Animal reservoirs for Nipah virus

Transcription

1 Animal reservoirs for Nipah virus Dr. D. T. Mourya ICMR-National Institute of Virology Pune , INDIA

2 Tracing the source of Infection ICMR-NIV, Pune has team of scientific experts and trained field staffs for capturing Pteropus giganteus bats. Team was deputed to capture the Pteropus bats Field team focused the areas from where the Index and primary case of Nipah virus were reported. Team successfully captured 52 Pteropus bats from Perambra Town: 4 bats from Kuttiady 48 bats from the Soopikada

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4 Phylogenetic analysis of NiV sequences from Bats & Human MH %Nucleotide divergence % amino acid divergence MH396625_NV/IN/HU/2018/Kerala_CS 0 0 MH523645_NV/IN/bat/2018/15/Kerala MH523644_NV/IN/bat/2018/Kerala 0 0 MH523643_NV/IN/bat/2018/Kerala MH523641_NV/IN/HU/2018/Kerala 0 0 MH523640_NV/IN/HU/2018/Kerala 0 0 MH423323_NV/IN/HU/2018/Kerala/H1087_urine 0 0 JN _NV/BD/HU/2010/Faridpur JN _NV/BD/HU/2008/RAJBARI JN _NV/BD/HU/2008/MA FN _NV/BD/HU/2008/RAJBARI FJ _NV/IN/HU/2007/FG AY _NV/BD/HU/2004/RA AY _NV/MY/HU/1999/UM AY _NV/MY/HU/1999/UM AJ _NV/MY/PI/1999/VRI AJ _NV/MY/HU/1999/UM AJ _NV/MY/PI/1999/VRI AJ _NV/MY/PI/1999/VRI High similarity found between NiV sequence from Pteropus giganteus & human NiV positive samples ( %) from Kerala, than other human NiV sequences ( %).

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6 Bat survey on the porous land borders; [Myanmar, Nepal, Bhutan]

7 Species of bats so far investigated in good numbers Rousettus leschenaulti Pteropus giganteus Cynopterus sphinx Pipistrellus tenuis mimus Megaderma lyra

8 Trapping of bats & field laboratory

9 Trapping of bats & field laboratory A. Robbers Cave B. Mist Net C. Cave & bat trapping A. Pteropus B. Organ collection C. Pteropus giganteus

10 Development of new bat cell line from Pipistrellus ceylonicus embryo

11 Susceptibility of new NIV-BtEPC cell line to various viruses as compared to Vero E-6 cells lines SL # Virus Virus susceptibility status in NIV-BtEPC Virus susceptibility status in Vero E-6 1 Chandipura Japanese encephalitis No growth + 3 Dengue 2 No growth + 4 West Nile No growth + 5 Chikungunya No growth + 6 CHIK AP No growth + 7 Chittoor No growth + 8 Ingwavuma No growth + 9 Ganjam No growth + 10 Umbre No growth + 11 Novel Adenovirus BtAdv-RLM + + Cell line found refractory for Alpha, flavi & Bunyavirus CPE only with Chandipura [Rhabdoviruses] & Adenovirus isolated from bats

12 Nipah virus from Pteropus giganteus, Siliguri, West Bengal Partial G gene HM Malaysia AJ /VRI-0626 Pig AY UMMC1 human AY UMMC2 human AJ /VRI-1413 Pig AJ /UM-0128 human AF human AF Pteropus bat NC_ AJ /VRI-2794 Pig AFJ Ind-Nipah-07-FG Human Ind-Nipah -10- Pteropus Bat AY Human Bangaladesh Malaysia India Nested PCR could amplify the partial G gene. All Indian isolates & Bangladesh are % identical & having a difference of 7.4% with Malaysia strains for nucleotides.

13 Blood smear examinations of Bats from Siliguri Area Sr # Observation Bat Species ID No. Remarks 1 Yersinia like organisms Megaderma lyra S-57 1/79= 1.26% 2 Rickettsial inclusions in lymphocytes Cynopterus sphinx (2), Megaderma lyra (1) S-80, S-100, S-98 3/79= 3.79% 3 Immature stages of Trypanosomiasis evident Megaderma lyra (1) Pipistrellus mimus (1) S-104 S-109 2/79= 2.53% Yersinia like organism

14 Yersinia like organisms in blood Smear [Megaderma lyra] Yersinia-species-isolatedfrom-Bats, Germany 2010, Emerging Infectious Diseases

15 Intracytoplasmic Inclusions [like Negri bodies] in the Neurons of brain of Bat

16 Parasites & parasitic eggs in the intestine of Bat

17 Rickettsial inclusions in lymphocytes [Cynopterus sphinx]

18 Trypanosomiasis in bats [Blood seam Megaderma & Pipistrellus] 1000x 100x

19 Isolated two novel viruses from the bats of Maharashtra EM image of Adenovirus from bat isolate infected Vero E6 cells at 3rd PID

20 New Phlebovirus: Malsoor virus isolated from Rousettus species of bats Isolation from Bat samples Mahabaleshwar NIV # Species Gender Sample details Rousettus leschenaulti Male Bat L/S Rousettus leschenaulti Female Bat L/S These two isolates were further pursued for identification & characterization

21 Phylogenetic relation with deadly Severe fever with thrombocytopenia syndrome (SFTS) & Heartland Virus Complete genome of KB sequenced kb (M), 6.4kb (L) and 1.78kb (S) segment are maximum identical at nucleotide of 66%, 59%, 60%, 47%f or L>M>N> NSs segments at amino acid level 71%, 58%, 58%, 40% respectively with Heartland virus. New virus clustered with Heartland and SFTS virus, within the SFTS group. This study reports for the first time the isolation and genetic characterization of a novel Bunyavirus from Rousettus bats in India

22 Tioman virus screening in bat specimens collected from Northeast, India [2015] Location of collection Jalpaiguri, West Bengal (08) Dhubri, Assam (21) Cooch Behar, West Bengal (39) Positivity/ Total samples tested Species [Collected no of Bats] P. giganteus (08) Unidentified (12) P. giganteus (21) Rousettus (4) Tioman RT-PCR (+ve/processed samples) 0/9 2/28 P. giganteus (39) 1/ /76 Total 1,113 pig serum samples screened for anti-nipah IgG antibody were found to be negative from 8 districts of Mizoram state.

23 Total number of bats processed for Nipah virus [2010 to 2014] Collection of specimens June 2010 Nov 2010 Feb 2014 Location Mahabaleshwar, Maharashtra Wageshwar, Maharashtra West Bengal state Mahabaleshwar, Maharashtra Number of bats captured Species PCR results Nipah virus Positive Total tested Ig G results Nipah virus Positive Total tested 152 Rousettus leschenaultii Pteropus giganteus Pteropus giganteus Cynopterus sphinx Megaderma lyra Rousettus leschenaultii

24 Total number of bats processed for Nipah virus [March-May 2015] Collection of specimens Location Number of bats captured Species PCR results Nipah virus Positive Total tested Ig G results Nipah virus Positive Total tested Jalpaiguri, West Bengal 8 Pteropus giganteus Cynopterus sphinx March- May 2015 Dhubri, Assam Cooch Behar, West Bengal 21 Pteropus giganteus Rousettus ND ND 39 Pteropus giganteus

25 Total number of bats processed for Nipah virus [June & Nov, 2015 and May 2018] Collection of specimens 2015 Nov Location Dhubri, Assam Number of bats captured Species Nipah virus Positive PCR results Total tested Nipah virus Positive Ig G results Total tested 39 Pteropus giganteus Rousettus leschenaulti ND ND 2015 June Karad, Maharashtra 47 Pteropus giganteus Rousettus leschenaulti May Kerala 52 Pteropus giganteus ND ND 12 Rousettus leschenaulti ND ND

26 Total number of bats processed for Nipah virus [ ] Bat species tested ( ) Number tested Number positive for Nipah virus IgG antibodies Number positive for Nipah virus by RT-PCR/qPCR Pteropus giganteus Rousettus leschenaulti Megaderma lyra Cynopterus sphinx Total Pigs NB: A total of 1,113 Pig serum samples from Mizoram state were tested and all were negative for anti-niv IgG antibodies.

27 Nipah virus positive bats ( ) PCR IgG 4% 1% Positive Negative Positive Negative 96% 99%

28 Nipah virus (NiV) screening of Birds [n=5] [These were caught in net while capturing bats hence tested for NiV] Location & no of Birds Location-1: Perambra, Near Rubber & Pineapple field [Sooppikada*] Location-2: Park residency wine and beer parlour, Kuttiady, Kerala Positive Bird s Throat swabs (TS) /Total tested by Realtime RT-PCR Positive Bird s Cloacal swabs (CS) /Total tested by Real-time RT- PCR Positive Bird s body organ / Total tested by Real-time RT-PCR Number of Birds positive /number screened 0/5 0/5 0/5 0/5 [0.0 %]

29 Necropsy specimens of single Rabbit (5 organs) from the index case s house [Joint Director, District Animal Husbandry Office, Kozhikode, Kerala] Sample received Village Specimens Brain Nipah Virus Real Time RT- PCR Negative 06 th June 2018 Perambra, Kozhikode Liver Lung Kidney Negative Negative Negative Spleen Negative

30 Pteropus giganteus

31 Common locations of fruit bat Rousettus leschenaultii

32 Common locations of fruit bat National Institute of Virology, Pune

33 Wrapping up Last 10 years Investigations in India show: So far Nipah virus has been detected only in Pteropus bats.

34 Thank you