Note: Descriptions are shown in the official language in which they were submitted.
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EQUINE HERPES VIRUS TEMPERATURE SENSITIVE MUTANT AND LIVE
VACCINE THEREOF
The present invention relates to an equine abortion virus mutant, a process
for the preparation of said mutant, use of said mutant and live vaccines
derived from
said mutant.
Equine abortion virus (EHV-1 ), a herpes virus, is a major equine pathogen
responsible for viral-induced abortion, neurological disease such as paresis,
infections of the upper respiratory tract, and neonatal foal disease (NFD).
NFD
results from close to term transplacental infection of fetuses, which are born
weak
to with severe respiratory disease and some with jaundice due to liver
infection by
EHV-1. These animals usually die within a few days after birth. Equine
rhinopneumonitis virus (EHV-4) is the major cause of acute respiratory tract
disease
("rhinopneumonitis") and infects most horses during their first two years of
life.
Rhinopneumonitis is characterized by fever, anorexia, and profuse serous nasal
IS discharge that later becomes mucopurulent. On rare occasions EHV4 infection
causes abortion in pregnant mares. Furthermore EHV1 and EHV4 establish
persistent, lifelong latent infections. Upon reactivation the viruses cause
recurrent
disease, accompanied by virus shedding and transmission to other animals.
Control of equine herpes virus infection and their diseases remain
2o inadequate, in particular against EHV1 mediated abortions, paresis and
neonatal foal
disease resulting from close to term transplacental infection of foetus.
Although
inactivated as well as modified live vaccines are available, neither vaccine
appears to
block infection sufficiently, nor do they prevent the establishment of latency
by wild-
type virus. Hence there is a great need for safe vaccines with improved
protection
z5 against field infections of these viruses, particularly against infections
caused by
EHV1.
The present invention provides for such vaccines.
In a first aspect the present invention provides for an EHV-1 Ts mutant as
deposited at the European Collection of Animal Cell Culture (ECACC),
Salisbury,
.o Wiltshire SP4 OJG, UK on 10 June 1999 under accession number V99061001, and
progeny thereof.
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The EHV-1 Ts mutants according to the invention are furthermore
phenotypically characterized in that
.they form small plaques when grown on several horse cell lines.
~ they have lost their ability to grow on rabbit kidney cells, in particular
RK13 cells,
and
.they are limited in their ability to cause viraemia ( that is, they are able
to ;
The EHV-1 Ts mutants according to the invention have the advantage that
replication is restricted to the upper respiratory tract of conventional
equidae with no
or limited ensuing viraemia. The Ts mutants are safe for pregnant mares while
giving
1o rise to significant immune stimulation following growth in the upper
respiratory tract.
The Ts mutants are not readily back-passaged form animal to animal thus
limited in
their potential for transmission and reversion.
For the purpose of this invention "progeny" is defined to include also all
strains obtained by further serial passage of the deposited EHV-1 Ts mutant.
15 For the purpose of this invention, a temperature sensitive mutant is
defined
as a mutant virus which has an impaired growth at or above a certain
temperature at
which the wild type has a normal growth. The EHV-1 Ts mutants according to the
present invention are characterized in that they are temperature sensitive at
the body
temperature of the host animal. The EHV-1 Ts mutants of the present invention
do
2o not replicate above a temperature of 38.5 to 39.0°C. Preferably the
EHV-1 Ts
mutants according to the invention do not replicate at a temperature of
38.5°C.
For the purpose of this invention, small plaques are defined as plaques that
are at least half to one third the size of the plaques formed by the wild-type
parent
strain in equine cells.
25 For the purpose of this invention the "limited abilty to cause viraemia" is
defined as the ability to cause no or low grade (that is, just detectable)
vireamia for 1
to 3 or 4 days in some animals with respect to the ability of the parent
strain to cause
viraemia.
Temperature sensitive EHV-1 mutants according to the invention can be
30 obtained by treatment of infected bovine, equine or other permissive cell
cultures at
34°C with non-toxic concentrations of a mutagens such as 5-bromo-2-
deoxy uridine,
azacytidine and the like during viral replication in vitro, followed by
biological cloning
of progeny virus from said treated cultures in bovine or equine or other
permissive
cell lines.
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The favorable properties of the Ts-mutants according to the invention makes
them very suitable for use in the preparation of a vaccine. Thus, in a second
aspect
the present invention provides for a composition, in particular a vaccine
composition,
comprising an EHV-1 Ts-mutant according to the invention, and a
pharmaceutically
acceptable carrier or vehicle. More specifically, a (vaccine) composition
according to
the invention comprises the EHV1 Ts-mutant deposited at the ECACC, Salisbury,
UK
having accession number V99061001 and/or progeny thereof. Pharmaceutical
acceptable carriers or vehicles that are suitable for use in a vaccine
according to the
invention are sterile water, saline, aqueous buffers such as PBS and the like.
In
to addition a vaccine according to the invention may comprise other additives
such as
adjuvants, stabilizers, anti-oxidants and others.
The vaccine compositions according to the invention are safe and can be
used to protect the equidae clinically and virologically against infections
with EHV-1
and to protect against virus-induced abortions and paresis. In addition the
vaccine
Is according to the invention was found to stop traps-placental infection,
thus protecting
the newborn foal from the effects of neonatal foal disease. The vaccine
composition
according to the present invention can be administered not only to horses but
also to
other animals that are susceptible to EHV-1 infection such as donkeys, zebra's
and
the like. Cattle which have been reported to be susceptible to EHV-1 and EHV-4
2o infection can also be treated with the vaccine according to the invention.
It was furthermore surprisingly found that vaccines comprising an EHV-1 Ts-
mutant according to the invention not only protect against EHV-1 infections
but also
against the disease and the associated virus shedding following EHV-4
infection.
Thus such a vaccine can be useful to obtain cross-protection in the vaccinated
25 equidae. Said vaccines give rise to improved protection thus effectively
blocking
infection with wild-type viruses.
Vaccine compositions according to the invention can be prepared following
standard procedures. A vaccine according to the invention preferably is a live
vaccine. For the preparation of the live vaccine, the seed virus of the EHV-Ts
mutant
;o can be grown on a cell culture, such as primary or secondary bovine kidney
or
equine cells. The viruses thus grown can be harvested by collecting the tissue
cell
culture fluids and/or cells. Optionally, during harvesting the yield of the
viruses can
be promoted by techniques that improve the liberation of the infective
particles from
the growth substrate, e.g. sonication. The live vaccine may be prepared in the
form
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of a suspension or may be lyophilized.
Pharmaceutical acceptable carriers that are suitable for use in a vaccine
according to the invention are sterile water, saline, aqueous buffers such as
PBS and
the like. In addition a vaccine according to the invention may comprise other
a additives such as adjuvants, stabilizers, anti-oxidants and others.
Suitable stabilizers are for example carbohydrates including sorbitol,
mannitol, starch, sucrose, dextran and glucose, proteins and degradation
products
thereof including but not limited to albumin and casein, protein-containing
agents
such as bovine serum or skimmed milk, and buffers including but not limited to
alkali
1o metal phosphates. In lyophilized vaccine compositions it is preferable to
add one or
more stabilizers.
Suitable adjuvants include but are not limited to aluminum hydroxide,
phosphate or oxide, amphigen, tocopherols, monophosphenyl lipid A, muramyl
dipeptide, oil emulsions, glucans, carbomers, block-copolymers, cytokines and
15 saponins such as Quil A. The amount of adjuvant added depends on the nature
of
the adjuvant itself.
EHV-1 Ts mutants according to the invention are preferably administered to
conventional, seronegative animals varying in ages from a few days to several
years,
including those in-foal. The vaccine can be administered to the animals via
non-
2o parenterally administration routes, including but not limited to
intradermal, oral,
spraying, aerosol, intra-ocular, and intranasal administration. Alternatively,
the
vaccine can be administered via parenteral administration routes. Preferably
the
vaccine is administered intradermally or intranasally.
In general the EHV-1 Ts mutant virus is administered in an amount that is
25 effective to induce protection against EHV-1 infection. The dose generally
will
depend on the route of administration, the time of administration, as well as
age,
health and diet of the animal to be vaccinated. The virus can be administered
in an
amount between 102 and 10~ pfu/dose per animal, preferably between 103 and 105
pfu/dose and more preferably at 10' pfu/dose per animal.
3o The vaccines according to the invention also may be given simultaneously or
concomitantly with other live or inactivated vaccines. These additional
vaccines can
be administered non-parenterally or parenterally. Preferably the additional
vaccines
are recommended for parenteral administration.
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EXAMPLES
1. Isolation and characterization of a temperature sensitive EHV 1 mutant
strain TS C147
Just confluent, day-old 75 cm~ monolayers of equine dermal (ED) cells were
infected at m.o.i. of 0.001 with EHV-1. Inoculum (2.0 ml) was adsorbed (1
hour,
37°C), removed and monolayers were washed with PBS and then re-fed with
tissue culture medium (25 ml) containing 40 ~g/ml of 5-bromo-2-deoxy uridine
and incubated at 34°C. At maximum CPE (7 days post inoculation), the
culture
was harvested (frozen at -40°C and then thawed at 37°C),
dialyzed overnight
to at 4°C against PBS, titrated for EHV-1 infectivity in ED cells at
37°C and
subsequently cloned at 34°C in ED cells grown in 96-well microtitration
plates.
Wells with single EHV-1 focus were identified, allowed to grow to maximum
CPE and then a small (20 ~I out of 200 ~I total) sample used for phenotyping
at
permissive (34°C) and restrictive (39°C) temperatures using ED
cells.
Temperature sensitive clones were further passaged in Bovine Kidney cells,
strain JCK (Jay's Calf Kidney- Intervet's strain) to produce the master and
working seeds.
2 Temperature sensitive phenot~ipe of EHV 1 strain TS C147
EHV-1 TS C147 strain at Master Seed Virus (MSV)+1 °level was
titrated in
2o parallel in Bovine Embryo Lung (strain BEL26 - Intervet's strain), Bovine
Kidney
(strain Jay's Calf Kidney, JCK - Intervet's strain), Equine Dermal (ED) cells,
Equine Dermal Clone W48 C10 (ED W48 C10 - Intervet's strain), and Equine
Dermal Clone W7 C5 (ED W7 C5 - Intervet's strain) at 37°C and
38.5°C. Virus
at MSV+1 ° passage level failed to grow at 38.5°C. Results are
given in Table 1.
3. EHV 1 strain TS C147 has EHV 4 like characteristics
A parameter for the differentiation between EHV-1 and EHV-4 is their ability
to
replicate in rabbit kidney (RK13) cells. EHV-1 strains replicate well in RK13
cells
but the cells are refractory to EHV-4 strains. EHV-1 strain TS C147 at MSV+1
°
level, its wild type parent EHV-1 strain, EHV-1 strain deficient in immediate
early
3o gene (EHV-1 IE), pathogenic EHV-1 strain CHLi and a field EHV-4 isolate
were
titrated in parallel at 37°C in RK13 cells and Equine dermal (ED)
cells. Results
given in Table 2 show that the 4 EHV-1 strains, including strain TS C147 and
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6
EHV-4 strain replicated in ED cells but EHV-1 strain TS C147 and EHV-4 strain
did not grow in RK13 cells.
TABLE 1: Relative titers of EHV-1 strain TS C147 at 37°and
38.5° in various
bovine and equine cell strains
Titers (log,o TCIDSO/ml) Virus Passage level TSC147
at 37C and MSV+1
38.5C in various bovine and
equine cell
types
37C
BELZ6: .2
38.5C <1.1
JCK: 37C 5.4
38.5C < 1.1
ED: 37C 5.4
38.5C <1.1
ED W48 C10: 37C 5.7
38.5C <1.1
ED W7 C5: 37C 5.7
38.5C <1.1
a) Titers after 5 days' incubation; Titters given as <1.1 log,o TCID<~/ml
represent no
EHV-1 foci detected in 4 x 200N1 of the lowest (10-') dilution of the virus
tested in the
titration
Table 2: Ability of rabbit kidney cells to support replication of EHV-1 strain
TS
C147
Virus Relative titer (log,o
TCIDSO/ml) at 37C
in RK13 & ED cells
RK13 cells ED cells
EHV-1 TS C147 MSV+1 <1.1a 5.7
EHV-1 040 5.7 5.7
EHV-1 IE- 6.0 6.2
EHV-1 CHLi 5.7 6.0
EHV-4 <1.1 3.7
is
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a - Titers given as <1.1 log,o TCIDSO/ml represent no EHV-1 CPE detected in 4
x
200u1 of the lowest dilution (10-') in the titration.
4. Clinical and viroloqical protection of conventional ponies acrainst
s infections by EHV 7 and EHV 4
Of 29 conventional ponies with low or no EHV-1 neutralizing (VN) antibody, 15
were vaccinated intranasally (IN) with a dose of 5.3 logo TCIDso of strain EHV-
1 TS C147 while 14 ponies were left unvaccinated to serve as unvaccinated
control. About a month following a single IN vaccination, 8 vaccinated and 8
1o unvaccinated (control) ponies were challenged IN with a field strain of EHV-
1
while a group of 7 EHV-1 vaccinated and 6 control animals were challenged IN
with a recent field isolate of EHV-4. Following vaccination and challenge,
animals were monitored for clinical reactions, virus shedding in nasal mucus,
infected leukocytes (viraemia) and EHV-1 neutralizing antibody.
15 Vaccine virus grew to low titers (nasal mucus peak titers 1.5 to 3.0 logo
TCIDSO/ml) for 1 to 8 days in 11/15 ponies and also resulted in low-grade
(just
detectable) leukocyte viraemia for 1 to 4 days in 7 of 15 animals. However all
15 ponies seroconverted. In contrast no EHV-1 was recovered from the nasal
mucus or the blood of 14 control ponies monitored daily for 10 or 14 days
2o respectively and the animals remained seronegative to EHV-1 until after
challenge infection. A similar level of pyrexia was seen in 10 animals in each
of the two (vaccinated and control) groups. These findings are summarized in
Table 3.
Following intranasal EHV-1 challenge, there was a significant reduction in
virus
25 excreted in nasal mucus by the vaccinated ponies relative to that recovered
from the control animals. Similarly a single vaccination prevented leukocyte
viraemia in 7 of 8 ponies while one pony was just virus positive for 1 day. In
contrast, however all 8 unvaccinated ponies became viraemic, 7 for 3 to 4 days
and 1 for 1 day. All 8 control ponies became moderately to highly febrile for
1
3o to 6 days but all 8 vaccinated animals remained normal. None of the 8
vaccinated animals responded anamnestically to the challenge infection while
all 8 control animals responded with a significant EHV-1 neutralizing
antibody.
These findings are summarized in Table 4.
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Following intranasal EHV-4 challenge, virus was recovered from the nasal
mucus of one of 7 vaccinated ponies on one occasion but all 6 control ponies
excreted virus at a significantly higher titer for 2 to 3 days, with one
exception.
None of the 7 EHV-1 vaccinated ponies became viraemic in contrast to 3 of 6
control ponies for 1-3 days. EHV-4 challenge infection resulted in pyrexia in
3
of 6 control animals for 2 to 3 days but none of the 7 vaccinated ponies were
affected. There was a slight (15 to 20 expirations/minute) increase in
respiration rate in 4 of 7 vaccinated and 5 of 6 control animals for 1 to 3
and 2
to 6 days respectively. These findings are summarized in Table 5.
to
TABLE 3: Results after vaccination
Result - No +ve/No Total (Peak activity range & duration)
Parameter Vaccinated Control
Virus shedding 11/15 (1.5-3.0 logo 0/14
in mucus TCIDSO/ml,
1-8 days)
Leukocyte viraemia7/15 (low grade, 1-4 0/14
days)
Seroconversion 15/15 0/14
Pyrexia (=38.5C) 10/15 (38.5-39.3, 1-3 10/14 (38.5-38.8,
(Between days 1-10)days) 1-3 days)
TABLE 4: After EHV-1 challenge
Result - No +ve/No Total (Peak activity range & duration)
Parameter Vaccinated Control
Virus shedding 5/8 (1.5 log,o/ml, 8/8 (2.2-3.4 logo
in mucus 1-2 days) TCIDSO/ml,
4-6 days)
Leukocyte viraemia1/8 (low grade, 1 day)8/8 (3-4 days; 1
day for 1
animal)
EHV-1 (VN) antibody0/8 (=4-fold rise) 8/8
rise)
Pyrexia 0/8 8/8 (38.9-41.0, 1-6
days)
Respiration 3/8 (15-20 expirations/min,2/8 (15-20 expirations/min
1 1
day) day)
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TABLE 5: Results after EHV-4 challenge
Result - No +ve/No Total (Peak activity range & duration)
Parameter Vaccinated Control
Virus shedding in 1/7 (1.5 log,o/ml, 6/6 (1.5-3.7 logo
mucus 1 day) TCIDSO/ml,
1-3 days)
Leukocyte viraemia 0/7 3/6 (1-3 days)
EHV-1 (VN) antibody1/7 (=4-fold rise) 5/6
rise)
Pyrexia 0/7 3/6 (38.6-38.8, 2-3
days)
Respiration 4/7 (15-20 expirations/min,5/6 (15-20 expirations/min
1-3 days) 2-6 days)
5. Protection of eguidae against paresis and abortions due to EHV 1
s infection
Of 12 pregnant mares with low or no EHV-1 neutralizing (VN) antibody, 6 were
vaccinated intranasally (IN) at about 6 months of gestation and then all 12
mares challenged IN with a pathogenic strain of EHV-1 at the critical stage of
gestation for EHV-1 abortions namely about 9 months of gestation. Following
1o vaccination and challenge, animals were monitored for clinical reactions,
virus
shedding in nasal mucus, infected leukocytes (viraemia) and EHV-1
neutralizing antibody.
Although no vaccine virus was recovered from nasal mucus from any of 6
vaccinated mares, low grade, transient (1 to 3 days) viraemia was detected in
15 5 of 6 mares and all 6 animals seroconverted with significant VN antibody
to
EHV-1. None of the 6 control mares, monitored in parallel to vaccinated
animals for 10 to 14 days, yielded EHV-1 from nasal mucus or leukocytes but 1
of 6 animals seroconverted some 2'/2 months later. These findings are
summarised in Table 6.
2o Following challenge, there was a significant (2 out of 6 compared to 5 of 6
and
1.5 to 1.7 logo TCIDSO/ml for 1-2 days compared to 2.4 to 3.7 logo TCIDSO/ml
for 1-6 days) reduction in virus excreted in nasal mucus by the vaccinated
mares. Similarly none of 6 vaccinated mares became viraemic in contrast to 5
of 6 unvaccinated control mares. In the control group 5 of 6 mares became
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febrile for 1 to 5 days, 3 also developed paresis accompanied by severe
jaundice and disintegration of the cervical plug in 2 mares with signs of
foetal
ejection. One of the two animals died while the 2"d had to be euthanased in
extremis. Both animals carried dead foals. Three further mares aborted.
Foetal tissues from all 5 foetuses were EHV-1 positive. In contrast however
all
6 vaccinated mares foaled normally. The only clinical reaction observed in
vaccinated mares was transient (1 day) pyrexia in one of 6 mares. The control
mare which foaled normally had in fact seroconverted just prior to challenge.
These findings are summarised in Table 7.
t0
TABLE 6: Results after vaccination
Result - No +ve/No Total (Peak activity range & duration)
Parameter Vaccinated (group 1 Control (group 2)a
)
Virus shedding 0/6 Not monitoreda
in mucus
Leukocyte viraemia5/6 (low, 1-3 days) 0/6
Pyrexia 1/6 (1 day) Not monitoreda
Seroconversion 6/6 (Month onwards after1 */6 (About 3 months
vaccination) after
vaccination)
a = Not monitored because animals kept in isolation away from the vaccinated
group
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TABLE 7: Results after challenge
Result - No +ve/No Total (Peak activity range & duration)
Parameter Vaccinated (groupControl (group 2)
1 )
Virus shedding in 2/6 (1.5-1.7 logo5/6 (2.4-3.7 logo TCIDSO/
mucus TCIDSo/ml, 1-2 ml, 1-6
days) days)
Leukocyte viraemia 0/6 5/6
EHV-1 VN antibody 0/6 5/6t
rise
Pyrexia 1/6 (1 day) 5/6 (1-5 days)
Paresis 0/6 3/6 (Terminal in 2 mares)
Jaundice 0/6 2/6
Death 0/6 2/6 (1 died, 1 euthanased
in extremis
with severe paralysis jaundice
and
rapid decrease in body
temperature
Abortion 0/6 5/6
t Control mare was seronegative in 3-monthly bleeds after vaccination of group
1 mares but
seroconverted prior to challenge.
6. Safety of EHV 1 TS C147 in pregnant mares
Four mares at about 9 months of gestation (critical stage for EHV-1 abortions)
were inoculated by the natural route with 10 times the protective dose and
monitored for abortions. Results given in Table 8 show that all 4 mares
seroconverted to EHV-1, one of 4 mares became transiently viraemic but
foaled normally. Three of 4 foals were EHV-1 VN antibody negative in blood
samples collected before suckling the respective dam while one foal was VN
antibody positive due to colostrum intake (born between monitoring intervals
in
the early hours). These results are summarised in Table 8.
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TABLE 8: An overdose safety for pregnant mares at the critical stage of
gestation
for EHV-1 abortions
Mare Shedding Viraemia VN antibody Foaling
No in to EHV-1 & antibodya
nasal mucus At dosing
& 3 wks
later
13 c -ve <2.0 3.5 Normal <2.0
14A c +ve (3 days)<2.0 6.0 Normal 4.0 b
15 c -ve <2.0 5.0 Normal <2.0
16 c -ve <2.0 6.0 Normal <2.0
a = EHV-1 neutralizing antibody at birth
b = Born between monitoring intervals in early hours and the foal was bled at
least 3 hours after birth
c - Pending, ie to be done
7. No transmission of EHV 1 TS C147 befween target species.
A back-passage study was performed in EHV-naive (all types) weaned foals
(specific pathogen free, SPF foals).
Two SPF foals were inoculated intranasally (IN) with 10 times protective dose
of EHV-1 strain TS C147 at Master Seed Virus+1 ° passage level and
virus
positive nasal mucus collected over several days used to similarly infect a
further pair of SPF foals. After IN inoculation, foals were monitored for (i)
virus shedding in nasal mucus, (ii) clinical reactions and (iii)
seroconversion to
EHV-1.
Foals given EHV-1 strain TS C147 at MSV+1 ° level excreted virus
in nasal
mucus and seroconverted. However, a pool of virus positive nasal mucus
samples failed to infect a further pair of EHV-naive foals as judged from the
failure to recover EHV-1 from their nasal mucus and the absence of
seroconversion to EHV-1. The results were confirmed by repeating the study
with a further 4 SPF foals, 2 inoculated with MSV+1 ° followed by a
further 2
given virus positive nasal mucus from the first two foals. Results are
summarised in Tables 9 and 10.
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TABLE 9: Backpassage of EHV-1 strain TS C147 in EHV-naive foals
(i) 5 PASSAGE ONE: Foals 1 & 2 inoculated intranasally with EHV-1 TS C147
(10x protective dose) at MSV+1 ° level.
Parameter Result (+i-, range & duration)
Virus shedding in nasal2; 2 ( 1.7-5.0 logo TCIDSO/ml, ~-~
mucus days)
Seroconversion to EHV-12/2 (bleed 2 weeks after inoculation
& by CF test)
(ii) PASSAGE TWO: Foals 5 & 6 inoculated intranasally with virus positive
nasal mucus from foals 1 & 2
Parameter Result (+:-, range & duration)
Virus shedding in nasal0/2
mucus
Seroconversion 0/2
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TABLE 10: Backpassage of EHV-1 strain TS C147 in EHV-naive foals
(i) PASSAGE ONE: Foals 7 & 8 inoculated intranasally with EHV-1 TS C147
(10x protective dose) at MSV+1 ° level.
Parameter Result (+/-, range & duration)
Virus shedding in nasal2/2 ( 1.5-3.7 logo TCIDs~,,%ml, 4-8
mucus days)
Seroconversion to EHV-12/2 (bleed 2 weeks after inoculation
& by CF test)
(ii) 1o PASSAGE TWO: Foals 9 & 10 inoculated intranasally with virus positive
nasal mucus from foals 7 8~ 8
Parameter Result (+/-, range & duration)
Virus shedding in 0/2
nasal mucus
Seroconversion to 0/2
EHV-1
