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

Strategy for the prevention and control of outbreaks

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