genome sequencing: fao's relevant activities in animal health

Genome Sequencing:

Relevant activities in Animal

Health

Gwenaelle DAUPHIN

Animal Health and Production Department

EMPRES Lab Unit CoordinatorOFFLU focal point for the FAO

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

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

Global initiatives

Linking influenza events and isolates

Other

sources(information

tracking, FAO

projects)

Peer-reviewed

publications

Sequence and meta data

Outbreak-isolate linkEpi data

Selected virus meta data

Virus information Epidemiological information

OpenFlu

Selected Epi meta-data

Clade 1 and derived

Clade 2.1 and derived



Clade 2.3.2 and derived

• 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)

1,769 H9N2 events created

17

OpenFMD - Browse

18

OpenFMD - Browse - Isolate details

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

0.01

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

Cadre réglementaire du PIP

Capacity building

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

29

Online ordering systemAn online ordering system of

sequence services, primers and

probes, since December 2014:https://shop.lgcgenomics.com/

- Each lab provided with a user nameand password.

- All individual accounts under an FAO master

account. FAO cannot see the data;

confidentially is ensured

https://shop.lgcgenomics.com/

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

POSITIVESend for full genome

sequencing

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

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

genome sequencing: fao's relevant activities in animal health

Education