EXPERIMENTAL INFECTION OF EXPERIMENTAL INFECTION OF BIG BROWN BATS (BIG BROWN BATS (Eptesicus Eptesicus

fuscusfuscus) WITH WEST ) WITH WEST CAUCASIAN BAT VIRUSCAUCASIAN BAT VIRUS

Kuzmin I.V., Franka R., Kuzmin I.V., Franka R., Rupprecht C.E.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:Background: The West Caucasian bat virus was The West Caucasian bat virus was

isolated in 2002 from the brain of isolated in 2002 from the brain of Shreiber’s bat (Shreiber’s bat (Miniopterus Miniopterus schreibersiischreibersii) in south-eastern Europe) in south-eastern Europe

Background:Background: As only one isolate is available, the As only one isolate is available, the

principal host species is unknown.principal host species is unknown. The distribution of The distribution of M. schreibersiiM. schreibersii is is

large, and several additional species of large, and several additional species of MiniopterusMiniopterus are distributed in the Old are distributed in the Old World tropics and subtropics.World tropics and subtropics.

Area of Miniopterus schreibersii

Phylogenetic position of WCBV according to the N gene sequence

50

8653YOU

93

39

ES

T

8

6

8

1

I

R

A

SA

DB

19

CVS9141RUS

8660GUI

88636HAV

AF467949

PA R89

ML5

AF006497

AF081020

9007FIN

9

0

1

8

H

O

L

8918FRA

8615POL

86132AS

W

CBV

99 100

100

56

6180

100

100

Genotype 6

Ethlag

8619NGA

Khuj

and

Arav

an

Irkut

Genotype 7

Genotype 1

Genotype 5

Genotype 4

Genotype 2

Genotype 3

BackgroundBackground::

138CVS :SADB19 :V006 :V267 :V020 :V552 :V550 :S59448 :V008 :V023 :V002 :V286 :V474 :V481 :Aravan :Khujand :Irkut :WCBV :

GK-SSEDKSTQTTGK-SSEDKSTQTTTQ-ATVSKPTQTDAK-ITDNKQTQTDPR-NLKSIQIQTEAR-KTKSVQIQTEAR-KTKSVQIQTEER-DTKSIQIQTENK-LFEDKSTQTVNK-LQADKSTQTTNK-LQADKSTQTTGK-STEDKSTQTPGK-TTENKSTQTTGK-TTESKSTQTTGK-SLADKSTQTSGK-STDDKSTQTVDK-ESAEKSTQTVPKPTTKDIAVQAD

LC8 binding site of the lyssavirus phosphoprotein (framed)

CVS : SADB19 : SHBRV : 8620CAR : 8619NGA : S59447 : 9020SA : 8918FRA : EBL1POL : 9018HOL : 9007FIN : AF006497 : AF081020 : Aravan : Khujand : Irkut : WCBV :

YKSVRTWYKSVRTWYKSVRTWYLRVDSWYLKVDNWYKRVDKWYKSVREWYKSVREWYKSVREWYKSIREWYKSIREWYNQVRTWYKSVRTWYKSVREWYKSIREWYKSIREWYIKVENW

333

AA 329-335 of the lyssavirus glycoprotein ectodomain

BackgroundBackground

Laboratory ICR mice – apathogenic.Laboratory ICR mice – apathogenic. Syrian hamsters – pathogenic.Syrian hamsters – pathogenic. Non-human primates (Non-human primates (MacacaMacaca sp.) – sp.) –

pathogenic.pathogenic. bats bats ??????

Comparative peripheral (intramuscular) pathogenicity of WCBV for different mammals:

Study: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

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.

Study:Study:

Results: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 (continued):

TissueTissue Bat #409Bat #409 Bat #433Bat #433 Bat #883Bat #883

BRBR ++ ++ ++

BFBF ++ -- ++

BLBL ++ ++ --

KIKI -- -- --

LULU ++ ++ ++

SGSG ++ ++ ++

Results of nRT-PCR for bat tissues

Results (continued):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)

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IRKV antibody dynamicsIRKV antibody dynamics For bats previously used

in the IRKV experiment. For one bat that died,

only the initial serum was available (positive; 1:12)

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ConclusionsConclusions 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):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.

AcknowledgementsAcknowledgementsAll staff of the Rabies Program, CDC.All staff of the Rabies Program, CDC.