Pandemic origins and a One Health approach to preparedness and prevention: Solutions based on SARS-CoV-2 and other RNA viruses.

COVID-19 is the latest zoonotic RNA virus epidemic of concern. Learning how it began and spread will help to determine how to reduce the risk of future events. We review major RNA virus outbreaks since 1967 to identify common features and opportunities to prevent emergence, including ancestral viral origins in birds, bats, and other mammals; animal reservoirs and intermediate hosts; and pathways for zoonotic spillover and community spread, leading to local, regional, or international outbreaks. The increasing scientific evidence concerning the origins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is most consistent with a zoonotic origin and a spillover pathway from wildlife to people via wildlife farming and the wildlife trade. We apply what we know about these outbreaks to identify relevant, feasible, and implementable interventions. We identify three primary targets for pandemic prevention and preparedness: first, smart surveillance coupled with epidemiological risk assessment across wildlife-livestock-human (One Health) spillover interfaces; second, research to enhance pandemic preparedness and expedite development of vaccines and therapeutics; and third, strategies to reduce underlying drivers of spillover risk and spread and reduce the influence of misinformation. For all three, continued efforts to improve and integrate biosafety and biosecurity with the implementation of a One Health approach are essential. We discuss new models to address the challenges of creating an inclusive and effective governance structure, with the necessary stable funding for cross-disciplinary collaborative research. Finally, we offer recommendations for feasible actions to close the knowledge gaps across the One Health continuum and improve preparedness and response in the future.

Nipah virus dynamics in bats and implications for spillover to humans.

Nipah virus (NiV) is an emerging bat-borne zoonotic virus that causes near-annual outbreaks of fatal encephalitis in South Asia-one of the most populous regions on Earth. In Bangladesh, infection occurs when people drink date-palm sap contaminated with bat excreta. Outbreaks are sporadic, and the influence of viral dynamics in bats on their temporal and spatial distribution is poorly understood. We analyzed data on host ecology, molecular epidemiology, serological dynamics, and viral genetics to characterize spatiotemporal patterns of NiV dynamics in its wildlife reservoir, Pteropus medius bats, in Bangladesh. We found that NiV transmission occurred throughout the country and throughout the year. Model results indicated that local transmission dynamics were modulated by density-dependent transmission, acquired immunity that is lost over time, and recrudescence. Increased transmission followed multiyear periods of declining seroprevalence due to bat-population turnover and individual loss of humoral immunity. Individual bats had smaller host ranges than other Pteropus species (spp.), although movement data and the discovery of a Malaysia-clade NiV strain in eastern Bangladesh suggest connectivity with bats east of Bangladesh. These data suggest that discrete multiannual local epizootics in bat populations contribute to the sporadic nature of NiV outbreaks in South Asia. At the same time, the broad spatial and temporal extent of NiV transmission, including the recent outbreak in Kerala, India, highlights the continued risk of spillover to humans wherever they may interact with pteropid bats and the importance of limiting opportunities for spillover throughout Pteropus's range.

Possibility for reverse zoonotic transmission of SARS-CoV-2 to free-ranging wildlife: A case study of bats.

The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (ß-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of ß-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of ß-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations.

Extreme mobility of the world's largest flying mammals creates key challenges for management and conservation.

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. CONCLUSIONS: The extreme inter-roost mobility reported here demonstrates the extent of the ecological linkages that nomadic flying-foxes provide across Australia's contemporary fragmented landscape, with profound implications for the ecosystem services and zoonotic dynamics of flying-fox populations. In addition, the extreme mobility means that impacts from local management actions can readily reverberate across jurisdictions throughout the species ranges; therefore, local management actions need to be assessed with reference to actions elsewhere and hence require national coordination. These findings underscore the need for sound understanding of animal movement dynamics to support evidence-based, transboundary conservation and management policy, tailored to the unique movement ecologies of species.

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.

Novel paramyxoviruses in Australian flying-fox populations support host-virus co-evolution.

Understanding the diversity of henipaviruses and related viruses is important in determining the viral ecology within flying-fox populations and assessing the potential threat posed by these agents. This study sought to identify the abundance and diversity of previously unknown paramyxoviruses (UPVs) in Australian flying-fox species (Pteropus alecto, Pteropus scapulatus, Pteropus poliocephalus and Pteropus conspicillatus) and in the Christmas Island species Pteropus melanotus natalis. Using a degenerative reverse transcription-PCR specific for the L gene of known species of the genus Henipavirus and two closely related paramyxovirus genera Respirovirus and Morbillivirus, we identified an abundance and diversity of previously UPVs, with a representative 31 UPVs clustering in eight distinct groups (100 UPVs/495 samples). No new henipaviruses were identified. The findings were consistent with a hypothesis of co-evolution of paramyxoviruses and their flying-fox hosts. Quantification of the degree of co-speciation between host and virus (beyond the scope of this study) would strengthen this hypothesis.

Isolation of multiple novel paramyxoviruses from pteropid bat urine.

Bats have been found to harbour a number of new emerging viruses with zoonotic potential, and there has been a great deal of interest in identifying novel bat pathogens to determine the risk to human and animal health. Many groups have identified novel viruses in bats by detection of viral nucleic acid; however, virus isolation is still a challenge, and there are few reports of viral isolates from bats. In recent years, our group has developed optimized procedures for virus isolation from bat urine, including the use of primary bat cells. In previous reports, we have described the isolation of Hendra virus, Menangle virus and Cedar virus in Queensland, Australia. Here, we report the isolation of four additional novel bat paramyxoviruses from urine collected from beneath pteropid bat (flying fox) colonies in Queensland and New South Wales during 2009-2011.

Molecular evidence of Ebola Reston virus infection in Philippine bats.

BACKGROUND: In 2008-09, evidence of Reston ebolavirus (RESTV) infection was found in domestic pigs and pig workers in the Philippines. With species of bats having been shown to be the cryptic reservoir of filoviruses elsewhere, the Philippine government, in conjunction with the Food and Agriculture Organization of the United Nations, assembled a multi-disciplinary and multi-institutional team to investigate Philippine bats as the possible reservoir of RESTV. METHODS: The team undertook surveillance of bat populations at multiple locations during 2010 using both serology and molecular assays. RESULTS: A total of 464 bats from 21 species were sampled. We found both molecular and serologic evidence of RESTV infection in multiple bat species. RNA was detected with quantitative PCR (qPCR) in oropharyngeal swabs taken from Miniopterus schreibersii, with three samples yielding a product on conventional hemi-nested PCR whose sequences differed from a Philippine pig isolate by a single nucleotide. Uncorroborated qPCR detections may indicate RESTV nucleic acid in several additional bat species (M. australis, C. brachyotis and Ch. plicata). We also detected anti-RESTV antibodies in three bats (Acerodon jubatus) using both Western blot and ELISA. CONCLUSIONS: The findings suggest that ebolavirus infection is taxonomically widespread in Philippine bats, but the evident low prevalence and low viral load warrants expanded surveillance to elaborate the findings, and more broadly, to determine the taxonomic and geographic occurrence of ebolaviruses in bats in the region.

Cedar virus: a novel Henipavirus isolated from Australian bats.

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin-B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-ß response than HeV.

The application of one health approaches to henipavirus research.

Henipaviruses cause fatal infection in humans and domestic animals. Transmission from fruit bats, the wildlife reservoirs of henipaviruses, is putatively driven (at least in part) by anthropogenic changes that alter host ecology. Human and domestic animal fatalities occur regularly in Asia and Australia, but recent findings suggest henipaviruses are present in bats across the Old World tropics. We review the application of the One Health approach to henipavirus research in three locations: Australia, Malaysia and Bangladesh. We propose that by recognising and addressing the complex interaction among human, domestic animal and wildlife systems, research within the One Health paradigm will be more successful in mitigating future human and domestic animal deaths from henipavirus infection than alternative single-discipline approaches.

Risk Factors for Nipah virus infection among pteropid bats, Peninsular Malaysia.

We conducted cross-sectional and longitudinal studies to determine the distribution of and risk factors for seropositivity to Nipah virus (NiV) among Pteropus vampyrus and P. hypomelanus bats in Peninsular Malaysia. Neutralizing antibodies against NiV were detected at most locations surveyed. We observed a consistently higher NiV risk (odds ratio 3.9) and seroprevalence (32.8%) for P. vampyrus than P. hypomelanus (11.1%) bats. A 3-year longitudinal study of P. hypomelanus bats indicated nonseasonal temporal variation in seroprevalence, evidence for viral circulation within the study period, and an overall NiV seroprevalence of 9.8%. The seroprevalence fluctuated over the study duration between 1% and 20% and generally decreased during 2004-2006. Adult bats, particularly pregnant, with dependent pup and lactating bats, had a higher prevalence of NiV antibodies than juveniles. Antibodies in juveniles 6 months-2 years of age suggested viral circulation within the study period.

Identification of diverse full-length endogenous betaretroviruses in megabats and microbats.

BACKGROUND: Betaretroviruses infect a wide range of species including primates, rodents, ruminants, and marsupials. They exist in both endogenous and exogenous forms and are implicated in animal diseases such as lung cancer in sheep, and in human disease, with members of the human endogenous retrovirus-K (HERV-K) group of endogenous betaretroviruses (ßERVs) associated with human cancers and autoimmune diseases. To improve our understanding of betaretroviruses in an evolutionarily distinct host species, we characterized ßERVs present in the genomes and transcriptomes of mega- and microbats, which are an important reservoir of emerging viruses. RESULTS: A diverse range of full-length ßERVs were discovered in mega- and microbat genomes and transcriptomes including the first identified intact endogenous retrovirus in a bat. Our analysis revealed that the genus Betaretrovirus can be divided into eight distinct sub-groups with evidence of cross-species transmission. Betaretroviruses are revealed to be a complex retrovirus group, within which one sub-group has evolved from complex to simple genomic organization through the acquisition of an env gene from the genus Gammaretrovirus. Molecular dating suggests that bats have contended with betaretroviral infections for over 30 million years. CONCLUSIONS: Our study reveals that a diverse range of betaretroviruses have circulated in bats for most of their evolutionary history, and cluster with extant betaretroviruses of divergent mammalian lineages suggesting that their distribution may be largely unrestricted by host species barriers. The presence of ßERVs with the ability to transcribe active viral elements in a major animal reservoir for viral pathogens has potential implications for public health.

Ecological aspects of hendra virus.

Hendra virus, a novel and fatally zoonotic member of the family Paramyxoviridae, was first described in Australia in 1994. Periodic spillover from its natural host (fruit bats) results in catastrophic disease in horses and occasionally the subsequent infection of humans. Prior to 2011, 14 equine incidents involving seven human cases (four fatal) were recorded. The year 2011 saw a dramatic departure from the sporadic incidents of the previous 16 years, with a cluster of 18 incidents in a single 3-month period. The fundamental difference in 2011 was the total number of incidents, the geographic clustering, and the expanded geographic range. The 2011 cluster more than doubled the total number of incidents previously reported, and poses the possibility of a new HeV infection paradigm. Epidemiologic evidence suggests that compelling additional host and/or environmental factors were at play.

The immune gene repertoire of an important viral reservoir, the Australian black flying fox.

BACKGROUND: Bats are the natural reservoir host for a range of emerging and re-emerging viruses, including SARS-like coronaviruses, Ebola viruses, henipaviruses and Rabies viruses. However, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. Massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. Although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. In this study, we describe the immune transcriptome of the Australian flying fox, Pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity. RESULTS: Towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from P. alecto. We identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. A small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts. CONCLUSIONS: This study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. In addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection.

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs.

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94 % identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery.

Experimental infection of horses with Hendra virus/Australia/horse/2008/Redlands.

Hendra virus (HeV) is a highly pathogenic zoonotic paramyxovirus harbored by Australian flying foxes with sporadic spillovers directly to horses. Although the mode and critical control points of HeV spillover to horses from flying foxes, and the risk for transmission from infected horses to other horses and humans, are poorly understood, we successfully established systemic HeV disease in 3 horses exposed to Hendra virus/Australia/Horse/2008/Redlands by the oronasal route, a plausible route for natural infection. In 2 of the 3 animals, HeV RNA was detected continually in nasal swabs from as early as 2 days postexposure, indicating that systemic spread of the virus may be preceded by local viral replication in the nasal cavity or nasopharynx. Our data suggest that a critical factor for reducing HeV exposure risk to humans includes early consideration of HeV in the differential diagnosis and institution of appropriate infection control procedures.

Urban habituation, ecological connectivity and epidemic dampening: the emergence of Hendra virus from flying foxes (Pteropus spp.).

Anthropogenic environmental change is often implicated in the emergence of new zoonoses from wildlife; however, there is little mechanistic understanding of these causal links. Here, we examine the transmission dynamics of an emerging zoonotic paramyxovirus, Hendra virus (HeV), in its endemic host, Australian Pteropus bats (fruit bats or flying foxes). HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. With models parametrized from field and laboratory data, we explore a set of probable contributory mechanisms that explain the spatial and temporal pattern of HeV emergence; including urban habituation and decreased migration-two widely observed changes in flying fox ecology that result from anthropogenic transformation of bat habitat in Australia. Urban habituation increases the number of flying foxes in contact with human and domestic animal populations, and our models suggest that, in addition, decreased bat migratory behaviour could lead to a decline in population immunity, giving rise to more intense outbreaks after local viral reintroduction. Ten of the 14 known HeV outbreaks occurred near urbanized or sedentary flying fox populations, supporting these predictions. We also demonstrate that by incorporating waning maternal immunity into our models, the peak modelled prevalence coincides with the peak annual spill-over hazard for HeV. These results provide the first detailed mechanistic framework for understanding the sporadic temporal pattern of HeV emergence, and of the urban/peri-urban distribution of HeV outbreaks in horses and people.

Australian Bat Lyssavirus: Analysis of National Bat Surveillance Data from 2010 to 2016.

Australian bat lyssavirus (ABLV) was first described in 1996 and has been regularly detected in Australian bats since that time. While the virus does not cause population level impacts in bats and has minimal impacts on domestic animals, it does pose a public health risk. For this reason, bats are monitored for ABLV and a national dataset is collated and maintained by Wildlife Health Australia. The 2010-2016 dataset was analysed using logistic regression and time-series analysis to identify predictors of infection status in bats and the factors associated with human exposure to bats. In common with previous passive surveillance studies, we found that little red flying-foxes (Pteropus scapulatus) are more likely than other species to be infected with ABLV. In the four Australian mainland species of flying-fox, there are seasonal differences in infection risk that may be associated with reproductive cycles, with summer and autumn the seasons of greatest risk. The risk of human contact was also seasonal, with lower risk in winter. In line with other studies, we found that the circumstances in which the bat is encountered, such as exhibiting abnormal behaviour or being grounded, are risk factors for ABLV infection and human contact and should continue be key components of public health messaging. We also found evidence of biased recording of some types of information, which made interpretation of some findings more challenging. Strengthening of "One Health" linkages between public health and animal health services at the operational level could help overcome these biases in future, and greater harmonisation nationally would increase the value of the dataset.