Functional comparisons of the virus sensor RIG-I from humans, the microbat Myotis daubentonii, and the megabat Rousettus aegyptiacus, and their response to SARS-CoV-2 infection.

IMPORTANCE: A common hypothesis holds that bats (order Chiroptera) are outstanding reservoirs for zoonotic viruses because of a special antiviral interferon (IFN) system. However, functional studies about key components of the bat IFN system are rare. RIG-I is a cellular sensor for viral RNA signatures that activates the antiviral signaling chain to induce IFN. We cloned and functionally characterized RIG-I genes from two species of the suborders Yangochiroptera and Yinpterochiroptera. The bat RIG-Is were conserved in their sequence and domain organization, and similar to human RIG-I in (i) mediating virus- and IFN-activated gene expression, (ii) antiviral signaling, (iii) temperature dependence, and (iv) recognition of RNA ligands. Moreover, RIG-I of Rousettus aegyptiacus (suborder Yinpterochiroptera) and of humans were found to recognize SARS-CoV-2 infection. Thus, members of both bat suborders encode RIG-Is that are comparable to their human counterpart. The ability of bats to harbor zoonotic viruses therefore seems due to other features.

Bartonella in bat flies from the Egyptian fruit bat in the Middle East.

In the family of fruit bats, Pteropodidae Gray, 1821, as in the third most diverse group of bats (Chiroptera), the bacterium of the genus Bartonella was detected in several species as well as in a few species of their insect ectoparasites in some tropical and sub-tropical regions of the Old World. The Egyptian fruit bat, Rousettus aegyptiacus (Geoffroy, 1810), is one of the most widespread fruit bats, occurring between South Africa, Senegal, and Pakistan. In this bat species, Candidatus Bartonella rousetti has been detected in three African populations in Nigeria, Kenya, and Zambia. This fruit bat, however, also occurs in the Palaearctic, an area isolating the species geographically and phylogenetically from the Afrotropical part of its distribution range. We screened the blood-sucking bat flies (family Nycteribiidae) from R. aegyptiacus for the presence of the Bartonella bacteria. A rich material of bat fly Eucampsipoda aegyptia (Macquart, 1850), a monoxenous ectoparasite of the Egyptian fruit bats, was collected at 26 localities in seven countries (Egypt, Iran, Jordan, Lebanon, Oman, United Arab Emirates, and Yemen) of the Middle East in 2007-2013. The DNA isolates from the bat flies were subjected to a three-marker (gltA, ssrA, and intergenic spacer region, ITS) multilocus sequence analysis. Based on the amplification of the fragment of ssrA gene by a real-time PCR, 65 E. aegyptia samples from 19 localities in all seven countries were positive for the bacteria. One to five Bartonella-positive individuals of E. aegyptia were collected per one individual of R. aegyptiacus. An analysis of the ITS and gltA genes indicated the presence of an uncultured Bartonella sp., belonging to the Cand. B. rousetti genogroup, identified from populations of the Egyptian fruit bat in Africa. These results support the hypothesis that Bartonella's diversity corresponds to its host's diversity (and phylogenetic structure). Specific lineages of pathogens are present in specific phylogenetic groups of bats.

Old World Fruit Bats (Pteropodidae, Chiroptera) from the Pleistocene of Vietnam.

Isolated teeth of a lesser short-nosed fruit bat Cynopterus brachyotis (Müller, 1838), a fulvous fruit bat Rousettus leschenaultii (Desmarest, 1820), and a dawn bat Eonycteris spelaea (Dobson, 1871) are described from the Middle Pleistocene Tham Hai cave locality in northern Vietnam (Lang Son Province). These are the first fossil findings of the Old World fruit bats in Vietnam. The Middle Pleistocene association of Pteropodidae from the Tham Hai locality may largely reflect the composition of species that roosted in local caves.

Micro‒Global Positioning Systems for Identifying Nightly Opportunities for Marburg Virus Spillover to Humans by Egyptian Rousette Bats.

Marburg virus disease, caused by Marburg and Ravn orthomarburgviruses, emerges sporadically in sub-Saharan Africa and is often fatal in humans. The natural reservoir is the Egyptian rousette bat (ERB), which sheds virus in saliva, urine, and feces. Frugivorous ERBs discard test-bitten and partially eaten fruit, potentially leaving infectious virus behind that could be consumed by other susceptible animals or humans. Historically, 8 of 17 known Marburg virus disease outbreaks have been linked to human encroachment on ERB habitats, but no linkage exists for the other 9 outbreaks, raising the question of how bats and humans might intersect, leading to virus spillover. We used micro‒global positioning systems to identify nightly ERB foraging locations. ERBs from a known Marburg virus‒infected population traveled long distances to feed in cultivated fruit trees near homes. Our results show that ERB foraging behavior represents a Marburg virus spillover risk to humans and plausibly explains the origins of some past outbreaks.

Retinal Ganglion Cell Topography and Spatial Resolution in Three Indian Pteropodid Bats.

Pteropodidae is the only phytophagous bat family that predominantly depends on visual and olfactory cues for orientation and foraging. During daytime, pteropodids of different species roost in sites with varying light exposure. Pteropodids have larger eyes relative to body size than insectivorous bats. Retinal topography has been studied in less than 10% of the approximately 200 pteropodid species, a behavioural estimation of spatial resolution is available only for Pteropus giganteus, and little is known about the relationship between their roost site preference and visual ecology. We present retinal ganglion cell topographic maps and anatomical estimates of spatial resolution in three southern Indian pteropodid species with different roosting preferences. Ganglion cell densities are between 1,000 and 2,000 cells/mm2 in the central retina and lower in the dorsal and ventral periphery. All three species have a temporal area in the retina with peak ganglion cell densities of 4,600-6,600 cells/mm2. As a result, the foliage-roosting Cynopterus sphinx and the cave-roosting Rousettus leschenaultii have similar anatomical resolution (2.7 and 2.8 cycles/degree, respectively). The anatomical estimate for the larger tree-roosting P. giganteus (4.0 cycles/degree) is higher than the spatial resolution determined earlier in behavioural tests. Like other pteropodids and unlike other vertebrates, all three species have choroidal papillae. Based on 15 pteropodid species studied to date, we find no relationship between roost type and eye size or visual acuity. For a general understanding of the sensory ecology of pteropodids that perform key ecosystem services in the tropics, it will be essential to study additional species.

Hearing, echolocation, and beam steering from day 0 in tongue-clicking bats.

Little is known about the ontogeny of lingual echolocation. We examined the echolocation development of Rousettus aegyptiacus, the Egyptian fruit bat, which uses rapid tongue movements to produce hyper-short clicks and steer the beam's direction. We recorded from day 0 to day 35 postbirth and assessed hearing and beam-steering abilities. On day 0, R. aegyptiacus pups emit isolation calls and hyper-short clicks in response to acoustic stimuli, demonstrating hearing. Auditory brainstem response recordings show that pups are sensitive to pure tones of the main hearing range of adult Rousettus and to brief clicks. Newborn pups produced clicks in the adult paired pattern and were able to use their tongues to steer the sonar beam. As they aged, pups produced click pairs faster, converging with adult intervals by age of first flights (7-8 weeks). In contrast with laryngeal bats, Rousettus echolocation frequency and duration are stable through to day 35, but shift by the time pups begin to fly, possibly owing to tongue-diet maturation effects. Furthermore, frequency and duration shift in the opposite direction of mammalian laryngeal vocalizations. Rousettus lingual echolocation thus appears to be a highly functional sensory system from birth and follows a different ontogeny from that of laryngeal bats.

Microbacterium chengjingii sp. nov. and Microbacterium fandaimingii sp. nov., isolated from bat faeces of Hipposideros and Rousettus species.

Four aerobic, Gram-stain-positive, rod-shaped bacteria (HY60T, HY54, HY82T and HY89) were isolated from bat faeces of Hipposideros and Rousettus species collected in PR China. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the four novel strains formed two separate but adjacent subclades close to Microbacterium agarici CGMCC 1.12260T (97.6-97.7 % similarity), Microbacterium humi JCM 18706T (97.3-97.5 %) and Microbacterium lindanitolerans JCM 30493T (97.3-97.4 %). The 16S rRNA gene sequence similarity was 98.3 % between strains HY60T and HY82T, and identical within strain pairs HY60T/HY54 and HY82T/HY89. The DNA G+C contents of strains HY60T and HY82T were 61.9 and 63.3 mol%, respectively. The digital DNA-DNA hybridization and average nucleotide identity values between each novel strain and their closest relatives were all below the 70 % and 95-96 % thresholds for species delimitation, respectively. All four novel strains contained anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0 as the main fatty acids, MK-11 and MK-12 as the major respiratory quinones, and diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid as the predominant polar lipids. The cell-wall peptidoglycan was of B type and contained alanine, glutamate, glycine and ornithine. The acyl type of the muramic acid was glycolyl. The whole-cell sugars were rhamnose and ribose. Based on the foregoing polyphasic analyses, it was concluded that the four uncharacterized strains represented two novel species of the genus Microbacterium, for which the names Microbacterium chengjingii sp. nov. [type strain HY60T (=CGMCC 1.17468T=GDMCC 1.1951T=KACC 22102T)] and Microbacterium fandaimingii sp. nov. [type strain HY82T (=CGMCC 1.17469T=GDMCC 1.1949T=KACC 22101T)] are proposed, respectively.

Evaluation of alfaxalone and midazolam with or without flumazenil reversal in Egyptian fruit bats (Rousettus aegyptiacus).

OBJECTIVES: To evaluate alfaxalone-midazolam anesthesia in Egyptian fruit bats (Rousettus aegyptiacus) and the effect of flumazenil administration on recovery time and quality. STUDY DESIGN: Randomized, blinded, crossover and controlled, experimental trial. ANIMALS: A total of 10 male Egyptian fruit bats. METHODS: Bats were anesthetized with alfaxalone (15 mg kg-1) and midazolam (2 mg kg-1) administered subcutaneously. During anesthesia, vital signs, muscle tone and reflexes were monitored every 10 minutes. Flumazenil (0.3 mg kg-1) or saline at an equal volume was administered subcutaneously 60 minutes after anesthetic administration. Time to induction, time to first movement and recovery time (flying) were measured. Quality of induction, anesthesia and recovery were assessed on a 1-3 scale (1, poor; 2, good; 3, excellent). RESULTS: Time to induction was 4.2 ± 1.9 minutes (mean ± standard deviation), with median quality score of 2 (range, 1-3). Anesthesia quality score was 3 (1-3). During anesthesia, heart rate and respiratory frequency decreased significantly and penis relaxation, indicating muscle tone, increased significantly. Administration of flumazenil significantly reduced mean recovery time compared with saline (10 ± 5 versus 45 ± 17 minutes, respectively), and significantly improved the quality of recovery [2.5 (2-3) versus 1 (1-2), respectively]. CONCLUSIONS AND CLINICAL RELEVANCE: Alfaxalone-midazolam anesthesia resulted in good induction, muscle relaxation and sufficient anesthesia to perform routine diagnostic and therapeutic procedures for approximately 40 minutes. Reversal of midazolam with flumazenil is recommended, resulting in quicker and better recovery.

Human-Pathogenic Kasokero Virus in Field-Collected Ticks.

Kasokero virus (KASV; genus Orthonairovirus) was first isolated in 1977 at Uganda Virus Research Institute from serum collected from Rousettus aegyptiacus bats captured at Kasokero Cave, Uganda. During virus characterization studies at the institute, 4 laboratory-associated infections resulted in mild to severe disease. Although orthonairoviruses are typically associated with vertebrate and tick hosts, a tick vector of KASV never has been reported. We tested 786 Ornithodoros (Reticulinasus) faini tick pools (3,930 ticks) for KASV. The ticks were collected from a large R. aegyptiacus bat roosting site in western Uganda. We detected KASV RNA in 43 tick pools and recovered 2 infectious isolates, 1 of which was derived from host blood-depleted ticks. Our findings suggest that KASV is maintained in an enzootic transmission cycle involving O. (R.) faini ticks and R. aegyptiacus bats and has the potential for incidental virus spillover to humans.

Shedding of Marburg Virus in Naturally Infected Egyptian Rousette Bats, South Africa, 2017.

We detected Marburg virus RNA in rectal swab samples from Egyptian rousette bats in South Africa in 2017. This finding signifies that fecal contamination of natural bat habitats is a potential source of infection for humans. Identified genetic sequences are closely related to Ravn virus, implying wider distribution of Marburg virus in Africa.

SURGICAL ORCHIECTOMY IN FRUIT BATS-DESCRIPTION OF TWO TECHNIQUES IN THE RUWENZORI LONG-HAIRED FRUIT BAT (ROUSETTUS LANOSUS) AND THE JAMAICAN FRUIT-EATING BAT (ARTIBEUS JAMAICENSIS).

Contraception is needed to prevent overpopulation and inbreeding in highly fecund captive bat colonies. Reports on surgical contraception in bats are limited. The objective of this study was to describe surgical castration techniques in a megachiropteran and a microchiropteran fruit bat species. Open orchiectomy by transfixing ligation of the spermatic cord was performed in 14 Ruwenzori long-haired fruit bats (Rousettus lanosus) (RL-LIG), and orchiectomy with radiosurgery alone was performed in 125 Jamaican fruit-eating bats (Artibeus jamaicensis) (AJ-RS) and one Ruwenzori bat (RL-RS). The surgical techniques were not compared in different species with the exception of the one Ruwenzori bat operated with RS. Anesthesia was induced and maintained with isoflurane in oxygen via facemask. Preoperatively, all bats received butorphanol and subcutaneous fluids. Meloxicam was administered postoperatively for the RL-LIG. For the RL-LIG, anesthesia lasted 49 ± 15 min (mean ± SD) with a total surgery time of 26 ± 12 min. In comparison, the RS was considerably shorter, lasting 10 ± 3 min for anesthesia and 5 ± 2 min for surgery. Complications were rare, with a morbidity rate of 6.7% with the RL-LIG (prolonged recovery [n = 1]) and of 4.8% with the RS (dyspnea [n = 3], hemorrhage [n = 2], and prolonged recovery [n = 1]). One of the cases of hemorrhage was in the single Ruwenzori bat castrated using the RS technique. Mortality rate was 1.6% (n = 2) with the RS. No mortality occurred with the RL-LIG. In conclusion, using radiosurgery alone appears to be a safe and rapid surgical technique in smaller species of fruit bats. For larger species, such as the Ruwenzori fruit bats, ligation or use of an advanced vessel sealing system is recommended.

Survey of Hepatocystis parasites of fruit bats in the Amurum forest reserve, Nigeria, identifies first host record for Rousettus aegyptiacus.

Parasites of the genus Hepatocystis are close relatives of Plasmodium that frequently infect epauletted fruit bats across West and East Africa. Our understanding of susceptible hosts and prevalence of infection of Hepatocystis remains fragmented. Non-invasive sampling of bat assemblages in representative habitats critically contribute to haemosporidian parasite distribution maps. Here, we report on a survey of Hepatocystis parasite infections in bats undertaken over two consecutive years in a protected area in Nigeria, where prevalence and diversity of bat-infecting haemosporidian parasites have not been studied. Microscopic examination of blood films in combination with PCR detection and sequencing revealed Hepatocystis infections with prevalences of 25% and 42% in the closely related epauletted fruit bats Epomophorus sp. and Micropteropus pusillus. For the first time, mature Hepatocystis gametocytes were identified in one Egyptian fruit bat (Rousettus aegyptiacus). This novel host record was confirmed by parasite and host genotyping and suggests that Hepatocystis parasites have a broader host distribution in African fruit bats than currently known.

Survey of Ebola Viruses in Frugivorous and Insectivorous Bats in Guinea, Cameroon, and the Democratic Republic of the Congo, 2015-2017.

To clarify the role of bats in the ecology of Ebola viruses, we assessed the prevalence of Ebola virus antibodies in a large-scale sample of bats collected during 2015-2017 from countries in Africa that have had previous Ebola outbreaks (Guinea, the Democratic Republic of the Congo) or are at high risk for outbreaks (Cameroon). We analyzed 4,022 blood samples of bats from >12 frugivorous and 27 insectivorous species; 2-37 (0.05%-0.92%) bats were seropositive for Zaire and 0-30 (0%-0.75%) bats for Sudan Ebola viruses. We observed Ebola virus antibodies in 1 insectivorous bat genus and 6 frugivorous bat species. Certain bat species widespread across Africa had serologic evidence of Zaire and Sudan Ebola viruses. No viral RNA was detected in the subset of samples tested (n = 665). Ongoing surveillance of bats and other potential animal reservoirs are required to predict and prepare for future outbreaks.

Isolated Case of Marburg Virus Disease, Kampala, Uganda, 2014.

In September 2014, a single fatal case of Marburg virus was identified in a healthcare worker in Kampala, Uganda. The source of infection was not identified, and no secondary cases were identified. We describe the rapid identification, laboratory diagnosis, and case investigation of the third Marburg virus outbreak in Uganda.

Genetically Diverse Filoviruses in Rousettus and Eonycteris spp. Bats, China, 2009 and 2015.

Genetically divergent filoviruses detected in Rousettus and Eonycteris spp. bats in China exhibited 61%-99% nt identity with reported filoviruses, based on partial replicase sequences, and they demonstrated lung tropism. Co-infection with 4 different filoviruses was found in 1 bat. These results demonstrate that fruit bats are key reservoirs of filoviruses.

REFRACTOMETRIC URINE SPECIFIC GRAVITY OF FREE-LIVING EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

In both human and veterinary medicine, urine specific gravity (USG) is commonly measured by refractometry to indirectly reflect the osmolality of urine to thereby evaluate the kidney's ability to concentrate or dilute urine according to physiologic need and certain disease conditions. However, for accurate interpretation of the significance of any value, knowledge of the expected USG for the healthy species in question is required. It is generally believed that fruit bats, and Egyptian fruit bats (Rousettus aegyptiacus) in particular, are unable to highly concentrate their urine. In this study, the USG was determined using a handheld urine refractometer in 43 free-living Egyptian fruit bats of both sexes. The calculated nonparametric 90% confidence interval for Egyptian fruit bats in this study was 1.006-1.050, with no association with capture site, sex, weight, or packed cell volume and total solids. Results suggest that free-living Egyptian fruit bats are able to highly concentrate their urine.

DISSEMINATED PROTOTHECOSIS IN A RUWENZORI LONG-HAIRED FRUIT BAT ( ROUSETTUS LANOSUS).

An adult male Ruwenzori long-haired fruit bat ( Rousettus lanosus) presented for lethargy and unthriftiness. Physical examination revealed cranial alopecia, mandibular ulceration, and dehydration. Supportive care and antibiotic therapy were initiated. The bat was found dead 3 days after presentation. Necropsy revealed alopecia on the head and body, exposed dried bone on the rostral tip of the mandible, and excessive clear pleural fluid. Lungs were congested and contained miliary white foci disseminated randomly throughout the parenchyma. Subcutaneous, intra-thoracic, and intra-abdominal adipose depots were minimal. Histologic examination of skin and lung revealed the presence of algal-like organisms morphologically consistent with Prototheca spp. Polymerase chain reaction amplification revealed >99% sequence identity match with Prototheca zopfii. Protothecosis has been previously reported in a single bat, Lyle's flying fox ( Pteropus lylei), in Switzerland, but definitive protothecal speciation was not possible.

A RETROSPECTIVE STUDY OF THE LESIONS ASSOCIATED WITH IRON STORAGE DISEASE IN CAPTIVE EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

Egyptian fruit bats (Rousettus aegyptiacus) are one of many species within zoologic collections that frequently develop iron storage disease. The goals of this retrospective multi-institutional study were to determine the tissue distribution of iron storage in captive adult Egyptian fruit bats and the incidence of intercurrent neoplasia and infection, which may be directly or indirectly related to iron overload. Tissue sections from 83 adult Egyptian fruit bats were histologically evaluated by using tissue sections stained with hematoxylin and eosin, trichrome, and Prussian blue techniques. The liver and spleen consistently had the largest amount of iron, but significant amounts of iron were also detected in the pancreas, kidney, skeletal muscle, and lung. Hepatocellular carcinoma (HCC; 11) was the most common neoplasm, followed by cholangiocarcinoma (4). Extrahepatic neoplasms included bronchioloalveolar adenoma (3), pulmonary carcinosarcoma (1), oral sarcoma (1), renal adenocarcinoma (1), transitional cell carcinoma of the urinary bladder (1), mammary gland adenoma (1), and parathyroid adenoma (1). There were also metastatic neoplasms of undetermined primary origin that included three poorly differentiated carcinomas, a poorly differentiated sarcoma, and a neuroendocrine tumor. Bats with hemochromatosis were significantly more likely to have HCC than bats with hemosiderosis (P = 0.032). Cardiomyopathy was identified in 35/77 bats with evaluable heart tissue, but no direct association was found between cardiac damage and the amount of iron observed within the liver or heart. Hepatic abscesses occurred in multiple bats, although a significant association was not observed between hemochromatosis and bacterial infection. To the authors' knowledge, this is the first publication providing evidence of a positive correlation between hemochromatosis and HCC in any species other than humans.

RETROSPECTIVE EVALUATION OF CASES OF NEOPLASIA IN A CAPTIVE POPULATION OF EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

Reports of neoplasia in Chiroptera species are rare. (6, 10) This retrospective study describes five types of neoplasia identified within a captive population of male Egyptian fruit bats (Rousettus aegyptiacus) housed in a zoo from 2004 through November of 2014. Tumor types identified include fibrosarcoma, cutaneous lymphoma, benign focal bronchioloalveolar neoplasm, anaplastic sarcoma, and sebaceous epithelioma. To the author's knowledge, aside from a recent report of focal brochioloalveolar adenoma, (8) these tumor types have not previously been described in the Rousettus species, nor in chiropterans in general. Based upon these findings and other recent publications regarding R. aegyptiacus, neoplasia does appear to be a significant cause of morbidity and mortality in captive members of this megachiropterid species.

Establishment of immortalized Egyptian Rousettus bat cell lines.

The Egyptian Rousettus bat (Rousettus aegyptiacus) is a common fruit bat species that is distributed mainly in Africa and the Middle East. Bats serve as reservoir hosts for numerous pathogens. Human activities, such as hunting bats for food, managing vermin, and causing habitat loss, elevate the likelihood of transmission of bat pathogens to humans and other animals. Consequently, bat cell lines play a crucial role as research materials for investigating viral pathogens. However, the inherent limitation of finite cell division in primary cells necessitates the use of immortalized cells derived from various bat tissues. Herein, we successfully established six fibroblast cell lines derived from an infant bat heart and lungs and an elderly bat heart. Three of the six cell lines, called K4DT cells, were transduced by a combination of cell cycle regulators, mutant cyclin-dependent kinase 4, cyclin D1, and human telomerase reverse transcriptase. The other three cell lines, named SV40 cells, were transfected with simian virus 40 large T antigen. Transgene protein expression was detected in the transduced cells. All three K4DT cell lines and one lung-derived SV40 cell line were virtually immortalized and nearly maintained the normal diploid karyotypes. However, the two other heart-derived SV40 cell lines had aberrant karyotypes and the young bat-derived cell line stopped proliferating at approximately 40 population doublings. These bat cell lines are valuable for studying pathogen genomics and biology.