Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.) Open Access

The Herpesviridae encompasses a large and diverse family of enveloped, double-stranded DNA viruses, divided into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae), infecting a wide range of mammal, bird, and reptile species (Davison et al. 2008). Herpesviruses display a shared morphology and a unifying biological feature of establishing latency after primary infection, with potential reactivation later in life (Boehmer and Nimonkar 2003). Most herpesvirus infections typically produce severe disease only in neonates, fetuses, immunocompromised individuals, or nondefinitive species (alternate host species). Transmission generally requires close contact, predominantly mucosal contact (MacLachlan and Dubovi 2017). Herpesviruses also may be transmitted from mother to progeny while in utero or during birth; this transmission route plays a considerable role in maintaining herpesviruses within wild and captive populations (MacLachlan and Dubovi 2017).

Since 1996, numerous herpesviruses have been discovered and classified in chiropteran species worldwide, predominantly from the beta- and gammaherpesvirus subfamilies (Tandler 1996; Pozo et al. 2016; Wada et al. 2018; James et al. 2020). To date the Pteropodidae are the only Chiropteran family known to host an alphaherpesvirus; this is primarily a result of limited screening during active infection or of tissue samples in which the virus establishes latent infection, such as the trigeminal ganglia (Razafindratsimandresy et al. 2009; Sasaki et al. 2014; Martins et al. 2019; Inagaki et al. 2020). Betaherpesviruses have only been classified so far in one Pteropodidae genus (Rousettus), and only two Pteropodidae gammaherpesviruses have been formally recorded (Jánoska et al. 2011; Anthony et al. 2013; Zheng et al. 2015; Pozo et al. 2016; Wada et al. 2018). These identified herpesviruses are probably an inadequate representation of the potential herpesviruses infecting Pteropus spp., resulting from limited investigation of species, age groups, sample selection, and methods that are biased towards gammaherpesviruses via primers that amplify the DNA polymerase (DPOL) gene (Anthony et al. 2015). Although herpesviruses have been documented in two subspecies of bent-winged bats, Miniopterus orianae bassanii and M. orianae oceanensis, from the Australian states of Victoria and South Australia (Holz et al. 2018), the presence of herpesvirus in Australian Pteropus spp. has not been investigated. We sought to examine the presence and prevalence of herpesviruses in these species.

The four mainland Australian megabats, or flying fox species (family Pteropodidae, Pteropus spp.), include the black flying fox (Pteropus alecto), little red flying fox (Pteropus scapulatus), grey-headed flying fox (Pteropus poliocephalus), and spectacled flying fox (Pteropus conspicillatus). Pteropus alecto, P. poliocephalus, and P. scapulatus have overlapping habitats in the states of Queensland and New South Wales, Australia. Pteropus conspicillatus has a more limited Australian distribution, in far northern Queensland, and overlaps only with P. alecto and P. scapulatus (Fig. 1).

Flying fox trapping and collection of blood, oropharyngeal swabs, fecal swabs, and urine samples were carried out in 2013, 2014, and 2015 as part of the National Hendra Virus Research Program (Biosecurity Queensland) as described (Edson et al. 2015). In brief, bats were caught in mist nets, before dawn, immediately transferred to cotton bags, and allowed to hang quietly. Bats were anesthetized using isoflurane in oxygen as described (Jonsson et al. 2004), and blood samples (2 mL) were taken from the cephalic (propatagial) vein using a 25G needle and syringe. One mL of blood was immediately placed in a 1.3 mL lithium heparin blood tube (Sarstedt, Nümbrecht, Germany) and 1 mL of blood placed in a 1.3 mL serum tube (Becton Dickinson, Franklin Lakes, New Jersey, USA) and allowed to clot. Following centrifugation, buffy coat from lithium heparin tubes was transferred to 2 mL cryovials (Sarstedt), and serum removed from the serum tube, retaining packed blood cells for analysis (Edson et al. 2015; McMichael et al. 2015). Oral and fecal swab samples were collected using 551C Minitip Size Nylon^® Flocked Swabs (Copan, Murrieta, Georgia, USA). Urine samples were collected by gentle transabdominal palpation of the bladder as described (Edson et al. 2015). Spleen samples were stored in RNALater (Qiagen, Hilden, Germany) upon necropsy of P. conspicillatus euthanased on welfare grounds in 2018 at the Tolga Bat Rescue and Research Inc., Queensland, Australia. All samples were stored at –80 C.

We extracted DNA from blood and spleen samples using the DNeasy Blood & Tissue Kit (Qiagen) and from oropharyngeal swabs, fecal swabs, and urine using the MagMax Viral/Pathogen Nucleic Acid Isolation Kit and KingFisher automated extraction system (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The DNA extraction efficiency was confirmed by cytochrome B gene amplification (Proboste 2020). Herpesvirus DPOL gene was amplified using a nested PCR, employing primers designed to the human herpesvirus 1 DPOL gene: primers DFA, ILK, and TGV align with nucleotides 2149, 2405, and 2440, respectively, and IYG and KG1 are complementary to nucleotides 2647 and 2857 of the HSV-1 DPOL gene sequence, GenBank accession number NC_001806, region 62807 to 66553 (VanDevanter et al. 1996). Amplicons (219–220 bp) were visualized with agarose gel electrophoresis (VanDevanter et al. 1996).

Sequencing of amplicons was prioritized by amplicon intensity, species, and sample type coverage to robustly examine routes of transmission between species: P. alecto (5/25), P. conspicillatus (3/17), P. scapulatus (7/9), and P. poliocephalus (9/12) PCR products were directly sequenced via Sanger sequencing by the Australian Genome Research Facility at the University of Queensland, St Lucia, Queensland, Australia. Sequences were subjected to Nucleotide BLAST (NCBI Resource Coordinators 2016) and MUSCLE alignment in MEGA-X, version 10.1.7 (Kumar et al. 2018). A phylogenetic tree was inferred using the maximum likelihood and Tamura-Nei model (Tamura and Nei 1993).

We detected herpesvirus DNA in blood, oropharyngeal or fecal swabs from 25/255 (10%) P. alecto, 12/108 (11%) P. poliocephalus, 9/53 (17%) P. scapulatus, and 4/43 (9%) P. conspicillatus individuals (Table 1). Five P. alecto and two P. poliocephalus had positive blood samples paired with negative oropharyngeal samples. The highest detection rate was found in P. conspicillatus spleen samples (17/55; 31%). The novel Pteropus gammaherpesvirus 1 sequence (PtGHV-1; MZ344568) amplified from P. alecto, P. conspicillatus, and P. poliocephalus blood or tissue samples showed 100% nucleotide identity between the species, and 90% sequence identity with Megabat gammaherpesvirus (LC268990; Fig. 2). Pteropus gammaherpesvirus 2 (PtGHV-2; MZ344569) amplified from P. poliocephalus blood and oropharyngeal swabs showed 96% identity with PtGHV-1. Pteropus gammaherpesvirus 3 (PtGHV-3; MZ344570) amplified from P. scapulatus blood samples showed only 85% identity with either PtGHV-1 and PtGHV-2. Pteropus gammaherpesvirus 4 (PtGHV-4; MZ344571) amplified from both P. poliocephalus and P. scapulatus fecal swabs showed 100% nucleotide identity between the two species, 79% identity with Pteropus giganteus herpesvirus 4 (KC692445), but only 55% identity with the three aforementioned Australian flying fox gammaherpesviruses. A betaherpesvirus sequence was amplified from P. scapulatus fecal swabs (Pteropus betaherpesvirus 1 [PtBHV-1, MZ344572]), with 99% nucleotide similarity to an Indonesian megabat betaherpesvirus isolate (LC268939). No alphaherpesvirus sequence was identified in any of the samples.

Our detection of herpesviruses in 9–17% of Australian flying fox blood and excreta samples is generally comparable to those recorded in various Asian Pteropus spp. with prevalence ranging from 0.06 to 20.7% (Anthony et al. 2013; Sasaki et al. 2014; Wada et al. 2018), but lower than that of the megabat gammaherpesvirus (MbGHV) recorded in Indonesian Pteropus species (36.23–55.74%; Wada et al. 2018).

We found similar prevalence of herpesviruses within the different sex cohorts in both P. alecto and P. poliocephalus, while P. scapulatus males and P. conspicillatus females had notably higher herpesvirus prevalence than their respective opposite sex (Table 1). Documenting herpesvirus prevalence in both sex cohorts and differing sample types may aid in the understanding of Australian Pteropus species herpesvirus epidemiology and routes of transmission.

Detections in oropharyngeal and fecal swabs support the notion that herpesvirus in Pteropus species is probably excreted in mucosal secretions. It is not surprising that paired samples produced a positive result in the blood but a negative result in oropharyngeal swabs; gammaherpesvirus are lymphotropic and establish latency in lymphocytes, and therefore a positive blood sample but negative oral swab may indicate a latent infection (Buschle and Hammerschmidt 2020).

Spleen samples, which were available only for P. conspicillatus, demonstrated a higher herpesvirus prevalence in comparison to blood samples, supporting the notion that gammaherpesvirus are lymphotropic (Subudhi et al. 2018) and indicating a high rate of latency in the population. We did not detect herpesvirus DNA in urine samples, although herpesviruses have the potential to be excreted in urine (Anthony et al. 2013; Inagaki et al. 2020).

The detection of the same gammaherpesvirus, PtGHV-1, in P. alecto and P. conspicillatus is not surprising, given that these two species are paraphylectic species with overlapping habitats (Almeida et al. 2009). The detection of PtGHV-1 also in P. poliocephalus may be explained by the species' overlapping habitat and coroosting behavior with P. alecto. Detection of PtGHV-4 in both P. poliocephalus and P. scapulatus species, with identical nucleotide sequences across the two species, is a plausible finding in species that have overlapping habitats and are known to coroost, although the two species are not very closely related (Almeida et al 2014). Conversely, the gammaherpesviruses PtGHV-2 and PtGHV-3, detected in P. poliocephalus and P. scapulatus, respectively, may be hypothesized to be species specific, as they were each detected only in a single species from the same sample site in Queensland. A larger sample size and additional sequencing would be required to confirm this.

Our finding of a betaherpesvirus in P. scapulatus with >99% nucleotide identity with an Indonesian fruit bat betaherpesvirus raises questions regarding long-range movements of flying fox species across land and sea borders increasing the likelihood of cross-species infection by one herpesvirus. Viral distribution across the Wallace Line, a bio-geographic barrier that delineates Australian and Southeast Asian fauna, remains a widely contentious topic within the areas of regional distribution and host specificity (Breed et al. 2013).

Further epidemiological research into herpesviruses in Australian Pteropus species is needed, both to elucidate the genetic relationships between Australian and bat herpesviruses on a global scale and to further investigate and understand herpesvirus transmission in Pteropus populations and the ability to produce disease or harbor co-infections, particularly in threatened bat species. Additionally, given that herpesviruses are shed during times of stress (Gerow et al. 2019), assessment of herpesvirus prevalence during physiologically stressful life cycle events, anthropogenic impacts, and climactic extremes would contribute to a holistic understanding of potential immunocompromised bat species.

This study was approved under University of Queensland Animal Ethics Committee (NEWMA AEC) approval numbers SVS/549/19 and SVS/ANRFA/238/19; Scientific Purposes Permit WA0019151; Queensland Department of Agriculture, Fisheries and Forestry Animal Ethics Committee Permit SA 2011/12/375; and Department of Environment, Heritage and Protection Scientific Purposes Permits WISP05810609, WISP14100614, and WA0019151. A portion of the bat samples were provided by courtesy of research funded by the State of Queensland, the State of New South Wales, and the Commonwealth of Australia under the National Hendra Virus Research Program (Biosecurity Queensland). The authors would like to acknowledge Heather Kiley for her graphic design skills. Lodged GenBank accession numbers are MZ2344568, MZ2344569, MZ2344570, MZ2344571, MZ2344572.