Transcript WITH WEST CAUCASIAN BAT VIRUS

EXPERIMENTAL INFECTION OF BIG
BROWN BATS (Eptesicus fuscus) WITH
WEST CAUCASIAN BAT VIRUS
Kuzmin I.V., Franka R., Rupprecht C.E.
Centers for Disease Control and Prevention,
1600 Clifton Rd. Atlanta, GA, 30329
Disclaimer: Use of trade names and commercial sources are for identification
only and do not imply endorsement by the U.S. Department of Health and
Human Services.The findings and conclusions in this report are those of the
authors and do not necessarily represent the views of the funding agency
Background:

The West Caucasian bat virus was isolated in
2002 from the brain of Shreiber’s bat
(Miniopterus schreibersii) in south-eastern Europe
Background:


As only one isolate is available, the principal
host species is unknown.
The distribution of M. schreibersii is large, and
several additional species of Miniopterus are
distributed in the Old World tropics and
subtropics.
Area of Miniopterus schreibersii
Background:
Genotype 6
L
O
NI
F
7
0
0
9
H
8
1
0
9
A
F
0
8
1
0
2
0
A
F0
06
49
7
Genotype 7
A
R
F
8
1
9
8
Genotype 5
L
8615PO
M
L5
86132AS
PA
R89
Genotype 4
100
61
80
56
AF46
7949
99
100
Ethlag
100
88636HAV
8619NGA
GUI
8660
100
Genotype 2
US
1R
914
VS
C
Phylogenetic
Genotype 1
position of
WCBV according
to the N gene
sequence
T
9
1
B
D
A
S
A
R
I
1
8
UO
Y3
56
8
S
E
9
3
3
9
6
8
50
Genotype 3
LC8 binding site of the
lyssavirus phosphoprotein
(framed)
AA 329-335 of the lyssavirus
glycoprotein ectodomain
138
CVS
SADB19
V006
V267
V020
V552
V550
S59448
V008
V023
V002
V286
V474
V481
Aravan
Khujand
Irkut
WCBV
: GK-SSEDKSTQTT
: GK-SSEDKSTQTT
: TQ-ATVSKPTQTD
: AK-ITDNKQTQTD
: PR-NLKSIQIQTE
: AR-KTKSVQIQTE
: AR-KTKSVQIQTE
: ER-DTKSIQIQTE
: NK-LFEDKSTQTV
: NK-LQADKSTQTT
: NK-LQADKSTQTT
: GK-STEDKSTQTP
: GK-TTENKSTQTT
: GK-TTESKSTQTT
: GK-SLADKSTQTS
: GK-STDDKSTQTV
: DK-ESAEKSTQTV
: PKPTTKDIAVQAD
333
CVS
SADB19
SHBRV
8620CAR
8619NGA
S59447
9020SA
8918FRA
EBL1POL
9018HOL
9007FIN
AF006497
AF081020
Aravan
Khujand
Irkut
WCBV
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
YKSVRTW
YKSVRTW
YKSVRTW
YLRVDSW
YLKVDNW
YKRVDKW
YKSVREW
YKSVREW
YKSVREW
YKSIREW
YKSIREW
YNQVRTW
YKSVRTW
YKSVREW
YKSIREW
YKSIREW
YIKVENW
Background
Comparative peripheral (intramuscular)
pathogenicity of WCBV for different
mammals:




Laboratory ICR mice – apathogenic.
Syrian hamsters – pathogenic.
Non-human primates (Macaca sp.) – pathogenic.
bats ???
Study:

The aim of the study was to assess the
susceptibility and general pathogenesis patterns
of WCBV in big brown bats (Eptesicus fuscus)
The species was selected because
of availability;
because this is one of the major
rabies virus reservoirs in North
America;
because experimental data for
other lyssaviruses in this species
are available for comparison
Study:

The bats, either recently captured or survivors from previous experiments
with Irkut virus, were inoculated intramuscularly into the masseter muscle
(7), the dorsal part of neck (8) or orally (6).

Bleeding was done on the day of WCBV inoculation (day 0) for baseline
antibody titers, and every two weeks for the next 6 months of observation.

Oral swabs were taken weekly during 1-3 months post inoculation, and
every two weeks during 4-6 months post inoculation.



Animals found dead, or euthanized during the terminal stage of disease,
were subjected to necropsy. Tissue samples from brain (BR),
submandibular and parotid salivary glands (SG), brown fat (BF), lung (LU),
kidney (KI) and bladder (BL) were tested.
All surviving bats were euthanized 6 months after the inoculation date. Only
BR and SG samples were taken from surviving animals post-mortem.
The tests included nested RT-PCR of tissues, swabs and blood pellets, and
virus isolation for all PCR-positive samples. Sera were tested in RFFIT with
WCBV; for bats previously inoculated with Irkut virus, this virus was
additionally used as the modified RFFIT virus challenge.
Results:




Three bats died or were euthanized during the lethargic stage of the
disease on days 10 to 18. The only sign of a disease observed a
few hours before this outcome was general weakness. The bats
were sluggish and unable to climb the cage walls. All three were
inoculated into the neck muscles.
Nested PCR demonstrated the presence of viral RNA in the BR, LU
and SG of all 3, and in the BF and BL of 2 of them. In contrast, KI
and blood pellets were negative in each. Isolation was successful
only from the BR of all 3.
For oral swabs, the single PCR-positive result was obtained for the
swab taken from one bat on the day of euthanasia. The isolation
attempt was negative.
The BR and SG from all survivors were PCR-negative, as well as all
available blood pellets (virus isolation not attempted).
Results (continued):
Results of nRT-PCR for bat tissues
Tissue
Bat #409
Bat #433
Bat #883
BR
+
+
+
BF
+
-
+
BL
+
+
-
KI
-
-
-
LU
+
+
+
SG
+
+
+
Results (continued):



WCBV-neutralizing antibodies were detected in 4 of 7
bats (57.5%) inoculated into masseter. Those that
succumbed to the disease demonstrated no serologic
response, perhaps due to the short incubation period.
The animals inoculated orally neither succumbed nor
developed antibodies.
IRKV-neutralizing antibody were detected in all bats
previously inoculated with this virus.
Either WCBV-neutralizing and IRKV-neutralizing
antibodies were detected up to the end of observation
period (6 months after WCBV challenge and 12 months
after IRKV challenge)
WCBV antibody dynamics
#877
231
211
191
171
151
131
111
91
71
51
31
11
Reciprocal dilutions
Reciprocal dilutions
#428
5/13
5/28
6/10
6/24
7/13
8/5
9/3
231
211
191
171
151
131
111
91
71
51
31
11
5/13
10/13 11/16
5/28
6/10
6/24
231
211
191
171
151
131
111
91
71
51
31
11
6/10
6/24
7/13
Dates
9/3
10/13 11/16
8/5
9/3
10/13 11/16
#884
Reciprocal dilutions
Reciprocal dilutions
#427
5/28
8/5
Dates
Dates
5/13
7/13
8/5
9/3
10/13 11/16
231
211
191
171
151
131
111
91
71
51
31
11
5/13
5/28
6/10
6/24
7/13
Dates
IRKV antibody dynamics
# 884
51
Dilution
For bats previously used
in the IRKV experiment.
For one bat that died,
only the initial serum was
available (positive; 1:12)
31
11
5/13/2005
7/13/2005
9/13/2005
11/13/2005
9/13/2005
11/13/2005
Date
# 887
51
Dilution
Dilution
# 877
31
11
5/13/2005
51
31
11
7/13/2005
9/13/2005
Date
11/13/2005
5/13/2005
7/13/2005
Date
Conclusions




WCBV was peripherally pathogenic for big brown bats.
However, the susceptibility of the animals was limited.
The infectious virus was isolated only from the bat brain,
although the presence of viral RNA was demonstrated in
a number of tissues.
Only one oral swab, taken at the lethargic stage from a
moribund bat, demonstrated the presence of viral RNA,
but not infectious virus.
None of animals inoculated orally developed either the
disease or serologic response, indicating that this route
was not invasive.
Conclusions (continued):

No suggestions for the carrier state were demonstrated.

No suggestions for viremia or presence of viral RNA in
the blood were demonstrated.


Antibody response was detected in 4/7 (57%) of bats
inoculated into masseter. The antibodies were
detectable up to the end of the observation period (6
months). For bats that came from the IRKV experiment,
the presence of IRKV-neutralizing antibodies was
demonstrated as well, indicating their circulation during
6-12 months post challenge.
As the big brown bat is not the principal WCBV host
(albeit from the same family Vespertilionidae),
pathogenesis and excretion patterns may be different.
Acknowledgements
All staff of the Rabies Program, CDC.