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1. Role of Disease Investigation Center Wates
on Influenza Virus Monitoring in Indonesia
Disease Investigation Center (DIC) Wates
Directorate General of Livestock and Animal Health Services
Ministry of Agriculture Indonesia
Jl. Wates KM 27, Kulon Progo, Yogyakarta
http://bbvetwates.ditjenpkh.pertanian.go.id/
Hendra Wibawa, DVM, MSc, PhD

2. DISEASE INVESTIGATION WATES (DIC) WATES
• Firstly established in 1983 as project collaboration between the Government of
Indonesia (Ministry of Agriculture) and the Canadian International Development
Agency (CIDA):
 Balai Penyidikan dan Pengujian Penyakit Hewan (Animal Disease Investigation
and Diagnostics Institute) from1984-2001.
 Balai Penyidikan dan Pengujian Veteriner (Veterinary Investigation and
Diagnostics Institute) from 2001-2003.
 Balai Besar Veteriner (Center of Veterinary Services = Disease Investigation
Center) from 2003 - now
HISTORY

3. DIC WATES WORKING AREA / COVERAGE
The area coverage of DIC Wates consiste 3 provinces in Java Islands, including:
East Java, Central Java, and Special Region of Yogyakarta
DIC Denpasar
DIC Maros
DIC Banjarbaru
DIC Wates
DIC Subang
DIC Lampung
DIC Bukittinggi
DIC Medan

4. LIVESTOCK DISTRIBUTION AND MOVEMENT
• DIC Wates has area coverage of East Java, Central Java and DI
Yogyakarta provinces. Compared to other provinces in Indonesia,
these three provinces have:
 larger livestock population and more households/farms
 higher livestock movements to western or eastern parts
(area/islands) of Indonesia.

5. The Indonesian Statistical Bureau
(BPS)
Area coverage of DIC Wates

6. https://www.isikhnas.com/
Monthly livestock movement
The majority of the source of animal movement from DIC Wates' coverage area

7. ROLE OF DIC WATES ON ANIMAL
HEALTH SURVEILLANCE
Disease outbreaks are more likely occur within area with a bigger population size and
higher livestock movement. Hence, DIC Wates plays a significant role on animal health
surveillance by providing:
• Early disease detection and outbreak investigation,
• Systematic monitoring and surveillance for animal and veterinary public health,
• Veterinary diagnostic assays and their results for animal health status and/or for
requirement of animal transportation,
• Mapping of animal health and related diseases,
• National reference laboratory for strategic animal diseases (Avian Influenza,
Salmonellosis, and Exotic Disease of Bovine Spongiform Encephalopathy)
• Recommendations for better farming practices and for local authorities & central
government related to controls and development livestock programs.

8. ROLE OF DIC WATES ON
SURVEILLANCE OF ZOONOTIC DISEASES
According decree from Ministry of Agriculture of Indonesia Number
89/Kpts/PD.620/1/2012, DIC WATES has been appointed as reference
laboratory for four strategic and contagious animal diseases (PHMS),
including:
1. Avian Influenza (AI)
2. Salmonellosis
3. SARS-CoV-2 on animals
4. Exotic disease of Bovine Spongiform Encephalopathy (BSE)
Those four have also been known as animal diseases that potentially could
infect or transmit from animal to humans (zoonosis).

9. DIC WATES CAPACITIES FOR
AVIAN INFLUENZA VIRUS (AIV)
SURVEILLANCE AND DIAGNOSTICS

10. TYPES OF AVIAN INFLUENZA VIRUS (AIV)
SURVEILLANCE CONDUCTED BY DIC WATES
1. Risk-based surveillance at live bird markets and live poultry traders
2. Survey representative-based surveillance in wild birds, including ducks
3. Survey representative-based surveillance in Poultry Sectors 3
4. Compartment/zone-based surveillance in poultry Sector 1-2
5. Investigation of disease outbreaks in poultry and other animals
6. Join investigation with public health sector for bird flu cases (poultry outbreak with
human cases)
7. Molecular surveillances (Influenza Virus Monitoring/IVM Network)

11. FLOW CHART LABORATORY
SAMPLES AND TEST
RESULTS AND DISEASE
REPORTING
iSIKHNAS is Indonesia's integrated real-time information system
for collecting, managing, reporting and using data to support
animal health and production. The data is sent directly from the
field to the integrated database for automated checking, analysis
and immediate reporting. (https://www.isikhnas.com)
Influenza Virus Monitoring (IVM) Online is a web-based animal health
laboratory network system in Indonesia that manages antigenic and
genetic data of the circulating avian influenza viruses in Indonesia. The
system represents the integrated and coordinated effort of its kind for
monitoring of influenza in animals at a national level and underpinned
by OFFLU recommendations. (https://www.ivmonlinewiki)
DICs
iSIKHNA
S
IVM
Online
Field
outbreak/c
ase
Active
surveillance
Passive
surveillance
Outbreak
investigation
Local/Central
Veterinary
Authorities
Source of samples
Reporting system
Respond of field outbreak
Data sharing
Isolate, antigenic and genetic data repository for AI

12. Molecular Surveillance for Avian Influenza:
the Influenza Virus Monitoring (IVM) Network

13. • Developed in collaboration with the Directorate General of PKH-Ministry of Agriculture, FAO-Indonesia, and
assisted by experts from ACDP Australia
• Since December 2017, real-time infolab data (particularly related to AI test results) from iSIKHNAS (National
Animal Health Information System) are shown automatically into IVM Online
IVM Online is web-based molecular
platform to accommodate data and analysis
to monitor influenza viruses.
IVM Online is web-based molecular platform to
accommodate data and analysis of influenza viruses
IVM Online

14. Sharing
Platform:
IVM Network – IVM Online
1. Members:
• Nine Disease Investigation Centers (DICs),
• National Center for Veterinary Biologics (Pusvetma),
• National Veterinary Drug Assay (BBPMSOH),
• Universities (Faculty of Veterinary Medicine)
• Private Companies (Local Vaccine Producers)
2. Registered users from IVM Members only can access and view
IVM Online Modules and its contain.
3. Registered users as Operator can upload, download, and edit
data including isolates, pre-screen antigenic, and HA sequences
and doing phylogenetic analysis but they are not allowed to edit
the data belonging other users.
4. Registered users as Observer can view isolate, pre-screen
antigenic and sequence data, but they cannot edit and download.
5. Registered users as Admin from IVM Focal Point (DIC Wates) can
view, upload, download, edit all data including isolates, pre-screen
antigenic, and HA and WGS sequences, and full-panel antigenic
cartography.

15. Workflow and Information flow
of Influenza Virus Monitoring
Identification of
variants or new virus
Update challenge
and/or vaccine strains
Review &
Recommendation
(DGLHS-MoA)
Samples from
active & passive
surveillances
(all DICs)
Diagnostic
tests for
samples:
VI and PCR
(all DIC)
Pre-Screening
AI Isolates
(all DIC)
Full-Panel HI
Testing
(DIC Wates)
Sequencing:
DIC Wates DIC
Bukittinggi
Pusvetma,
BBPMSOH
Bioinformatic
s Analysis
(DIC Wates))
IVM
ONLIN
E
Sample/
isolate data
Surveillance
data
Antigenic
data
Genetic
data
Bioin-
formatics
data
Pre-screen
HI data
Biological testing of
candidate strains
(BBPMSOH):
-Viability
- Pathogenicity
- Stability
Epi
data
Preskreen
data
1
2
3
4
5
6
7
8
Workflow
Information/Data flow

16. DEVELOPMENT OF NEXT GENERATION SEQUENCING:
CHANGE OF THE PROCESS OF CHARACTERIZATION
(GENETIC  ANTIGENIC)
• Propagasi
• Viable
Virus
Isolate/
Sample
Ori
3-4 Ref.
Antisera
Antigenic
pre-
screen
PCR
Genome
NGS
pre-
screen
• Whole
Genome
• Full Gen
(HA-NA)
NGS
Antigenic
Mapping
Full Panel
Antigenic
Kartografi
Viable virus with high HA titre (> 64)
• Full genome or at
least full HA-NA
• Predicted
antigenic distance

17. Avian Influenza Investigation: Laboratory Algorithm
Surveillance (Healthy flock):
Swab
(Sampling frame/Strategy)
Prepare Samples
# Do not pool >5 animals
** PCR (M)
e.g. † AAHL primers & probes
PCR (e.g. H5, H7 or H9, also
clade specific PCRs e.g. H5 2.3.2
e.g. † recommended regional SOPs
Virus Isolation (3 passages)
& HA
H Typing HI full titration
* (Specific H type e.g. H5 or H7)
& ND
Sick Animal or In-contact
flock (Swab / Tissue)
Differential Diagnosis
(ND, IBD, DVE)
‡ H & N typing (Confirmation)
‡ HA & N Gene Sequencing
Whole genome Sequencing
(Optional)
REFERENCE LAB
Report
Diagnosis – use one or both tests, with
virus isolation positives also tested by PCR
Surveillance – start with PCR, then
isolate virus from positive sample
Report
Report
H typing
† PCR/Sequencing
Further
Characterisation
H & N typing HI
(H & N typing by
PCR/Sequencing is the
preferred method)
Further
Characterisation
NEG POS POS NEG
NEG POS POS/NEG NEG/POS
N typing † † .
† PCR/Sequencing
Further
Characterisation
Differential Diagnosis
(IBD, DVE)
* Where screening for
H5 or H7 the antigens
and antisera in the test
must match the
circulating H5 or H7
clade or strain. The
antiserum used must
be specific for the H
type (Hyperimmune
serum allows detection
of all clades with the H
type)
# Avoid pooling samples in the field whenever
possible; where it is required for testing purposes, it is
best done at the laboratory by combining a
maximum of 5 similar samples per pool from the
same sample type, species, and epidemiologic unit
**Screening flocks for
all influenza viruses
using PCR (M) is
recommended where
possible. Specific virus
PCR can be used first
e.g. H5 or H7 where a
diagnosis is required for
a specific virus in an
emergency.

18. Laboratory Virology - Related to AIV
• Isolation and identification AIV
through inoculation of field
samples to chicken embryonic
eggs following by HA/HI test,
• Development of AIV isolation
using tissue cultures,
• Antigenic pre-screen and
cartography for AIV.
• Provide national- AIV antigens
control

19. Laboratory Serology - Related to AIV
• Detection antibodies to
AIV using:
- ELISA (not often)
- HI Test (routine)
• Antigenic pre-screen
and cartography for
AIV.

20. Laboratory Pathology - Related to AIV
• Post-mortem for gross
pathology
• Histopathology
• Detection AIV antigen using
immunohistochemistry

21. Molecular Lab - Related to AIV
• AIV detection using
conventional and realtime PCR
(Type A, H5N1, H9N2, HxNx),
• Primer and probe designs for
AIV detection,
• Provide national-AIV PCR
controls,
• Development of AIV-whole
genome sequencing using
NGS technique.

22. Sequencing Techniques used in Veterinary Labs (MoA)
1st Gen Sequencing : Sanger-based
HA Sequencing
Fx: Rapid detection HPAI/LPAI,
Labs:
- BBVF Pusvetma (2012-now)
- BBPMSOH (2012-now)
- BVet Bukittinggi (2012-2016)
2nd Gen Sequencing : Illumina
Whole Genome Sequencing
Fx: Full Genetic Characterization
Lab:
BBVet Wates (2016-now)
3rd Gen Sequencing : Nanopore
Fragment/Whole Genome Sequencing
Fx: Quick Identification and
Genetic Characterization
Labs:
- BBVet Wates (start in August-23)
- BBMSOH (development)
- BVet Lampung (development)

23. Detection Reassortant H5N1 Clade 2.3.2.1c viruses using
Next Generation Sequencing Technique at DIC Wates (an example)
Multisegments RT-PCR (Zhou et al., 2009)
NGS: Principal and Workflow
Phylogenetic Tree
Schematic Diagram of H5N1 Reassortant
Bioinformatics Analyses

24. Molecular Surveillance through IVM
RESULTS AND BENEFIT

25. Phylogenetic analysis 2012-2013
Identification of a new clade (2.3.2.1) H5N1 virus incursion into Indonesia through
bioinformatics analyses (phylogenetics and antigenic cartography) lead to the
successful and timely development H5N1 clade 2.3.2.1 vaccine that produced locally
Antigenic cartography 2012-2013 isolates
The Benefits of IVM Network for AI Surveillance & Vaccination
2.1.3.2
2.3.2.1
2.3.2.1. vaccine
Output and Outcome IVM (2012-2013):

26. Updated challenge strains for vaccine efficacy due to mutation of HPAI H5N1 clade
2.3.2.1c and clade 2.1.3.2 and rapid detection and identification of LPAI H9N2 from egg
drop production disease syndrome in layer farm (2017) followed by development of
H9N2 local vaccine
The Benefits of IVM Network for AI Surveillance & Vaccination
Output and Outcome IVM (2014-2017):
2.1.3.2 challenge strain candidate:
A/chicken/Barru/BBVM 41-13/2013
2.3.2.1 challenge strain candidates:
A/chicken/Semarang/0401225-07/2014
Sbg29/ 07
LPAI H9N2
Y-280
G1
Genepool
H9N2
Indonesia

27. • Provided recommendation to update master seed vaccine due to significant
mutations (genetically and antigenically) of HPAI H5N1 clade 2.3.2.1c
The Benefits of IVM Network for AI Surveillance & Vaccination
Output and Outcome IVM (2018-2021):

28. Results of Challenge Study
of HPAI H5N1 Clade 2.3.2.1c
(Source: NVDAL - IVM Network)
Vaccine
Seed Strain
(Clade
2.3.2.1c)
Challenge Strain(Clade 2.3.2.1c)
TGM/1076/2017 50-KT/0376/2018 SMG/1225/2014
Dead/
Survive
Shedding
Dead/
Survive
Shedding
Dead/
Survive
Shedding
SKH/1428/
2012
0/10
Yes
(Day
3-6)
0/10
Yes
(Day
3-6)
1/9
Yes
(Day 3-
6)
50-
KT/0376/2
018
0/10 No 0/10 No 1/9
Yes
(Day 3-
6)
TGM/1076
/2017
0/10 No 0/10 No 0/10 No
From the process of virus characterization through IVM Network,
IVM Team recommended to MoA to update vaccine seed strain for H5N1
clade 2.3.2.1c : A/chicken/Tanggamus/031711076-65/2017 (TGM/1076/2017)
The Benefits of IVM Network for AI Surveillance & Vaccination

29. Detection of HPAI H5N1 clade 2.3.4.4b in Indonesia

30. Impact of detection of H5N1 clade
2.3.4.4b for AIV diagnostics?
Update Algorithm Influenza Virus Monitoring (IVM)

31. THANK YOU