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Research priorities for Nipah Virus Disease in India
ICMR-NIV mandate: Research on viruses of Public health Importance
-Laboratory based research/-Diagnostic preparedness
-Training & capacity building for NiV diagnosis in India
-Point of care tests
Dr. Pragya D Yadav, M.Sc., PhD,Scientist 'E’, ICMR –National Institute of
Virology, Sus Road, Pashan, Pune, India
Conventional Nipah virus diagnostic methods
Molecular assays– Real-time RT-PCR
– Conventional RT-PCR and Sanger sequencing
Serological assays– Anti Nipah Human IgM [Sample screening]
– Anti Nipah Human IgG ELISA [serosurvey or diagnosis]
– Anti Nipah Bat IgG ELISA [Bat serosurvey]
– Anti Nipah Swine IgG ELISA [Swine serosurvey]
Virus isolation and characterisation
Antigen detection– Immunofluorescence
– Immunohistochemistry
Commercial assays : None
• Require Containment Laboratory for handling specimen2
Commonly used
Diagnosticmethods
3
Day Post illness
NiV RNA positive/samples
(%positivity)
IgM positive/Total samples
(%positivity)
IgG positive/Total samples
(%positivity)
Day 2 2/2 (100) 1/2 (50) 0/2 (0)
Day 3 3/3 (100) 1/3 (33) 1/3 (33)
Day 5 5/5 (100) 4/5 (80) 0/5 (0)
Day 6 5/5 (100) 3/5 (60) 2/5 (40)
Day 7 2/2 (100) 2/2 (100) 0/2 (0)
Day 9 1/1 (100) 1/1 (100) 1/1 (100)
Detection of Nipah viral RNA and antibody of patients of Kerala Outbreak till 9th POD (n=18)
Detection of Nipah antibody of patients of Malaysia Outbreak, 1999
4
Day Post illness
IgM % positivity
IgG % positivity
Day 1 44%-50% 27%-31%
Day 4 60%-71% 7%-10%
Day 12 100% 40%-44%
Day 17 100% 100%
3 months 95% 100%
Total Number of patients tested: 176Reference: Ramasundrum, et al., Neurol J Southeast Asia, 2000
Cumulative seropositive
response
IgM seropositivity from Day of
illness
IgG seropositivity from Day of illness
25% Day 5 Day 8
50% Day 11 Day 18
75% Day 15 Day 34
95% Day 50 Day 193
Cumulative IgM seropositive response
Cumulative IgG seropositive response
NiV Seropositivity in patient [Bangladesh and India Outbreaks]
5
Day Post illness
IgM positive/Total
samples (%positivity)
IgG positive/Total
samples (%positivity)
Day 1-4 3/5 (60%) 1/5 (20%)
Day 5-10 22/24 (92%) 10/24 (42%)
Day 11-14 9/9 (100%) 6/9 (67%)
Day 15-30 4/4 (100%) 4/4 (100%)
Day 45-60 3/4 (75%) 4/4 (100%)
>1.5 years 0/4 (0%) 4/4 (100%)
Day Post illness
IgM positive/Total
samples(% positivity)
IgG positive/Total
samples(% positivity)
Day 1-3 2/5 (40%) 3/5 (60%)
Day 5 2/2 (100%) 2/2 (100%)
Day 6 1/1 (100%) 1/1 (100%)
Day 7 0/2 (0%) 0/2 (0%)
Day 8 1/2 (50%) 1/2 (50%)
Day 9 1/2 (50%) 1/2 (50%)
Day 10 2/3 (67%) 2/3 (67%)
Bangladesh Outbreak, 1999Total Number of patients tested: 50
Hossain, et al., Clinical Infectious Diseases, 2008
India Outbreak, 2001Total Number of patients tested: 17Chadha, et al., Emerging Infectious
Diseases, 2006
NiV isolations in different Nipah outbreaks
POD NiV isolation from TS*
/Total samples (% positivity)
NiV isolation from NS*
/Total samples
(% positivity)
NiV isolation from urine/Total
samples(% positivity)
1 1/1 (100) 0/1 (0) 0/1 (0)
2 3/5 (60) 1/5 (20) 2/5 (40)
3 0/2 (0) 0/2 (0) 1/2 (50)
4 1/5 (20) 0/5 (0) 1/5 (20)
5 1/4 (25) 0/4 (0) 0/4 (0)
7 0/3 (0) 0/3 (0) 0/3 (0)
Reference: Chua, et al., Journal of Infection, 2001
Malaysia Outbreak, 1999
POD NiV isolation from TS*/Total
samples (% positivity)
NiV isolation from CSF*/Total
samples(% positivity)
3 1/1 (100) 0/1 (0)
5 0/0 0/1 (0)
6 1/2 (50) 0/2 (0)
8 0/1 (0) 0/1 (0)
9 0/1 (0) 0/1 (0)
Reference: Rahman, et al., Vector-Borne and Zoonotic Diseases, 2012
Bangladesh Outbreak, 2008
Reference: Chua, et al., The Lancet, 1999
* TS: Throat Swab, NS: Nasopharyngeal Swab, CSF: Cerebrospinal Fluid
Two NiV isolates were obtained from CSF of two index death cases on Day 4 and 16
No isolate was obtained from 18 serum samples and 6 urine samples from NiV-positive samples
Siliguri, India Outbreak, 2001
Nipah Virus isolation from Kerala Outbreak [2018] in VeroCCL81 cells
Only one NiV isolate was obtained from throat swab (POD:3) Total number of samples 23 processed of 9 NiV positive cases (7-fatal, 2-survival)
Control Vero CC81 cells Infected cells at 1st PID Infected cells at 2nd PID
Cytopathic effects in throat swab Infected VeroCCL81 cells at 2nd PID
Commercial Nipah diagnostic assays (?)
No commercial Serodiagnostic assays are available for detection of Anti Nipah Human IgM antibodyAnti Nipah Human IgG antibodyAnti Nipah Bat IgG antibodyAnti Nipah Pig IgG antibody
Krishgen Biosystems, Mumbai, India: Purchased ELISA kit for Nipah Human IgM and IgG detection but failed to detect positive controls
Pen-side strip test for the detection of Nipah virus in swine [Canadian Science Centre for Human and Animal Health]
Sensitivity: NiV stock: 1000PFU/ml, NiV in Swine Nasal aspirate: 158 TCID50/ml
Name of assay OrganismAssays
developedsensitivity/ specificity
validated (n)
Anti-Nipah IgM ELISA
Humans 2 2
Pigs 2 2
Bats 2 2
Anti-Nipah IgG ELISA
humans 3 2
Pigs 8 4
Bats 3 3
Antigen Capture ELISA
Humans, Bats, Pigs 3 1
Neutralization Humans, Bats, Pigs 1 1
Plaque Assay Humans, Bats, Pigs 1 1
Luminex Based multiplex
microsphere assayHumans, Bats, Pigs 2 1
Nipah Virus Non-Commercial Serological Diagnosis
9
A large number of papers are published on development of Nipah virus screening methods but none of them reached to commercialization stage ???
Nipah Virus Non-Commercial Molecular Assays
Name of assayNumber of assays
developedsensitivity/ specificity
validated (Number)
Taqman qRT-PCR 3 2
Taqman qRT-PCR array card 1 1
SYBR Green qRT-PCR 2 1
Conventional RT-PCR 3 1
Center for Disease Control and Prevention , USA
DVS, Malaysia , Chinese Nat. Diagn., Nat. Inst. Inf. Dis., Japan(Validated assay), Institute of Tropical Medicine, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan,1 (Validated assay)
CSIRO, Australia (Under validation)
CSIRO, Australia
CDC, USA & Nat. Inst. Inf. Dis., Japan
Nat. Inst. of Animal Health, Japan
Institute Pasteur, UK , Inst. of Zoology, UK , Chulalongkorn Uni. Hosp., Thailand and CDC, USA
National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, Canadian Science Centre for Human and Animal Health
Adapted from: http://www.who.int/blueprint/priority-diseases/key-action/WHO_NIPAH_baseline_situation_analysis_27Jan2018.pdf
10
Organization or Laboratory involved
Nipah real-time assays comparison with in-vitro transcribed RNA on Indian NiV
y = -3.600x + 44.25R² = 0.996
0
5
10
15
20
25
30
35
40
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Ct V
alu
es
Log10 of copy number
CDC primers
y = -3.610x + 44.62R² = 0.989
0
5
10
15
20
25
30
35
40
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Ct V
alu
es
Log10 of copy number
Guillaume et al, 2004
y = -3.348x + 41.31R² = 0.999
0
5
10
15
20
25
30
35
40
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Ct V
alu
es
Log10 of copy number
ABI Primers/probe
1010 109 108 107 106 105 104
Primers provided by CDC, USA & ABI were 10-fold more sensitive than published primers (Guillaume et al., 2004) 11
Nipah virus complete genome sequencing from clinical samples by Next Generation Sequencing [NGS]
12
➢Nipah virus is a negative-sense enveloped RNA with ~18.2 Kb genome size.
➢Genome encodes six genes : nucleocapsid, phosphoprotein, matrix, fusion
protein, glycoprotein, and polymerase.
➢Two different genotypes of Nipah virus:
➢B genotype Bangladesh and India
➢M genotype Malaysia and Cambodia
Image adapted from: https://www.jle.com/en/revues/vir/e-docs/les_virus_nipah_et_hendra_des_agents_pathogenes_zoonotiques_emergents_276204/article.phtml?tab=images
Nipah virus isolate and throat swabs [n=3] of Nipah cases were
sequenced using NGS and retrieved complete genome
Also positive Pteropus bats were sequenced by RT-PCR
Phylogenetic analysis of complete genome [n=4] NiV human sequences
18,100nt = 4991AA
13Indian Nipah virus clusters with the B genotype
2018 strains (Kerala outbreak)
B genotype
M genotype
2007 Indian (West Bengal) strain
Phylogenetic analysis of NiV sequences from bat and human samples [N gene (region: 1293 to 1608) length = 316nt. ]
Similarity between
NiV sequence from
Pteropus giganteus
(fruit bat) and human
sample from Kerala :
99.7-100%
Similarity between
human NiV sequences:
85.14 - 96.15% from
other countries
2018 Kerala Strain
B genotype
M genotype
2007 West Bengal strain
Percent nucleotide divergence with respect to Kerala human NiV sequence
15
99.7-100% identity between the human and bat NiV sequence indicates the source of infection was Pteropus giganteus bats
Amino acid
position in
nucleoprotein
MH523640
Human
MH523645
Bat
MH523644
Bat
MH523643
Bat
Indian
FJ513078
Bangladesh
AY988601
Malaysia
AF212302
413 A A A P A A A
429 V V V V V V I
432 E E E E E E G
457 D D D D D D N
503 N N N N S S S
505 R R R R K K R
506 D D D D D D T
508 R R R R R R G
Country Year Organism% Nucleotide
divergence
% amino acid
divergence
Kerala, India 2018 Human 0 0
Kerala, India 2018 Bat 0-0.63 0-1.05
Bangladesh 2004-2010 Human 1.9-9.81 3.16-22.11
Nucleoprotein gene (1,599nt=533 AA)
16
Country N Protein
ND (%) AD (%)
Malaysia 6.3-6.4 1.7-2.1
West Bengal,
India 1.4 0.8
Bangladesh 1.3-6.2 0.6-1.1
Phosphoprotein gene (2,130nt= 710AA)
(polymerase cofactor, enhancing polymerase processivity & allowing the encapsidation of
the newly synthesized viral genomes)
Country P protein
ND(%) AD(%)
Malaysia 8.5 9.3-9.4
West Bengal, India 2.2 2.4
Bangladesh 1.8-9.31.8-
10.7
*ND: Nucleotide Difference, $AD: Amino acid difference
Fusion gene (1,641nt = 547AA)
(Viral Entry)
17
CountryFusion
ND(%) AD(%)
Malaysia 6.6-6.7 1.3-1.5
West Bengal, India 1.7 0.4
Bangladesh 1.5-6.6 0.2-1.5
Matrix gene (1,059nt =353AA)
Country Matrix
ND(%) AD(%)
Malaysia 6.9-4.0 0.9-1.1
West Bengal,
India 1.2 0.3
Bangladesh 1.1-6.6 0-0.9
Glycoprotein gene (1,809nt = 603AA)
18
Country L gene
ND(%) AD(%)
Malaysia 7.5-7.6 4.5
West Bengal, India 2.0 1.2
Bangladesh 1.8-7.3 1-4.2
Mut
L gene (6,735nt=2,245AA)(catalyzes the transcription of viral mRNAs,
their capping and polyadenylation.)
Country L gene
ND(%) AD(%)
Malaysia 6.7-6.8 1.4-1.6
West Bengal,
India 1.6 0.1
Bangladesh 1.6-6.8 0.2-1.6
Research priorities
20
1. Development of anti-Nipah human IgM antibody detection ELISA
2. Development of anti-Nipah human IgG antibody detection ELISA
3. Development of anti-Nipah Bat IgG antibody detection ELISA
4. Development of anti-Nipah Swine IgG antibody detection ELISA
5. Development of Nipah antigen capture ELISA
6. Generation and characterization of monoclonal antibodies against Nipah virus
7. Preparedness and laboratory capacity building of VRLD network
Early detection saves lives of human Controlling spread of highly infectious agent require rapid identification of infected
pigs or bats. For this purpose various serological procedures which could either directly or
indirectly detect evidence of the Nipah virus infection in pigs or bats will be useful.
DHR/ICMR Virus Research Diagnostic Laboratory Network across the country - 55 functional
A total 82 VRDLs (5 Regional, 15, State Level & 62 Medical college Level Labs) have been approved So far.
55 VRDLs have become functional and carrying out day today diagnosis.
Other VRDLs are either in the process of procurement of equipment or in the process of staff appointment.
The staff of these labs are trained at NIV, Pune for all the concept of Virology research including Bio-risk mitigation
Preparedness of VRLD network
Strengthening Biorisk mitigation training
➢Creating awareness for high–risk pathogens and its handling
➢Training of personal protective equipment's for handling of specimen, packaging and transport
Preparedness for enhancing capacity of laboratory diagnosis
A study of ICMR –NIV, Pune Evaluation of certain [chaotropic] compounds for inactivation of viruses of
public health importanceViruses tested in the study:
• Flaviviridae (JEV, KFDV, DENV)
• Alphviridae (CHIKV)
• Rhabdoviridae (CHPV)
• Nairoviridae (CCHFV)
• Orthomyxoviridae (Influenza virus)
1
• MTT assay and dose selection
• Use different concentration of chaotropic agent S1, S2 and S3 alone and in combinations
2
• Inactivate known titer virus for 8 days
• Spiking of virus in blood samples in EDTA or gel tubes
3
• Checking stability of virus at 40C for 8 days at different time points
• in-vivo (cell culture) and in-vitro (ELISA and RT-PCR) methods
Technology patented &
Transfer to industry by ICMR , HQ [20018]
Effect of inactivation compound on Hematology and biochemical parameters in normal and diseased patients
Blood samples from twenty normal subjects and 54 CHIKV and 9 DENV
positive cases were analyzed
Hematology: Hb, TLC, DC and Platelet count
Biochemistry: Liver function Test, Kidney Function Test, Markers of tissue
damage, Serum electrolytes
24
Blood parameters were unaffected in normal and infected subjects by treating with inactivation compound.
ICMR-NIV Pune has applied for patent for indigenous kit for inactivation of viruses
The same technology can be used for inactivation of Nipah virus belonging to family Paramyxoviridae in clinical samples of affected subjects
How this is useful for VRDL network???
25
Practical applications:
1. This will enhance the laboratory capacities of VRDLs
2. Supply of positive controls to all VRDLs
3. It might save Crores of rupees by allowing any lab to perform
ELISA and Molecular tests even at medical colleges and no
issues of infections (Biorisk is taken care)
4. In investigations of outbreaks where maintaining cold chain for
sending samples to main Lab Pune is difficult will provide safe
means of samples transportation
Conclusion
• The virus inactivation technology developed at NIV, Pune was used
successfully to inactivate number of viruses like JEV, KFDV, DENV, CHIKV,
CHPV, CCHFV, Influenza blood samples/Throat swab of subjects
• The virus was found to be stable in ELISA and also viral RNA was stable for
performing RT-PCR and nested RT-PCR at various time-points
• Blood hematology and serum biochemistry parameters were unaffected
using this inactivation compound in normal and diseased patients
• The current technology is being tested for Nipah virus inactivation and seems
working fine for molecular assays
• This can be made available at the referral diagnostic labs, pathology labs and
hospitals
26
Acknowledgements!
28
Grants North east Task Force, ICMR, New
Delhi, India [2015-1] GDD funding, CDC, USA [2009-10] GHSA Funding , CDC, USA [2015-
20]
CDC , USA
Percent nucleotide divergence with respect to Kerala human NiV
Kerala NiV is closer to the Bangladesh strain under B genotype but make a separate clade
Country
N P F M G LComplete
genome
ND
1,599*
AD
533$
ND
2,130*
AD
710$
ND
1,641*
AD
547$
ND
1,059*
AD
353$
ND
1,809*
AD
603$
ND
6,735*
AD
2,245$
ND*
18,100
AD$
4991
Malaysia 6.3-6.4 1.7-2.1 8.5 9.3-9.46.6-
6.71.3-1.5 6.9-4.0 0.9-1.1 7.5-7.6 4.5 6.7-6.8 1.4-1.6 10.1 22
West Bengal,
India 1.4 0.8 2.2 2.4 1.7 0.4 1.2 0.3 2.0 1.2 1.6 0.1 2.5 5.3
Bangladesh 1.3-6.2 0.6-1.1 1.8-9.3 1.8-10.71.5-
6.60.2-1.5 1.1-6.6 0-0.9 1.8-7.3 1-4.2 1.6-6.8 0.2-1.6 1.9-9.8
3.2-
22.1
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