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EXPERIMENTAL INFECTION OF BIG BROWN BATS ( Eptesicus fuscus ) WITH WEST CAUCASIAN BAT VIRUS

EXPERIMENTAL INFECTION OF BIG BROWN BATS ( Eptesicus fuscus ) WITH WEST CAUCASIAN BAT VIRUS. Centers for Disease Control and Prevention, 1600 Clifton Rd. Atlanta, GA, 30329

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EXPERIMENTAL INFECTION OF BIG BROWN BATS ( Eptesicus fuscus ) WITH WEST CAUCASIAN BAT VIRUS

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  1. EXPERIMENTAL INFECTION OF BIG BROWN BATS (Eptesicus fuscus) WITH WEST CAUCASIAN BAT VIRUS 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 Kuzmin I.V., Franka R., Rupprecht C.E.

  2. Background: • The West Caucasian bat virus was isolated in 2002 from the brain of Shreiber’s bat (Miniopterus schreibersii) in south-eastern Europe

  3. 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

  4. Genotype 6 Khujand L O N I H F 8 7 1 0 0 0 Aravan 9 9 A F 0 Irkut 8 1 0 2 0 A Genotype 7 F 0 0 6 A Genotype 5 4 R 9 F 7 8 1 9 8 L O P 5 1 6 8 M L 5 S A 2 3 1 6 8 P A R89 Genotype 4 100 61 80 56 A F 99 4 6 7 9 4 100 9 Ethlag 100 8 8 6 3 6 H A V 8619NGA I U G 0 6 6 8 100 Genotype 2 S U R 1 4 1 9 U S O V Y 3 C 5 6 8 T 9 S 1 A E B 9 D R I 3 A 3 1 Genotype 1 S 9 8 6 8 Genotype 3 50 WCBV Background: Phylogenetic position of WCBV according to the N gene sequence

  5. LC8 bindingsite of the lyssavirus phosphoprotein (framed) AA 329-335 of the lyssavirus glycoprotein ectodomain

  6. Background Comparative peripheral (intramuscular) pathogenicity of WCBV for different mammals: • Laboratory ICR mice – apathogenic. • Syrian hamsters – pathogenic. • Non-human primates (Macaca sp.) – pathogenic. • bats ???

  7. 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

  8. 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.

  9. 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).

  10. Results (continued): Results of nRT-PCR for bat tissues

  11. 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)

  12. WCBV antibody dynamics #877 #428 231 231 211 211 191 191 171 171 151 151 131 131 Reciprocal dilutions Reciprocal dilutions 111 111 91 91 71 71 51 51 31 31 11 11 5/13 5/28 6/10 6/24 7/13 8/5 9/3 10/13 11/16 5/13 5/28 6/10 6/24 7/13 8/5 9/3 10/13 11/16 Dates Dates #427 #884 231 231 211 211 191 191 171 171 151 151 131 131 Reciprocal dilutions Reciprocal dilutions 111 111 91 91 71 71 51 51 31 31 11 11 5/13 5/28 6/10 6/24 7/13 8/5 9/3 10/13 11/16 5/13 5/28 6/10 6/24 7/13 8/5 9/3 10/13 11/16 Dates Dates

  13. IRKV antibody dynamics # 884 For bats previously used in the IRKV experiment. For one bat that died, only the initial serum was available (positive; 1:12) 51 Dilution 31 11 5/13/2005 7/13/2005 9/13/2005 11/13/2005 Date # 887 # 877 51 51 Dilution Dilution 31 31 11 11 5/13/2005 7/13/2005 9/13/2005 11/13/2005 5/13/2005 7/13/2005 9/13/2005 11/13/2005 Date Date

  14. 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.

  15. 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.

  16. Acknowledgements All staff of the Rabies Program, CDC.

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