), which have been classified at the highest biosafety level (BSL4) due to their high pathogenicity for humans. Meanwhile, CedV is apathogenic for humans and animals. As such, it is often used as a model virus for the highly pathogenic henipaviruses HeV and NiV. In this study, we challenged eight Rousettus aegyptiacus fruit bats of different age groups with CedV in order to assess their age-dependent susceptibility to a CedV infection. Upon intranasal inoculation, none of the animals developed clinical signs, and only trace amounts of viral RNA were detectable at 2 days post-inoculation in the upper respiratory tract and the kidney as well as in oral and anal swab samples. Continuous monitoring of the body temperature and locomotion activity of four animals, however, indicated minor alterations in the challenged animals, which would have remained unnoticed otherwise.

Attempted Transmission of Marburg Virus by Bat-Associated Fleas Thaumapsylla breviceps breviceps (Ischnopsyllidae: Thaumapsyllinae) to the Egyptian Rousette Bat (Rousettus aegyptiacus).

Egyptian rousette bats (ERBs) are implicated as reservoir hosts for Marburg virus (MARV), but natural mechanisms involved in maintenance of MARV in ERB populations remain undefined. A number of hematophagous ectoparasites, including fleas, parasitize bats. Subcutaneous (SC) inoculation of ERBs with MARV consistently results in viremia, suggesting that infectious MARV could be ingested by blood-sucking ectoparasites during feeding. In our study, MARV RNA was detected in fleas that took a blood meal during feeding on viremic bats on days 3, 7, and 11 after SC inoculation. Virus concentration in individual ectoparasites was consistent with detectable levels of viremia in the blood of infected host bats. There was neither seroconversion nor viremia in control bats kept in close contact with MARV-infected bats infested with fleas for up to 40 days post-exposure. In fleas inoculated intracoelomically, MARV was detected up to 14 days after intracoelomic (IC) inoculation, but the virus concentration was lower than that delivered in the inoculum. All bats that had been infested with inoculated, viremic fleas remained virologically and serologically negative up to 38 days after infestation. Of 493 fleas collected from a wild ERB colony in Matlapitsi Cave, South Africa, where the enzootic transmission of MARV occurs, all tested negative for MARV RNA. While our findings seem to demonstrate that bat fleas lack vectorial capacity to transmit MARV biologically, their role in mechanical transmission should not be discounted. Regular blood-feeds, intra- and interhost mobility, direct feeding on blood vessels resulting in venous damage, and roosting behaviour of ERBs provide a potential physical bridge for MARV dissemination in densely populated cave-dwelling bats by fleas. The virus transfer might take place through inoculation of skin, mucosal membranes, and wounds when contaminated fleas are squashed during auto- and allogrooming, eating, biting, or fighting.

Jamaican fruit bat (Artibeus jamaicensis) insusceptibility to mucosal inoculation with SARS-CoV-2 Delta variant is not caused by receptor compatibility

The ancestral sarbecovirus giving rise to SARS-CoV-2 is posited to have originated in bats. While SARS-CoV-2 causes asymptomatic to severe respiratory disease in humans, little is known about the biology, virus tropism, and immunity of SARS-CoV-2-like sarbecoviruses in bats. SARS-CoV-2 has been shown to infect multiple mammalian species, including various rodent species, non-human primates, and Egyptian fruit bats. We show that SARS-CoV-2 can utilize Jamaican fruit bat (Artibeus jamaicensis) ACE2 spike for entry in vitro. Therefore, we investigate the Jamaican fruit bat as a possible in vivo model to study reservoir responses. We find that SARS-CoV-2 Delta does not efficiently replicate in Jamaican fruit bats in vivo. We observe infectious viruses in the lungs of only one animal on day 1 post-inoculation and find no evidence of shedding or seroconversion. This is possibly due to host factors restricting virus egress after aborted replication. Furthermore, we observe no significant immune gene expression changes in the respiratory tract but do observe changes in the intestinal metabolome after inoculation. This suggests that, despite its broad host range, SARS-CoV-2 is unable to infect all bat species, and Jamaican fruit bats are not an appropriate model to study SARS-CoV-2 reservoir infection.

Neural representation of human experimenters in the bat hippocampus

Here we conducted wireless electrophysiological recording of hippocampal neurons from Egyptian fruit bats in the presence of human experimenters. In flying bats, many neurons modulated their activity depending on the identity of the human at the landing target. In stationary bats, many neurons carried significant spatial information about the position and identity of humans traversing the environment. Our results reveal that hippocampal activity is robustly modulated by the presence, movement and identity of human experimenters.

Leptospira infection in bats in Vietnam

Bats from three provinces in Vietnam (Lai Chau, Son La, and Dong Thap) were examined for the presence of pathogenic Leptospira or specific antibodies using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and microscopic agglutination test (MAT). Tissue specimens from 298 bats belonging to 11 species were analyzed using a real-time PCR assay specific for leptospires of pathogenic species. Leptospiral DNA was identified in 40 bats from following species: Rousettus amplexicaudatus (5/9; 55.5 %), Rousettus leschenaultii (17/42; 40.4 %), Myotis hasseltii (8/25; 32 %), Taphozous longimanus (3/12; 25 %), and Eonycteris spelaea (7/32; 21.9 %). Based on secY phylogeny, sequences from M. hasseltii bore a strong resemblance to L. borgpetersenii. Sequences from other species revealed unique lineages: one of them resembled Leptospira sp., previously identified in Rousettus madagascariensis (Madagascar) and Rousettus aegyptiacus (South Africa); the second lineage showed close relation to L. kirshneri; and the third held an intermediary position between L. noguchii and L. interrogans. Through ELISA, anti-Leptospira antibodies were found in 83 of 306 bats, with the highest seroprevalence observed in R. leschenaultii (44/48; 91.6 %), R. amplexicaudatus (6/8; 75 %), and E. spelaea (19/25; 76 %). 66 of these ELISA-positive samples were tested using MAT; 41 of them were confirmed in MAT as positive. The predominant serogroups in our study were Tarassovi and Mini.

Human macrophages infected with Egyptian Rousette bat-isolated Marburg virus display inter-individual susceptibility and antiviral responsiveness

Marburg virus (MARV) is a highly pathogenic filovirus and a causative agent of sporadic zoonotic viral hemorrhagic fever outbreaks with high case fatality rates. In humans, filoviruses like MARV and Zaire Ebola virus (EBOV) target, among others, innate immune cells like dendritic cells and macrophages (MΦs). Filovirus-infected dendritic cells display impaired maturation and antigen presentation, while MΦs become hyper-activated and secrete proinflammatory cytokines and chemokines. Our current understanding of human macrophage responses to MARV remains limited. Here, we used human monocyte-derived macrophages (moMΦs) to address how their phenotype, transcriptional profile, and protein expression change upon an in vitro infection with a bat isolate of MARV. Confirming its tropism for macrophages, we show that MARV induces notable shifts in their transcription distinct from responses induced by lipopolysaccharide (LPS), marked by upregulated gene expression of several chemokines, type I interferons, and IFN-stimulated genes. MARV infection also elicited pronounced inter-individually different transcriptional programs in moMΦs, the induction of Wnt signaling-associated genes, and the downregulation of multiple biological processes and molecular pathways.

Comparative study of the ciliary body and iris morphology in the anterior eye chamber of five different vertebrate classes.

One crucial component of the optical system is the ciliary body (CB). This body secretes the aqueous humour, which is essential to maintain the internal eye pressure as well as the clearness of the lens and cornea. The histological study was designed to provide the morphological differences of CB and iris in the anterior eye chambers of the following vertebrate classes: fish (grass carp), amphibians (Arabian toad), reptiles (semiaquatic turtle, fan-footed gecko, ocellated skink, Egyptian spiny-tailed lizard, Arabian horned viper), birds (common pigeon, common quail, common kestrel), and mammals (BALB/c mouse, rabbit, golden hamster, desert hedgehog, lesser Egyptian jerboa, Egyptian fruit bat). The results showed distinct morphological appearances of the CB and iris in each species, ranging from fish to mammals. The present comparative study concluded that the morphological structure of the CB and iris is the adaptation of species to either their lifestyle or survival in specific habitats.

Cross-species spill-over potential of the H9N2 bat influenza A virus

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus–like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Bat-borne H9N2 influenza virus evades MxA restriction and exhibits efficient replication and transmission in ferrets

Influenza A viruses (IAVs) of subtype H9N2 have reached an endemic stage in poultry farms in the Middle East and Asia. As a result, human infections with avian H9N2 viruses have been increasingly reported. In 2017, an H9N2 virus was isolated for the first time from Egyptian fruit bats (Rousettus aegyptiacus). Phylogenetic analyses revealed that bat H9N2 is descended from a common ancestor dating back centuries ago. However, the H9 and N2 sequences appear to be genetically similar to current avian IAVs, suggesting recent reassortment events. These observations raise the question of the zoonotic potential of the mammal-adapted bat H9N2. Here, we investigate the infection and transmission potential of bat H9N2 in vitro and in vivo, the ability to overcome the antiviral activity of the human MxA protein, and the presence of N2-specific cross-reactive antibodies in human sera. We show that bat H9N2 has high replication and transmission potential in ferrets, efficiently infects human lung explant cultures, and is able to evade antiviral inhibition by MxA in transgenic B6 mice. Together with its low antigenic similarity to the N2 of seasonal human strains, bat H9N2 fulfils key criteria for pre-pandemic IAVs.

Sosuga Virus Detected in Egyptian Rousette Bats (Rousettus aegyptiacus) in Sierra Leone.

Sosuga virus (SOSV), a rare human pathogenic paramyxovirus, was first discovered in 2012 when a person became ill after working in South Sudan and Uganda. During an ecological investigation, several species of bats were sampled and tested for SOSV RNA and only one species, the Egyptian rousette bat (ERBs; Rousettus aegyptiacus), tested positive. Since that time, multiple other species have been sampled and ERBs in Uganda have continued to be the only species of bat positive for SOSV infection. Subsequent studies of ERBs with SOSV demonstrated that ERBs are a competent host for SOSV and shed this infectious virus while exhibiting only minor infection-associated pathology. Following the 2014 Ebola outbreak in West Africa, surveillance efforts focused on discovering reservoirs for zoonotic pathogens resulted in the capture and testing of many bat species. Here, SOSV RNA was detected by qRT-PCR only in ERBs captured in the Moyamba District of Sierra Leone in the central region of the country. These findings represent a substantial range extension from East Africa to West Africa for SOSV, suggesting that this paramyxovirus may occur in ERB populations throughout its sub-Saharan African range.

Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.