1.
Genome Sequencing:
Relevant activities in Animal
Health
Gwenaelle DAUPHIN
Animal Health and Production Department
EMPRES Lab Unit Coordinator
OFFLU focal point for the FAO

2.
Background
• Priorities: mostly virus pathogens – shorter genomes
• PCR and sequencing is a great alternative to virus isolation
• Huge funding for avian influenza
• As of today, our work supports Sanger sequencing and not
specifically whole genome sequencing
• Some developed countries: start using NGS in routine (US, NL,
Germany,…) to get quick and complete confirmatory diagnosis
and advanced characterization (urgent culling). Quality control
issues.
• Discussions within OIE on sequence information
• Presentation of a few examples of where FAO could play a role
in such topic
2

3.
Avian Influenza : Asia region - 2014-2015 subtypes
2.3.2.1.A
2.3.2.1.C
2.3.4.2
1.1/2.3.2.1.C
2.3.2.1.C
2.1.3.2 / 2.3.2.1.C
2.3.4.4
2.3.4.4
2.3.4.4
2.3.4.4

4.
Global initiatives

5.
5

6.
Linking influenza events and isolates

7.
Other
sources
(information
tracking, FAO
projects)
Peer-reviewed
publications
Sequence and meta data
Outbreak-isolate link
Epi data
Selected virus meta data
Virus information Epidemiological information
OpenFlu
Selected Epi meta-data

8.
11

9.
Clade 1 and derived
Clade 2.1 and derived
Clade 2.2 and derived
Clade 2.3 and derived
Clade 2.3.2 and derived

10.
• Visualize thousands of
viruses’ genetic distances
on the same map
• Overlay epidemiological
data onto the map
• Detect reassortment
events
• Could be used as a risk
assessment tool
Sequence Similarity Maps (SSM)

11.
1,769 H9N2 events created

12.
OpenFMD - Browse

13.
OpenFMD - Browse - Isolate details

14.
H5N1
Nomenclature
3, 4, 5, 6,
7, 8, 9
1
2-3-4
2-3-2
2-2
2-1
0
2-2
2-5
2-4
2-3-2
2-3-3
2-3-1
2-3-4
2-1
1
8 & 9
7
5 & 6
3
4
3
0
0
0
A/goose/Guangdong/1/96 0
2.3.4.6
2.3.4.4
2.3.4.1
2.3.4.2
2.3.4.3
2.3.4.0
2.3.4.5

15.
Cadre réglementaire du PIP

16.
24

17.
Capacity building

18.
27

19.
Initiative on provision of access to
sequencing services
• Satisfactory PCR testing capacities in most animal
health units of national vet labs; yet rare access to
sequencing capabilities.
• Scope of the initiative: increase the scientific
knowledge on pathogens genetics
• Selection of 10 African countries
• 3 FAO Reference Centres to help for validation of
PCR protocols with the same commercial kits and for
training 28

20.
29
Online ordering system
An online ordering system of
sequence services, primers and
probes, since December 2014:
https://shop.lgcgenomics.com/
- Each lab provided with a user name
and password.
- All individual accounts under an FAO master
account. FAO cannot see the data;
confidentially is ensured

21.
INDIVIDUAL CHICKEN SAMPLE
M-GENE PCR
Differential Diagnosis
(NDV, IBR, DVE)
RNA extraction
sample is Flu A POSITIVEsample is Flu A NEGATIVE
POSITIVENEGATIVE
Perform 3 HA-GENE PCRs
(H5, H7, H9)
sample is Flu A POSITIVE
But NOT H5, H7, H9
sample is H5 or H7 or H9
POSITIVE
Send for full genome
sequencing

22.
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)
e.g. † recommended regional
SOPs e.g. AAHL/FLI
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 (Conformation)
‡ 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.
† Use
recommended
regional PCR
primers &
probe
‡ For
confirmation of
H & N type the
isolate will need
sequencing
(e.g. H7N9
from China or
H5 clade
2.3.4.6)
PCR (e.g.N9, N6 or
N8) e.g. † CNIC
primers & probes
POS POS
NEG NEG

23.
33

24.
Conclusions
• Sequence databases
– Support to some developments (global and national)
– Linkage with EMPRES-i
• Support to sequencing for advanced testing +
compilation of sequence data (thru national labs and Ref
centres)
• Building capacities in generating and using sequence
information
– Training in bioinformatics
– Users’ interfaces
• How will these activities/initiative evolve with NGS?
– Collaborations with COMPARE 34