Post on 14-Feb-2017
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
22nd Joint Annual Meeting of the National Laboratories
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)
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 2
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 3
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 4
Week
Nu
mb
er
of
su
sp
icio
ns
H5 HP positive negative
Breeding flock surveillance
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 5
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 6
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 7
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)
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 8
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 9
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 10
- 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.
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 11
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 12
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)
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 13
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)
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 14
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%
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 15
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 16
Experimental infection/transmission studies (2)
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 17
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 18
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
22nd Joint Annual Meeting of the National Laboratories
for AI and ND of EU Member States Copenhagen, 19-21 April 2016 19
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.