Download Experimental infection of sheep with bovine herpesvirus

Veterinary Microbiology 66 (1999) 89±99
Experimental infection of sheep with bovine
herpesvirus type-5 (BHV-5): acute
and latent infection
A.M. Silvaa, R. Weiblena, L.F. Irigoyenb, P.M. Roehec,
H.J. Surd, F.A. Osorioe, E.F. Floresa,*
a
Departamento de Medicina VeterinaÂria Preventiva e Departamento de Microbiologia e Parasitologia,
Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil
b
Departamento de Patologia, Universidade Federal de Santa Maria, Santa Maria 97105-900, Brazil
c
Instituto de Pesquisas DesideÂrio Finamor (IPVDF), Eldorado do Sul, Brazil and Departamento de
Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
d
Plum Island Animal Disease Center, Agricultural Research Center, USDA, Greenport NY 11944-0848, USA
e
Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln NE 68583-0905, USA
Received 2 July 1998; accepted 2 December 1998
Abstract
We demonstrated that sheep are susceptible to acute and latent infection by bovine herpesvirus
type-5 (BHV-5). Lambs inoculated intranasally with two South American BHV-5 isolates replicated
the virus with titers up to 107.1 TCID50/ml for up to 15 days and showed mild signs of rhinitis. Four
lambs in contact with the inoculated animals acquired the infection and excreted virus for up to
seven days. One lamb developed progressive signs of neurological disease and was euthanized in
extremis. Clinical signs consisted of tremors of the face, bruxism, ptyalism, incoordination, lateral
flexion of the neck and head, circling, walking backwards, recumbency and paddling. The virus was
detected in the anterior and posterior cerebrum, dorso- and ventro-lateral cortex, cerebellum, pons,
midbrain and olfactory bulb. Viral nucleic acids were demonstrated in neurons and astrocytes of the
anterior and ventro-lateral cortex by in situ hybridization. Histological changes consisting of nonsuppurative meningitis, perivascular mononuclear cuffing, focal gliosis, neuronal necrosis and
intranuclear inclusions were observed in the anterior cerebrum, ventro-lateral cortex and midbrain.
Dexamethasone treatment at Day 50 pi resulted in reactivation of the latent infection and virus
shedding in 13/16 (81%) of the lambs. Together with previous reports of BHV-5 antibodies in
sheep, these findings show that sheep are fully susceptible to BHV-5 suggesting that infection by
BHV-5 in sheep may occur naturally. # 1999 Elsevier Science B.V. All rights reserved.
Keywords: Bovine herpesvirus type-5; Experimental infection; Sheep-viruses; Latency; Encephalitis
* Corresponding author. Tel.: +5555-220-8055; fax: +5555-220-8742; e-mail: [email protected]
0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 1 3 5 ( 9 8 ) 0 0 3 0 5 - 8
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A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
1. Introduction
Bovine herpesvirus type-5 (BHV-5), also known as bovine encephalitis herpesvirus
(BEHV), is an alphaherpesvirus associated with fatal meningoencephalitis in young cattle
(Studdert, 1989; Roizman, 1992). Antigenically, genetically and biologically, BHV-5 is
closely related to the respiratory and genital BHV-1 strains (Bagust, 1972; Metzler et al.,
1986; Bulach and Studdert, 1990; Bratanich et al., 1991). The major difference between
BHV-5 and BHV-1 is the distinct ability of the former to cause neurological disease in
cattle (Bagust, 1972; Studdert, 1989; Belknap et al., 1994). Isolation of BHV-1 has been
largely restricted to cases of respiratory and genital disease, whereas BHV-5 has been
almost exclusively associated with meningoencephalitis (Studdert, 1989). Clinical signs
of BHV-5-associated neurological disease include tremors, circling, bruxism, incoordination, nystagmus, recumbency followed by convulsions, paddling and inevitably death
(Hall et al., 1966; Bagust and Clark, 1972; Carrillo et al., 1983).
The natural history, serology and geographic distribution of BHV-5 infections are
largely unknown, partially due to the historic unprecise taxonomic classification of the
virus. Only recently, serologic and virologic means of identifying and differentiating
BHV-5 from BHV-1 on a field scale have been developed (Lindner et al., 1993a, b;
d'Offay et al., 1995). Severe outbreaks of meningoencephalitis by BHV-5 have been
frequently described in well-defined geographical locations, mainly in Argentina and
southern Brazil (Carrillo et al., 1983; Schudel et al., 1986; Riet-Correa et al., 1989;
Weiblen et al., 1989, 1996; Roehe et al., 1997; Odeon, 1998; Salvador et al., 1998).
Elsewhere, BHV-5-associated neurological disease has been only sporadically reported
(Johnston and McGavin, 1962; Bartha et al., 1969; Eugster et al., 1974). Cross-protection
by naturally occurring or vaccine-induced BHV-1 antibodies has been suggested as a
possible explanation for the rare occurrence of BHV-5-associated disease in BHV-1
endemic areas (d'Offay et al., 1995). The rare occurrence and high fatality of BHV-5induced neurological disease have led to speculations about the origin of the virus. It has
been suggested that BHV-5 might be a virus naturally infecting zebu cattle or another
closely related ruminant species (Studdert, 1989; Bulach and Studdert, 1990).
In this study, we investigated the susceptibility of sheep to infection with two South
American BHV-5 isolates. Our results demonstrate that sheep are fully susceptible to
acute and latent BHV-5 infection and may even develop neurologic disease. Taken
together with recent reports of BHV-5 antibodies in sheep (Lindner et al., 1993a, b), these
findings suggest that natural infections by BHV-5 in this species may occur.
2. Material and methods
2.1. Experimental design
Two inoculations were performed independently to evaluate the susceptibility of sheep
to BHV-5 infection. Lambs were inoculated intranasally with either of two South
American BHV-5 isolates (A663 and EVI-88) and submitted to clinical and virological
monitoring. In the first experiment (isolate A663), nine lambs were inoculated and three
A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
91
remained as non-inoculated controls. In the second experiment (isolate EVI-88), 12 lambs
were inoculated and four non-inoculated lambs were maintained in close contact for
monitoring virus transmission. Fifty days after inoculation, the lambs from both
experiments were treated with dexamethasone during five days and monitored thereafter.
Nasal swabs collected after virus inoculation and dexamethasone treatment were
examined for infectivity. Necropsy was performed in one animal that developed
neurological signs and was euthanized in extremis. Tissue samples were submitted to
virus detection and quantitation, histological examination and in situ hybridization.
2.2. Cell culture and viruses
The BHV-5 strain A663 was isolated in Argentina and has been extensively
characterized (Carrillo et al., 1983; Schudel et al., 1986; Bratanich et al., 1991). The
BHV-5 isolate EVI-88 was isolated from calves with clinical meningoencephalitis in
Brazil and characterized by Roehe et al. (1997). Pestivirus-free Madin-Darby bovine
kidney cells (MDBK, American Type Culture Collection, CCL-22) were used for all
procedures of virus multiplication, quantitation and isolation from nasal swabs and
tissues. Cells were routinely maintained in Eagle's minimal essential medium (MEM)
containing penicillin (1.6 mg/l), streptomycin (0.4 mg/l), supplemented with fetal calf
serum (Gibco, BRL). Lambs were inoculated with 5 106 TCID50 of isolate A663 or
5 107 TCID50 of isolate EVI-88.
2.3. Animals, virus inoculation and dexamethasone treatment
Two to three-month-old, BHV-5 seronegative, recently weaned Corriedale lambs
(weighing approximately 10±12 kg) were used for experimental inoculation. Lambs were
maintained in collective pens (3±4 animals Each) (Experiment No. 1) or in a yard with
native grass (Experiment No. 2). The animals were inoculated with 5 ml of viral
suspension divided in the two nostrils, with the help of a cotton swab. Beginning at Day
50 pi, the animals were treated with dexamethasone (Azium, Schering) intramuscularly
during five days (2 mg per animal per day) and monitored thereafter.
2.4. Animal monitoring, sample collection and processing
Lambs were monitored clinically and nasal swabs for viral isolation were collected on
a daily basis up to Day 21 post-inoculation (dpi) and Day 15 post dexamethasone
treatment (dpt). Nasal swab specimens (0.5 ml) were inoculated onto MDBK cells grown
in 24-well plates and monitored for cytopathic effect (CPE) for 5 days. Negative samples
were further inoculated onto fresh MDBK cell monolayers and monitored for five
additional days. Samples positive for CPE were subsequently quantitated by inoculating
ten-fold dilutions onto MDBK cells cultivated in 96-well plates. Virus titers were
calculated according to Reed and Muench (1938) and expressed as log10TCID50/ml.
Blood for serology was collected from all inoculated lambs before virus inoculation, at
Days 15 and 30 pi and 15 days after DX treatment. Serum samples were submitted to a
standard microtiter virus-neutralizing (VN) assay (Bratanich et al., 1991), using two-fold
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A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
dilutions of serum against a fixed dose of virus (100±200 TCID50/well). Tissue samples
for viral isolation, histologic examination and ISH were collected at the necropsy of the
animal which developed neurological disease (lamb No. 60). Different sections of the
CNS (Table 2) were aseptically collected and submitted to virus isolation. Tissue samples
for virus isolation were processed by preparing a 10% (w/v) homogenized suspension and
inoculated onto MDBK cell monolayers. Monitoring of CPE and quantitation of
infectivity were performed as described above. Tissues for histopathologic examination
were fixed in 10% formalin, embedded in paraffin, sectioned and stained with
hematoxylin±eosin (HE) using routine methods. In situ hybridization (ISH) for BHV-5
nucleic acids was performed in formalin-fixed tissue sections essentially as described by
Chowdhury et al. (1997). Briefly, sections were deparaffinized, rehydrated, treated with
proteinase K (Sigma; 20 mg/ml in PBS) and hybridized with a 1.7 kb DNA probe
corresponding to the BHV-5 gC coding region (Dr. S. Chowdury, Kansas State
University). The probe was obtained by digestion of the plasmid pGEM3Z with BamHI/
AflII. The digestion fragment was gel-purified and labelled by nick translation with
digoxigenin-dUTP (DIG; Boehringer Mannheim, Indianapolis, IN). The hybridized DNA
probe was detected with the DIG-alkaline phosphatase conjugate and colour substrate
nitroblue tetrazolium salt (Boehringer Mannhein) as previously described (Sur et al.,
1996).
3. Results
3.1. Acute infection
Lambs inoculated with BHV-5 shed virus in nasal secretions from Day 1 to Day 16 pi
(Table 1). Peaks of virus shedding were observed between Days 3 and 5 pi, declining
progressively thereafter (data not shown). Maximum individual titers reached 107.1 and
105.9 TCID50/ml for isolates EVI-88 and A663, respectively. Some lambs inoculated with
isolate EVI-88 shed virus in titers above 106 TCID50/ml for two to three days. The extent
of virus shedding was capable of spreading virus to lambs maintained in close contact: all
four non-inoculated contact controls (sentinels) acquired the infection and shed virus
from Day 2 pi up to 7 days (Table 1). Most lambs inoculated with isolate A663 did not
show any clinical alteration. Only two lambs showed very mild signs of upper respiratory
tract infection, characterized by mild hyperemia of the nasal mucosa. Nine lambs (out of
12) inoculated with the isolate EVI-88 and two in-contact controls showed transient and
moderate hypertermia, mild to moderate hiperemia of the nasal mucosa and serous to
mucopurulent nasal discharge. These signs lasted for three to four days and
spontaneously subsided. Starting at Day 10 pi, one lamb (No. 60) showed intermittent
neurological signs consisting of depression, bruxism, tremors of the face, ptyalism,
incoordination, circling, backwards walking, lateral flexion of the neck and recumbency.
These signs were progressive, in both intensity and frequency, and this animal was
euthanized in extremis at Day 13 pi. All 13 inoculated lambs kept for latency studies
serocoverted to BHV-5 after acute infection, with VN titers ranging from 1 : 4 to 1 : 128
at Day 30 pi.
A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
93
Table 1
Viral shedding in nasal secretions during acute infection and after dexamethasone treatment in lambs
experimentally infected with bovine herpesvirus type-5 (BHV-5)
Isolate
Titer
Viral shedding
Acute infection
A663
EVI-88
Sentinels
(EVI88)
Total
a
b
c
5 106
5 107.64
After dexamethasone
a
b
No.
Duration
(days)
Titer
(max)
9/9
12/12
4/4
12.7 (11±15)
12.8 (11±16)
5.0 (3±7)
2.7 (5.89) 2/3
3.3 (7.11) 8/10
2.4 (3.11) 3/3
25/25
No.
Start
(dpt)c
Durationa
(days)
Titer
(max)
8
4
9
2.5 (2±3)
7.0 (3±11)
3.3 (2±5)
2.38 (2.96)
1.58 (4.81)
1.83 (1.98)
13/16 (81.2%)
Values in parenthesis indicate min and max days.
Virus titers were calculated according to Reed and Muench (1938) and expressed as log10TCID50/ml.
Day post-treatment with dexamethasone.
Infectious BHV-5 was recovered from several regions of the brain of the lamb No. 60.
The distribution and levels of infectivity detected in the brain of this animal are shown in
Table 2. The highest virus titers were demonstrated in the cerebral cortex, mainly in the
dorso- and ventro-lateral hemispheres and anterior cerebrum. Nonetheless, other areas of
the brain also harbored moderate levels of infectivity (Table 2). Infectivity was not
detected in non-neural tissues such as lungs, bronchial lymph nodes, kidneys and liver.
3.2. Histopathology
No gross lesions were observed in the brain of this lamb. Microscopic examination
revealed histological changes of varied severity in several areas of the brain, but mostly in
the cerebral cortex (anterior, ventro- and dorso-lateral). These changes were characterized
by mononuclear perivascular cuffing, focal gliosis, occasional neuronal degeneration,
mononuclear meningitis (Table 2 and Fig. 1). Large intranuclear inclusions were
observed in neurons and astrocytes of the anterior cerebrum (Fig. 2).
3.3. In situ hybridization
In situ hybridization using a BHV-5 gC-specific DNA probe revealed positive staining
in neurons and astrocytes of the anterior cerebrum and ventro-lateral cortex (Fig. 1). The
number of reactive cells was significantly higher in the anterior cerebrum, agreeing with
the highest viral titers and the more severe histological changes observed in this location.
No reactive cells were observed in brain sections of a BHV-5 seronegative animal
obtained in a BHV-5 seronegative lamb used as negative control (not shown).
3.4. Latent infection
Administration of dexamethasone to the lambs, starting at Day 50 pi, was followed by
reactivation of latent infection and viral shedding in 13 out of 16 (81%) BHV-5-infected
94
Anterior cerebrum
Infectivityc
Mononuclear meningitis
Perivascular cuffing
Gliosis
Neuronal necrosis
Intranuclear inclusions
a
Ra
Lb
4.67
‡‡d
‡‡‡
‡‡‡
‡‡
‡‡
3.88
‡‡‡
‡‡‡
‡‡‡
‡‡
‡‡
Ventro-lateral
Cortex
R
3.81
‡‡‡
‡‡‡
‡‡
‡
ÿ
Dorso-lateral
Cortex
L
R
L
4.52
‡
‡
‡
ÿ
ÿ
5.88
‡‡
ÿ
ÿ
ÿ
ÿ
4.50
ÿ
ÿ
ÿ
ÿ
ÿ
Midbrain
Posterior
cerebrum
Pons
Medulla
oblongata
Cerebellum
Olfactory
bulb
3.31
‡‡
‡
‡
ÿ
ÿ
2.50
‡
ÿ
ÿ
ÿ
ÿ
2.88
ÿ
ÿ
ÿ
ÿ
ÿ
3.01
ÿ
ÿ
ÿ
ÿ
ÿ
1.88
ÿ
ÿ
ÿ
ÿ
ÿ
3.11
ÿ
ÿ
ÿ
ÿ
ÿ
Right.
Left.
c
Infectivity expressed as log10TCID50/g of tissue.
d
Histological changes: severe (‡‡‡); moderate (‡‡); mild (‡); no histological alteration (-).
b
A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
Table 2
Histological changes and infectivity in the brain of the lamb that developed neurological disease following intranasal inoculation with bovine herpesvirus type-5
(BHV-5)
A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
95
Fig. 1. Anterior cerebrum of lamb No. 60. In situ detection of viral nucleic acids in the cerebral cortex. Black
foci represent virus-infected neurons and astrocytes detected by in situ hybridization with a BHV-5 glycoprotein
gC coding region probe. Methyl green counterstain. (40).
Fig. 2. Anterior cerebrum of lamb No. 60. Intranuclear inclusions in astrocytes of the cerebral cortex (arrows).
HE. 320.
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A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
lambs, including the in-contact controls (Table 1). The animals excreted virus in nasal
secretions from day four post-treatment up to 11 days. Reactivation of infection was not
followed by any evident clinical recrudescence. Viral shedding was intermittent in many
animals and occurred at lower titers and for a shorter period than in acute infection. Nine
inoculated lambs showed an increase (two- and four-fold) in the VN titers when tested at
Day 15 post-dexamethasone treatment. Four inoculated lambs plus the in-contact controls
did not show detectable increase in the VN titers following dexamethasone treatment.
4. Discussion
The present study was conducted to investigate the susceptibility of sheep to infection
with bovine herpesvirus type-5 (BHV-5). Recent studies have reported the natural
occurrence of BHV-5 antibodies in sheep (Lindner et al., 1993a, b). In many regions,
including some of those experiencing frequent outbreaks of meningoencephalitis due to
BHV-5, cattle and sheep are raised together, sharing pastures and facilities. These mixed
herds may provide conditions for interspecies transmission of pathogens. Under such
conditions, if they were susceptible to BHV-5, sheep could be naturally infected and
potentially serve as occasional reservoirs of the virus.
Our results demonstrated that sheep are fully susceptible to BHV-5 acute and latent
infection and may even develop neurological disease upon experimental inoculation. To
our knowledge, these are the first direct evidences of the susceptibility to BHV-5 of a
ruminant species different than cattle. Previous reports of BHV-5 infection in this species
were limited to detection of positive serology in sheep flocks (Lindner et al., 1993a, b). In
our study, intranasal inoculation of seronegative lambs with two South American BHV-5
isolates was followed by efficient virus replication in the nasal mucosa. The duration and
levels of virus shedding during acute infection were even superior to values reported in
some experimental infections with BHV-5 in cattle (Hall et al., 1966; Belknap et al.,
1994) and with BHV-1 in sheep (Shankar and Yadav, 1987; Giuliani and Sharma, 1995;
Hage et al., 1997). Most lambs inoculated with the isolate EVI-88 developed mild signs
of upper respiratory tract infection. Mild signs of rhinitis have also been reported in sheep
experimentally inoculated with respiratory BHV-1 strains (Shankar and Yadav, 1987;
Giuliani and Sharma, 1995; Hage et al., 1997) and in calves inoculated with BHV-5 (Hall
et al., 1966; Belknap et al., 1994). These results demonstrated that seronegative lambs are
susceptible to BHV-5 infection following intranasal inoculation. Moreover, the extent and
duration of virus shedding was capable of spreading the virus to other animals. All the incontact controls were infected, indicating that transmission of the virus among sheep
during acute infection can occur. The ability of sheep to transmit the virus to cattle was
not investigated in this experiment, yet this will be necessary as to ascertain the potential
of sheep-to-cattle transmission of BHV-5.
Although the isolate EVI-88 was isolated from cases of meningoencephalitis in cattle
(Roehe et al., 1997), this virus has subsequently demonstrated a limited ability to
reproduce the neurological disease in calves upon experimental inoculation (Roehe, P.M.,
personal communication). In addition, no previous report of neurological disease caused
by BHV-5 in a ruminant non-bovine species had been published to date. For these
A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
97
reasons, the development of neurological disease by lamb No. 60 was somewhat
unexpected. Nevertheless, the clinical, histological and virological findings associated
with this clinical case were very similar to the observations described in natural and
experimental BHV-5 infections of cattle (Bagust and Clark, 1972; Schudel et al., 1986;
Riet-Correa et al., 1989; Belknap et al., 1994).
In order to investigate whether acute BHV-5 infection in sheep is followed by latent
infection, infected lambs were treated with dexamethasone at Day 50 pi and monitored
thereafter. Since the ability to establish and reactivate latent infections is crucial for the
perpetuation of alphaherpesviruses in nature, a potential role for sheep in the transmission
of BHV-5 should necessarily include their ability to harbor the virus in a latent state.
Dexamethasone administration was followed by reactivation of latent infection and viral
excretion in 13 out of 16 lambs, with virus shedding lasting up to 11 days in some
animals. These results are consistent with previous observations of BHV-5 latent infection
in calves (Belknap et al., 1994) and of BHV-1 latent infection in sheep and goats (Shankar
and Yadav, 1987; Tolari et al., 1990; Hage et al., 1997). Moreover, these are the first
observations of BHV-5 latent infection in sheep. Further studies are underway to ascertain
the capacity of BHV-5 latently infected sheep to spread virus to other sheep and cattle due
to virus shedding occurring upon reactivation of latent infection.
Evidence of natural infection by BHV-5 in a non-bovine ruminant species have been
limited to positive serology in sheep (Lindner et al., 1993a, b). Nevertheless, natural
infections by a closely related alphaherpesvirus, BHV-1, either by detection of antibodies
or by direct identification of the virus, have been reported in several domestic and wild
ruminants (Mohanty et al., 1972; Taylor et al., 1977; Legrottagile and Mancianti, 1982;
Tolari et al., 1990; Clark et al., 1993; Rosadio et al., 1993; Suresh and Suribabu, 1993;
Frolich, 1996; Pituco and Del Fava, 1998). Likewise, experimental studies have
demonstrated the susceptibility of sheep to acute and latent infection by BHV-1 and
sheep-to-sheep and sheep-to-cattle transmission of the virus (Shankar and Yadav, 1987;
Giuliani and Sharma, 1995; Hage et al., 1997). In a study by Rosadio et al. (1993), the
prevalence of antibodies cross-reacting with BHV-1 in sheep and goats was found to be
higher in herds where these species were raised together with BHV-1 seropositive cattle.
Although the epidemiological significance of these observations remains uncertain, these
findings are at least suggestive that cattle may not be the only natural host for BHV-1 and
that natural infections in other species may occur. As a consequence, a potential role for
these species in the epidemiology of BHV-1 should not be discounted (Shankar and
Yadav, 1987; Tolari et al., 1990; Suresh and Suribabu, 1993). The potential implication of
other ruminants in the epidemiology of this closely related virus, in addition to the recent
reports of BHV-5 positive serology in sheep (Lindner et al., 1993a, b), led us to consider a
possible involvement of sheep in the transmission of BHV-5 in nature. In this sense, the
epidemiological meaning of our findings that sheep are susceptible to BHV-5 infection
should be subject of further investigation.
In summary, our results demonstrated that sheep are fully susceptible to acute and
latent BHV-5 infection. These findings represent the first direct evidence of the
susceptibility of a ruminant species other than cattle to infection with BHV-5. In addition,
these results may serve to reinforce the hypothesis that other ruminant species may
occasionally participate in the transmission of BHV-5 in nature. Indeed, the degree of
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A.M. Silva et al. / Veterinary Microbiology 66 (1999) 89±99
susceptibility taken together with previous reports of BHV-5 antibodies in sheep (Lindner
et al., 1993a, b) are suggestive that natural infections by BHV-5 in this species possibly
occur. Since raising cattle and sheep together is a common practice in many regions,
opportunities for interspecies transmission of BHV-5 in the field are likely to occur. If so,
a potential role for sheep in the epidemiology of BHV-5 infection should be considered.
In addition, the susceptibility of sheep to BHV-5 may also render this species a suitable
animal model in which to characterize BHV-5 infection and disease.
Acknowledgements
This work was supported by a MCT, CNPq, CAPES and FINEP grant (PRONEX em
Virologia VeterinaÂria, 215/96). E.F. Flores (352386/96), R. Weiblen (520161/97-1) and
L.F. Irigoyen (523134/96-7) have scholarships from the Brazilian Council for Research
(CNPq). F.A. Osorio's participation was partially supported by a fellowship by the
Fullbright Commission in Brazil (ComissaÄo Fullbright Brazil) and by a grant from
USDA/FAS/ICD/RSED of the United States of America.
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