Hendra virus (HeV) causes highly lethal disease in horses and humans in the eastern Australian states of Queensland (QLD) and New South Wales (NSW), with multiple equine cases now reported on an annual basis. Infection and excretion dynamics in pteropid bats (flying-foxes), the recognised natural reservoir, are incompletely understood. We sought to identify key spatial and temporal factors associated with excretion in flying-foxes over a 2300 km latitudinal gradient from northern QLD to southern NSW which encompassed all known equine case locations. The aim was to strengthen knowledge of Hendra virus ecology in flying-foxes to improve spillover risk prediction and exposure risk mitigation strategies, and thus better protect horses and humans. Monthly pooled urine samples were collected from under roosting flying-foxes over a three-year period and screened for HeV RNA by quantitative RT-PCR. A generalised linear model was employed to investigate spatiotemporal associations with HeV detection in 13,968 samples from 27 roosts. There was a non-linear relationship between mean HeV excretion prevalence and five latitudinal regions, with excretion moderate in northern and central QLD, highest in southern QLD/northern NSW, moderate in central NSW, and negligible in southern NSW. Highest HeV positivity occurred where black or spectacled flying-foxes were present; nil or very low positivity rates occurred in exclusive grey-headed flying-fox roosts. Similarly, little red flying-foxes are evidently not a significant source of virus, as their periodic extreme increase in numbers at some roosts was not associated with any concurrent increase in HeV detection. There was a consistent, strong winter seasonality to excretion in the southern QLD/northern NSW and central NSW regions. This new information allows risk management strategies to be refined and targeted, mindful of the potential for spatial risk profiles to shift over time with changes in flying-fox species distribution.

Pteropid bats or flying-foxes (Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species (Pteropus alecto, P. poliocephalus and P. scapulatus) were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples (urine and serum, and urogenital, nasal, oral and rectal swabs) were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples (>serum, packed haemocytes >faecal >nasal >oral), identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus (n = 1168 animals; n = 2958 samples) or P. scapulatus (n = 262 animals; n = 985 samples), suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.

Background: Effective conservation management of highly mobile species depends upon detailed knowledge of movements of individuals across their range; yet, data are rarely available at appropriate spatiotemporal scales. Flying-foxes (Pteropus spp.) are large bats that forage by night on floral resources and rest by day in arboreal roosts that may contain colonies of many thousands of individuals. They are the largest mammals capable of powered flight, and are highly mobile, which makes them key seed and pollen dispersers in forest ecosystems. However, their mobility also facilitates transmission of zoonotic diseases and brings them in conflict with humans, and so they require a precarious balancing of conservation and management concerns throughout their Old World range. Here, we analyze the Australia-wide movements of 201 satellite-tracked individuals, providing unprecedented detail on the inter-roost movements of three flying-fox species: Pteropus alecto, P. poliocephalus, and P. scapulatus across jurisdictions over up to 5 years. Results: Individuals were estimated to travel long distances annually among a network of 755 roosts (P. alecto, 1427-1887 km; P. poliocephalus, 2268-2564 km; and P. scapulatus, 3782-6073 km), but with little uniformity among their directions of travel. This indicates that flying-fox populations are composed of extremely mobile individuals that move nomadically and at species-specific rates. Individuals of all three species exhibited very low fidelity to roosts locally, resulting in very high estimated daily colony turnover rates (P. alecto, 11.9 ± 1.3%; P. poliocephalus, 17.5 ± 1.3%; and P. scapulatus, 36.4 ± 6.5%). This indicates that flying-fox roosts form nodes in a vast continental network of highly dynamic "staging posts" through which extremely mobile individuals travel far and wide across their species ranges.

Within host-parasite communities, viral co-circulation and co-infections of hosts are the norm, yet studies of significant emerging zoonoses tend to focus on a single parasite species within the host. Using a multiplexed paramyxovirus bead-based PCR on urine samples from Australian flying foxes, we show that multi-viral shedding from flying fox populations is common. We detected up to nine bat paramyxoviruses shed synchronously. Multi-viral shedding infrequently coalesced into an extreme, brief and spatially restricted shedding pulse, coinciding with peak spillover of Hendra virus, an emerging fatal zoonotic pathogen of high interest. Such extreme pulses of multi-viral shedding could easily be missed during routine surveillance yet have potentially serious consequences for spillover of novel pathogens to humans and domestic animal hosts. We also detected co-occurrence patterns suggestive of the presence of interactions among viruses, such as facilitation and cross-immunity. We propose that multiple viruses may be interacting, influencing the shedding and spillover of zoonotic pathogens. Understanding these interactions in the context of broader scale drivers, such as habitat loss, may help predict shedding pulses of Hendra virus and other fatal zoonoses.