key: cord-0939088-8dzhibb4 authors: Yuen, Ka Y.; Fraser, Natalie S.; Henning, Joerg; Halpin, Kim; Gibson, Justine S.; Betzien, Lily; Stewart, Allison J. title: Hendra virus: Epidemiology dynamics in relation to climate change, diagnostic tests and control measures date: 2020-12-21 journal: One Health DOI: 10.1016/j.onehlt.2020.100207 sha: 7bf861093e040ba1f5b001899549ab5204b98177 doc_id: 939088 cord_uid: 8dzhibb4 Hendra virus (HeV) continues to pose a serious public health concern as spillover events occur sporadically. Terminally ill horses can exhibit a range of clinical signs including frothy nasal discharge, ataxia or forebrain signs. Early signs, if detected, can include depression, inappetence, colic or mild respiratory signs. All unvaccinated ill horses in areas where flying foxes exist, may potentially be infected with HeV, posing a significant risk to the veterinary community. Equivac® HeV vaccine has been fully registered in Australia since 2015 (and under an Australian Pesticides and Veterinary Medicines Authority special permit since 2012) for immunization of horses against HeV and is the most effective and direct solution to prevent disease transmission to horses and protect humans. No HeV vaccinated horse has tested positive for HeV infection. There is no registered vaccine to prevent, or therapeutics to treat, HeV infection in humans. Previous equine HeV outbreaks tended to cluster in winter overlapping with the foaling season (August to December), when veterinarians and horse owners have frequent close contact with horses and their bodily fluids, increasing the chance of zoonotic disease transmission. The most southerly case was detected in 2019 in the Upper Hunter region in New South Wales, which is Australia's thoroughbred horse breeding capital. Future spillover events are predicted to move further south and inland in Queensland and New South Wales, aligning with the moving distribution of the main reservoir hosts. Here we (1) review HeV epidemiology and climate change predicted infection dynamics, (2) present a biosecurity protocol for veterinary clinics and hospitals to adopt, and (3) describe diagnostic tests currently available and those under development. Major knowledge and research gaps have been identified, including evaluation of vaccine efficacy in foals to assess current vaccination protocol recommendations. Pteropid bats (also known as flying foxes or fruit bats) have been identified as the reservoir host. The virus circulates between asymptomatic flying foxes, and is maintained in these species, providing a common source of infection to horses. Serological surveys of over 5000 sera samples from 46 animal species detected neutralizing HeV antibodies in all four main species of flying foxes in eastern Australia: Pteropus conspicillatus (spectacled fruit bat), P. alecto (black fruit bat), P. scapulatus (little red flying fox), and P. poliocephalus (grey-headed flying fox) [26, 27] . Viral RNA has been detected in a range of tissues in both naturally and experimentally infected P. alecto and P. poliocephalus [28] [29] [30] . Spatial analysis and molecular studies showed that P. alecto and P. conspicillatus are the two main natural reservoir hosts and are likely to be responsible for spillover events [31] [32] [33] [34] . Horses act as an amplifying host and are the only known mammalian species that has been infected directly from bats. To date, two asymptomatic dogs have been reported to have been infected due to a natural infection, arising from exposure to infected horses [35, 36] . Experimental infections have been successful in dogs [36] , pigs [37] , hamsters [38] , guinea pigs [30, 39, 40] , ferrets [41] , African green monkeys [42] , cats [40, 43] and horses [17] . Notwithstanding the very high viral doses used in experimental inoculations, dogs, cats, guinea pigs, ferrets and pigs housed outdoors are plausible susceptible hosts of HeV infection and may conceivably transmit the virus to humans. Horse-to-horse and horse-to-human transmissions are likely via contact with infected bodily fluids, especially nasal or oral secretions, from an infected horse during all stages of disease from preclinical to post-mortem [17] . The veterinary profession is at a particularly high risk with four infected people belonging to this profession and an additional one being the husband of a veterinarian who assisted with a necropsy on a HeV infected horse. Therefore, extra precautions should be taken especially when performing diagnostic, treatment or necropsy procedures involving the upper/lower airway and oral cavity of horses. Risk factors related to horse management J o u r n a l P r e -p r o o f After decades of research, the exact mode of transmission remains inferred from field data. Viral isolation from liver and lung of aborted flying fox fetuses and uterine fluid from aborting female flying fox suggests possible transmission to horses via direct contact with a recently aborted fetus or associated fetal fluid during parturition [29] . Another hypothesis is oro-nasal mucosal contact with infected flying fox urine, or less likely with other fluids such as blood, feces, nasal discharge and saliva, via contaminated pasture or feed, especially when horses are feeding under trees where flying foxes are foraging or roosting [32, 33] . Considering HeV survival is relatively short (likely to be less than 96 hours) and very sensitive to environmental conditions [44] [45] [46] , direct contact of the mucosal membranes (such as conjunctiva) with fresh infectious flying fox urine is more likely to allow HeV transmission to occur. Interestingly, studies demonstrated higher prevalence of HeV genetic material in P. alecto and P. conspicillatus as compared to the two other species [32] [33] [34] 47] suggesting they are more important in maintaining and transmitting infection both within-species and cross-species. Spatial analysis studies showed that previous spillover events were associated with close proximity and denser populations of P. alecto and P. conspicillatus roosts [31, [48] [49] [50] . A recent study suggested that immunologically naïve P. alecto sub-adults may be important in maintaining HeV infection within-species at a population level [47] . The larger population size and geographic distribution of P. alecto makes P. alecto potentially the most important species in transmission and spillover of HeV, followed by P. conspicillatus, P. poliocephalus and P. scapulatus. While densities of dominant flying fox species in a region can be affected by urbanization [51, 52] , P. alecto territories are expanding and could potentially be replacing or dominating current P. conspicillatus territory in northern and eastern Australia [53] , increasing the risk of spillover events to additional areas in northern Queensland (QLD). Likewise, P. alecto is expanding its territories further south, with implications for southern New South Wales (NSW), Victoria and South Australia. Hendra virus infection dynamics vary spatio-temporally. HeV excretion from flying fox urine fluctuates within-year and peaks in autumn and winter especially along the eastern coast (southern QLD and northern NSW) and central NSW [34, 47, 54] . This coincides with many mid-year spillover events in these locations. This is especially concerning to veterinarians as foaling season commences infection prevalence in pteropid bats may be a main driver for horse infection [34] , spillover events are rare and sporadic. In addition, HeV antibodies were first detected in archived samples from flying foxes collected at least 10 years before the first recorded equine HeV case [27] . It is unknown if unidentified equine cases occurred throughout this time, but HeV appears to be a true emerging infectious disease of horses and humans. Around 20% of cases occurred in spring and summer months, therefore, there are still some drivers within the complex multifactorial HeV transmission cycle remain unidentified. Flying fox distributions are highly dependent on food sources, a combination of nectar, pollen and fruit especially those produced by Eucalyptus trees in woodlands and open forests [55] [56] [57] . Flying foxes have an important role in maintaining a balanced ecosystem through pollination and seed dispersion [55] . Seasonal climatic triggers (e.g. normal changes in temperature) are one of the many factors that influences eucalypts flowering patterns [58, 59] . Extreme dry conditions are associated with poor flowering [59] . Therefore, a lack of food resources due to tree clearance and climate change may disperse flying foxes outside of typical territories with resultant increased levels of stress. Studies using urine cortisol concentrations to measure physiological stress in pteropid bats have demonstrated lower winter temperatures increase cortisol concentrations which is associated with HeV excretion in flying foxes [60, 61] . Body condition score is a proxy of nutritional status. Poor body condition is associated with increased seroconversion and HeV infection risk in flying foxes. This increases urinary excretion of viable virus, with increased risk of transmission to horses [47, 62] . HeV prevalence in bats and winter HeV spillover events [34, 50, 57] . Although most HeV outbreaks tend to cluster in cooler months, individual equine cases present all year round. Studies showed that flying foxes excrete HeV throughout the year and it is possible for spillover events to peak in other seasons [50, 54] . El Niño and drought events are predicted to occur more frequently in the future due to climate change [64, 65] . Therefore, these extreme weather conditions coupled with the cascading effects to flying foxes may increase the risk and frequency of HeV spillover events/outbreaks and expansion into novel areas. The territory of P. alecto, the main reservoir host is expanding southward, and it was predicted equine HeV infections would occur within the high horse density Hunter Valley, the Thoroughbred racehorse breeding capital of Australia [53] . A year after this prediction, the first and southernmost case of HeV was recorded in June 2019 in the Upper Hunter Valley. HeV spillover events have been expanding southward along the eastern coast and inland ( Figure 1 ). Equivac® HeV vaccine is a subunit-based vaccine, manufactured by Zoetis Australia, containing recombinant HeV soluble G (sG) glycoprotein and thiomersal (adjuvant) [66, 67] . The primary immunization protocol requires two doses administered as an intramuscular injection three to six weeks apart, then another dose 6 months later. An annual booster is required thereafter to maintain immunity to HeV. J o u r n a l P r e -p r o o f Hendra virus vaccine uptake has been low [75] , due to a wide range of reasons, including owner's perceived knowledge of HeV and vaccine safety and efficacy concerns [69, 76, 77] . This is possibly due to the effects of amplified reports of anecdotal adverse events from social media. However, a Queensland state government parliamentary enquiry concluded that the vaccine was the best way to prevent HeV infections in horses and humans [78, 79] . Veterinarians should build rapport and educate horse owners with scientific evidence about HeV and the importance of vaccination as a public health initiative [69, 80, 81] . HeV vaccination remains to be the most effective, reliable and direct approach to prevent infection in horses and therefore transmission to humans. Other management strategies, such as targeted tree clearance, are possible to implement but can be overwhelmingly complex, operationally challenging and would disturb existing ecosystems. As the risk of HeV spillover events are likely to expand to novel areas, it is important for veterinarians in areas where flying foxes are endemic to establish a biosecurity plan to protect horses, horse owners, veterinarians and veterinary nurses/technicians. The authors propose the following strict biosecurity protocol 1 for individual veterinary clinics and hospitals to adopt to minimize risk ( Figure 2) . A less rigorous protocol that allows routine procedures to be performed on unvaccinated or unexcluded horses with use of personal protective equipment (PPE) at the discretion of the veterinarians ( Figure 3) is also presented. These models may also be suitable to adapt for other infectious diseases. For a horse to be considered adequately HeV vaccinated its vaccination status must be recorded on an Horses > 6 months of age that have not been appropriately vaccinated and foals born from unvaccinated mares should be considered as unvaccinated. To minimize risk to veterinary hospital personnel, in the strict policy (Figure 2) Although HeV has not been studied in foals, a foal born from a vaccinated mare is assumed to be protected by passive transfer of immunity before 6 months of age if colostrum intake was adequate. Currently it is recommended that foals from unvaccinated mares commence HeV vaccination at 4month-of-age, and vaccination be delayed until 6-month-of-age for foals born to vaccinated mares. However, these recommendations are based on studies from other diseases and there are no published studies on the persistence of HeV-specific maternal antibody and HeV vaccination responses in foals. HeV infection, foals should be considered as susceptible hosts. Therefore, a cross-sectional longitudinal study of HeV-specific antibody concentrations in foals is warranted. Veterinary institutions and some privately owned veterinary clinics have workplace health and safety responsibilities to equine and veterinary students rotating through the equine hospitals for practical J o u r n a l P r e -p r o o f experiences. This makes a strict biosecurity protocol ( Figure 2 ) even more crucial to protect all stakeholders. It is advisable that students should not be in-contact with any unvaccinated horse before a negative exclusion test result is obtained. The assigned HeV exclusion test stalls should be physically away from the hospital vicinity and only accessible by trained staff members. While fruit trees and eucalypts should be removed from equine paddocks to discourage flying foxes foraging in the area, solid overhead covering should be installed to provide shade for horses to fulfil fundamental animal welfare responsibilities. Water troughs and feed should be placed away from trees in open areas or under a solid covering to prevent contamination from flying fox excreta. A range of diagnostics tests are available through government veterinary laboratories, ranging from viral isolation, serology and molecular testing (Table 1, Figure 4 ). Diagnostic sensitivity (Se) and specificity (Sp) have been included where available. Veterinarians can submit swabs and blood samples to the state veterinary laboratories for molecular TaqMan real-time reverse transcription polymerase chain reaction (qRT-PCR) assay specific for the matrix (M) and nucleoprotein (N) gene of HeV [82] [83] [84] . This highly sensitive molecular test detects HeV-specific ribonucleic acid, and can provide results within four hours [83] . A negative result should be interpreted within the clinical and epidemiological context of that case. Positive results indicate presence of viral genome. As HeV is a notifiable disease in Australia [85] , when a positive exclusion test is detected, relevant government officials/department are notified by the state laboratory and a series of biosecurity responses will be triggered. Submitted samples will be transported to Australian Centre for Disease Preparedness (ACDP) in Victoria for further testing, namely PCR and viral isolation [86] , for a definitive diagnosis. As HeV is a biosafety level 4 agent, any laboratory activities involving live HeV, Indirect enzyme-linked immunosorbent assay (iELISA) and serum neutralization tests (SNT, also known as virus neutralization tests) are used to detect antibodies. A new iELISA assay was recently developed as a screening test (Se = 84.2%, Sp = 97.1%), using a recombinant-expressed HeVsG glycoprotein [67] , which has improved specificity compared to the previous HeV iELISA which uses inactivated virus, and both tests can be performed in a PC2/3 laboratory [84, 88, 89] . A positive result (S/P ratio > 0.4) indicates presence of HeV-specific antibodies [88] . All inconclusive and positive results from HeVsG iELISA are subjected to SNT as a confirmatory test, which is highly specific, and allows for the determination of HeV-specific antibody titer [84, 90] . Furthermore, due to the biological nature of the SNT which uses live virus, repeatability is an issue, making test results difficult to compare across runs. Bead-based fluorescent microsphere immuno-assays (Luminex®) allow detection and differentiation of HeV and NiV specific antibodies in one test via a total antibody-binding format, and pseudo-viral neutralization using recombinant proteins via a restricted-receptor blocking format [86, 90] . Both assays utilize recombinant sG proteins of HeV or NiV and require the use of Bio-Plex® Protein Array System and software for data acquisition and analysis. The result of the binding assay is recorded as median fluorescent intensity (MFI) of 100 beads which can then be transformed to percent positive relative to the MFI from positive control, while the blocking assay is reported as percent inhibition [84, 86, 90] . Luminex® is highly sensitive and specific (blocking assay Se = 95.24%, Sp = 100%; binding assay Se = 95.24%, Sp = 99.64%) [86] . As no live HeV is used, a PC2 laboratory is adequate. Although the blocking assay is designed as a surrogate of viral neutralization, it does not provide an antibody titer value. For this reason, further investigation is required to determine if there is a correlation between the test results of SNT and Luminex®. To overcome this, an approach differentiating infected and vaccinated animals (also known as DIVA) using ELISA is currently being developed specific for the N protein of HeV [86] . Loop-mediated isothermal amplification (LAMP) assay first described by Notomi et al. [91] , is an emerging rapid point-of-care diagnostic tool for a number of animal diseases, such as footrot caused by Dichelobacter nodosus [92] , foot and mouth disease [93] , bovine viral diarrhea virus [94] and other OIE notifiable diseases [95] . Currently, all diagnostic tests for HeV require at least several hours to days and require specialized equipment. Some assays also require a PC4 laboratory. LAMP for HeV detection is currently under development. Once validated and commercially available, veterinarians may be able to carry a portable device to perform preliminary HeV testing in the field. However, LAMP is not likely to become a reference standard for confirmatory diagnosis. Taqman qRT-PCR assay performed in a laboratory should remain the gold standard for HeV exclusion testing. Nevertheless, LAMP technology can be used for early detection of HeV thus guiding implementation of biosecurity protocols to protect human and animal lives. HeV continues to pose a serious public health threat, animal welfare concerns and wildlife conservation issues. While it is important to understand the ecology and transmission of HeV, these studies are time consuming and further research is required to reduce human and horse infections. Luminex® and SNT would result in reduced turnaround time for reporting, address workplace health and safety concerns, and technical difficulties due to the need for a PC4 laboratory for SNT. SNT titer cut-off at which annual booster could be safely delayed (due to previous vaccination reactions) requires further study. Development of point-of-care diagnostics will improve biosecurity response, thus animal and human health. Lastly, while this review focused on the specific characteristics of HeV epidemiology, especially in regards to diagnostics and biosecurity, human behaviors and attitudes towards HeV vaccination and associated side effects and adverse events are likely to play a major role in HeV prevention. This review did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. The authors declare no competing interests. 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