Introduction

Historically, infectious diseases were believed to be of minimal significance in species population declines. An International Union for Conservation of Nature (IUCN) report listed 833 species extinctions over the past 500 years and attributed only 3.7% of these to infectious disease [1]. This low percentage of extinctions caused by infectious disease may be partly due to the past use of less sophisticated diagnostic techniques to detect infectious agents, as it is now becoming apparent that the effects of urbanization, human population growth, altered land use, deforestation, reduced biodiversity and global trade are leading to an increase in infectious disease incidence and impact [2]. These same factors are also increasing the frequency of disease transmission between humans, domestic animals and wildlife [2, 3] resulting in a dramatic rise in both emerging infectious diseases and zoonoses [4].

Bats, often seen as carriers of many infectious disease agents [5], have also been impacted by this increase in infectious disease incidence, as demonstrated by the recent large scale mortalities caused by white nose syndrome in North America [6]. Other infectious agents have also impacted bat populations. Of particular note is an event involving the mass mortality of thousands of bent-winged bats (Miniopterus schreibersii) in France, Spain and Portugal in 2002 [7]. This was thought to have been caused by infection with a novel filovirus, termed Lloviu virus [8], which was only found in dead bats. A betaherpesvirus was also found in the same affected bats. Its significance in the mortality event was unclear, however herpesviruses have been found asymptomatically in a number of different bat species, in addition to bent-winged bats [9–14].

Bent-winged bats are small, cave-roosting, insectivorous bats, weighing between 10 and 19 grams [15]. In south-eastern Australia there are two subspecies of large bent-winged bat (M. orianae) that form separate maternity colonies [16]. The southern bent-winged bat (M. orianae bassanii) occurs only in south-western Victoria and south-eastern South Australia (SA). There are two maternity caves, one near Warrnambool (38.3687° S, 142.4982° E) in Victoria and the other near Naracoorte (36.9602° S, 140.7413° E) in SA. In the last 50 years the size of the Naracoorte population of southern bent-winged bats has declined from an estimated 200,000 in the 1950s to 20,000 in 2009 [17]. The Warrnambool population declined from 15,000 to 10,000 over the same time period [17]. The subspecies was listed as critically endangered under the Environment Protection and Biodiversity Conservation Act in 2007. The eastern bent-winged bat (M. orianae oceanensis) is more common and widespread, being distributed along the east coast of Australia [15]. Although numbers appear to be stable, the subspecies is listed as vulnerable in Victoria due to the use of just a single maternity site [17].

In south-eastern Australia, disease of unknown aetiology has been identified as a possible cause for the dramatic declines in southern bent-winged bat populations [17]. Despite this, there has been only one published disease investigation of this subspecies, at Naracoorte, which examined skin nodules caused by a species of nematode [18]. There has been no systematic survey for viruses to determine if they may be playing a role in the decline.

In addition to herpesviruses and filoviruses, lyssaviruses and adenoviruses are also known to cause disease in bats. Australian bat lyssavirus (ABLV), causes a fatal disease in bats and humans. It is carried by all four species of Australian flying foxes (Pteropus spp.), as well as the yellow-bellied sheath-tailed bat (Saccolaimus flaviventris) [19]. While previous surveys of bent-winged bats in Australia failed to find any serologically positive animals [19, 20] a serosurvey of common bent-winged bats (M. schreibersii) in the Philippines found that 36% were positive for neutralizing antibodies against ABLV [21]. Bat-associated adenovirus infections have been mostly asymptomatic [22–25]. The virus was found in the gut of three common pipistrelles (Pipistrellus pipistrellus) in Germany with gastrointestinal disease but causation was not established [24].

Bats act as asymptomatic reservoir hosts for a number of important zoonotic viruses that can cause disease or death in humans. This includes the coronavirus that cause Severe Acute Respiratory Syndrome (SARS) [26]. Alpha- and beta-coronavirus RNA and antibodies have been identified in different species of bent-winged bats from northern Australia [27], Thailand [28], Hong Kong [29], China [30] and Bulgaria [31]. Henipaviruses, such as Hendra and Nipah viruses, can also cause severe disease and death in humans [19]. No serological evidence of Hendra virus infection was detected in 62 common bent-winged bats sampled in Queensland, Australia [19], but bent-winged bats that were seropositive for Nipah-like viruses have been identified in China [32]. Filoviruses, including Ebola and Marburg viruses, can cause severe haemorrhagic fevers in humans [33]. More recent publications have identified the presence of a different filovirus (Reston ebolavirus) in three apparently healthy bent-winged bats (M. schreibersii) in the Philippines [34] and have found serological evidence of infection in two bent-winged bats (M. schreibersii) in China [35]. The status of Australian bent-winged bats with respect to this viral group is unknown.

The aim of this study was to characterise the virus diversity of the two subspecies to investigate the possibility of viral disease contributing to the decline of the southern bent-winged bat by comparing viruses detected in southern bent-winged bats with those from the more common eastern bent-winged bats. Consequently, we tested populations of southern and eastern bent-winged bats in south-eastern Australia for ABLV, adenoviruses, filoviruses, herpesviruses, coronaviruses and henipaviruses.

Materials and methods

Results

All the initial samples (a total of 213) collected from Christmas Hills, Allansford and Naracoorte (January 2016) were negative for the presence of adenoviruses and RNA viruses (i.e. ABLV, filoviruses, coronaviruses and henipaviruses). Therefore, no further testing for these viruses was undertaken on subsequently collected samples.

In contrast, herpesvirus DNA was detected in 146 (i.e. 31%; n = 467) bats examined. All PCR negative control reactions, and extraction negative control reactions, were negative for the presence of herpesvirus DNA. All positive extraction controls and PCR positive control samples were positive for the presence of the herpesvirus DNA. Herpesvirus DNA prevalence was higher in eastern bent-winged bats (41%; n = 116) than in southern bent-winged bats (27%, n = 351), with the lowest prevalence at Naracoorte (Table 1).

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Table 1. Prevalence of herpesvirus DNA in oropharyngeal swabs, including proportion of sequence-confirmed positive swabs (confirmed), collected from populations of southern and eastern bent-winged bats from south-eastern Australia.

All bats are adults unless otherwise indicated.

https://doi.org/10.1371/journal.pone.0197625.t001

All the dead bats tested negative for herpesvirus, except for two southern bent-winged bats from Naracoorte, one from a liver sample and another from a lung sample. However, the DNA was of insufficient quality to confirm the positive results by sequencing. All oral swabs collected from dead bats tested negative for herpesvirus DNA.

Herpesvirus DNA suitable for sequencing was obtained from 89/146 of the PCR positive samples. Analysis of the herpesvirus sequence data from these 89 samples revealed six different consensus viral sequences or sequence types (S1 Table), including one gammaherpesvirus (referred to as N7050-like viruses) and five betaherpesviruses (referred to as D15, NG46, CH20, E22 and CH6-like viruses). The viral sequences were compared with bat herpesviruses in GenBank and the maximum percentage predicted amino acid identity to other known bat herpesviruses was found to range from 61% (for CH20-like viruses) to 96% (for CH6-like viruses) (Table 2). This region of the genome is highly conserved in gammaherpesviruses (e.g. 93% amino acid pairwise identity between the closely related equine gammaherpesviruses 5 and 7, GenBank accession numbers AF141886.1 and AKE07652.1, respectively) and betaherpesviruses (e.g. 98% amino acid pairwise identity between the closely related human betaherpesviruses 6A and 6B, GenBank accession numbers APO39176.1 and AB283024.1, respectively), suggesting that the detected viruses may be novel viruses, although further genome sequencing is required to confirm this. A phylogenetic tree demonstrating the relationship of the six virus sequence types with each other and with other known herpesviruses is presented in Fig 1. A similarity matrix of aligned amino acid sequences and a nucleotide phylogenetic tree are available as supplementary information (S2 Table and S1 Fig). This analysis, despite the small fragment of protein sequence available for assessment and low posterior support values for some internal branches, highlighted that the detected viruses clustered within the Herpesviridae subfamilies, Gammaherpesvirinae and Betaherpesvirinae.

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Fig 1. Phylogenetic tree demonstrating the relationship between six bent-winged bat herpesviruses detected in southern and eastern bent-winged bats from south-eastern Australia using PCR, and a selection of viruses from the Alpha- (red), Beta- (blue) and Gammaherpesvirinae (purple) subfamilies.

The tree was generated from the 45 amino acid long alignment using MrBayes v3.2.6 [47] with four heated chains, a chain length of 1,000,000, sampling every 10,000 iterations, and a burn in of 10%. GenBank accession numbers are located to the right of each virus.

https://doi.org/10.1371/journal.pone.0197625.g001

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Table 2. Comparison of bent-winged bat (BWB) herpesvirus sequences, detected by PCR analysis, from southern and eastern bent-winged bats from south-eastern Australia, with the bat herpesvirus in GenBank with the closest identity.

https://doi.org/10.1371/journal.pone.0197625.t002

After univariable screening, all predictors, except ticks and bat flies, were significant at the p < 0.20 level (Table 3). In the final, multivariable model, location group (p < 0.001), season (p < 0.001), and sex (p = 0.024) were significant predictors of betaherpesvirus shedding (Table 4). Both eastern bent-winged bats and Victorian southern bent-winged bats were significantly more likely to be shedding betaherpesvirus than southern bent-winged bats in South Australia (Odds Ratio, OR = 7.1 and 23.3, respectively), and Victorian southern bent-winged bats were significantly more likely to be shedding than eastern bent-winged bats (OR = 3.3). Bats were more likely to be shedding betaherpesvirus in summer or autumn, and least likely in spring. Males were more likely to be shedding than females (OR = 1.8) (Table 4). Body mass, age and presence of parasites were not significant predictors in the multivariable model.

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Table 3. Predictor variables screened using univariable logistic regression models for an association with betaherpesvirus infection in southern (SB) and eastern (EB) bent-winged bats from Victoria (Vic) and South Australia (SA), and found to be significant at the p < 0.20 level.

https://doi.org/10.1371/journal.pone.0197625.t003

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Table 4. Multivariable logistic regression model for betaherpesvirus infection in southern (SB) and eastern (EB) bent-winged bats from Victoria (Vic) and South Australia (SA).

https://doi.org/10.1371/journal.pone.0197625.t004

There was geographical and subspecies-specific variation in the distribution of the different viral sequence types (Fig 2). The D15-like betaherpesvirus was found in both bat subspecies and at all locations sampled. In addition, NG46-like and CH20-like betaherpesviruses were found in both subspecies, although not at every location. The N7050-like gammaherpesvirus was only found in southern bent-winged bats, in eight juveniles from Naracoorte, while E22-like and CH6-like betaherpesviruses were only recorded, in small numbers, in eastern bent-winged bats. The eastern bent-winged bats from Christmas Hills contained the largest number of different herpesviruses (four), while the southern bent-winged bats from Allansford only contained one herpesvirus.

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Fig 2. Distribution of herpesviruses detected in each of the seven bent-winged bat locations sampled across Victoria and South Australia (southern bent-winged bat: Naracoorte, Portland 1 & 2, Allansford; and eastern bent-winged bat: Christmas Hills, Eildon and Lakes Entrance).

Numbers represent the number of viruses of each type present in the population. D15-like viruses = green. NG46-like viruses = orange. N7050-like viruses = blue. CH20-like viruses = maroon. E22-like viruses = lilac. CH6-like viruses = salmon.

https://doi.org/10.1371/journal.pone.0197625.g002

Discussion

This is the most comprehensive viral survey of Australian bent-winged bats. No RNA viruses were found in any of the bats tested. All four RNA virus families tested (ABLV, filoviruses, coronaviruses and henipaviruses) include significant human pathogens. While no serious zoonoses were detected in any of the bats examined in the present study, only a small percentage of the total bat population was sampled, and further research and larger sample sizes are required before concluding that there is no risk to public health.

PCR based assays, as used in this study, generally only detect viruses in animals that have an active infection and are shedding virus at the time of testing [19], while serology will also detect animals that have experienced infections in the past. Due to the small size of bent-winged bats it was not possible to collect sufficient blood to screen for exposure to viruses using the serological tools that were available to us. The small amount of blood that was collected in this study (a maximum of 90 μL per bat) was prioritised for haematological and biochemical testing (as a component of a related study) rather than for virological screening. The use of oral swab samples combined with molecular methods to screen for shedding of viruses has been successfully used in bats for European bat lyssavirus, a virus closely related to ABLV, detecting virus in 15 of 71 samples [53] and consistent with lyssaviruses being shed in the saliva of infected animals [54]. While the present study also used this method to screen for adenoviruses, coronaviruses, filoviruses and henipaviruses, which can all be found in the respiratory and gastrointestinal tracts, testing faeces, urine or blood may have increased the chances of finding these viruses [8, 19, 22, 26, 55].

Six new herpesviruses were detected in the bats examined. This is consistent with recent reports from China and Europe which have revealed a great diversity of herpesviruses in bats, including both betaherpesviruses and gammaherpesviruses [9–14]. Five of the bent-winged bat viruses detected in this study were betaherpesviruses, while one virus, found only in juvenile Naracoorte bats, was a gammaherpesvirus, (N7050-like). This virus had an amino acid sequence identity of 92% to a virus found in Chinese bent-winged bats (Bat Gammaherpesvirus ALH21060.1) [13].

The percentage of bats sampled that were PCR positive for herpesvirus DNA varied widely between the different populations in this study, from 7% to 82%, but this is comparable with other studies of herpesviruses in bats, where the percentage of herpesvirus detection ranged from 3% to 72% [12, 13]. The high prevalence of betaherpesviruses detected in this study may be explained, in part, by the type of samples (oropharyngeal swabs) that were collected. Betaherpesviruses become latent in secretory glands, particularly salivary glands, and would be expected to appear in saliva when excreted [12]. By contrast, a Chinese study that tested bat faecal samples, found a greater proportion of gammaherpesviruses [13]. Faecal samples were not tested in this study but could be considered for future work.

Little is known regarding the extent of movement between the three bent-winged bat populations, although there does appear to be some interchange between them [17]. The different viral diversity profiles present within the bat populations are consistent with restricted, rather than continual, movement of animals between the different locations. Two herpesviruses (CH6-like and E22-like) were detected only in eastern bent-winged bats, whilst another herpesvirus (N7050-like) was detected only in southern bent-winged bats, raising the possibility that these viruses are specific for each of the sub-species of bent-winged bats, although this requires further investigation with larger numbers of animals and samples collected over different times of the year. Three herpesviruses were detected across both bat subspecies, including D15-like herpesvirus, which was found at all seven locations. Herpesviruses have a low rate of nucleotide substitution and have co-evolved with their hosts over many millions of years [56], thus, even in the presence of relatively limited movement of bats between the locations included in the present study, it is expected that the same or similar viruses would occur due to some herpesviruses being present before sub-speciation of the bats, or before bat populations became relatively isolated from each other.

Betaherpesvirus shedding was higher in summer and autumn and lower in spring. Similar patterns have been found in marsupials, humans and cattle [57–59]. When herpes simplex virus was grown in tissue culture, 35°C was found to be the optimum temperature for growth, which ceased below 25°C [60]. As temperatures within the caves occupied by the bats are below 25°C year round [61] and bats go into torpor over winter, lowering their body temperatures to within a few degrees of ambient [62], this would create unfavourable conditions for virus growth and replication. As the bats’ body temperatures and activity levels reach a maximum during the summer months, conditions at this time of year would be more conducive to viral replication and transmission.

Male bats were more likely to be shedding betaherpesviruses than female bats, a pattern which has also been observed in marsupials [57], cats [63] and deer [64]. This may be due to behavioural differences between the sexes affecting rates of transmission, but potential mechanisms for this are unknown.

None of the bats surveyed in this study showed any obvious external signs of ill health or malnutrition. Ideally, ill or recently dead bats would be surveyed as they may contain infectious agents not found in healthy bats, as demonstrated by the European filovirus outbreak [8]. Unfortunately, dead and dying bats are rapidly removed from caves by rats, foxes and other scavengers and rarely found in a state that allows for disease investigation. Based on these results, and the absence of disease or any obvious lesions attributable to herpesvirus infection in any of the bats sampled, it seems likely that the detected herpesviruses are well adapted to their hosts and that bent-winged bats usually carry these herpesviruses with no apparent ill effects.

Latency is an important characteristic of all herpesviruses studied to date and is typically punctuated by intermittent, recurrent viral activation and shedding in response to compromised immunity caused by some form of stress [52]. As southern bent-winged bat numbers have recently declined, it could be assumed that this population is labouring under some form of stress, which may be absent from the eastern bent-winged bat population, which would result in a greater number of herpesvirus positive southern bent-winged bats. While Victorian southern bent-winged bats did have a higher prevalence of infection than eastern bent-winged bats, the opposite was true for the South Australian bats. Similarly, presumably due to an immature immune system [65], more juveniles than adults tend to excrete herpesviruses [52]. This was observed in bats from Naracoorte (southern bent-winged bat), but not those from Lakes Entrance (eastern bent-winged bat).

The PCR used in the present study is able to detect herpesviruses that are being actively shed by the host. However the ability of this PCR to detect latent virus in oropharyngeal swabs cannot be ruled out, as the same nested PCR has been used to detect latent virus genomes in other species [39]. Detection of herpesvirus DNA from both active and latent infection would confound associations between stress/immune status and herpesvirus status because virus detection would not necessarily correlate with virus shedding.

In conclusion, a sample of southern and eastern bent-winged bats was tested by PCR for the presence of six viral families. All bats were negative for adenoviruses, ABLV, filoviruses, coronaviruses and henipaviruses. Six new herpesviruses were identified across all sampled locations. No clinical effects could be attributed to these infections. However, it may be worthwhile conducting further surveys, encompassing a broader range of viruses, because European bent-winged bats (M. schreibersii) were found to harbour rotaviruses, astroviruses, picornaviruses and bufaviruses [66–69], while other bat species have tested positive for papillomaviruses, polyomaviruses, retroviruses and poxviruses [55, 70].

Permits

Samples were collected with approval from the Faculty of Veterinary and Agricultural Science Animal Ethics Committee, University of Melbourne, Victoria (ethics approval 1513456.1), Department of Environment, Land, Water and Planning, Victoria (permit number 0007644), Wildlife Ethics Committee, South Australia (permit number 37/2015) and the Department of Environment, Water and Natural Resources, South Australia (permit number Q26488-1).

Supporting information

Acknowledgments

The authors would like to thank the Holsworth Wildlife Research Endowment, Wildlife Disease Association Australasia, Australian Government’s Threatened Species Discretionary Grants Program, Department of Environment, Land, Water and Planning Victoria, Karst Conservation Fund and David Middleton for providing generous financial support for this project. The lead author is supported by an Australian Postgraduate Award scholarship. The authors acknowledge the valuable assistance provided by Amanda Bush, Mauricio Coppo, David McLelland, Tony Mitchell, Terry Reardon, Penelope Steer-Cope and the numerous volunteers involved with the bat trapping and sampling trips.