Animal reservoirs for Nipah virus Dr. D. T. Mourya ICMR-National Institute of Virology Pune 411021, INDIA
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
Phylogenetic analysis of NiV sequences from Bats & Human MH523642 %Nucleotide divergence % amino acid divergence MH396625_NV/IN/HU/2018/Kerala_CS 0 0 MH523645_NV/IN/bat/2018/15/Kerala 0.32 1.05 MH523644_NV/IN/bat/2018/Kerala 0 0 MH523643_NV/IN/bat/2018/Kerala 0.63 1.05 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 JN808864.1_NV/BD/HU/2010/Faridpur 1.9 3.16 JN808863.1_NV/BD/HU/2008/RAJBARI 2.85 6.32 JN808857.1_NV/BD/HU/2008/MA 2.85 6.32 FN869553.1_NV/BD/HU/2008/RAJBARI 9.81 22.11 FJ513078.1_NV/IN/HU/2007/FG 2.53 5.26 AY988601.1_NV/BD/HU/2004/RA1 2.22 4.21 AY029768.1_NV/MY/HU/1999/UM2 10.13 22.11 AY029767.1_NV/MY/HU/1999/UM1 10.13 22.11 AJ627196.1_NV/MY/PI/1999/VRI-0626 10.13 22.11 AJ564623.1_NV/MY/HU/1999/UM-0128 10.13 22.11 AJ564622.1_NV/MY/PI/1999/VRI-1413 10.13 22.11 AJ564621.1_NV/MY/PI/1999/VRI-2794 10.13 22.11 High similarity found between NiV sequence from Pteropus giganteus & human NiV positive samples (99.7-100%) from Kerala, than other human NiV sequences (85.14-96.15%).
Bat survey on the porous land borders; [Myanmar, Nepal, Bhutan]
Species of bats so far investigated in good numbers Rousettus leschenaulti Pteropus giganteus Cynopterus sphinx Pipistrellus tenuis mimus Megaderma lyra
Trapping of bats & field laboratory
Trapping of bats & field laboratory A. Robbers Cave B. Mist Net C. Cave & bat trapping A. Pteropus B. Organ collection C. Pteropus giganteus
Development of new bat cell line from Pipistrellus ceylonicus embryo
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 + + 2 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
Nipah virus from Pteropus giganteus, Siliguri, West Bengal Partial G gene HM545086.1 Malaysia AJ627196.1 99/VRI-0626 Pig AY029767.1 UMMC1 human AY029768.1 UMMC2 human AJ564622.1 99/VRI-1413 Pig AJ564623.1 99/UM-0128 human AF212302.2 human AF376747.1 Pteropus bat NC_002728.1 AJ564621.199/VRI-2794 Pig AFJ513078.1 Ind-Nipah-07-FG Human 1056660 Ind-Nipah -10- Pteropus Bat AY988601.1 Human Bangaladesh Malaysia India Nested PCR could amplify the partial G gene. All Indian isolates & Bangladesh are 99.3-99.9% identical & having a difference of 7.4% with Malaysia strains for nucleotides.
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
Yersinia like organisms in blood Smear [Megaderma lyra] Yersinia-species-isolatedfrom-Bats, Germany 2010, Emerging Infectious Diseases
Intracytoplasmic Inclusions [like Negri bodies] in the Neurons of brain of Bat
Parasites & parasitic eggs in the intestine of Bat
Rickettsial inclusions in lymphocytes [Cynopterus sphinx]
Trypanosomiasis in bats [Blood seam Megaderma & Pipistrellus] 1000x 100x
Isolated two novel viruses from the bats of Maharashtra EM image of Adenovirus from bat isolate infected Vero E6 cells at 3rd PID
New Phlebovirus: Malsoor virus isolated from Rousettus species of bats Isolation from Bat samples Mahabaleshwar NIV # Species Gender Sample details 1050639 Rousettus leschenaulti Male Bat L/S 1050650 Rousettus leschenaulti Female Bat L/S These two isolates were further pursued for identification & characterization
Phylogenetic relation with deadly Severe fever with thrombocytopenia syndrome (SFTS) & Heartland Virus Complete genome of 11.65 KB sequenced. 3.46 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
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) 1/39 84 3/76 Total 1,113 pig serum samples screened for anti-nipah IgG antibody were found to be negative from 8 districts of Mizoram state.
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 00 152 00 152 24 Pteropus giganteus 0 24 00 24 31 Pteropus giganteus 01 31 01 31 30 Cynopterus sphinx 00 30 00 30 79 Megaderma lyra 00 79 00 79 51 Rousettus leschenaultii 00 51 00 51
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 00 08 00 08 11 Cynopterus sphinx 00 11 00 11 March- May 2015 Dhubri, Assam Cooch Behar, West Bengal 21 Pteropus giganteus 03 21 02 21 4 Rousettus 00 04 ND ND 39 Pteropus giganteus 06 39 01 05
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 02 39 01 30 26 Rousettus leschenaulti 00 26 ND ND 2015 June Karad, Maharashtra 47 Pteropus giganteus 00 47 01 47 10 Rousettus leschenaulti 00 10 00 10 2018 May Kerala 52 Pteropus giganteus 12 52 ND ND 12 Rousettus leschenaulti 00 12 ND ND
Total number of bats processed for Nipah virus [2010-2018] Bat species tested (2010-2018) Number tested Number positive for Nipah virus IgG antibodies Number positive for Nipah virus by RT-PCR/qPCR Pteropus giganteus 261 6 24 Rousettus leschenaulti 255 0 0 Megaderma lyra 79 0 0 Cynopterus sphinx 41 0 0 Total 636 6 24 Pigs NB: A total of 1,113 Pig serum samples from Mizoram state were tested and all were negative for anti-niv IgG antibodies.
Nipah virus positive bats (2010-2018) PCR IgG 4% 1% Positive Negative Positive Negative 96% 99%
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 %]
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
Pteropus giganteus
Common locations of fruit bat Rousettus leschenaultii
Common locations of fruit bat National Institute of Virology, Pune
Wrapping up Last 10 years Investigations in India show: So far Nipah virus has been detected only in Pteropus bats.
Thank you