April 26, 2017 Zagreb, Croatia

Wild as drivers for the emergence and re-emergence of highly pathogenic avian influenza virus H5N8 in Europe, 2014-2017 Poen MJ, MSc (DVM), PhD-candidate Bestebroer TM, Vuong O, Scheuer RD, Van Der Jeugd HP, Kleyheeg E, Van Der Vliet S, Lexmond P, Eggink WD, Nolet B, Müskens GJDM, Majoor FA, Slaterus R, Koopmans MPG, Kuiken T, Fouchier RAM

HPAI H5N8 2014-2017: Introduction

. Global emerge in 2014 . SE Asia  Europe/USA . 2015: a few detections in Asia . June 2016: HPAI H5N8 in water birds at Ubsu-Nur Lake in Russia . September 2016: FAO releases a warning about the spread

. Outbreaks in Europe from late October Figure: Verhagen et al. Science 347:616-617, 2015 . Second wave of intercontinental movement of HPAI H5N8 viruses  spread by migratory wild birds (Global consortium H5N8, Science 2016)

. Different virus from the 2014 HPAI H5N8

HPAI H5N8 2014-2017: Phylogeny of HA

Phylogenetics by: Dr. C. Russell, University of Cambrigde Dr. W.D. Eggink, University of Amsterdam/ Erasmus MC HPAI H5N8 2014-2015: The Netherlands

. Wave 1: . Intesified suveillance EMC in wild birds . 14 Nov 2014 – 13 May 2015: 5387 birds

. First detection: November 2014 . 3 wild birds (EW) . 5 commercial poultry holdings . Last detection: Februari 2015

HPAI H5N8 2015-2016: The Netherlands

. Suveillance EMC in wild birds . 14 May 2015 – 31 Jan 2016: 7337 birds . Increased surveillance: Sep 2015 – 31 Dec 2015

. No detections of HPAI H5

HPAI H5N8 2016-2017: The Netherlands

. Wave 2: . First detection: November 2016 . 10.000s wild birds . Commercial holdings and backyard poultry

. Intesified suveillance EMC in wild birds . 10 Nov 2016 – 31 Dec 2016: 3054 birds . 08 Feb 2017 – 20 Feb 2017: 1442 birds

10 Nov – 31 Dec 2016: H5 RRT-PCR results

HPAI H5 # samples # # Dead # Alive confirmed Black-headed Gull 6 3 3 0 Y Common Buzzard 1 1 0 1 Y Common Eider 2 1 0 1 Y Common Pochard 2 1 0 1 N Eurasian Herring Gull 4 2 0 2 Y Eurasian Teal 1 1 0 1 N (LPAI) Eurasian Wigeon 56 39 6 33 Y Gadwall 1 1 0 1 N Great Black-backed Gull 10 8 8 0 Y Great Crested Grebe 2 1 1 0 Y Lesser Black-backed Gull 1 1 1 0 Y 11 8 2 6 Y Mew Gull 1 1 1 0 Y Peregrine Falcon 1 1 1 0 Y Tufted 2 1 1 0 Y Grand Total 101 70 24 46

H5+: 2.3% C+P samples takes

Detections in live birds 2016/2017

Species Location Status Date No of animals

Eurasian Wigeon Echtenerburg Dropping 23-Nov-2016 1

Eurasian Wigeon Klemweg Warder Live without clinical signs 4-Dec-2016 3

Common Buzzard Stroeërkoog, Hippolytushoef Live without clinical signs 7-Dec-2016 1

Eurasian Wigeon Oud Alblas Live without clinical signs 8-Dec-2016 1

Eurasian Wigeon Oud Alblas Live without clinical signs 12-Dec-2016 1

Eurasian Wigeon Nijkerk Dropping 12-Dec-2016 3

Eurasian Wigeon Oud Alblas Live without clinical signs 13-Dec-2016 1

Eurasian Wigeon Nijkerk Dropping 14-Dec-2016 2

Eurasian Wigeon Nijkerk Dropping 21-Dec-2016 3

Mallard Oud Alblas Live without clinical signs 7-1-2017 7

Mallard Oud Alblas Live without clinical signs 9-1-2017 4

Mallard Oud Alblas Live without clinical signs 10-1-2017 2

Mallard Oud Alblas Live without clinical signs 11-1-2017 2 HPAI H5N8 2014-2017: The Netherlands II

2014/2015

. 2016: . 76 samples (40 †) . 55 individuals . 12 species

HPAI H5N8 2016-2017: Infected species Europe (FAO 06FEB2017)

. Domestic species affected: Chicken, Duck, Turkey, Goose, Guinea fowl . Wild bird species affected: Wild bird species affected: Armenian Gull, Bean Goose, Black-headed Gull, Black Swan, Canada Goose, Cattle Egret, Black-winged Stilt, Columbidae, Common Barn-Owl, Common Buzzard, Common Coot, Common Crane, Common Goldeneye, Common Kestrel, Common Magpie, Common Moorhen, Common Pheasant, Common Pochard, Common Raven, Common ringed plover, Common Shelduck, Common Teal, Common Tern, Crow, Curlew, Eider, Emu, Eurasian Blackbird, Eurasian Collared Dove, Eurasian Curlew, Eurasian Eagle-Owl, Eurasian Sparrowhawk, Eurasian Spoonbill, Eurasian Wigeon, Ferruginous Pochard, Glossy Ibis, Great Cested Grebe, Great Cormorant, Great Egret, Greater White-fronted Goose, Greylag Goose, Gadwall, Great black-backed Gull, Greater Flamingo, Greater Rhea, Green Sandpiper, Grey Heron, Gull, Herring Gull, Hooded Crow, Kentish plover, Indian Peafowl, Lesser Black-backed Gull, Little Egret, Little ringed plover, Little stint, Long Eared Owl, Little Grebe, Mew Gull, Mallard, Marbled teal, Munia, Muscovy Duck, Mute Swan, , Northern Shoveler, Owl, Painted Stork, Peacock, Pelican, Peregrine Falcon, Pheasant, Pied avocet, Pygmy Cormorant, Red-crested Pochard, Red-footed Falcon, Ruff, Stork, Tufted Duck, White Stork, White Tailed Eagle, White-winged Black Tern, Whooper swan, Wild Duck, Wood Sandpiper, Yellow-legged Gull. HPAI H5N8 2016-2017: Infected species Europe (FAO 06FEB2017)

. Domestic bird species affected: Chicken, Duck, Turkey, Goose, Guinea fowl . Wild bird species affected: Wild bird species affected: Armenian Gull, Bean Goose, Black-headed Gull, Black Swan, Canada Goose, Cattle Egret, Black-winged Stilt, Columbidae, Common Barn-Owl, Common Buzzard, Common Coot, Common Crane, Common Goldeneye, Common Kestrel, Common Magpie, Common Moorhen, Common Pheasant, Common Pochard, Common Raven, Common ringed plover, Common Shelduck, Common Teal, Common Tern, Crow, Curlew, Eider, Emu, Eurasian Blackbird, Eurasian Collared Dove, Eurasian Curlew, Eurasian Eagle-Owl, Eurasian Sparrowhawk, Eurasian Spoonbill, Eurasian Wigeon, Ferruginous Pochard, Glossy Ibis, Great Cested Grebe, Great Cormorant, Great Egret, Greater White-fronted Goose, Greylag Goose, Gadwall, Great black-backed Gull, Greater Flamingo, Greater Rhea, Green Sandpiper, Grey Heron, Gull, Herring Gull, Hooded Crow, Kentish plover, Indian Peafowl, Lesser Black-backed Gull, Little Egret, Little ringed plover, Little stint, Long Eared Owl, Little Grebe, Mew Gull, Mallard, Marbled teal, Munia, Muscovy Duck, Mute Swan, Northern Goshawk, Northern Shoveler, Owl, Painted Stork, Peacock, Pelican, Peregrine Falcon, Pheasant, Pied avocet, Pygmy Cormorant, Red-crested Pochard, Red-footed Falcon, Ruff, Stork, Tufted Duck, White Stork, White Tailed Eagle, White-winged Black Tern, Whooper swan, Wild Duck, Wood Sandpiper, Yellow-legged Gull. Phylogenetics Dutch WB HPAI H5 Eurasian wigeon Duck spp Mute swans Gull spp Serological assay: validation

- Development of serological assays to detect clade 2.3.4.4-specific HA antibodies in wild birds

Antiserum raised against Characteristics Species Haemagglutination inhibition assay Virus neutralisation assay Viruses Viruses LPAI HPAI clade HPAI clade 1a 2.1b 2.2c 2.3d 2.3.4.4e 2.1b 2.3d 2.3.4.4e

A/Mallard/Netherlands/3/1999 LPAI H5N2 Ferret 160 < 10 < 10 < 10 < 10 < 10 ND ND ND

A/Viet Nam/1194/2004 HPAI H5N1 Ferret < 10 80 < 10 < 10 < 10 < 10 ND ND ND clade 1 A/Indonesia/5/2005 HPAI H5N1 Ferret < 10 < 10 120 < 10 60 < 10 80 < 10 < 10 clade 2.1 A/Turkey/Turkey/1/2005 HPAI H5N1 Ferret < 10 < 10 < 10 1,280 60 < 10 ND ND ND clade 2.2 A/Anhui/1/2005 HPAI H5N1 Ferret < 10 < 10 < 10 20 320 < 10 < 10 160 < 10 clade 2.3 A/Chicken/Netherlands/EMC-3/2014 HPAI H5N8 Ferret < 10 < 10 < 10 < 10 < 10 160 < 10 < 10 40 clade 2.3.4.4 Turkey/Germany/AR2487/2014 HPAI H5N8 Domestic < 10 < 10 < 10 < 10 < 10 160 ND ND ND clade 2.3.4.4 duck Turkey/Germany/AR2487/2014 HPAI H5N8 Domestic < 10 < 10 < 10 < 10 < 10 80 ND ND ND clade 2.3.4.4 goose Order Family Species Number of individuals sampled Before first outbreak During first outbreak After first outbreak During second outbreak (before 2014) (14 Nov 2014–13 May (1 Sep 2015–31 Dec (10 Nov 2016 – 31 Dec 2015) 2015) 2016) AnseriformesSerology 2016Common teal (Anas crecca) 0 15 111 22 Egyptian goose (Alopochen aegyptiaca) 9 62 28 10

Eurasian wigeon (Anas penelope) 0 78 46 53 (+69) Gadwall (Anas strepera) 1 3 1 5 Mallard (Anas platyrhynchos) 21 93 18 49 (+23) Mandarin duck ( galericulata) 1 2 0 0 Northern pintail (Anas acuta) 0 0 1 6 Northern shoveler (Anas clypeata) 0 2 3 0 Ruddy shelduck (Tadorna ferruginea) 1 0 0 0 Tufted duck (Aythya fuligula) 0 0 0 1 Geese Barnacle goose (Branta leucopsis) 20 19 0 0 Bean goose (Anser fabalis) 5 0 0 0 Brent goose (Branta bernicla) 0 19 0 0 Greylag goose (Anser anser) 0 2 0 0 Lesser white-fronted goose (Anser erythropus) 0 3 0 0

Pink-footed goose (Anser brachyrhynchus) 0 1 0 0

Greater white-fronted goose (Anser albifrons) 20 77 0 0 Swans Bewick's swan (Cygnus columbianus bewickii) 0 20 0 20 Mute swan (Cygnus olor) 10 90 29 24 Whooper swan (Cygnus cygnus) 0 1 0 3 Charadriiformes Gulls Black-headed gull (Chroicocephalus ridibundus) 20 262 31 88

Caspian gull (Larus cachinnans) 0 6 3 1 Common gull (Larus canus) 12 34 17 7 Great black-backed gull (Larus marinus) 0 1 0 0 Herring gull (Larus argentatus) 7 61 28 15 Lesser black-backed gull (Larus fuscus) 1 3 8 0 Mediterranean gull (Ichthyaetus melanocephalus) 2 1 0 0

Yellow-legged gull (Larus michahellis) 0 0 1 0 Gruiformes Rails Common coot (Fulica atra) 19 84 24 35 Moorhen (Gallinula chloropus) 0 6 0 0 Water rail (Rallus aquaticus) 0 0 0 16 Passeriformes Pica Eurasian magpie (Pica pica) 0 0 0 1 Pelicaniformes Ardeidae Grey heron (Ardea cinerea) 0 0 0 1 Total 149 945 349 374 (+69+23) Serology results: HI assays

Species 2014/2015 Sep-Dec 2015 March 2016 Nov-Dec 2016 Feb 2017 pos/total Percentage pos/total Percentage pos/total Percentage pos/total Percentage Pos/total Percentage Eurasian wigeon 12/78 15.4% 3/45 6.7% 3/28 10.7% 3/104 2.9% Lesser white- fronted goose 1/3 33.3% 0/0 Mute swan 29/88 33.0% 5/24 20.8% 2/24 8.3% Common coot 1/84 1.2% 1/22 4.5% 0/35 Black-headed gull 0/262 0/31 1/88 1.1% Mallard 0/93 0/18 1/49 0.1% 10/23 43,5% Egyptian goose 0/62 1/28 3.6% 0/10

Total 43/940 4.6% 10/319 3.0% 3/28 10.7% 7/408 1.7% Early 2016: Evidence for decline in HPAI H5N8-specific antibody prevalence after 2014-2015 winter (Poen et al., Euro Surveill. 2016;21(38))

Late 2016: No rise in antibody prevalence compared to late 2015 - A lot of the infected birds died - Timing of serology: during outbreaks Early 2017: Incidence in high up Serology results: VN assays

. 2014/2015: Confirmation of . 2015/2016: Confirmation of . 9/9 EW . 3/3 EW . 10/26 MS . 1/5 MS . 0/1 CC . 1/1 CC . 0/1 LWFG . 0/1 EG . No cross-reactivity between clades . No cross-reactivity between clades

. 2016/2017: Confirmation of . 1/3 and 2/3 EW . 1/1 BHG . 1/1Mallard . 0/2 MS . Mallards Feb 2017: pending . Cross-reactivity between clades Conclusions

• Genetically and antigenetically different virus 2014 vs. 2016

• 2016/2017: Detection 55 HPAI H5N8 infected wild birds of 12 species, only HPAI H5N8

• No increase in antibody incidence early during the 2016 outbreak in comparison to 2015 and 2014 based on HI assays

• The addition of serology to virological surveillance will increase the chance to identify potential host species and has added value to monitor the 2016/2017 outbreak

• Further validate serological assays: add value to early warning surveillance

• Worldwide collaboration between ecologists/ornithologists and virologist is needed Future plans

. H13/H16 modelling . Combining sequence data, ecological data, and additional metadata, with phylogenetic analyses will lead for the first time to the development of quantitative epidemiology models for AIV in gulls, that can subsequently be applied and validated for influenza viruses in other wild bird species such as ducks. . Full set NGS . Antibody detection . Designing an epidemiological model describing H13/H16 dynamics in black-headed gulls . Apply model to multiple black-headed gull colonies in the United States of America and Georgia. . Longitunal Goose . Finalise H5N8 viro/sero 2016/2017 . Sequencing (DIGs)