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Hendra and Nipah Virus Infections
EMERGING INFECTIOUS DISEASES
0749-0739/00 $15.00
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HENDRA AND NIPAH VIRUS INFECTIONS Peter T. Hooper, BVSc, MACVSc, PhD, and Mark M. Williamson, BVSc, MVS, MACVSc, PhD
Tn September 1994, infection with a previously undescribed member of the Paramyxoviridae family caused the deaths of 14 horses and one human from acute respiratory disease. " 211 The causal virus, which was known initially as equine morbillivirus, w as renamed Hendra virus (HeV) after Hendra, a suburb of Brisbane, Australia with numerous racing stables and site of the first disease outbreak. 2 1, 27 In October ]995, a fanner in Makay, northern Queensland, developed fatal encephalitis as a result uf HeV infection,' 7 which was attributed to exposure from 2 HeV-infected horses that had died more than 1 year earlier? " A third case of the disease was recorded in a horse near Cairns, Australia in early 1999. 2 S
HENDRA VIRUS In the original outbreak, HeV was isolated from a range of tissues from horses and from the kidney of the deceased horse trainer.'" Initial sequence determinations of the matrix and fusion genes and part of the P gene confirmed that the virus belonged to the family Paramyxoviridae. 13 On the basis of limited sequence data on the matrix protein, comparisons with known Paramyxoviridae matrix proteins revealed a 50'X, similarity with the morbilli virus matrix proteins; this increased to 80'Yo if conservative amino acid substitutions were used. The virus was considered to be more closely related to members of the genus Mor/Jiilivirus than to other genera in the Paramyxoviridae family; thus, the virus was given the provisional name of equine morbillivirus. 3 Subsequent studies have determined that the HeV genome is 18.2 kilobases (18,234 nucleotides), with large intcrgenic regions,'" and that it is significantly
From the Australian Animal Health Laboratory, Division of Animal Health, CSIRO, Geelong, Victoria, Australia (PTH, MMW); a nd presently, the Department of Veterinary Pathology, College of Veterinary Medicin e, Iowa State University, Ames, Iowa (MMW)
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larger than other previous known members of the ParamyxoviridaeY Sequence comparisons have confirmed that although the virus is a member of the Paramyxovirinae subfamily, there is only low-level homology with respiroviruses and morbilli viruses, negligible homologies with rubulaviruses,2 " 27 and negligible immunologic cross-reactivity with a range of antisera to other members of the Paramyxoviridae, 14 On ultrastructural examination, the virus has a distinct double fringe, which has not been observed in other members of the Paramyxoviridae,K Because the virus could not be readily classified in any of the existing genera in the family Paramyxoviridae, and because it is not really closely related to morbilli viruses, it was renamed HeV '4, 21
EPIDEMIOLOGY Transmission
In September 1994, 14 horses died or were euthanatized with acute respiratory disease at a training stable in the Brisbane suburb of Hendra in Australia, Another 7 horses were affected and recovered, and a further 9 horses remained unaffected and seronegative for antibodies to HeV 's With the exception of 1 affected horse on a neighboring premise and a horse that became ill after transportation, there was no spread of the disease from the original affected stable, A large number of horses and humans were subsequently tested, and all were seronegative, 12, 22 In spite of the fact that as many as 21 horses were infected in the first known outbreak, the disease did not spread from the original focus , Likewise, there was little spread in the other two recorded outbreaks, In one of these, infection was confined to the index horse, 1 other horse, and one human who had close contact with both affected animals, IH In the other, the disease was confined to the index case; a second horse and persons who had contact with the affected animal remained free of the disease ,' This minimal level of contagiousness of HeY was confirmed by specific experiments designed to maximize the chances of spread by aerosol transmission , Despite optimizing the opportunities for spread, there was no transmission from HeV-inoculated horses to in-contact horses or cats," This correlated with the virus isolation results; HeY was not isolated from the nasal cavities or trachea of infected horses, The only proven route of shedding of HeY was into the urinary tract of infected animals, It was isolated from the kidneys of six acutely infected experimental horses and the urine of four of six acutely infected horses at necropsy but not from the conjunctivae, nasal cavities, feces, or mouth. 2s It was also present in the urine of cats,>3 and urine from a cat may have caused infection in a horse in one of the experimental studies. 25 It should be pOinted out that cases of experimental HeV infection in horses differed from those in the field in that there was no frothy nasal discharge. In cases of naturally acquired infection, where affected animals exhibited frothing from the nostrils, virus-rich fluid derived from the lungs could be a source of infection for humans coming into close contact with it (I. Douglas, MVSc, personal communication, 1999). In the case of the original outbreak, the trainer was known for the intimate care with which he attended to his animals, This close contact could have inadvertently resulted in the spread of the virus to the large number of horses and to himself. The significant number of affected horses and the coincidental fatal human infection triggered an intensive investigation that was successful in identifying this previously undescribed disease. Without
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the ironic and tragic train of events that took place, the virus probably would not have been discovered at the time. Virus Reservoirs
There is serologic or virologic e vidence that fruit bats (flying foxes, or pteropodidae) are likely wildlife reservoirs of HeV:, 26 An overall HeV antibody prevalence of 42'Yo has been found in wild caught fruit bats in Australia and Papua New Guinea,l ll The bats occur naturally throughout southeast Asia, southern Africa, and along the northern coast and eastern seaboard of Australia,!6 HeV was found in uterine fluids,· and there is experimental evidence of vertical transmission to the fetus, 2. Fruit bats congregate in colonies of thousands of bats, and the male bats are known to lick the body, wings, and genitals of the female bats throughout the breeding season. 16 It is postulated that the source of the field outbreaks in horses could have been the ingestion of HeV-infected fruit bat placentae, or food or water contaminated with infective fetal fluids, CLINICAL SIGNS
The incubation period in experimentally infected horses ranges from 5 to 10 days, irrespective of the dose of virus or its route of administration,6 25 Typically, the course of the disease in fatal field and experimental cases is short, lasting only about 2 days. Clinical recovery with seroconversion to HeV occurred in 7 of 21 horses in the original outbreak at Hendra; 1 of 14 experimentally inoculated horses recovered even though it developed severe disease of 6 days' duration. 2s Clinical signs observed in affected horses have been consistent and comprise severe pneumonia, a high respiratory rate, an elevated body temperature, a progressively increased heart rate, lethargy, and anorexia. In light of the significant respiratory involvement, the disease caused by HeV has been termed acute equine respiratory syndrome, Facial edema has been observed in some cases, and jaundiced mucous membranes have also been reported. Frothy nasal discharges have been described in some terminal field cases, but this clinical sign has not been seen in experimentally infected animals. Two of the horses that recovered during the Hendra outbreak exhibited mild neurologic signs,1" The latter may have been caused by lesions in the brain such as those associated with vascular disease with localized cerebral infarction, or they may be symptomatic of the meningoencephalitis seen in experimental horses,25 and in one human. 17 PATHOLOGY
The most significant gross lesions in horses were dilated pulmonary lymphatics, severe pulmonary edema, and congestion." Other less frequent gross lesions included edema of the mesentery, increased pleural and pericardial fluids, and congested lymph nodes. In many field but not experimental cases, the airways were filled with thick foam, which was occasionally blood tinged. The predominant histologic findings in horses were interstitial pneumonia and severe vascular degeneration, with serofibrinous alveolar edema, alveolar macrophages, hemorrhage, dilated lymphatics, thrombosis, and necrosis of the alveolar walls 6 There was generalized fibrinoid degeneration of small blood
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vessels; this was seen in the lungs, kidneys, heart, skeletal muscle, alimentary tract, spleen, lymph nodes, bladder, and m en inges.'" 25 The characteristic histologic lesion of HeV infections is the presence of endothelial syncytial cells, which are commonly observed in pulmonary capillaries and arterioles, and in experimental cases, can also be seen in the lymph nodes, spleen, brain, stomach, heart, and kidney.ls Using the indirect immunofluorescence and immunoperoxidase tests, viral antigen was readily detected in blood vessels, especially in endothelial cells. 6 On electron microscopic examination, viral nucleocapsids typical of the family Paramyxoviridae have been observed in the cytoplasm of endothelial syncytial cells. Free nucleoca psids have been demonstrated in horse pulmonary homogenates using gold labeling and immunoelectron microscopy." There was a nonsuppurative encephalitis characterized by perivascular cuffing of lymphocytes, neuronal necrosis, and focal gliosis in the human case in Mackay.l? This has also been reported in some horses. 05 Based on immunohistochemical examination carried out in these cases, H eY was shown to be neurotropic, affecting neurons as well as blood vessels. DIAGNOSIS
HeV infection should be considered in a horse affected with a high fever and signs of acute lower respiratory disease in a region where the virus has been known to occur or is suspected to have been introduced . Laboratory testing is essential for confirming a diagnosis of acute equine respiratory syndrome. This is based on virus isolation or detection of viral nucleic acid in blood, tissue, or body fluids, or demonstration of HeY-specific antibody in serum. For isolation of the virus, fresh specimens of the lung, spleen, and kidney should be collected aseptically at necropsy examination of acutely ill horses. If the affected animal displayed neurologic signs before death, the brain should also be removed, and samples should be collected for virus isolation. Although the virus can be isolated in a variety of cell cultures, MDCK, BHK-21, RK-13, LLC-MK2' and MRC5 cells, Yero cells are most commonly used. " A syncytial type of cytopathic effect develops in infected cell culture monolayers in about 3 days. The identity of a virus isolate can be confirmed by electron microscopy, serologic, and molecular testing. Blood for serum antibody determination should be collected in the acute and convalescent stages of the disease. The serum neutralization test or an indirect enzyme-linked immunosorbent assay25 is used for antibody determination . A range of tissues, especially of the lung and kidney, should also be collected from necropsy cases for histologiC examination. Evidence of endothelial syncytial cells and severe necrotizing vasculitis is highly indicative of HeV infection. The virus specificity of these lesions can be confirmed by indirect immunoperoxidase staining using rabbit polyclonal antibodies to inactivated HeY or mouse monoclonal antibodies to the viral nucleocapsid proteins. Differential Diagnosis
Infection with HeY should be differentiated from other causes of sudden death in horses, especially those associated with severe pneumonia. Among the bacterial agents that need to be considered are Pasteurella spp, Bllcilllls IlntiJracis, Yersinia spp, Legionella spp, Pseudomonas spp, and Streptobacillus moniliformis. Viral diseases such as African horse sickness, equine influenza, and peracute
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equine herpesvirus 1 infection as well as chemical and plant poisoning such as that caused by crofton weed (Eupatorium adenophorum and E. riparium) must also be ruled out. CONTROL
There is no evidence of HeV antibodies in the equine/ 2 human,12 or wildlife populations of Australia other than in fruit bats. 1O Evidence from field and experimental studies indicates that the virus is not highly contagious. Only isolated outbreaks of disease in horses have been detected even though there has been a vigorous surveillance program in place since the original outbreak was diagnosed in 1994. The few outbreaks of disease in horses have been dealt with by slaughter of serologically positive horses and the imposition of movement controls on horses within a defined zone. It would appear that fruit bats are the known reservoir of HeY, and considering the rarity of equine or human infection, it would not be justified to effect any control measures against them other than avoiding contact, especially with urine and uterine fluids. Appropriate measures to prevent human exposure to HeV should be taken during clinical and necropsy examination of suspect cases of the disease; these should include the wearing of gloves and protective glasses. NIPAH VIRUS INFECTION
In 1998, an outbreak of a serious and often fatal disease of humans and pigs was reported in peninsular Malaysia. Initially, it was thought to be due to Japanese encephalitis virus, which had been known to occur in Malaysia since 1935. Most human cases were seen in pig farmers. In March 1999, a new paramyxovirus was isolated from the cerebrospinal fluid of a number of human cases.' It is now evident that this virus was responsible for widespread morbidity and mortality in pigs, which was characterized by pneumonia and, occasionally, encephalitis. It is likely that infected pigs were the source of infection for humans, dogs, cats, and several horses. The new virus was named Nipah virus (NiV) after the Malaysian village, Sungai Nipah, where the first human case was recorded. Although NiV is antigenically related to HeV, it is clearly a different virus. Preliminary results of viral genome sequencing of the M and N proteins indicate that there is a 10% difference between the two viruses at the amino acid level. The P and V proteins are less conserved, with a 15% to 20% difference in amino acids.' 9 Antigenically, there is some cross-reactivity between the two viruses. NiV should be considered a member of the family Paramyxoviridae belonging to the same genus as HeY. HeV and NiV differ in terms of the range of species they infect and the manner and ease with which each virus seems to be transmitted. HeV is not readily contagious between animals except for fruit bats. Conversely, NiV seems to spread easily between pigs and can also be transmitted from pigs to humans and probably to other species. In light of the information on HeV infection in horses in Australia, there was concern about the potential for NiV to cause infection and disease in horses in Malaysia. In an extensive survey of equine sera in which more than 3200 serum samples were tested by the serum neutralization test (SNT) and enzymelinked immunosorbent assay, only 5 samples were found to be positive.lI Two of these were from polo ponies that had been pastured near an infected pig farm . After euthanasia and necropsy examination, no evidence of current infection was
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found in either animal. An archival equine case from 1998 that had histologic evidence of meningitis (S. Shahirudin, BVSc, MRCVS, and P. Daniels BVSc, PhD, personal communication, 1999) was positive on immunohistochemical examination for NiV antigen in the meninges and tunica media of small blood vessels in the meninges (P. Hooper, BVSc, MACVSc, PhD, unpublished data). Other tissue samples from this animal were not available for examination. It would seem reasonable to assume that NiV infection in horses is similar to NiV or HeV infection in other species, namely, a generalized vasculitis with the possibility of localization in the lung or brain. Likewise, it is also probable that the few cases of infection in horses may have originated from pigs-similar to the situation in humans, dogs, and cats. The original source of NiV infection in pigs was likely similar to that of HeV, namely, fruit bats. This is supported by serologic evidence of NiV infection in two larger and two smaller species of fruit bats, Pteropus vampyrus and P. hypomelanus, and Eonycteris spelaea and Cynopterus brachyotis, respectively, in Malaysia." References 1. Chua KB, Goh KJ, Wong KT, et al: Fatal encephalitis du e to Nipah virus among pigfarmers in Malaysia. Lancet 354:1257, 1999 2. Field HE, Hughes R, Barratt J, et al: A fatal case of Hendra virus (equine morbillivirus) infection in a horse in North Queensland-an incident report with an epidemiological perspective. Aust Vet J 78:279, 2000 3. Gould AR: Comparison of the deduced matrix and fusion protein sequences of equine morbilli virus with cognate genes of the Paramyxoviridae. Virus Res 43:17, 1996 4. Halpin K, Young PL, Field H, et al: Newly discovered viruses of flying foxes. Vet Microbiol 68:83, 1999 5. Hooper PT, Gould AR, Hyatt AD, e t al: The laboratory diagnosis and molecular characterisation of a new case of Hendra virus infection in a horse in Queensland. Aust Vet J 78:281, 2000 6. Hooper PT, Ketterer pJ, Hyatt AD, et al: Lesions of experimental equine morbilli virus pneumonia in horses. Ve t Pathol 34:312, 1997 7. Hooper PI, Gould AR Russell GM, et al: The retrospective diagnosis of a second outbreak of equine morbillivirus infection. Aust Vet ]74:244, 1996 8. Hyatt AD, Selleck PW: Ultrastructure of equine morbilli virus. Virus Res 43:1, 1996 9. Johara MY, Field H, Sohayati AR et al: Preliminary investigation of probable reservoir host of Nipah virus. In Proceedings of the National Congress of Animal Health and Production, Alor Gajah, Malaysia, September 3-4, 1999 10. Mackenzie JS: Emerging viral diseases: An Australian perspective. Emerg Infect Dis 5:1, 1999 11. Mahendran R Naseem M, Shahirudin, S, et al: A preliminary study on the seroprevalence of Nipah virus infection in horses in Malaysia. II! Proceedings of the National Congress of Animal Health and Production, Alor Gajah, Malaysia, September 3-4, 1999 12. McCormack JG, Allworth AM, Selvey LA, et al: Transmissibility from horses to humans of a novel paramyxovirus, equine morbillivirus (EMV). J Infect 38:22, 1999 13. Murphy FA, Fauquet CM, Bishop DHL, et al: Classification and nomenclature of viruses. II! Sixth Report of the International Committee on Taxonomy of Viruses. New York, Springer-Verlag, 1995, p 268 14. Murray PK, Eaton B, Hooper P, et al: Flying foxes, horses and humans: A zoonosis caused by a new member of the Paramyxoviridae. III ScheId WM, Armstrong 0, Hughes JM (eds): Emerging Infections 1. Washington, DC, ASM Press, 1998, p 43 15. Murray PK, Selleck PW, Hooper PT, et al: A morbillivirus that caused fatal disease in horses and humans. Science 268:94, 1995 16. Nelson JE: Pteropodidae. 111 Walton OW, Richardson BJ (eds): Fauna of Australia, vol lB. Mammalia. Canberra, Australian Gove rnment Publishing Service, 1989, p 836
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17. O'Sullivan lD, Allworth AM, Paterson DL, et al: Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet 349:93, 1997 18. Rogers RL, Douglas Ie, Baldock, Fe, et aI: Investigation of a second focus of equine morbillivirus infection in coastal Queensland. Alist Vet 1 74:243, 1996 19. Rota P, Harcourt B, Rollin P, et al: III Proceedings of the XI International Congress on Virology, Sydney, 1999, 20. Selvey LA, Wells RM, McCormack lG, et al: Infections of humans and horses by a newly described morbillivirus. Med J Alist 162:642, 1995 21. Wang L-F, Michalski W, Yu M, et al: A novel P IV IC gene in a new Paralllyxoviridae virus which causes lethal infection in humans, horses and other animals. J Virol 72:1482, 1998 22. Ward MP, Black PF, Childs AJ, et al: Negative findings from serological studies of equine morbi11ivirus in the Queensland horse population. Alist Vet J 74:241, 1996 23. Westbury HA, Hooper PT, Brouwer SL, et al: Susceptibility of cats to equine morbi11ivirus. Aust Vet J 74:132, 1996 24. Williamson MM, Hooper PT, Selleck PW, et al: Experimental Hendra virus infection in pregnant fruit bats and guinea pigs. J Comp Pathol 122:201, 2000 25. Williamson MM, Hooper PT, Selleck PW, et al: Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 76:813, 1998 26. Young PL, Halpin K, Selleck P, et al: Serological evidence for the presence in Pterol'lIs bats of a paramyxovirus related to equine morbillivirus. Emerg Infect Dis 2:239, 1996 27. Yu M, Hansson E, Shiell B, et al: Sequence analysis of the Hendra virus nucleoprotein gene: Comparison with other members of the subfamily Paramyxoviril1ae. J Gen Virol 79:1775, 1998
Address reprillt requests to Pctcr T. Hooper, BVSc, MACVSc, PhD CSIRO Australian Animal Health Laboratory PO Box 24 Geelong, Victoria Australia 3220
0749-0739/00 $15.00
+ .00
HENDRA AND NIPAH VIRUS INFECTIONS Peter T. Hooper, BVSc, MACVSc, PhD, and Mark M. Williamson, BVSc, MVS, MACVSc, PhD
Tn September 1994, infection with a previously undescribed member of the Paramyxoviridae family caused the deaths of 14 horses and one human from acute respiratory disease. " 211 The causal virus, which was known initially as equine morbillivirus, w as renamed Hendra virus (HeV) after Hendra, a suburb of Brisbane, Australia with numerous racing stables and site of the first disease outbreak. 2 1, 27 In October ]995, a fanner in Makay, northern Queensland, developed fatal encephalitis as a result uf HeV infection,' 7 which was attributed to exposure from 2 HeV-infected horses that had died more than 1 year earlier? " A third case of the disease was recorded in a horse near Cairns, Australia in early 1999. 2 S
HENDRA VIRUS In the original outbreak, HeV was isolated from a range of tissues from horses and from the kidney of the deceased horse trainer.'" Initial sequence determinations of the matrix and fusion genes and part of the P gene confirmed that the virus belonged to the family Paramyxoviridae. 13 On the basis of limited sequence data on the matrix protein, comparisons with known Paramyxoviridae matrix proteins revealed a 50'X, similarity with the morbilli virus matrix proteins; this increased to 80'Yo if conservative amino acid substitutions were used. The virus was considered to be more closely related to members of the genus Mor/Jiilivirus than to other genera in the Paramyxoviridae family; thus, the virus was given the provisional name of equine morbillivirus. 3 Subsequent studies have determined that the HeV genome is 18.2 kilobases (18,234 nucleotides), with large intcrgenic regions,'" and that it is significantly
From the Australian Animal Health Laboratory, Division of Animal Health, CSIRO, Geelong, Victoria, Australia (PTH, MMW); a nd presently, the Department of Veterinary Pathology, College of Veterinary Medicin e, Iowa State University, Ames, Iowa (MMW)
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larger than other previous known members of the ParamyxoviridaeY Sequence comparisons have confirmed that although the virus is a member of the Paramyxovirinae subfamily, there is only low-level homology with respiroviruses and morbilli viruses, negligible homologies with rubulaviruses,2 " 27 and negligible immunologic cross-reactivity with a range of antisera to other members of the Paramyxoviridae, 14 On ultrastructural examination, the virus has a distinct double fringe, which has not been observed in other members of the Paramyxoviridae,K Because the virus could not be readily classified in any of the existing genera in the family Paramyxoviridae, and because it is not really closely related to morbilli viruses, it was renamed HeV '4, 21
EPIDEMIOLOGY Transmission
In September 1994, 14 horses died or were euthanatized with acute respiratory disease at a training stable in the Brisbane suburb of Hendra in Australia, Another 7 horses were affected and recovered, and a further 9 horses remained unaffected and seronegative for antibodies to HeV 's With the exception of 1 affected horse on a neighboring premise and a horse that became ill after transportation, there was no spread of the disease from the original affected stable, A large number of horses and humans were subsequently tested, and all were seronegative, 12, 22 In spite of the fact that as many as 21 horses were infected in the first known outbreak, the disease did not spread from the original focus , Likewise, there was little spread in the other two recorded outbreaks, In one of these, infection was confined to the index horse, 1 other horse, and one human who had close contact with both affected animals, IH In the other, the disease was confined to the index case; a second horse and persons who had contact with the affected animal remained free of the disease ,' This minimal level of contagiousness of HeY was confirmed by specific experiments designed to maximize the chances of spread by aerosol transmission , Despite optimizing the opportunities for spread, there was no transmission from HeV-inoculated horses to in-contact horses or cats," This correlated with the virus isolation results; HeY was not isolated from the nasal cavities or trachea of infected horses, The only proven route of shedding of HeY was into the urinary tract of infected animals, It was isolated from the kidneys of six acutely infected experimental horses and the urine of four of six acutely infected horses at necropsy but not from the conjunctivae, nasal cavities, feces, or mouth. 2s It was also present in the urine of cats,>3 and urine from a cat may have caused infection in a horse in one of the experimental studies. 25 It should be pOinted out that cases of experimental HeV infection in horses differed from those in the field in that there was no frothy nasal discharge. In cases of naturally acquired infection, where affected animals exhibited frothing from the nostrils, virus-rich fluid derived from the lungs could be a source of infection for humans coming into close contact with it (I. Douglas, MVSc, personal communication, 1999). In the case of the original outbreak, the trainer was known for the intimate care with which he attended to his animals, This close contact could have inadvertently resulted in the spread of the virus to the large number of horses and to himself. The significant number of affected horses and the coincidental fatal human infection triggered an intensive investigation that was successful in identifying this previously undescribed disease. Without
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the ironic and tragic train of events that took place, the virus probably would not have been discovered at the time. Virus Reservoirs
There is serologic or virologic e vidence that fruit bats (flying foxes, or pteropodidae) are likely wildlife reservoirs of HeV:, 26 An overall HeV antibody prevalence of 42'Yo has been found in wild caught fruit bats in Australia and Papua New Guinea,l ll The bats occur naturally throughout southeast Asia, southern Africa, and along the northern coast and eastern seaboard of Australia,!6 HeV was found in uterine fluids,· and there is experimental evidence of vertical transmission to the fetus, 2. Fruit bats congregate in colonies of thousands of bats, and the male bats are known to lick the body, wings, and genitals of the female bats throughout the breeding season. 16 It is postulated that the source of the field outbreaks in horses could have been the ingestion of HeV-infected fruit bat placentae, or food or water contaminated with infective fetal fluids, CLINICAL SIGNS
The incubation period in experimentally infected horses ranges from 5 to 10 days, irrespective of the dose of virus or its route of administration,6 25 Typically, the course of the disease in fatal field and experimental cases is short, lasting only about 2 days. Clinical recovery with seroconversion to HeV occurred in 7 of 21 horses in the original outbreak at Hendra; 1 of 14 experimentally inoculated horses recovered even though it developed severe disease of 6 days' duration. 2s Clinical signs observed in affected horses have been consistent and comprise severe pneumonia, a high respiratory rate, an elevated body temperature, a progressively increased heart rate, lethargy, and anorexia. In light of the significant respiratory involvement, the disease caused by HeV has been termed acute equine respiratory syndrome, Facial edema has been observed in some cases, and jaundiced mucous membranes have also been reported. Frothy nasal discharges have been described in some terminal field cases, but this clinical sign has not been seen in experimentally infected animals. Two of the horses that recovered during the Hendra outbreak exhibited mild neurologic signs,1" The latter may have been caused by lesions in the brain such as those associated with vascular disease with localized cerebral infarction, or they may be symptomatic of the meningoencephalitis seen in experimental horses,25 and in one human. 17 PATHOLOGY
The most significant gross lesions in horses were dilated pulmonary lymphatics, severe pulmonary edema, and congestion." Other less frequent gross lesions included edema of the mesentery, increased pleural and pericardial fluids, and congested lymph nodes. In many field but not experimental cases, the airways were filled with thick foam, which was occasionally blood tinged. The predominant histologic findings in horses were interstitial pneumonia and severe vascular degeneration, with serofibrinous alveolar edema, alveolar macrophages, hemorrhage, dilated lymphatics, thrombosis, and necrosis of the alveolar walls 6 There was generalized fibrinoid degeneration of small blood
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vessels; this was seen in the lungs, kidneys, heart, skeletal muscle, alimentary tract, spleen, lymph nodes, bladder, and m en inges.'" 25 The characteristic histologic lesion of HeV infections is the presence of endothelial syncytial cells, which are commonly observed in pulmonary capillaries and arterioles, and in experimental cases, can also be seen in the lymph nodes, spleen, brain, stomach, heart, and kidney.ls Using the indirect immunofluorescence and immunoperoxidase tests, viral antigen was readily detected in blood vessels, especially in endothelial cells. 6 On electron microscopic examination, viral nucleocapsids typical of the family Paramyxoviridae have been observed in the cytoplasm of endothelial syncytial cells. Free nucleoca psids have been demonstrated in horse pulmonary homogenates using gold labeling and immunoelectron microscopy." There was a nonsuppurative encephalitis characterized by perivascular cuffing of lymphocytes, neuronal necrosis, and focal gliosis in the human case in Mackay.l? This has also been reported in some horses. 05 Based on immunohistochemical examination carried out in these cases, H eY was shown to be neurotropic, affecting neurons as well as blood vessels. DIAGNOSIS
HeV infection should be considered in a horse affected with a high fever and signs of acute lower respiratory disease in a region where the virus has been known to occur or is suspected to have been introduced . Laboratory testing is essential for confirming a diagnosis of acute equine respiratory syndrome. This is based on virus isolation or detection of viral nucleic acid in blood, tissue, or body fluids, or demonstration of HeY-specific antibody in serum. For isolation of the virus, fresh specimens of the lung, spleen, and kidney should be collected aseptically at necropsy examination of acutely ill horses. If the affected animal displayed neurologic signs before death, the brain should also be removed, and samples should be collected for virus isolation. Although the virus can be isolated in a variety of cell cultures, MDCK, BHK-21, RK-13, LLC-MK2' and MRC5 cells, Yero cells are most commonly used. " A syncytial type of cytopathic effect develops in infected cell culture monolayers in about 3 days. The identity of a virus isolate can be confirmed by electron microscopy, serologic, and molecular testing. Blood for serum antibody determination should be collected in the acute and convalescent stages of the disease. The serum neutralization test or an indirect enzyme-linked immunosorbent assay25 is used for antibody determination . A range of tissues, especially of the lung and kidney, should also be collected from necropsy cases for histologiC examination. Evidence of endothelial syncytial cells and severe necrotizing vasculitis is highly indicative of HeV infection. The virus specificity of these lesions can be confirmed by indirect immunoperoxidase staining using rabbit polyclonal antibodies to inactivated HeY or mouse monoclonal antibodies to the viral nucleocapsid proteins. Differential Diagnosis
Infection with HeY should be differentiated from other causes of sudden death in horses, especially those associated with severe pneumonia. Among the bacterial agents that need to be considered are Pasteurella spp, Bllcilllls IlntiJracis, Yersinia spp, Legionella spp, Pseudomonas spp, and Streptobacillus moniliformis. Viral diseases such as African horse sickness, equine influenza, and peracute
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equine herpesvirus 1 infection as well as chemical and plant poisoning such as that caused by crofton weed (Eupatorium adenophorum and E. riparium) must also be ruled out. CONTROL
There is no evidence of HeV antibodies in the equine/ 2 human,12 or wildlife populations of Australia other than in fruit bats. 1O Evidence from field and experimental studies indicates that the virus is not highly contagious. Only isolated outbreaks of disease in horses have been detected even though there has been a vigorous surveillance program in place since the original outbreak was diagnosed in 1994. The few outbreaks of disease in horses have been dealt with by slaughter of serologically positive horses and the imposition of movement controls on horses within a defined zone. It would appear that fruit bats are the known reservoir of HeY, and considering the rarity of equine or human infection, it would not be justified to effect any control measures against them other than avoiding contact, especially with urine and uterine fluids. Appropriate measures to prevent human exposure to HeV should be taken during clinical and necropsy examination of suspect cases of the disease; these should include the wearing of gloves and protective glasses. NIPAH VIRUS INFECTION
In 1998, an outbreak of a serious and often fatal disease of humans and pigs was reported in peninsular Malaysia. Initially, it was thought to be due to Japanese encephalitis virus, which had been known to occur in Malaysia since 1935. Most human cases were seen in pig farmers. In March 1999, a new paramyxovirus was isolated from the cerebrospinal fluid of a number of human cases.' It is now evident that this virus was responsible for widespread morbidity and mortality in pigs, which was characterized by pneumonia and, occasionally, encephalitis. It is likely that infected pigs were the source of infection for humans, dogs, cats, and several horses. The new virus was named Nipah virus (NiV) after the Malaysian village, Sungai Nipah, where the first human case was recorded. Although NiV is antigenically related to HeV, it is clearly a different virus. Preliminary results of viral genome sequencing of the M and N proteins indicate that there is a 10% difference between the two viruses at the amino acid level. The P and V proteins are less conserved, with a 15% to 20% difference in amino acids.' 9 Antigenically, there is some cross-reactivity between the two viruses. NiV should be considered a member of the family Paramyxoviridae belonging to the same genus as HeY. HeV and NiV differ in terms of the range of species they infect and the manner and ease with which each virus seems to be transmitted. HeV is not readily contagious between animals except for fruit bats. Conversely, NiV seems to spread easily between pigs and can also be transmitted from pigs to humans and probably to other species. In light of the information on HeV infection in horses in Australia, there was concern about the potential for NiV to cause infection and disease in horses in Malaysia. In an extensive survey of equine sera in which more than 3200 serum samples were tested by the serum neutralization test (SNT) and enzymelinked immunosorbent assay, only 5 samples were found to be positive.lI Two of these were from polo ponies that had been pastured near an infected pig farm . After euthanasia and necropsy examination, no evidence of current infection was
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found in either animal. An archival equine case from 1998 that had histologic evidence of meningitis (S. Shahirudin, BVSc, MRCVS, and P. Daniels BVSc, PhD, personal communication, 1999) was positive on immunohistochemical examination for NiV antigen in the meninges and tunica media of small blood vessels in the meninges (P. Hooper, BVSc, MACVSc, PhD, unpublished data). Other tissue samples from this animal were not available for examination. It would seem reasonable to assume that NiV infection in horses is similar to NiV or HeV infection in other species, namely, a generalized vasculitis with the possibility of localization in the lung or brain. Likewise, it is also probable that the few cases of infection in horses may have originated from pigs-similar to the situation in humans, dogs, and cats. The original source of NiV infection in pigs was likely similar to that of HeV, namely, fruit bats. This is supported by serologic evidence of NiV infection in two larger and two smaller species of fruit bats, Pteropus vampyrus and P. hypomelanus, and Eonycteris spelaea and Cynopterus brachyotis, respectively, in Malaysia." References 1. Chua KB, Goh KJ, Wong KT, et al: Fatal encephalitis du e to Nipah virus among pigfarmers in Malaysia. Lancet 354:1257, 1999 2. Field HE, Hughes R, Barratt J, et al: A fatal case of Hendra virus (equine morbillivirus) infection in a horse in North Queensland-an incident report with an epidemiological perspective. Aust Vet J 78:279, 2000 3. Gould AR: Comparison of the deduced matrix and fusion protein sequences of equine morbilli virus with cognate genes of the Paramyxoviridae. Virus Res 43:17, 1996 4. Halpin K, Young PL, Field H, et al: Newly discovered viruses of flying foxes. Vet Microbiol 68:83, 1999 5. Hooper PT, Gould AR, Hyatt AD, e t al: The laboratory diagnosis and molecular characterisation of a new case of Hendra virus infection in a horse in Queensland. Aust Vet J 78:281, 2000 6. Hooper PT, Ketterer pJ, Hyatt AD, et al: Lesions of experimental equine morbilli virus pneumonia in horses. Ve t Pathol 34:312, 1997 7. Hooper PI, Gould AR Russell GM, et al: The retrospective diagnosis of a second outbreak of equine morbillivirus infection. Aust Vet ]74:244, 1996 8. Hyatt AD, Selleck PW: Ultrastructure of equine morbilli virus. Virus Res 43:1, 1996 9. Johara MY, Field H, Sohayati AR et al: Preliminary investigation of probable reservoir host of Nipah virus. In Proceedings of the National Congress of Animal Health and Production, Alor Gajah, Malaysia, September 3-4, 1999 10. Mackenzie JS: Emerging viral diseases: An Australian perspective. Emerg Infect Dis 5:1, 1999 11. Mahendran R Naseem M, Shahirudin, S, et al: A preliminary study on the seroprevalence of Nipah virus infection in horses in Malaysia. II! Proceedings of the National Congress of Animal Health and Production, Alor Gajah, Malaysia, September 3-4, 1999 12. McCormack JG, Allworth AM, Selvey LA, et al: Transmissibility from horses to humans of a novel paramyxovirus, equine morbillivirus (EMV). J Infect 38:22, 1999 13. Murphy FA, Fauquet CM, Bishop DHL, et al: Classification and nomenclature of viruses. II! Sixth Report of the International Committee on Taxonomy of Viruses. New York, Springer-Verlag, 1995, p 268 14. Murray PK, Eaton B, Hooper P, et al: Flying foxes, horses and humans: A zoonosis caused by a new member of the Paramyxoviridae. III ScheId WM, Armstrong 0, Hughes JM (eds): Emerging Infections 1. Washington, DC, ASM Press, 1998, p 43 15. Murray PK, Selleck PW, Hooper PT, et al: A morbillivirus that caused fatal disease in horses and humans. Science 268:94, 1995 16. Nelson JE: Pteropodidae. 111 Walton OW, Richardson BJ (eds): Fauna of Australia, vol lB. Mammalia. Canberra, Australian Gove rnment Publishing Service, 1989, p 836
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17. O'Sullivan lD, Allworth AM, Paterson DL, et al: Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet 349:93, 1997 18. Rogers RL, Douglas Ie, Baldock, Fe, et aI: Investigation of a second focus of equine morbillivirus infection in coastal Queensland. Alist Vet 1 74:243, 1996 19. Rota P, Harcourt B, Rollin P, et al: III Proceedings of the XI International Congress on Virology, Sydney, 1999, 20. Selvey LA, Wells RM, McCormack lG, et al: Infections of humans and horses by a newly described morbillivirus. Med J Alist 162:642, 1995 21. Wang L-F, Michalski W, Yu M, et al: A novel P IV IC gene in a new Paralllyxoviridae virus which causes lethal infection in humans, horses and other animals. J Virol 72:1482, 1998 22. Ward MP, Black PF, Childs AJ, et al: Negative findings from serological studies of equine morbi11ivirus in the Queensland horse population. Alist Vet J 74:241, 1996 23. Westbury HA, Hooper PT, Brouwer SL, et al: Susceptibility of cats to equine morbi11ivirus. Aust Vet J 74:132, 1996 24. Williamson MM, Hooper PT, Selleck PW, et al: Experimental Hendra virus infection in pregnant fruit bats and guinea pigs. J Comp Pathol 122:201, 2000 25. Williamson MM, Hooper PT, Selleck PW, et al: Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 76:813, 1998 26. Young PL, Halpin K, Selleck P, et al: Serological evidence for the presence in Pterol'lIs bats of a paramyxovirus related to equine morbillivirus. Emerg Infect Dis 2:239, 1996 27. Yu M, Hansson E, Shiell B, et al: Sequence analysis of the Hendra virus nucleoprotein gene: Comparison with other members of the subfamily Paramyxoviril1ae. J Gen Virol 79:1775, 1998
Address reprillt requests to Pctcr T. Hooper, BVSc, MACVSc, PhD CSIRO Australian Animal Health Laboratory PO Box 24 Geelong, Victoria Australia 3220