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Rev. sci. tech. Off. int. Epiz., 2000, 19 (1), 151-159 Hendra virus disease in horses H.A. Westbury Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australian Animal Health Laboratory, P.O. Bag 24, Geelong, Victoria 3220, Australia Summary The author provides an account of the discovery of a previously undescribed disease of horses and a description of the studies involved in determining the aetiology of the disease. The causative virus, now named Hendra virus (HeV), is the reference virus for a proposed n e w genus within the virus family Paramyxoviridae. The virus is a lethal zoonotic agent able to cause natural disease in humans and horses and experimentally induced disease in cats, guinea-pigs and mice. The virus also naturally infects species of the family Megachiroptera, mainly subclinically, and such animals are the natural host of HeV. The virus appears to transmit readily between species of Megachiroptera, but not readily between horses under natural and experimental conditions, or from horses to humans. The method of transmission from bats to horses is not known. Three incidents of HeV disease in horses have been recorded in Australia - t w o in 1994 which caused the death of two humans and fifteen horses and one in 1999 which involved the death of a single horse. Hendra virus is related to Nipah virus, the virus that caused disease and mortality in humans, pigs, dogs and cats in Malaysia during 1998 and 1999. Keywords Australia - Bats - Biosecurity level 4 - Equine morbillivirus - Hendra virus - Horses Menangle virus - Nipah virus - Pteropus - Public health - Zoonoses. Introduction In August 1994, two horses d i e d suddenly o n a property in Mackay, Queensland, Australia. T h e owners, a veterinarian and her h u s b a n d , p e r f o r m e d p o s t - m o r t e m examinations o n the horses, and s a m p l e s w e r e submitted for laboratory examination. T h e cause of death was attributed to a v o c a d o toxicity and snakebite, respectively, based on histopathological examination, and n o further tests into the cause of death w e r e undertaken. T e n days after the death of the s e c o n d horse, the h u s b a n d was admitted to hospital with meningitis, from w h i c h h e m a d e an apparently successful recovery. T h e s e incidents w o u l d h a v e b e e n consigned to the family history e x c e p t for a totally u n e x p e c t e d event fourteen months later. One m o n t h after the incident in Mackay, thirteen of twenty thoroughbred h o r s e s d i e d o r w e r e euthanised at a stable in Hendra, Brisbane, Queensland (approximately 8 0 0 k m south of Mackay), with the deaths occurring over a p e r i o d of two weeks. T h e owner/trainer and a stable worker, b o t h of w h o m h a d contact with the affected h o r s e s , b e c a m e ill with a n influenza-like disease, from w h i c h the trainer d i e d after six days in intensive care. A link b e t w e e n the illness and death in h u m a n s and the virulent respiratory disease in the horses was not considered initially b e c a u s e the physician suspected Legionella infection as the likely cause of illness in the trainer. Similarly, the clinical signs and lesions in the horses w e r e suggestive of acute African h o r s e sickness, or p e r h a p s acute equine influenza (diseases exotic to Australia), and investigations w e r e c o n d u c t e d o n this basis. However, these potential diagnoses w e r e eliminated rapidly, as w e r e bacterial diseases, plant intoxications and poisoning. A n o v e l paramyxo-like virus was isolated from the affected horses, and from the trainer o n p o s t - m o r t e m examination. This virus r e p r o d u c e d the disease s y n d r o m e w h e n inoculated into horses, and specific antibodies w e r e detected in the trainer and stable worker, and in all the r e c o v e r e d horses. T h e gross and microscopic lesions detected at p o s t - m o r t e m in the naturally and experimentally infected horses and in the trainer w e r e similar, and the convalescent h u m a n sera reacted strongly in immunofluorescent and i m m u n o p e r o x i d a s e tests o n tissues from affected horses. Furthermore, this virus induced similar clinical signs and lesions in cats inoculated with the virus, and similar m i c r o s c o p i c lesions in experimentally infected guinea-pigs. T h e virus was therefore 152 considered to b e the causative agent of the disease and a n e w zoonotic pathogen. T h e n a m e equine morbillivirus was suggested initially b e c a u s e the virus was m o r e closely related to the morbillivirus genus of the virus family Paramyxoviridae than to the other virus groups in the family (12). However, subsequent research demonstrated that the virus was unique and p r o b a b l y best categorised as the first virus of a n e w genus within the Paramyxoviridae family (16). It h a s n o w b e e n n a m e d Hendra virus (HeV). In October 1995, the m a n w h o assisted with the post-mortems of the horses in Mackay was again admitted to hospital with clinical signs of a disease of the central nervous system (CNS). He d i e d following an illness w h i c h lasted twenty-five days, characterised b y seizures and worsening paralysis. Extensive microbiological testing for recognised causes of s u c h disease did not reveal the cause of death. In desperation, tests for HeV infection w e r e requested b e c a u s e of the connection with horses and the earlier death of the h o r s e trainer in Brisbane, although the presenting signs of this disease w e r e different. Hendra virus antigen was demonstrated in the brain using immunofluorescence and i m m u n o e l e c t r o n m i c r o s c o p y , and typical HeV nucleocapsids w e r e s e e n in ultra-thin sections of neuronal cells (6, 9). T h e polymerase chain reaction (PCR) was u s e d to detect HeV in cerebrospinal fluid and brain tissue, and the nucleotide s e q u e n c e of PCR product was h o m o l o g o u s with that of HeV isolated in 1994. A significant rise in specific neutralising antibody titre to HeV was detected during the course of the disease (from 1:16 at admission to 1:5,792 just before death). T h e virus was not isolated, p e r h a p s b e c a u s e of the virus-antibody complexing. T h e s e results incriminated HeV as the cause of the disease, especially as the m i c r o s c o p i c lesions (vascular thrombosis and occasional multinucleated giant cells in the e n d o t h e l i u m of b l o o d vessels) w e r e also consistent with HeV i n d u c e d disease. Furthermore, a neutralising antibody titre of 1:4 was detected in a s e r u m sample collected from the patient s o o n after his recovery from the e p i s o d e of meningitis in August 1994. This result suggested that the source of infection with HeV m a y h a v e b e e n the horses that d i e d in 1994, e v e n t h o u g h these w e r e p r e s u m e d to h a v e d i e d from a v o c a d o poisoning and s n a k e bite. Retrospective testing of stored s p e c i m e n s from b o t h horses, using specific fluorescent antibody and PCR tests, revealed that b o t h h o r s e s w e r e infected with HeV and, despite the absence of k e y tissue samples s u c h as lung, the histopathological lesions w e r e consistent with HeV disease (6). Thus, in 1994, two outbreaks of HeV disease occurred in Queensland, o n properties approximately 8 0 0 k m apart and b e t w e e n w h i c h n o apparent direct or indirect contact existed (3). A 'think-tank' of scientists within the Queensland Department of Primary Industries conjectured that birds or bats m a y b e h a v e b e e n the source of the virus and the link b e t w e e n the two affected properties. Serological testing of birds and bats k n o w n to inhabit b o t h Brisbane and Mackay Rev. sci. tech. Off. int. Epiz.,19(1) was therefore undertaken. Specific s e r u m neutralising antibody was s o o n detected and HeV isolated in bats of the four species of the genus Pteropus found in Australia. Specific antibody was not detected in other fauna tested, or in horses, following an intensive structured serological survey in Queensland and elsewhere in Australia. This data p o i n t e d to fruit bats of the genus Pteropus as b e i n g the natural reservoir h o s t of HeV (21), and these animals w e r e the p r o b a b l e source of infection for the horses, although the m o d e of infection is unknown. A further case of HeV disease in a h o r s e occurred in north Queensland in 1999. This was the first r e c o r d e d case since 1994, despite intensive on-going surveillance b y government animal health agencies and private veterinary practitioners. This history suggests that transmission of virus from the natural h o s t to h o r s e s is a sporadic and occasional event, a concept supported b y a failure to detect HeV infection during retrospective examination of stored lung s a m p l e s collected from horses with p n e u m o n i c lesions. N i p a h virus, a virus closely related to HeV, caused a dramatic outbreak of disease in h u m a n s and pigs, and s o m e other animal species, in Malaysia in 1998 and 1999. Affected h u m a n s exhibited clinical signs of neurological disease following exposure to pigs with respiratory and/or CNS disease caused b y the virus (1). N i p a h virus transmits naturally and easily b e t w e e n pigs o n a farm and b e t w e e n pig farms (by m o v e m e n t of pigs). Transmission from pigs, and p e r h a p s other animals, to h u m a n s requires close contact (e.g. as during obstetrical interventions), and transmission b e t w e e n h u m a n s d o e s not occur readily, if at all. Serum neutralising antibody to N i p a h virus h a s b e e n detected in bats and, as with HeV, these are suspected to b e the natural h o s t of the virus. Nipah virus is related morphologically, antigenically and genetically to HeV, and is p r o b a b l y the s e c o n d m e m b e r of the putative megamyxovirus genus of the family Paramyxoviridae. Nipah virus is distinguishable from HeV using serological and molecular tests (1). T h e finding that bats of the genus Pteropus, family Megachiroptera, m a y b e the natural host of these viruses, as well as the possible h o s t of other n e w viruses s u c h as Menangle virus (14) and n e w strains of lyssavirus (4), points to a n e e d for a better understanding of the microbiological status of these creatures. Importance for animal and public health Hendra and N i p a h viruses are previously unrecognised viruses w h i c h can cause fatal disease in h u m a n s and animals. Hendra virus e m e r g e d in 1994, and N i p a h virus in 1998, although there are n o w indications that N i p a h virus m a y have b e e n present in pigs in Malaysia b e f o r e that time. E a c h has caused variable clinical manifestations in animals, ranging 153 Rev. sci. tech. Off. int. Epiz., 19 (1) from severe to m i l d disease in the respiratory tract, in addition to disease in the CNS. T h e s a m e s y m p t o m s h a v e also b e e n observed in h u m a n s , s o m e t i m e s with relapses following a primary CNS disease e p i s o d e , in o n e instance (with HeV), fourteen m o n t h s after the first occurrence (13). H u m a n s b e c o m e infected as a result of handling infected animals. N o instances of h u m a n - t o - h u m a n transmission of HeV or Nipah virus h a v e b e e n r e c o r d e d to date. Infected animals m a y include the natural host, thought to b e fruit bats of the Megachiroptera family, and unusual hosts, s u c h as horses for HeV, or pigs, d o g s and cats for Nipah virus. T h e m e c h a n i s m of transfer of the virus from the usual to the unusual animal host is n o t understood, nor is the m o d e of transmission to humans, although close contact with the infected animal seems to b e necessary. N o vaccines are currently available to control these diseases in animals or h u m a n s and, at the time of writing, n o d o c u m e n t e d information exists regarding the effectiveness of antivirals, although these h a v e b e e n tested in Malaysia. T h e relative l a c k of information about the viruses and the e p i d e m i o l o g y of the diseases they cause, together with the unavailability of vaccines and antivirals with assessed effectiveness as therapeutic c o m p o u n d s in h u m a n s , h a v e caused regulatory agencies to require w o r k with these viruses to b e p e r f o r m e d at the highest possible level of microbiogical security, i.e. biosecurity level 4 (BL4). Thus, extreme care is required in defining p r o c e d u r e s for field staff working with infected animals and o n contaminated premises, and laboratory staff working b o t h in vitro and in vivo, within the confines of BL4. The virus Hendra virus was originally isolated from h o r s e tissues using Vero (African green m o n k e y k i d n e y ) m o n o l a y e r cell cultures in w h i c h the virus i n d u c e d a focal syncytial cytopathogenic effect (CPE) after approximately three days. T h e h u m a n virus was isolated in LLC-MK2 and MRC5 cell monolayers in which it i n d u c e d focal CPE after approximately twelve days. However, the h o r s e trainer h a d detectable levels of s e r u m neutralising antibody at the time of death, and thus the amount of virus present in the tissues m a y h a v e b e e n quite small. T h e virus w a s isolated f r o m the k i d n e y only. Hendra virus will g r o w o n a remarkably w i d e range of cell culture systems, although Vero cells are m o s t c o m m o n l y used. Morphological characteristics place HeV within the family Paramyxoviridae, but it cannot, o n defining morphological, biological and g e n o m i c grounds, b e easily categorised into the existing genera of the family (i.e., rubulavirus, morbillivirus, respirovirus, pneumovirus and metapneumovirus). W a n g et al. have suggested that HeV is the first virus of a n e w genus within the family (16). This suggestion is currently being considered b y the International C o m m i t t e e for the T a x o n o m y of Viruses. Nipah virus is p r o b a b l y the s e c o n d m e m b e r of this p r o p o s e d n e w genus within the family. Hendra virus h a s unique m o r p h o l o g i c a l characteristics that are useful for diagnostic purposes. Electron micrographs of negatively stained virus reveal p l e o m o r p h i c e n v e l o p e d particles containing characteristic paramyxovirus nucleocapsids. T h e e n v e l o p e is very characteristically c o v e r e d with surface projections of 10 n m and 18 n m in length, giving the particle a unique 'double-fringed' appearance. Such a double-fringed arrangement is n o t a feature of previously described m e m b e r s of the Paramyxoviridae family. Free-lying h e r r i n g b o n e - s h a p e d nucleocapsids, w h i c h w e r e 18 n m w i d e and h a d a periodicity of 5 n m , w e r e frequently s e e n in negatively stained preparations (9). T h e virus is e n v e l o p e d and consequently, inactivation is possible using c o m p o u n d s that disrupt this e n v e l o p e . In the laboratory, o n e of the following is normally used: 3 % lysol for 3 m i n at 20°C; 2 % s o d i u m d o d e c y l sulphate (SDS) for 2 m i n at 100°C; 1% glutaraldehyde for 10 m i n at 20°C; 100% acetone for 15 m i n at 20°C; pure m e t h a n o l for 10 m i n at 0°C; or gamma-ray irradiation for diverse virus inactivation requirements, in addition to f o r m a l d e h y d e fumigation of HeV contaminated animal r o o m s . The disease T h e clinical course in b o t h naturally and experimentally infected horses is short, with h o r s e s usually dying within 3 6 hours of the onset of clinical signs. Affected horses are febrile (>40°C), d e p r e s s e d and h a v e an increased respiratory and heart rate. An increase in the heart rate, in particular, is an indicator of imminent death. Ataxia, h e a d pressing and r e c u m b e n c y , together with a frothy nasal discharge, occur as the disease progresses. T h e incubation p e r i o d in experimentally infected horses is six to twelve days. Only o n e of eight experimentally infected horses has r e c o v e r e d following the onset of clinical disease. Seven h o r s e s recovered or were subclinically infected during the field o u t b r e a k at Hendra (all d e v e l o p e d detectable specific s e r u m antibody to the virus). T w o of these horses subsequently d e v e l o p e d tonic spasms of the muscles of the n e c k and h i n d limbs (15). T h e m o s t significant gross lesions are found in the lungs w h i c h are congested, firm and fluid filled with dilated lymphatics, particularly in the ventral portions. S o m e lungs have fibrin tags o n the pleura. S o m e naturally occurring cases have a thick, foamy haemorrhagic exudate in the airways, and s o m e also h a v e approximately 100 m l of serous fluid in the pericardial sac. Interstitial p n e u m o n i a with proteinaceous alveolar o e d e m a associated with h a e m o r r h a g e , dilated lymphatics, alveolar thrombosis and necrosis of the wall of small b l o o d vessels are the m a i n microscopic lesions. However, p e r h a p s the m o s t remarkable microscopic lesion is the presence of syncytial 154 Rev. sci. tech. Off. int. Epiz., 19 (1) giant cells in b l o o d vessel walls, especially in the endothelium. T h e s e are c o m m o n in lung capillaries and arterioles, t h o u g h they are also present in b l o o d vessels i n l y m p h n o d e s , spleen, brain, s t o m a c h , heart a n d kidney. T h e s e syncytial giant cells react strongly in indirect fluorescent and i m m u n o p e r o x i d a s e tests using specific HeV antiserum. Cytoplasmic inclusion b o d i e s containing nucleocapsid structures c a n b e s e e n i n these syncytial giant cells w h e n e x a m i n e d under the electron microscope. T h e case in Cairns involved a single nine-year-old t h o r o u g h b r e d h o r s e w h i c h shared a p a d d o c k with another horse. T h e affected h o r s e s u c c u m b e d quickly with clinical signs of depression, o e d e m a of the face, lips and n e c k and an elevated heart rate. Rectal temperature and respiratory rate w e r e not r e c o r d e d . T h e disease was diagnosed o n the basis of microscopic pathology, immunostaining, molecular virological tests and electron m i c r o s c o p y . T h e s e c o n d horse was not detectably infected, b a s e d o n clinical examination and serological testing, although the animal shared a feed b i n with the affected h o r s e . Epidemiology T h e disease is n o t highly contagious u n d e r natural or experimental conditions. A five-kilometre radius around the infected premises in the Brisbane outbreak e n c o m p a s s e d m a n y other training stables, as the area lies b e t w e e n two major t h o r o u g h b r e d racing and training tracks. Therefore, m a n y h o r s e s could h a v e b e c o m e infected if the virus was readily transmissible. However, n o spread occurred to other stables contiguous with the infected premises, and not all h o r s e s o n the premises b e c a m e infected. Similarly, h o r s e s in the infected premises p l a c e d in u n c l e a n e d stalls that previously contained h o r s e s that s u c c u m b e d to the disease, did n o t b e c o m e infected. In addition, other h o r s e s o n the infected premises at Mackay and Cairns w e r e n o t infected. T h e index case in the Brisbane outbreak was a pregnant mare k e p t at a spelling p a d d o c k approximately six kilometres from the stable. T h e trainer n o t e d that the m a r e was ill, and m o v e d the animal, together with another healthy h o r s e , to the stable w h e r e treatment was c o m m e n c e d . T h e m a r e died within two days, showing typical clinical signs. T h e h o r s e transported with the mare never b e c a m e ill or detectably infected. A further twelve h o r s e s d i e d within a p e r i o d of seventeen days (ten h o r s e s in the stable, o n e in the neighbouring stable, and another that was transported from the m a i n stable to a p a d d o c k 150 k m away). Seven other h o r s e s w e r e infected, s o m e sub-clinically (four in the m a i n stable a n d three others, all c o n n e c t e d i n s o m e way with the m a i n stable or the neighbouring stable). T h e s e s e v e n h o r s e s w e r e euthanised b y order of veterinary authorities as uncertainty existed about w h e t h e r the h o r s e s w e r e persistently infected with the virus and, if s o , w h e t h e r they might periodically excrete the virus. Fourteen other horses, c o n s i d e r e d b y animal health professionals as likely to h a v e b e e n e x p o s e d to infected horses, did not b e c o m e detectably infected with the virus (2). T w o horses died during the Mackay incident (15). T h e affected horses were h e l d in adjoining p a d d o c k s , but only for a period of less than 2 4 h o u r s after onset of clinical signs in the first horse. Clinical signs in this h o r s e , a pregnant mare, w e r e reported to b e severe respiratory disease, ataxia and m a r k e d swelling of the h e a d , particularly the infra-orbital fossa and c h e e k s . T h e s e c o n d h o r s e , a stallion, reportedly s h o w e d aimless pacing, muscle trembling and haemorrhagic nasal discharge, and the lungs w e r e full of b l o o d at p o s t - m o r t e m . In b o t h instances, death occurred within 2 4 h o u r s of the onset of clinical signs. T h e s e c o n d h o r s e h a d brief contact with the carcass of the first h o r s e , w h i c h was lying slightly inside the dividing fence b e t w e e n the adjoining p a d d o c k s . T h e eleven day interval b e t w e e n the deaths is consistent with the incubation p e r i o d of b e t w e e n six and twelve days, s e e n i n experimental HeV disease and during the o u t b r e a k in Brisbane. N o other h o r s e s o n the property, or i n Queensland, w e r e found to b e infected following epidemiological investigations a n d serological testing (17), n o r w e r e other animal species found to h a v e detectable levels of specific antibody (15). Williamson et al. w e r e unable to demonstrate transmission of the virus b e t w e e n infected a n d in-contact h o r s e s under experimental conditions, although the infected horses d e v e l o p e d the e x p e c t e d disease (albeit without the frothy nasal discharge s e e n in the Brisbane o u t b r e a k ) (20). However, o n e h o r s e did apparently b e c o m e infected following exposure to experimentally infected cats k e p t in close contact with the horses. Similarly, Westbury et al. found transmission of the virus and disease to only o n e of two cats cohabiting with affected cats, but not to cats in contiguous cages (19). Therefore, natural transmission d o e s occur but s e e m s to require very close contact. T h e l o w transmissibility of HeV b e t w e e n h o r s e s and cats contrasts vividly with that of Nipah virus w h e r e the virus spreads readily and rapidly b e t w e e n infected and in-contact pigs u n d e r experimental conditions (H.A. Westbury and D. Middleton, u n p u b l i s h e d data). Nipah virus is not only excreted in the urine of infected pigs, but is also undoubtedly present in aerosols generated during coughing (a c o m m o n clinical sign of the disease), as the virus multiplies in cells lining the u p p e r a n d l o w e r respiratory tracts and h a s b e e n detected in exudate present in the airways of affected pigs. Virological and serological testing suggests that bats of the Megachiroptera family are the natural reservoirs of HeV (21, 2 2 ) although the m e t h o d of transmission from bats to horses is not k n o w n at this time. Similarly, little is k n o w n about the biology of the virus in bats. 155 Rev. sci. tech. Off. int. Epiz., 19 (1) Pathogenesis Horses can b e experimentally infected with HeV following parenteral challenge, and b y the oronasal route ( 1 2 , 2 0 ) . Virus has b e e n isolated from the b u c c a l cavity, b l o o d , brain, spleen, lungs, bronchial and prescapular l y m p h n o d e s , k i d n e y s of experimentally infected horses, and perhaps most significantly, from urine (20). T h e virus h a s also b e e n isolated from the urine of experimentally infected cats (18, 19), this therefore appears to b e an important route of excretion of the virus from infected animals. Williamson et al. speculate that consumption of f o o d material contaminated b y urine containing HeV, c o u l d b e the m e t h o d of transmission u n d e r natural conditions, although virus was not recovered in urine swabs from the floor of stalls containing horses with h i g h titres of virus in b l a d d e r urine at p o s t - m o r t e m (20). T h e virus has also b e e n isolated from saliva of two experimentally infected horses, but not from the rectum or faeces (20). Detailed sequential studies into the pathogenesis of HeV disease in h o r s e s h a v e not b e e n u n d e r t a k e n b e c a u s e s u c h w o r k is expensive and must b e p e r f o r m e d u n d e r current Australian standards, at BL4 (i.e. staff w o r k i n g in fully encapsulating suits: a requirement that m a k e s working with horses difficult). Infected cats provide an excellent m o d e l animal for HeV disease as the underlying respiratory disease process is the s a m e in horses and cats (7, 8). N i p a h virus induces the s a m e disease p r o c e s s in experimentally infected cats (H.A. W e s t b u r y and D. Middleton, unpublished data) and, in contrast to HeV, N i p a h virus h a s also b e e n found to naturally infect cats. Therefore, s o u n d reasons exist for using cats in the study of these n e w zoonotic diseases. C o m p r e s s i o n cages and rapidly acting anaesthetics m a k e the handling of cats u n d e r BL4 conditions practical and safe, and s o these animals offer the opportunity to discover m o r e about the pathogenesis of Hendra and N i p a h viruses. Hendra virus and bats Six of eight susceptible bats inoculated with a d o s e of HeV k n o w n to infect and i n d u c e disease in horses, cats and guinea-pigs, d e v e l o p e d detectable levels of specific antibody to the virus, without exhibiting o b v i o u s clinical signs of disease (20). Only two of these animals h a d m i c r o s c o p i c vascular lesions suggestive of HeV infection w h e n e x a m i n e d twenty-one days after challenge. T h e vascular lesions observed w e r e similar to t h o s e s e e n in experimentally infected guinea-pigs and c o u l d b e specifically stained using immunohistochemical techniques. N o virus was isolated from samples collected at p o s t - m o r t e m w h i c h is p e r h a p s not surprising as the bats h a d d e v e l o p e d specific antibody at the time of virus sampling. Additional experimental studies also induced sub-clinical infection in bats, including animals that were pregnant. During these studies, the virus was isolated from the foetuses of experimentally infected pregnant bats and specific immunostaining was o b s e r v e d in the placenta (M.M. Williamson, personal communication). Studied in combination, these results indicate that HeV d o e s not induce disease in bats, at least u n d e r the conditions of the experiments, but is responsible for a sub-clinical infection, except p e r h a p s in pregnant animals w h e r e the virus might induce congenital disease following transplacental transmission. I n d e e d , Halpin et al. describe the isolation of HeV from uterine discharges of a bat that h a d miscarried twin foetuses, and from three other bats (5). A range of issues concerning HeV infection of bats requires further study. Little is k n o w n of the b i o l o g y of the virus in bats including the m o d e of transmission b e t w e e n bats, w h e n transmission occurs and, p e r h a p s m o s t importantly, h o w the virus is transmitted to its unusual host, the horse. T h e virus infects the four species of p t e r o p i d bats (flying foxes) present in Australia and h a s b e e n found across the entire geographic range of these bats. Serological data also suggests infection of bats in Papua N e w Guinea and N e w Britain (11). This, together with the fact that infection s e e m s to b e subclinical in bats, suggests that the virus is part of the natural microbiological e x p e r i e n c e of bats. W h y infection h a s only apparently e m e r g e d recently is a frequently a s k e d question and opinions and answers are diverse. Diagnosis and surveillance Experience indicates that HeV disease in horses in Australia is u n c o m m o n and sporadic, although this provides n o grounds for c o m p l a c e n c y as there is widespread appreciation of the importance of the disease from b o t h an animal and h u m a n health perspective. T h e possibility of HeV disease in horses should b e considered in cases of s u d d e n death, particularly if there is premonitory respiratory disease, and if the case occurs in an area or region w h e r e fruit bats are c o m m o n . Clinical signs of the onset of disease are a rise in rectal temperature, increased respiratory and heart rates and, occasionally, signs of CNS involvement. Field veterinarians n e e d to take stringent microbiological security precautions when handling potentially infected horses and during autopsy of s u c h horses. T h e s e include, as a m i n i m u m , effective eye, n o s e and m o u t h protection from fomites and aerosols, c o m p l e t e covering of the b o d y with overalls, and d o u b l e gloving with gloves tied b y adhesive tape to the cuffs of the overalls/shirt. In the Australian Animal Health Laboratory of the C o m m o n w e a l t h Scientific and Industrial Research Organisation (CSIRO), all in vivo and in vitro w o r k with live virus and infected animals is p e r f o r m e d at BL4. T h e possibility of b o n e - s t i c k injuries and injuries from other sharp e q u i p m e n t u s e d during autopsy n e e d s to b e in the forefront of planning for s u c h w o r k . The gross pathology in the lungs of affected horses is highly suggestive, but is not p a t h o g n o m o n i c . Not all naturally and experimentally infected horses h a v e exhibited the typical lesions of pulmonary o e d e m a and dilation of the ventral lymphatic of the lung, together with h a e m o r r h a g e and froth in Rev. sci. tech. Off. int. Epiz., 19 (1) 156 the trachea, b r o n c h i and bronchioles. Histologically, syncytial giant cells in b l o o d vessel walls, particularly i n the e n d o t h e l i u m of lung capillaries and arterioles are very characteristic of the disease. T h e s e syncytial cells can also b e s e e n in l y m p h n o d e s , spleen, brain, s t o m a c h , heart and k i d n e y (7, 8). T h e syncytial cells can b e immunostained with specific antibody using fluorescent or i m m u n o p e r o x i d a s e staining techniques. T h e virus grows well in a range of cell culture systems, though Vero cells are c o m m o n l y used. It induces CPE of the syncytial type after approximately three days in Vero cells in the first passage, rarely requiring blind passaging. Electron m i c r o s c o p y (EM) can b e used to visualise the virus b y negative staining in ultrathin sections of infected cell culture material. T h e virus can b e similarly s e e n in tissue sections from affected animals (9). T h e unique 'double fringe' of the virus can b e s e e n i n this way, and the identity of the virus can b e further confirmed b y i m m u n e EM using gold labelled p r o b e s . Detection using PCR, and nucleotide s e q u e n c e characterisation h a s b e e n p e r f o r m e d using cell culture propagated virus and fresh and formalin-fixed tissues from affected animals (6). Specific antibody is detected using an enzyme-linked i m m u n o s o r b e n t assay (ELISA) or virus neutralisation tests (12, 15). Epidemiological investigations and structured serological surveys w e r e c o n d u c t e d in Queensland and other parts of Australia, following the detection of HeV infected h o r s e s in Brisbane and Mackay (2, 15). Similar investigations w e r e undertaken b y the Queensland State Government animal health authorities following the diagnosis of the disease in a single h o r s e in Cairns in 1999 (K.J. Dunn, personal communication). T h e s e studies demonstrated that n o spread of HeV h a d occurred b e y o n d the k n o w n infected premises. In Australia, the m e d i a and professional awareness p r o g r a m m e s h a v e b e e n u s e d to create a h i g h awareness a m o n g government and private veterinarians, and all t h o s e w h o k e e p horses, of the possibility that horses can b e c o m e infected with this lethal zoonotic virus. T h e disease is officially notifiable u n d e r regulations in all States of Australia and all animal carers h a v e an obligation to report suspicions of unusual animal disease to animal disease control authorities. Prophylaxis and treatment N o vaccines are available to control HeV disease and n o antivirals are suitable for use in infected animals, e v e n if this w e r e a financially viable option. T h e o u t b r e a k of the disease in Brisbane was controlled b y a stamping-out p r o g r a m m e involving slaughter of all k n o w n infected horses, quarantine of premises, controls o n the m o v e m e n t of h o r s e s within a defined disease control zone, and serological surveillance to determine the extent of infection. Serological testing revealed n o other k n o w n infected premises. Similarly, serological testing was u s e d to determine the extent of infection o n the infected premises and elsewhere in the incidents in Mackay and Cairns. N o other infected horses w e r e identified o n the premises, suggesting that infection did not spread b e y o n d the horses affected initially (two h o r s e s o n the Mackay property and o n e h o r s e o n the Cairns property). Perspectives T h e isolation of three previously unrecognised zoonotic viruses in Australia in the 1990s (HeV, Australian bat lyssavirus and Menangle virus), the natural hosts of w h i c h appear to b e bats of the family Megachiroptera, and p e r h a p s Microchiroptera, was totally u n e x p e c t e d and p r o v i d e d an Australian perspective to the w o r l d - w i d e c o n c e p t of emerging diseases. T h e recognition of these viruses b e g a n with the s p e e d y discovery of HeV as the cause of an unusual disease in horses w h i c h was zoonotic (12). This was followed rapidly b y the demonstration of the role of bats as the p r o b a b l e natural h o s t of the virus (21), in addition to the disturbing finding of the variable s y m p t o m s in infected h u m a n s and the possible delayed sequelae to infection (10). Subsequently, research a i m e d at acquiring a better understanding of the b i o l o g y of HeV in bats l e d to the discovery of Australian bat lyssavirus (4) and the recognition that Menangle virus was p r o b a b l y also a bat virus (14). Each of these zoonotic viruses w e r e n e w to h u m a n and veterinary m e d i c i n e and presented challenges to disease control authorities not only b e c a u s e the viruses were n e w , but also b e c a u s e a co-ordinated effort was required b e t w e e n h u m a n and animal health authorities and institutions. In addition, it was neccesary to d e v e l o p means and resources for studying a wildlife species w h i c h is difficult to access. Hendra virus disease was not only n e w to h u m a n and veterinary m e d i c i n e but the virus was also n e w to virology. Analysis b y Hyatt and Selleck (9), Yu et al. (23) and W a n g et al. (16) found that the virus was unique and p r o b a b l y was the first recognised m e m b e r of a n e w genus within the Paramyxoviridae family. This p r o m p t e d thoughts about w h e t h e r other m e m b e r s of this putative genus exist, a question answered with the e m e r g e n c e of Nipah virus. Given the extensive distribution of Megachiroptera in Asia, Australia and the surrounding islands, additional m e m b e r s of this p r o p o s e d genus m a y b e discovered i n the future. T h e p r o b l e m of natural zoonotic virus infections in bats is not easily solved. Awareness of these infections is important, as is ensuring that the k n o w l e d g e and m e a n s exist to prevent, or at least significantly diminish, opportunities for 'spillover' of such viruses from the natural h o s t into h u m a n s and other animal populations. H o w 'spillover' of HeV occurs is not k n o w n , although b a s e d o n the Australian experience, it is sporadic and u n c o m m o n . Current data suggest that additional natural hosts of HeV are unlikely to exist in Australia. Consequently, 'spillover' of the virus m u s t occur from bats to horses, although w h e t h e r this is direct, or through intermediaries, requires further study. S o m e time 157 Rev. sci. tech. Off. int. Epiz., 19 (1) may elapse b e f o r e this can b e determined, as the conjunction of events leading to 'spillover' s e e m to occur infrequently. It is also apparent that HeV d o e s not transmit readily b e t w e e n horses. Opportunities for m o r e widespread transmission b e t w e e n h o r s e s existed during the incidents in Brisbane, Mackay and Cairns, but did not occur. In the incident in Brisbane, disease spread to only o n e contiguous property and this s e e m e d to involve direct horse-to-horse contact. T h e explosive o u t b r e a k of disease o n the index property in Brisbane m a y b e inconsistent with this concept, although the sequence of events relating to the management and husbandry of the h o r s e s b e f o r e and during the early stages of the o u t b r e a k is not k n o w n . T h e r e are suspicions that iatrogenic spread of the virus m a y h a v e occurred. Nipah virus, in contrast to HeV, spreads readily b e t w e e n pigs, p e r h a p s b y aerosols generated during coughing, as the virus multiplies in the respiratory epithelium and is present in exudate in the airways of affected pigs (H.A. Westbury and D. Middleton, unpublished data). This feature h a s neither b e e n o b s e r v e d in horses naturally infected with HeV, nor in horses, cats or guinea-pigs experimentally infected with the virus. W h e t h e r this is characteristic of the respective viruses or of the host (pig or horse), is not k n o w n at this time. However, a strain of HeV with the propensity to spread a m o n g horses in the way that Nipah virus spreads in pigs w o u l d significantly change current perspectives of HeV. syncytial cells are present in the e n d o t h e l i u m of b l o o d vessels. T h e s e cells can b e immunostained, as can virus or viral c o m p o n e n t s in ultrathin sections of affected tissues u s e d for EM. T h e virus grows readily in a range of cell cultures and rapidly induces a syncytial-type CPE. It can b e readily visualised in these s a m p l e s b y EM, following negative staining. T h e virus can b e d e t e c t e d in infected cell cultures or fresh, formalin-fixed and paraffin e m b e d d e d tissue s a m p l e s from affected h o r s e s using molecular virological techniques. Thus, presumptive diagnosis of the disease can b e m a d e using conventional histopathological or irrrmunohistochemical techniques at regional animal health laboratories with confirmation of the diagnosis m a d e at central laboratories with access to virus isolation, EM and molecular virological techniques. T h e k e y c o m p o n e n t to diagnosis, as with m o s t diagnoses, is the awareness of the disease b y veterinarians and other animal health professionals w o r k i n g i n the field. This h a s b e e n a c h i e v e d with HeV disease b e c a u s e the significant zoonotic potential of the virus h a s d e m a n d e d changes in the handling of horses and disease diagnosis in h o r s e s , particularly w h e r e respiratory disease is present, and in areas inhabited b y bats. Diagnosis of HeV disease is relatively uncomplicated. T h e gross pathology in the lungs is suggestive, and the microscopic pathology is distinctive especially if giant Infection due ay virus Hendra chez les équidés H.A. Westbury Résumé L'auteur retrace la découverte d'une nouvelle maladie des équidés, non décrite auparavant, et fait le point sur les études consacrées à son étiologie. L'agent causal, qui porte aujourd'hui le nom de virus Hendra, est le virus de référence pour la constitution d'un nouveau genre proposé au sein de la famille des Paramyxoviridae. Agent de zoonoses létales, il est à l'origine d'infections naturelles chez l'homme et les équidés ainsi que d'infections expérimentales chez le chat, le cobaye domestique et la souris. Par ailleurs, ce virus est responsable d'infections naturelles, dans la plupart des cas sans signes cliniques apparents, chez les mégachiroptères, qui en sont l'hôte naturel. Le virus se transmet facilement entre les différentes espèces de mégachiroptères ; il se transmet plus difficilement entre chevaux, dans des conditions naturelles et expérimentales, ainsi qu'à l'homme. On ignore encore le mode de transmission entre les chauves-souris et les équidés. Trois épisodes dus au virus Hendra ont été signalés chez des équidés en Australie - deux en 1994, entraînant la mort de 15 chevaux ainsi que deux décès humains, et un en 1999, causant la mort d'un seul Rev. sci. tech. Off. int Epiz., 19 (1) 158 cheval. Le virus Hendra est apparenté au virus Nipah, responsable d'infections et de morts chez l'homme, mais aussi chez des porcs, des chiens et des chats en Malaysia pendant les années 1998 et 1999. Mots-clés Australie - Chauves-souris - Chevaux - Morbillivirus équin - Niveau 4 de biosécurité Pteropus - Santé publique - Virus Hendra - Virus Menangle - Virus Nipah - Zoonoses. Enfermedad causada por virus Hendra en el caballo H.A. Westbury Resumen El autor da cuenta del descubrimiento de una enfermedad equina hasta ahora no descrita y explica los estudios realizados para determinar la etiología de la enfermedad. El virus causante de esa enfermedad, denominado ahora "virus Hendra", es el virus de referencia de un nuevo género que se ha propuesto añadir dentro de la familia Paramyxoviridae. Se trata de un agente zoonótico letal, capaz de infectar en condiciones naturales al hombre y el caballo y en condiciones experimentales a gatos, cobayas y ratones. En condiciones naturales el virus también infecta a los megaquirópteros (en general de forma subclínica), que constituyen su huésped natural. Este virus, que parece transmitirse fácilmente entre especies de megaquirópteros, no presenta la misma facilidad de transmisión entre caballos, tanto en condiciones naturales como experimentales, ni tampoco del caballo al ser humano. Se desconoce el mecanismo por el cual se transmite de los murciélagos al caballo. En Australia se han registrado tres incidentes de enfermedad equina causada por el virus Hendra, dos en 1994 que causaron la muerte de quince caballos y de dos seres humanos, y uno en 1999 que se saldó con la muerte de un solo caballo. El virus Hendra guarda parentesco con el virus Nipah, el agente que provocó brotes de enfermedad y mortalidad en seres humanos, cerdos, perros y gatos en Malasia en 1998 y 1999. Palabras clave Australia - Caballos - Nivel 4 de segundad biológica - Morbillivirus equino - Murciélagos - Pteropus - Salud pública - Virus Hendra - Virus Menangle - Virus Nipah - Zoonosis. • References Queensland, Australia. In Proc. Commonwealth Veterinary Association Conference, 7-10 November, Singapore. Commonwealth Veterinary Association, Singapore, 57-61. 1. Anon. (1999). - Outbreak of Hendra-like virus. - Malaysia and Singapore, 1998-1999. Morb. Mort. weekly Rep., 48 (13), 265-269. 2. Baidock F.C., Douglas I.C., Halpin K., Field H., Young P.L., Black P.F. & Lim M.G.B. (1996). - Epidemiological investigations into the 1994 equine morbillivirus outbreak in 3. Douglas L, Baidock F. & Black P. (1997). - Outbreak investigation of an emerging disease (equine morbillivirus). Epidemiol. Santé anim., 4 (8), 1-3. 159 Rev. sci. tech. Off.int.Epiz., 13(1) 4. Fraser G.C., Hooper P.T., Lunt R.A., Gould A.R., Gleeson L.J., Hyatt A.D., Russell G.M. & Kattenbelt J.A. (1996). - Encephalitis caused by a Lyssavirus in fruit bats in Australia. Emerg. infect. Dis., 2 (4), 327-331. 5. Halpin K., Young P.L. & Field H. (1996). - Identification of likely natural hosts for equine morbillivirus. Communicable disease intelligence. Aust. Dept. Health Aged Care (Canberra), 20, 476. 6. Hooper P.T., Gould A.R., Russell G.M., Kattenbelt J.A. & Mitchell G. (1996). - The retrospective diagnosis of a second outbreak of equine morbillivirus infection. Aust. vet. J., 74 (3), 244-245. 15. Rogers R.J., Douglas I.C., Baidock F.C., Glanville R.J., Seppanen K.T., Gleeson L.J., Selleck P.N. & Dunn K.J. (1996). - Investigation of a second focus of equine morbillivirus infection in coastal Queensland. Aust. vet. J., 74 (3), 243-244. 16. Wang L.F., Michalski W.P., Yu M., Pritchard L.I., Crameri G., Shiell B. & Eaton B.T. (1998), - A novel P/V/C gene in a new member of the Paramyxoviridae family which causes lethal infection in humans, horses and other animals. J. Virol, 72 (2), 1482-1490. 17. Ward M.P., Black P.F., Childs A.J., Baidock F.C., Webster W.R., Rodwell B.J. & Brouwer S.L. (1996). Negative findings from serological studies of equine morbillivirus in Queensland horse population. Aust. vet. J . , 74 (3), 241-243. Westbury H.A., Hooper P.T., Selleck P.W. & Murray P.K. (1995). - Equine morbillivirus pneumonia: susceptibility of laboratory animals to the virus. Aust. vet. J., 72 (7), 278-279. 7. Hooper P.T., Ketterer P.J., Hyatt A.D. & Russell G.M. (1997). - Lesions in experimental equine morbillivirus pneumonia in horses. Vet. Pathol., 34 (4), 312-322. 8. Hooper P.T., Westbury H.A. & Russell G.M. (1997). - The lesions of experimental equine morbillivirus disease in cats and guinea pigs. Vet. Pathol, 34 (4), 323-329. 18. 9. Hyatt A.D. & Selleck P.W. (1996). - Ultrastructure of equine morbillivirus. Virus Res., 4 3 (1), 1-15. 19. Westbury H.A., Hooper P.T., Brouwer S.L. & Selleck P.W. (1996). - Susceptibility of cats to equine morbillivirus. Aust. vet. J., 74 (2), 132-134. 10. McCormack J.G., Allworth A.M., Selvey L.A. & Selleck P.W. (1999). - Transmissibility from horses to humans of a novel paramyxovirus, equine morbillivirus (EMV). J. Infect., 38 (1), 22-23. 11. 12. 13. 14. 20. Williamson M.M., Hooper P.T., Selleck P.W., Gleeson L.J., Daniels P.W., Westbury H.A. & Murray P.K. (1998). Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust. vet. J., 76 (12), 813-818. MacKenzie J.S. (1999). - Emerging viral disease: an Australian perspective. Emerg. infect Dis., 5 (1), 1-8. 21. Murray K., Selleck P., Hooper P., Hyatt A., Gleeson L., Westbury H., Hiley L., Selvey L., Rodwell B. & Ketterer P.J. (1995). - A morbillivirus that caused fatal disease in horses and humans. Science, 2 6 8 (5207), 94-97. Young P.L., Halpin K., Selleck P.W., Field H., Gravel J.L., Kelly M.A. & Mackenzie J.S. (1996). - Serological evidence for the presence in Pteropus bats of a paramyxovirus related to equine morbillivirus. Emerg. infect. Dis., 2 (3), 239-240. 22. Young P.L., Halpin K., Field H., Mackenzie J.S. & Asche V. (1997). - Finding the wildlife reservoir of equine morbillivirus. Recent Adv. Microbiol, 5, 1-12. 23. Yu M., Hansson E., Shiell B., Michalski W., Eaton B.T. & Wang L.F. (1998). - Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxovirinae. J. gen. Virol, 79 (7), 1775-1780. O'Sullivan J.D., Allworth A.M., Paterson D.L., Snow T.M., Boots R., Gleeson L.J., Gould A.R., Hyatt A.D. & Bradfield J. (1997). - Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet, 349 (9045), 93-95. Philbey A.W., Kirkland P.D., Ross A.D., Davis R.J., Gleeson A.B., Love R.J., Daniels P.W., Gould A.R. & Hyatt A.D. (1998). - An apparently new virus (family Paramyxoviridae) infectious for pigs, humans and fruit bats. Emerg. infect. Dis., 4 (2), 269-271.