h5 hpai outbreaks in poultry in france

22nd Joint Annual Meeting of the National Laboratories

for avian influenza and Newcastle disease

of European Union Member States

Copenhagen, 19-21 April 2016

H5 HPAI outbreaks in poultry in France:

update on the current situation


for AI and ND of EU Member States Copenhagen, 19-21 April 2016 1

76 H5 HPAI outbreaks since 24/11/2015

Localisation of the 76 H5 HPAI

outbreaks in France

on March 15, 2016

Outbreaks

&

&

Restriction zone

Restriction zone (18 dpts)

established 17/12/2015

(last declared

H5 HPAI outbreak)

http://www.plateforme-esa.fr/



AI surveillance

authorized local labs network

virology (n=12) + serology (n=9)

+ NRL

1) Clinical surveillance in poultry

2) Programmed surveillance: - Epidemiological link investigation / outbreaks

- Restocking of holdings / outbreaks

- Derogation on movement restrictions

- Breeding flock / game bird flock surveillance

- Annual sero. survey virological follow-up



AI surveillance – outcome

CONTEXT

of INVESTIGATION

nb of H5 HPAI

outbreaks (n=76)

Clinical Surveillance

28

Programmed Surveillance

epidemiological links

/ outbreaks

3

restocking of holdings

/ outbreaks

3

derogation on

movement restrictions

36

breeding flock

surveillance

3

2015 sero. survey 3

March 29, 2016

Clinical surveillance

Poultry type total H5 HP pos.

Galliform 19 3

Waterfowl 21 15

Gall. + Wfowl 7 6

Backyard 35 4

Total 82 28



Week

Nu

mb

er

of

su

sp

icio

ns

H5 HP positive negative


Breeding flock surveillance



Zone Poultry type Nb of flocks

to be inspected

Nb of

inspected flocks

Restriction Zone

(RZ)

Galliform 119 46 (39%)

Waterfowl 254 201 (79%)

Outside RZ

Galliform 679 378 (56%)

Waterfowl 699 174 (25%)

Galliform: 20 blood samples / flock

Waterfowl: 20 blood samples + 20 oro. & 20 clo. swabs / flock

ISOLATES : H5N1 (1x) H5N2 (2x) H5N9 (2x)

NGS Sequencing : H5N1 (1x) H5N2 (2x) H5N9 (2x)

ASSOCIATED SUBTYPES (n= 76 outbreaks)

Plateforme génomique (Unité GVB Anses-Ploufragan)

H5N1 H5N2 H5N9 H5NX H5N1+

H5N9

H5N2+

H5N9



AI surveillance – detected viruses


H5 cleavage site sequence analysis

HP cleavage site acquired by mutation

No insertions of basic amino acids (ie: Lineage Gs/Gd/1/96-Like (H5N1)) (PQRERRRKKRGLF)

Unique described cleavage site



Confirmed by IVPI= 2.9

Position (AA) 337 338 339 340 341 342 343 344 345 Pathotype

closely related H5 LP

AA P Q R E T R G L F LP

nt C C T C A A A G A G A A A C A A G A G G A C T A T T T

H5 HP France 2015 (150169a)

nt C A T C A A A G G A G A A A A A G A G G A C T A T T T

AA H Q R R K R G L F HP

Protein Amino acid substitution % of European Avian Influenza viruses

sharing same residue as 150169a Comments

HA

D94N N94 (62.5) Increased binding to alpha 2-6 receptor

S159N; T160A N159 (81.4); A160 (97.6) Increased binding to alpha 2-6 receptor

S239P P239 (99.4) Slight increased binding to alpha 2-6 receptor (aa 239 corresponds to aa 235 in H5 numbering)

T160A A160 (97.6) Increased airborne transmission in ferrets ; Increased binding to alpha2-6 receptor

(aa 160 corresponds to aa 156 in H5 numbering)

Multibasic cleavage site

HQRRKR/GLF /

Multiple basic residues in H5 viruses that are highly pathogenic for avian hosts (but unique sequence)

PB2

I63T I63 (99.2) Decreased pathogenicity in mice in association with PB1 T677M

L89V; G309D ; T339K; R477G; I495V; A676T

V89 (99.7); D309 (99.5); K339 (91.7); G477 (100); V495 (99.4); T676 (98.3)

Increased polymerase activity in mouse cells.

R368Q; Q447H R368 (91.9); Q447 (99.9) Reduced virulence (lethality in mice) and conferred histologic alteration in the lungs, liver and brain of ferrets.

PB1

K207R K207 (100) Decreased polymerase activity.

Y436H Y436 (99.9) Decreased virulence in ducks, mice and ferrets

V473L V473 (99.5) Decreased polymerase activity in mammalian cells and mice

T677M T677 (99.9) Increased polymerase activity in vitro. Reduced replication efficiency. Decreased virulence in mice in association with PB2 I63T

PB1-F2 N66S S66 (15.1) Increased replication efficiency in mice

H5N1 HPAI virus: analysis of virulence markers (1)

Increased pathogenicity, virulence and replication in mammal hosts

No major markers for increased α2,6 receptor affinity (HA)

No major markers of increased replication in mammals (polymerase complex: PB2 PB1 PA)



Anses opinion 15/12/2015 : https://www.anses.fr/fr/system/files/SANT2015sa0241.pdf

Protein Amino acid substitution % of European Avian Influenza viruses

sharing same residue as 150169a Comments

PA T515A T515 (99.8) Decreased polymerase activity

R266H; T515S R266 (99.8) T515 (99.8) Reduced polymerase activity in vitro.

M1 N30D D30 (99.9) Increased virulence in mice

T215A A215 (99.9) Increased virulence in mice

NS1

P42S S42 (66.0) Increased virulence (lethality in mice and the systemic spread of infection). Affected IFN pathway.

E92D D92 (99.8) Cytokine resistance using antiviral activity assay

L103F; I106M F103 (65.7); M106 (99.8) Increased virulence compared to WT in mice

N205S S205 (64.9) Implicated in high virulence in ferrets

227-230 (presence of PDZ ligand domain)

ESEV (>80) ESEV increased virulence and pathogenicity in mice

NS2 T48A A48 (66.2) Implicated in high virulence in ferrets

Markers of increased virulence or host response antagonism in mammals (NS)

Anses opinion 15/12/2015 : https://www.anses.fr/fr/system/files/SANT2015sa0241.pdf

All identified markers are also present in majority of European avian influenza

viruses

except: N66S on PB1-F2



H5N1 HPAI virus: analysis of virulence markers (2)

Increased pathogenicity, virulence and replication in mammal hosts

Chronophylogenetic study of complete H5 sequences (n=5)

H5 HP FR

Analyses of time of the Most Recent Common Ancestor : tMRCA

Eurasian

lineage

H5 AIV

Belongs to Eurasian lineage but not directly related to Gs/Gd/1/96-like (H5N1) Estimated age of H5 HP common ancestor

≈ emergence of H5 HP cleavage site ≈ beginning 2014 ± 1yr

≈ 2014,4 ± 1

≈ 2009,8 ± 1



- in wild birds, followed by transmission to poultry?

- in poultry?

Study of reassortment events

At least 4 reassortment events since the H5 HP cleavage site emerged (to be complemented and refined with other virus sequences)

Implies many co-infections with different avian influenza viruses + reassortment

For each segment, sequences are considered to belong to a common genogroup (identical color) based on relatedness by NJ method with bootsrap support > 75.



A/chicken/France/

150169a/2015

A/duck/France/

150233/2015

A/chicken/France/

160013g/2016

A/duck/France/

150236/2015

A/duck/France/

150289/2015

H5N1 H5N2 H5N2 H5N9 H5N9

PB2

PB1

PA

HA

NP

NA

M

NS Allele B

2014 2013 2015 2012

at least 4 detected

reassortment events,

generating H5 HPAIV diversity



Serological data (1)

Verification of serological diagnostic tools

Panel of sera tested :

- 103 sera from five confirmed H5 HP infected holdings (duck)

- 30 sera from experimental infection with H5N9 HP

(Muscovy ducks): at 3, 6, 7 and 8 days post infections

- 7 reference chicken sera (H5)



Serological data (2)

Comparison of methods:

- HI using European antigens:

• H5N3 A/teal/England/7394-2805/2006

• H5N1 A/chicken/Scotland/1959

- HI using French antigens from LP viruses:

• H5N3 A/muscovyduck/France/070090b/2007

• H5N2 A/chicken/France/03426a/2003

- HI using French antigens from recent HP H5 viruses:

• H5N1 A/duck/France/150169/2015

• H5N9 A/duck/France/150236b/2015

- H5 ELISA (IdVet-ELISA ID Screen® Influenza H5 Antibody

Competition kit v2.0, IdVet, Montpellier France)



Serological data (3) Comparison of results using different methods or antigens

(% individual positivity):

Conventionnal duck batches: simultaneous blood samples and virological samples

(detection of HPH5)

- HI test: equivalent batch diagnosis whatever antigens used

- H5 ELISA: more sensitive? consistent with previous study

(Schmitz et al., J.Virol.Meth., 2013)

rRT-PCR H5 results

of virological

samples

nb of

tested

sera

HI using

European Ag

HI using

French LP Ag

HI using

recent H5HP Ag H5 ELISA

batch n°1 36.8 Ct 40 38% 68% 53% 100%

batch n°2 37,5 Ct 19 11% 26% 42% 89%

batch n°3 26-34.8 Ct 20 0% 0% 0% 20%

batch n°4 34.8 Ct 18 61% 83% 72% 100%

batch n°5 35.6 Ct 6 100% 100% 100% 100%

expal sera / 30 0% 0% 0% 17%

ref. pos. sera / 7 100% 100% 100% 100%



planned in collaboration with EURL

3-week-old SPF Muscovy ducks

BSL2+ / BSL3 facilities,

straw litter,

8 m² rearing area

Experimental infection/transmission studies (1)

D0 : 6 inoculated ducks

106 EID50 / duck

ocular instillation

H5N9 A/duck/France/150236b/2015

D1 : introduction of 12 contact ducks



Experimental infection/transmission studies (2)



Preliminary virus shedding results:

- oro. + clo. shedding by all infected and contact ducks,

from D1 to D4 (except 3 contacts at D2 = oro. only),

- all infected and contact ducks still shed virus

at D7 and D9, but oro. shedding not constant,

- at D14: 4 infected and 6 contact ducks still shedding

by clo. route (only 1 contact also oro. shedding).

HI vs homologous antigen:

- at D7: 1 infected duck HI titre = 16,

- at D14 and D20: all infected and contact ducks positive

Laboratory Activities

Screening laboratory activities: • 511 batches in rRT-PCR M / H5 / H7 (20 oro + 20 cl swabs)

• 424 batches in H5 / H7 HI ( 20 blood samples)

• 375 batches in AGID ( 20 blood samples)

NRL activities: confirmation tests for:

• 108 batches in RT-PCR H5

• 158 batches for subtyping M+ / H5- / H7- cases (in progress)

• 105 batches in HI tests (since 2016-02-01)

15 isolation assays, 5 H5 HPAI viruses isolated

production of 2 new H5 HPAI haemagglutinating antigens

production of 1 new anti H5N9 monospecific anti serum

production and supply of antigens, positive and negative control (serum,

extraction and PCR positive control, …) for screening and confirmation

analysis to French laboratories

modifying working hours (Monday to Sunday) + system of on-call duty



NRL Activities

Complementary studies (ongoing / pending):

Further isolation assays

Further full genome sequencing

Phylogenetic and reassortments studies

Antigenicity assays

Serum production

In vivo studies (in collaboration with EURL)

Study of virus persistence in duck manure



Acknowledgements:

Briand FX., Schmitz A., Ogor K., Le Prioux A., Guillou-Cloarec C.,

Guillemoto C., Allée C., Le Bras M.O., Cherbonnel-Pansart M.,

Lemaître E., Courtillon C., Eterradossi N., Jestin V.

Hirchaud E., Touzain F., Quenault H., Blanchard Y.

Huneau A., Lebouquin-Leneveu S.

van der Werf S.

Gares H.

LDAR24 staff

Daniel P.

LPL40 staff

Fediaevsky A.

Brown I., Essen S., Banks J.

http://www.pasteur.fr/fr


h5 hpai outbreaks in poultry in france

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