Mia Kim Torchetti, DVM MS PhD on behalf of many!

US Department of Agriculture, APHIS VS STAS

National Veterinary Services Laboratories, Ames IA

Nvsl.dvl.avian@aphis.usda.gov

Ph. 515-337-7551

Molecular Epidemiology of the 2015-2016 HPAIV Outbreaks

Avian Influenza

• Influenza A viruses with high pathogenicity (HPAI) and H5 and H7 subtypes with low pathogenicity (H5/H7 LPAI) are reportable worldwide.

• Other non-H5/H7 subtypes are known as “influenza A of birds” (aka fluA).

• Waterfowl are natural reservoir hosts for all influenza A subtypes, but not usually HPAI.

• Influenza A viruses tend to circulate within flyways and wax/wane in multiyear cycles; geographic origin may often be gleaned from the genome.

• The only HPAI virus recognized to circulate in natural reservoir hosts emerged in domestic poultry in Asia (H5N1) and likely spilled back into the wild reservoir; this lineage continues to circulate and a related virus made its way to North America in late 2014.

2

Influenza viruses can rapidly EVOLVE• Antigenic Drift: mutations

that cause minor antigenic changes to H protein– Ongoing process

– Limits efficacy of protective & vaccine induced immunity

• Antigenic Shift: “Shift Happens”– Replacement of H or N

– Genetic re-assortment (human and animal viruses)

– Insertion of large section of extraneous RNA near the HA cleavage site – virulence shift

• Both mechanisms can contribute to emergence of novel (pandemic) strains

3

H

Host range

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H5N6, H7N9H10N8…

Adapted from: Animal Influenza Viruses: Gap Analysis Workshop Report. 2014. U.S. Department of Agriculture, Agricultural Research Service, Washington, DC. http://go.usa.gov/KpGP

AI Epidemiology

Adapted from M. Pantin-Jackwood, USDA ARS Southeast Poultry Research

. 5

Asian H5N1 HPAIRe-adaptation

Mammals

fluA | LPAI H5/H7

H5N8/H5N2/H5N1

H5

HPAI (H5/H7)HA Mutation H5/H7

ExposureAdaptation

fluA | LPAI H5/H7

Over 200 avian species are susceptible hosts

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HPAILPAI

Cleavage by trypsin-like enzymes

Respiratory &GI epitheliumOccasionally other epithelium

Furin-like enzymes Ubiquitous in many

cell types (trypsin not needed)

Pathogenicity Determination is Specific to the Species Tested – e.g. chickens

LPAI (local) vs HPAI (systemic)

HPAI Reported Worldwide1890’s-1950’s numerous fowl plague outbreaks, subtypes not known1924-25 North east US?1927 New Jersey ?1934 Germany, H7N1 1959 Scotland, H5N1 1961 South Africa, H5N31963 England, H7N31966 Canada, H5N91975 Australia, H7N71979 Germany, H7N71979 England, H7N71983-84 USA, H5N21983 Ireland, H5N81985 Australia, H7N71991 England, H5N11992 Australia, H7N31994 Australia, H7N31994-95 Mexico, H5N21995 Pakistan, H7N31997 Australia, H7N41997 Italy, H5N21996-present Asia/Europe/ Africa, H5N11999-2000 Italy, H7N1

2002 Chile, H7N32003 Netherlands, H7N72004 USA, H5N2 – per cleavage site2004 Canada, H7N32004 Pakistan H7N32004 South Africa, H5N22005 North Korea, H7N7 2006 South Africa H5N22007 Canada, H7N32008 England, H7N72009 Spain, H7N72011 South, Africa H5N22012-2013 Taipei, H5N22012-2013 South Africa, H7N1, H5N22012 Australia, H7N7, H7N22012 Mexico H7N32013 Italy H7N72014-2015 Japan, Korea, Germany, Netherlands, Hungary, Italy, UK, Sweden, Romania H5N82014 Russia, H5N12014 Canada H5N22014-2015 H5 icA, H5N2 US 2015 H5Nx, China, Chinese Taipei ,South Korea2016 H7N8 US

Outbreaks in red are ongoing

7 Courtesy E.Spackman, USDA ARS Southeast Poultry Research

AI Surveillance in the United States

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>2 million tests/year

Laboratory Services and CollaborationsConfirmatory diagnostic lab, assay development, and research

• Updated assay validation (H5/H7)

• Training for NPIP, FADDL, NAHLN, LBM

• OIE FAO Reference & Collaborating Centres

• Collaboration with Public Health

NAHLN and NPIP protocols for serology and antigen detection (>50 labs)

•PT and standardized reagent distribution for AGID•Harmonized rRT-PCR protocols (NAHLN labs only)

AGID, ELISA, and antigen capture immunoassay

• PT and standardized reagent distribution for AGID

• PT for ELISA using USDA-licensed kits available for 2014

Federal Laboratories

• APHIS NVSL Diagnostic Virology Lab

• ARS Southeast Poultry Research Lab

• CDC

NAHLN / State -University / NPIP-approved labs

NPIP approved labs

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Molecular / Sequencing

• Both screening and confirmation

• fluA, H5, H7, and H5 2.3.4.4 specific assay

• Subtyping direct from samples using Sanger methods

Virus isolation

• Molecular and antigenic characterization

• In vivo pathotyping

Serologic assays

• fluA, subtype specific confirmatory tests

Pen-side tests

• Screening test for clinically ill/dead birds – requires

confirmation

Current Diagnostics

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Monitoring influenza A in natural reservoir hosts

H5 in US prior to late 2014

•North American lineage influenza A–H5N2 LPAI historically one of the most common subtypes–LPAI to HPAI mutation in poultry

•1982-83 H5N2 PA•2004 H5N2 TX – LBM; disease not severe, cleavage site

consistent w/HPAI –North American lineage H5N8 LPAI – different virus from Eurasian

H5N8

•Wild bird surveillance for Eurasian H5 2006-2011–No Eurasian H5 detected in nearly 500,000 samples

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P

P

P

P P P P

W

W

W

W

P

H5N3 in wild birds

H5N3 in poultryP

W

H5N2 in poultry

H5N2 in wild birds

P

W H5N8 in wild birds

H5N8 in poultryP

WH5N1 in wild birds

H5N1 in poultryP

W H5N6 in wild birds

H5N6 in poultryP

W

H5 HPAI reports from East AsiaSeptember 2014 through February 2015 (OIE)

Eastern Asia

Reports of H5 HPAI subsided in the region during summer

2014. Then in September, outbreaks of H5N1, H5N2,

H5N6, H5N8 HPAI occurred in China.

Original H5N8 outbreak occurred January-April, 2014,

most intensely in S. Korea and Japan. After 5 months with

no reported cases, H5N8 was detected again, 24

September, in S. Korean commercial poultry. It was

detected again in Japan in November.

H5N2, H5N3, H5N8 outbreaks occurred in Taiwan in early

2015.

P

W

W

P

P

P

In Korea – 2 distinct groups of viruses were recognized

Group B viruses were closely related across all 8 genes to the H5N8 viruses from China late 2013Group A viruses predominated and subsequently spread along migratory bird pathways

‒ “Intercontinental Group A” (icA) now contains Chinese, Russian, South Korean, Japanese, European, and North American viruses

13 Adapted from: Center for Food Safety and Public Health, Iowa State University

Introduction of H5 icA

14

Lee DH, Torchetti MK, Winker K, Song CS, Swayne D. Intercontinental Spread of Asian-origin H5N8 to North America through Beringia by Migratory BirdsJ. Virol. JVI.00728-15; epub ahead of print 8 April 2015, doi:10.1128/JVI.00728-15

Detections in Europe late 2014

EA/AM H5N2 BC Nov 2014

EA H5N8 and EA/AM H5N2 WA Dec 2014

Early detection in the US thanks to wild bird surveillance efforts

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• Captive gyrfalcon mortality • Wild bird mortality event

fluA IHC of gyrfalcon A3465 brainCourtesy of T. Baszler, WADDL

NOPI with aspergillosisCourtesy of V. Shearn-Bochsler,

NWHC

Mortality not due to influenza

Mortality was due to influenza

Reassortment of H5 icA

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12/8/2015 to 6/17/2015 H5 icA HPAIV in wild bird, backyard poultry and

commercial poultry

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• 89K samples tested from poultry; ~58K from wild birds• Nine States with 211 affected commercial premises • Eleven States with 21 backyard flocks • Efforts to control HPAI resulted in the destruction of 7.5 million turkeys

and 42.1 million egg-layer and pullet chickens• Cost to Federal taxpayers estimated in excess of $950 million

H5 icA in North America

• First avian Eurasian H5 virus detected in North America

• Circulates as a highly pathogenic virus for poultry but early viruses adapted to

ducks

– BID50:100-1,000 times higher than previous H5N1 HPAIVs

– Inefficient transmission to contacts except when placed with high challenge dose group

• Earliest viruses better adapted to ducks than to gallinaceous poultry (turkey >

chicken) – later viruses demonstrated adaptations to poultry

– Longer mean death times, poor transmissibility

– High infectious dose based upon laboratory studies

• Although distantly related to Asian H5N1; the North American viruses

characterized represented low risk to human health

Adapted from M. Pantin-Jackwood, USDA ARS Southeast Poultry Research19

Mallards > Pheasants, Partridges, GF, Pekin, geese > Quail > Turkeys > Chickens

What can the virus tell us?

• Viruses sequenced by next generation methods• Consensus sequence data used to generate 3-gene and 8-gene (full

genome) analyses

• Outcome of analysis used to help inform epidemiologic investigations

• NOTE: The outcomes of molecular analysis need to be interpreted in context of all available virus and epidemiologic information and should not be used to directly infer transmission

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How was the data used?

Pacific Flyway Findings

• 3 different subtypes detected; the H5N2 viruses predominated in wild birds–There were no H5N2 detections in commercial poultry

•H5N8 was detected in both poultry and wild bird populations in the Pacific flyway –The H5N8 viruses have wholly Eurasian gene constellations except

two from OR (Jan2015) with two North American internal genes (PB1,PA)

• Long branches observed by network analysis for all H5 icA viruses in the Pacific flyway are suggestive of point source introductions –Findings are consistent with both the movement of the virus in wild

bird flyways and the low infectivity in gallinaceous poultry

Refer to H5 September 2015 Epi and Virus Report22

Midwest EA/AM H5N2 Findings

• The Midwest viruses cluster into two major groups; wild bird-type viruses present in each

• Some subgroups span several states and counties and are suggestive of point source introductions with limited evidence of lateral spread

• Data for other subgroups contain evidence for point source introductions and lateral spread concurrently

Refer to H5 September 2015 Epi and Virus Report23

0

20

40

60

80

100

120

140

backyard layer, commercial turkey,commercial

wild bird

Nu

mb

er o

f se

qu

ence

s

Lower virus diversity

from layers

Greater virus diversity from

turkeys

13

11

8

12,16, 15 1

710

914

1

4

6

753

2

8-gene network: 17 detections spanning 5 states and 16 counties from27 February to 20 April 2015; long branches suggest largely point source introductions with limited evidence of lateral spread

Refer to H5 September 2015 Epi and Virus Report24

H5N2 Midwest Example 1

Network analysis with D. Lee USDA/ARS/SEPRLMapping with USDA/CEAH

Refer to H5 September 2015 Epi and Virus Report25

H5N2 Midwest Example 2

8-gene network: 18 detections in single state across 4 counties from 26 March to 14 May 2015; highly similar viruses and shorter branches consistent with lateral or secondary spread

Network analysis with D. Lee USDA/ARS/SEPRLMapping with USDA/CEAH

Summary of Key Events during the 2014–2015 HPAI Outbreak

26 Available at: https://www.aphis.usda.gov/animal_health/emergency_management/downloads/hpai/presentation/finalreport14-15_shortppt.pdf

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December 2014 – June 2016 Last Updated: 6/30/2016Total birds sampled: 52,633HPAI positive cases : 65 (none since June 2015)PCR detection only: 35 (2 since June 2015)

Total birds sampled by flyway Atlantic: 11,663 Mississippi: 15,871Central: 9,741 Pacific: 15,242American Oceania : 116

2016 Events–H7N8 HPAI/LPAI of North American wild bird lineage, January 2016 in turkeys; molecular and epi data suggest a single introduction with limited spread; mutation to HPAI occurred in a single flock.

–H5N1 LPAI of North American wild bird lineage, April 2016 in turkeys; molecular and epi data indicate single introduction with no further spread.

–H5N2 LPAI of North American wild bird lineage,June 2016 in LBMs; molecular and epi data suggest single source introduction.

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Overview• Investments in diagnostics and research have paid off

with more rapid virus characterization and ability to push information from the virus to the field in real time.

• More rapid response upon detection with agreement of State and Federal officials.

• Investigation into whole house pathogen detection methods continue.

•To date, the 2016 detections originate from ‘native’ North American LPAI and are not related to the 2014-15 Eurasian H5 icA viruses

• There have been no confirmed Eurasian H5 wild bird or poultry detections since June 2015

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Helpful References

• USDA APHIS Avian Influenza Disease–Epidemiology and Virus reports for EA H5 September 2015, and

–AM H7N8 March 2016 are available at this site

• HPAI FAD PReP Materials and MANY helpful references– Including Avian Sample Collection WI-AV-0020

Other general avian influenza resources:

• CIDRAP Avian Influenza Lessons

• National Accreditation Module 6: Exotic Avian Diseases

• http://viralzone.expasy.org/all_by_species/6.html

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Special thanks to the avian team and our partners!