Marburg Virus Persistence on Fruit as a Plausible Route of Bat to Primate Filovirus Transmission.

Marburg virus (MARV), the causative agent of Marburg virus disease, emerges sporadically in sub-Saharan Africa and is often fatal in humas. The natural reservoir for this zoonotic virus is the frugivorous Egyptian rousette bat (Rousettus aegyptiacus) that when infected, sheds virus in the highest amounts in oral secretions and urine. Being fruit bats, these animals forage nightly for ripened fruit throughout the year, including those types often preferred by humans. During feeding, they continually discard partially eaten fruit on the ground that could then be consumed by other Marburg virus susceptible animals or humans. In this study, using qRT-PCR and virus isolation, we tested fruit discarded by Egyptian rousette bats experimentally infected with a natural bat isolate of Marburg virus. We then separately tested viral persistence on fruit varieties commonly cultivated in sub-Saharan Africa using a recombinant Marburg virus expressing the fluorescent ZsGreen1. Marburg virus RNA was repeatedly detected on fruit in the food bowls of the infected bats and viable MARV was recovered from inoculated fruit for up to 6 h.

Longitudinal Secretion of Paramyxovirus RNA in the Urine of Straw-Coloured Fruit Bats (Eidolon helvum).

The straw-coloured fruit bat (Eidolon helvum) is widespread in sub-Saharan Africa and is widely hunted for bushmeat. It is known to harbour a range of paramyxoviruses, including rubuloviruses and henipaviruses, but the zoonotic potential of these is unknown. We previously found a diversity of paramyxoviruses within a small, captive colony of E. helvum after it had been closed to contact with other bats for 5 years. In this study, we used under-roost urine collection to further investigate the paramyxovirus diversity and ecology in this colony, which had been closed to the outside for 10 years at the time of sampling. By sampling urine weekly throughout an entire year, we investigated possible seasonal patterns of shedding of virus or viral RNA. Using a generic paramyxovirus L-gene PCR, we detected eight distinct paramyxovirus RNA sequences. Six distinct sequences were detected using a Henipavirus-specific PCR that targeted a different region of the L-gene. Sequence detection had a bi-annual pattern, with the greatest peak in July, although different RNA sequences appeared to have different shedding patterns. No significant associations were detected between sequence detection and birthing season, environmental temperature or humidity, and no signs of illness were detected in any of the bats in the colony during the period of sample collection.

Persistence of Multiple Paramyxoviruses in a Closed Captive Colony of Fruit Bats (Eidolon helvum).

Bats have been identified as the natural hosts of several emerging zoonotic viruses, including paramyxoviruses, such as Hendra and Nipah viruses, that can cause fatal disease in humans. Recently, African fruit bats with populations that roost in or near urban areas have been shown to harbour a great diversity of paramyxoviruses, posing potential spillover risks to public health. Understanding the circulation of these viruses in their reservoir populations is essential to predict and prevent future emerging diseases. Here, we identify a high incidence of multiple paramyxoviruses in urine samples collected from a closed captive colony of circa 115 straw-coloured fruit bats (Eidolon helvum). The sequences detected have high nucleotide identities with those derived from free ranging African fruit bats and form phylogenetic clusters with the Henipavirus genus, Pararubulavirus genus and other unclassified paramyxoviruses. As this colony had been closed for 5 years prior to this study, these results indicate that within-host paramyxoviral persistence underlies the role of bats as reservoirs of these viruses.

Co-Circulation and Excretion Dynamics of Diverse Rubula- and Related Viruses in Egyptian Rousette Bats from South Africa.

The Egyptian rousette bat (Rousettus aegyptiacus) has previously been implicated as the natural host of a zoonotic rubulavirus; however, its association with rubulaviruses has been studied to a limited extent. Urine, spleen, and other organs collected from the R. aegyptiacus population within South Africa were tested with a hemi-nested RT-PCR assay targeting a partial polymerase gene region of viruses from the Avula- and Rubulavirus genera. Urine was collected over a 14-month period to study the temporal dynamics of viral excretion. Diverse rubulaviruses, including viruses related to human mumps and parainfluenza virus 2, were detected. Active excretion was identified during two peak periods coinciding with the host reproductive cycle. Analysis of additional organs indicated co-infection of individual bats with a number of different putative rubulaviruses, highlighting the limitations of using a single sample type when determining viral presence and diversity. Our findings suggest that R. aegyptiacus can harbor a range of Rubula- and related viruses, some of which are related to known human pathogens. The observed peaks in viral excretion represents potential periods of a higher risk of virus transmission and zoonotic disease spill-over.

Hematology and Plasma Biochemistry of Wild Spectacled Flying Foxes ( Pteropus conspicillatus) in Australia.

The spectacled flying fox ( Pteropus conspicillatus) is listed as vulnerable to extinction in Australia. The species' restricted population is in decline, putatively attributed to decreasing habitat, climatic extremes, anthropogenic activities, and more recently, mass mortality events associated with tick paralysis and neonatal cleft palate syndrome. Knowledge of fundamental physiologic parameters of the species is limited. To address this knowledge gap, we sampled 50 wild-caught adult spectacled flying foxes in June (winter) in Far North Queensland, Australia. Hematologic and plasma biochemistry reference ranges were established, and a suite of urine biochemistry analytes were measured. Analyte values were compared within spectacled flying fox sex cohorts and between the spectacled flying fox and the paraphyletic black flying fox ( Pteropus alecto). Significant differences in multiple analytes (including erythrocyte, leucocyte, plasma, and urine biochemistry) were found between spectacled flying fox sex cohorts. The majority of spectacled flying fox analyte values did not differ significantly from black flying fox values. Of those analytes that differed between species (erythrocyte, platelet, eosinophil, liver enzyme, and triglyceride levels), the majority were plausibly explained by intraerythrocyte parasite burden and food resource type. Our findings provide baseline data essential to measure and meaningfully interpret flying fox population health in ecologic, conservation, and epidemiologic contexts.

HEMATOLOGY, PLASMA BIOCHEMISTRY, AND URINALYSIS OF FREE-RANGING GREY-HEADED FLYING FOXES ( PTEROPUS POLIOCEPHALUS) IN AUSTRALIA.

The grey-headed flying fox ( Pteropus poliocephalus) is a species endemic to coastal eastern Australia. This study presents a comprehensive set of biochemistry, hematology, and urinalysis biomarkers from which reference values were derived. Blood samples collected from free-ranging P. poliocephalus were submitted for hematology ( n = 140) and plasma biochemistry ( n = 161) and urine for urinalysis ( n = 95). The values for P. poliocephalus were broadly consistent with those values published for other Australian Pteropus species. Statistically significant within-species age and sex effects were observed: adult P. poliocephalus had higher mean corpuscular volume, mean corpuscular hemoglobin, urea, creatinine, bilirubin, alanine transferase (ALT), protein, globulin, urinary specific gravity, and urinary ketones, whereas subadults had higher mean red blood cell, white blood cell (WBC), lymphocyte, and monocyte counts, and juveniles had higher mean neutrophil count and alkaline phosphatase; male P. poliocephalus had higher mean reticulocyte count, alanine transferase, glucose, and urinary ketones, whereas females had higher mean WBC, lymphocyte, and monocyte counts. The findings inform both clinical and research scenarios for P. poliocephalus in captivity or rehabilitation and for health assessments of free-living populations.

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.

Physiological stress and Hendra virus in flying-foxes (Pteropus spp.), Australia.

Pteropid bats (flying-foxes) are the natural reservoir of Hendra virus, an emergent paramyxovirus responsible for fatal infection in horses and humans in Australia. Pteropus alecto (the Black flying-fox) and the paraphyletic P. conspicillatus (the Spectacled flying-fox) appear to be the primary reservoir hosts. Previous studies have suggested that physiological and ecological factors may underpin infection dynamics in flying-foxes, and subsequent spillover to horses and in turn humans. We sought to examine temporal trends in urinary cortisol concentration in wild Australian flying-fox populations, to elucidate the putative relationship between Hendra virus infection and physiological stress. Pooled and individual urine samples were non-invasively collected from under roosting flying-foxes at two latitudinally disparate regions in the eastern Australian state of Queensland. Hendra virus detection, and (in individual urine samples) sex and species determination were PCR-based. Urinary cortisol measurement used a validated enzyme immunoassay. We found no direct correlation between increased urinary cortisol and Hendra virus excretion, but our findings do suggest a biologically plausible association between low winter temperatures and elevated cortisol levels in P. alecto in the lower latitude Southeast Queensland roosts. We hypothesize an indirect association between low winter temperatures and increased Hendra virus infection and excretion, mediated by the physiological cost of thermoregulation. Our findings and our approach are directly relevant to elaboration of the disease ecology of Nipah virus and other emerging henipaviruses in bats. More broadly, they inform investigation of emerging disease infection dynamics across the wildlife/livestock/human interface.

Progesterone transfer among cohabitating female big brown bats (Eptesicus fuscus).

Experiments using female mice and bats have demonstrated that tritium-labeled 17ß-estradiol (3H-E2) can be absorbed via cutaneous and intranasal routes and distributed to reproductive and neural tissues. Radioactivity has also been measured in tissues of untreated females after 48h cohabitation with 3H-E2 injected males. The present study was designed to quantify steroid transfer among female bats. Radioactive quantification via liquid scintillation counting revealed absorption of tritium-labeled progesterone (3H-P4) in adult females 1h after cutaneous and intranasal application (10µCi). Subsequently, pairs of mature females were each housed for 48h with a single mature female that had been administered 3H-P4 (50µCi) via intraperitoneal injection. Radioactivity was observed in all collected tissues of all non-injected females at levels significantly greater than the control group. Following the same paradigm, radioactivity was not observed in the tissues of untreated female bats that were housed with stimulus females treated with 3H-E2 (50µCi). Enzyme immunoassays revealed measurable levels of unconjugated progesterone and estradiol in the urine of female bats, suggesting urine as a vector for steroid transfer. Given that bats of this species live in predominantly female roosts in very close contact, progesterone transfer among individuals is likely to occur in natural roosts.

Flying-fox roost disturbance and Hendra virus spillover risk.

Bats of the genus Pteropus (flying-foxes) are the natural host of Hendra virus (HeV) which periodically causes fatal disease in horses and humans in Australia. The increased urban presence of flying-foxes often provokes negative community sentiments because of reduced social amenity and concerns of HeV exposure risk, and has resulted in calls for the dispersal of urban flying-fox roosts. However, it has been hypothesised that disturbance of urban roosts may result in a stress-mediated increase in HeV infection in flying-foxes, and an increased spillover risk. We sought to examine the impact of roost modification and dispersal on HeV infection dynamics and cortisol concentration dynamics in flying-foxes. The data were analysed in generalised linear mixed models using restricted maximum likelihood (REML). The difference in mean HeV prevalence in samples collected before (4.9%), during (4.7%) and after (3.4%) roost disturbance was small and non-significant (P = 0.440). Similarly, the difference in mean urine specific gravity-corrected urinary cortisol concentrations was small and non-significant (before = 22.71 ng/mL, during = 27.17, after = 18.39) (P= 0.550). We did find an underlying association between cortisol concentration and season, and cortisol concentration and region, suggesting that other (plausibly biological or environmental) variables play a role in cortisol concentration dynamics. The effect of roost disturbance on cortisol concentration approached statistical significance for region, suggesting that the relationship is not fixed, and plausibly reflecting the nature and timing of disturbance. We also found a small positive statistical association between HeV excretion status and urinary cortisol concentration. Finally, we found that the level of flying-fox distress associated with roost disturbance reflected the nature and timing of the activity, highlighting the need for a 'best practice' approach to dispersal or roost modification activities. The findings usefully inform public discussion and policy development in relation to Hendra virus and flying-fox management.

Isolation and identification of a natural reassortant mammalian orthoreovirus from least horseshoe bat in China.

BACKGROUND: Mammalian orthoreoviruses (MRVs) have a wide geographic distribution and can infect virtually all mammals. Infections in humans may be either symptomatic or asymptomatic. This study describes the isolation and identification of a natural reassortant MRV from least horseshoe bats (Rhinolophus pusillu) in China, referred to as RpMRV-YN2012. METHODS AND RESULTS: The RpMRV-YN2012 was obtained from urine samples of Rhinolophus pusillus by cell culture. Negative-staining electron microscopy revealed that RpMRV-YN2012 was a non-enveloped icosahedral virus with ∼75 nm in diameter. Polyacrylamide gel electrophoresis (PAGE) migration patterns of the genome segments showed that RpMRV-YN2012 contained 10 segments in a 3:3:4 arrangement. The whole genome sequence of RpMRV2012 was determined. The consensus terminal sequences of all segments of 5'-GCUAh…yUCAUC-3' (h = A, U or C; y = C or U) were similar to the MRV species within the genus Orthoreovirus. Its evolution and evidence of genetic reassortment were analyzed by sequence comparison and phylogenetic analysis. The results showed that RpMRV-YN2012 is a novel serotype 2 MRV that may have originated from reassortment among bat, human, and/or pig MRV strains which associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in animals and humans. CONCLUSIONS: RpMRV-YN2012 is a novel bat reassortant MRV, which may have resulted from a reassortment involving MRVs known to infect humans and animals. It is necessary to identify whether RpMRV-YN2012 is associated with diarrhea, acute gastroenteritis and necrotizing encephalopathy in clinical patients. In addition, we should carefully monitor its evolution and virulence in real time.

Measuring physiological stress in Australian flying-fox populations.

Flying-foxes (pteropid bats) are the natural host of Hendra virus, a recently emerged zoonotic virus responsible for mortality or morbidity in horses and humans in Australia since 1994. Previous studies have suggested physiological and ecological risk factors for infection in flying-foxes, including physiological stress. However, little work has been done measuring and interpreting stress hormones in flying-foxes. Over a 12-month period, we collected pooled urine samples from underneath roosting flying-foxes, and urine and blood samples from captured individuals. Urine and plasma samples were assayed for cortisol using a commercially available enzyme immunoassay. We demonstrated a typical post-capture stress response in flying-foxes, established urine specific gravity as an attractive alternative to creatinine to correct urine concentration, and established population-level urinary cortisol ranges (and geometric means) for the four Australian species: Pteropus alecto 0.5-305.1 ng/mL (20.1 ng/mL); Pteropus conspicillatus 0.3-370.9 ng/mL (18.9 ng/mL); Pteropus poliocephalus 0.3-311.3 ng/mL (10.1 ng/mL); Pteropus scapulatus 5.2-205.4 ng/mL (40.7 ng/mL). Geometric means differed significantly except for P. alecto and P. conspicillatus. Our approach is methodologically robust, and has application both as a research or clinical tool for flying-foxes, and for other free-living colonial wildlife species.

Continent-wide panmixia of an African fruit bat facilitates transmission of potentially zoonotic viruses.

The straw-coloured fruit bat, Eidolon helvum, is Africa's most widely distributed and commonly hunted fruit bat, often living in close proximity to human populations. This species has been identified as a reservoir of potentially zoonotic viruses, but uncertainties remain regarding viral transmission dynamics and mechanisms of persistence. Here we combine genetic and serological analyses of populations across Africa, to determine the extent of epidemiological connectivity among E. helvum populations. Multiple markers reveal panmixia across the continental range, at a greater geographical scale than previously recorded for any other mammal, whereas populations on remote islands were genetically distinct. Multiple serological assays reveal antibodies to henipaviruses and Lagos bat virus in all locations, including small isolated island populations, indicating that factors other than population size and connectivity may be responsible for viral persistence. Our findings have potentially important public health implications, and highlight a need to avoid disturbances that may precipitate viral spillover.

Hendra virus infection dynamics in Australian fruit bats.

Hendra virus is a recently emerged zoonotic agent in Australia. Since first described in 1994, the virus has spilled from its wildlife reservoir (pteropid fruit bats, or 'flying foxes') on multiple occasions causing equine and human fatalities. We undertook a three-year longitudinal study to detect virus in the urine of free-living flying foxes (a putative route of excretion) to investigate Hendra virus infection dynamics. Pooled urine samples collected off plastic sheets placed beneath roosting flying foxes were screened for Hendra virus genome by quantitative RT-PCR, using a set of primers and probe derived from the matrix protein gene. A total of 1672 pooled urine samples from 67 sampling events was collected and tested between 1 July 2008 and 30 June 2011, with 25% of sampling events and 2.5% of urine samples yielding detections. The proportion of positive samples was statistically associated with year and location. The findings indicate that Hendra virus excretion occurs periodically rather than continuously, and in geographically disparate flying fox populations in the state of Queensland. The lack of any detection in the Northern Territory suggests prevalence may vary across the range of flying foxes in Australia. Finally, our findings suggest that flying foxes can excrete virus at any time of year, and that the apparent seasonal clustering of Hendra virus incidents in horses and associated humans (70% have occurred June to October) reflects factors other than the presence of virus. Identification of these factors will strengthen risk minimization strategies for horses and ultimately humans.

Ammonia excretion increased and urea excretion decreased in urine of a new world nectarivorous bat with decreased nitrogen intake.

We determined the effect of water and nitrogen intake on nitrogenous waste composition in the nectarivorous Pallas's long-tongued bat Glossophaga soricina (Phyllostomidae) to test the hypothesis that bats reduce excretion of urea nitrogen and increase the excretion of ammonia nitrogen as nitrogen intake decreases and water intake decreases. Because changes in urine nitrogen composition are expected only in animals whose natural diets are low in nitrogen and high in water content, we also measured maintenance nitrogen requirements (MNR). We hypothesized that, similar to other plant-eating vertebrates, nectarivorous bats have low MNR. Our nitrogen excretion hypothesis was partly proved correct. There was an increase in the proportion of N excreted as ammonia and a decrease in the proportion excreted as urea in low-nitrogen diets. The proportion of N excreted as ammonia and urea was independent of water intake. Most individuals were ureotelic (n = 28), and only a few were ureo-ammonotelic (n = 3) or ammonotelic (n = 2). According to our nitrogen requirement hypothesis, apparent MNR (60 mg kg(-0.75) d(-1)) and truly digestible MNR (54 mg N kg(-0.75) d(-1)) were low. A decrease in urea excretion in low-nitrogen diets may result from urea recycling from liver to the gut functioning as a nitrogen salvage system in nectarivorous bats. This mechanism probably contributes to the low MNR found in Pallas's long-tongued bats.

Isolation of Waddlia malaysiensis, a novel intracellular bacterium, from fruit bat (Eonycteris spelaea).

An obligate intracellular bacterium was isolated from urine samples from 7 (3.5%) of 202 fruit bats (Eonycteris spelaea) in peninsular Malaysia. The bacterium produced large membrane-bound inclusions in human, simian, and rodent cell lines, including epithelial, fibroblastlike, and lymphoid cells. Thin-section electron microscopy showed reticulate bodies dividing by binary fission and elementary bodies in the inclusions; mitochondria surrounded the inclusions. The inclusions were positive for periodic acid-Schiff stain but could not be stained by fluorescein-labeled anti-Chlamydia trachomatis major outer membrane protein monoclonal antibody. The bacterium was resistant to penicillin and streptomycin (MICs > 256 mg/L) but susceptible to tetracycline (MIC = 0.25 mg/L) and chloramphenicol (MIC = 0.5 mg/L). Sequence analysis of the 16SrRNA gene indicated that it was most closely related to 2 isolates of Waddlia chondrophila (94% and 96% identity). The 16S and 23S rRNA gene signatures were only 91% identical. We propose this novel bacterium be called W. malaysiensis.

Role of urea in the postprandial urine concentration cycle of the insectivorous bat Antrozous pallidus.

Insectivorous bats, which feed once daily, produce maximally concentrated urine only after feeding. The role of urea as an osmolyte in this process was investigated in pallid bats (Antrozous pallidus) in the laboratory. Following a 24-h fast, plasma and urine were sampled before and 2 h after feeding in postprandial (PP) animals and before and 2 h after similar treatment without feeding in nonfed (NF) animals. Food consumption by PP animals and handling of NF animals had no effect on blood water content as measured by hematocrit and plasma oncotic pressure. Food consumption increased both plasma osmolality (P(osm)) and plasma urea (P(urea)) by as much as 15%. Food consumption also increased urine osmolality (U(osm)) and urine urea (U(urea)) by 50-100%. Feeding increased U(osm) regardless of changes in P(osm), and elevation of U(osm) resulted primarily from increased U(urea). In NF bats, P(osm) and P(urea) were unchanged, while U(osm) and U(urea) increased by as much as 25%. Again, increased U(osm) resulted primarily from increased U(urea). The PP urine concentration cycle of pallid bats resulted from increased urea excretion in response to apparent rapid urea synthesis. Bats rapidly metabolized protein and excreted urea following feeding when body water was most plentiful.

A novel approach for collecting samples from fruit bats for isolation of infectious agents.

During the outbreak of Nipah virus encephalitis involving pigs and humans in peninsular Malaysia in 1998/1999, a conventional approach was initially undertaken to collect specimens from fruit bats by mist-netting and shooting, as an integral part of wildlife surveillance of the natural reservoir host of Nipah virus. This study describes a novel method of collecting fruit bats' urine samples using plastic sheets for isolation of Nipah virus. This novel approach resulted in the isolation of several other known and unidentified infectious agents besides Nipah virus.

Isolation of Nipah virus from Malaysian Island flying-foxes.

In late 1998, Nipah virus emerged in peninsular Malaysia and caused fatal disease in domestic pigs and humans and substantial economic loss to the local pig industry. Surveillance of wildlife species during the outbreak showed neutralizing antibodies to Nipah virus mainly in Island flying-foxes (Pteropus hypomelanus) and Malayan flying-foxes (Pteropus vampyrus) but no virus reactive with anti-Nipah virus antibodies was isolated. We adopted a novel approach of collecting urine from these Island flying-foxes and swabs of their partially eaten fruits. Three viral isolates (two from urine and one from a partially eaten fruit swab) that caused Nipah virus-like syncytial cytopathic effect in Vero cells and stained strongly with Nipah- and Hendra-specific antibodies were isolated. Molecular sequencing and analysis of the 11,200-nucleotide fragment representing the beginning of the nucleocapsid gene to the end of the glycoprotein gene of one isolate confirmed the isolate to be Nipah virus with a sequence deviation of five to six nucleotides from Nipah virus isolated from humans. The isolation of Nipah virus from the Island flying-fox corroborates the serological evidence that it is one of the natural hosts of the virus.

Could bats act as reservoir hosts for Rift Valley fever virus?

The inter-epizootic reservoir host of Rift Valley fever virus (RVFV) remains unknown, although the namaqua rock rat, Aethomys namaquensis, as well as bats have been implicated. Bats can be asymptomatically infected with rabies, as well as several arboviruses; the possibility that they can act as host for RVFV therefore exists. To examine this possibility, 350 different samples (brain, liver, salivary glands and brown fat) obtained from 150 bats (comprising seven species) were tested for RVFV antigen using an enzyme linked immunosorbent assay (ELISA). None of the samples tested positive, but the ELISA proved to have limited sensitivity (> or = 10(3) TCID50/ml). In order to determine whether bats could be infected with RVFV, one Miniopterus schreibersii and two Eptesicus capensis bats were inoculated by the oral or intramuscular route with 100 ml and 30 ml, respectively, of a RVFV suspension with a titre of 10(6) TCID50/ml. None of the bats developed any clinical signs. A low concentration of RVFV antigen was found in the liver and urine of M. schreibersii, but not in brain tissue. A third E. capensis bat was inoculated by the intramuscular route and sacrificed on day 18. A low level of antigen was detected in the brown fat. These results demonstrate that bats can be infected with RVFV, and that further studies should be done to determine the potential of different bat species to act as reservoir hosts for RVFV during inter-epizootic periods.