Download Hendra virus disease in horses

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
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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
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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.
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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.
•
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