Management and Control of Important Transboundary Animal Diseases in the Asian Pacific Region

Management and Control of ImportantTransboundary Animal Diseases in the Asian Pacific RegionEvolution and Patho-biological Features of Avian Influenza VirusesChing-Ho WANGNational Taiwan UniversityJuly 26, 2016, 1:30 2:15at Bay Leaf Hotel, Manila, Philippines (Food and Fertilizer Technology Center for the Asian and Pacific Region, FFTC)

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Outline Avian influenza virus Evolution of H5N1 AIV Virus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region2

Influenza virus: Structure and Gene Type ASubtypes HA: H1 to H18 NA: N1 to N113

Highly pathogenic avian influenzaOIE definition (Office International des Epizotics)Intravenous pathogenicity index (IVPI) greater than 1.2 at least 75 percent mortalityormultiple basic amino acids at the cleavage site of the haemagglutinin molecule (HA0)[OIE code]4

5Cleavage of HA0 is necessary for virus Entry: HPAIV has multiple basic amino acid at the HA cleavage site but LPAIV doesnt

LPAIV HA1 Trypsin HA2

Respiratory and enteric tractsLocal infection

HPAIV protease

Systemic infectionFusion peptideRRKRRR

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The HA cleavage is hindered by carbohydrate moleculesGlycosylation at 11th amino acidis close toHA cleavage site

N-glycosylation at 11st of HA protects cleavage siteMERIVIAFAIISIVTG- signal peptide 11 12 13 NXT, NXSDRICIGYHANNSTKQVDTIMEKNVTVTHAQDILEKEHNGRLCSLKGVKPLILKD-

327HA2 -NVPQKKKR/GLFG

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Disappearance of N-glycosylation loses cleavage site protectionMERIVIAFAIISIVTG- signal peptide 11 12 13 DRICIGYHANNSKKQVDTIMEKNVTVTHAQDILEKEHNGRLCSLKGVKPLILKD-

HA2 -NVPQKKKR/ GLFG

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9The number of basic amino acids at the cleavage site of HA affects pathogenicity StrainA. a. PathogenicityMost avirulentRETR/GLF AvirulentUSA avirulentRKTR/GLF Avirulent (BBXR)A/Penn/1/83 (H5N2)KKKR/GLF AvirulentPenn/1370/83 (H5N2)KKKR/GLF VirulentScotland/59 (H5N1)RKKR/GLF VirulentItaly/00 (H7)

Texas/04 (H5N2)KKKKR/GLF VirulentKRRRR/GLFQRKKR/GLF HPAI (USDA) LPAI (Texas)

Clin Microbiol Rev 14:129

131211-CHO10

Outline Avian influenza virus Evolution of H5N1 AIVRisk factors for virus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region10

Strain Gs/Gd is the ancestor of recent H5N111

H5N1 evolution in China12

http://www.ncbi.nlm.nih.gov/

A/goose/Guangdong/1/1996A/Henan/09/2015

H5N1 persistence in Asia13

[WHO, 2014]

AIVs transmitted by migrating birds, wild bird trade, and poultry14

Outline Avian influenza virus Evolution of H5N1 AIVVirus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region15

AIV-circulation can be significantly reduced by closing the live poultry market

Temporary LPM closurePeriodic rest days Market depopulation overnight Improved hygiene and disinfection.

16[Offeddu, 2016][Mario Ruckh]

Impact of incoming poultry prevalence on the effectiveness of rest days in LPMs. 17

[Offeddu, 2016:55]

Pahtologic changes from H5N2 LPAIV to HPAIV in chicken farms

PQREKRPQRKKRPQRRKR/)

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DateCountyPoultryvirusHA0 cleavage/IVPI2003.12ChanghLayer0312-REKR-IVPI=0.02012.1.4ChanghLayer12010RKKR-IVPI=2.012012.2.9TainanBreederA1997-RRKR-IVPI=2.532012.5.8YunlinNative1205-RRKR-,IVPI=2.91

Pathogenicity of H5N2/TW viruses19

H5N2 AIV increases pathogenicity after embryo passagesPassage ICPIIVPIIVPI HA cleavage sequence00.190.0REKR50.710.01REKR101.640.45REKR201.64NTRRKKR301.61NTRRKKR401.611.45REKR501.881.92REKR

[Cheng, Wang, 2011]20

Plaque forms and increases in size by H5N2 after embryo passagesPassage Plaque size with trypsin Plaque size without trypsin01.6051.00101.41.0202.01.0302.01.0402.01.2502.01.2

[Cheng, Wang, 2011]21

Vaccines might push higher H5N1 mutationCountryDate Vaccine usedGenetic cladeEvolutionary rateSelected sitesEgypt2006-10Yes 2.2.15.369Egypt A2007-10Yes 2.2.14.074Egypt B2007-10Yes 2.2.18.875Indonesia 2003-07Yes 2.16.136Nigeria2006-08No2.25.202Turkey2005-08No2.24.040Thailand2004-08No12.523

[Cattoli, 2011:9368]Evolutionary rate: 10*-3 sub/site/year. P = 0.1722

Outline Avian influenza virus Evolution of H5N1 AIVVirus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region23

The appearance of H5N8: a novel H5 HPAIV HA clade 2.3.4.424

[de Vries, 2015:842]HA reassorts with different NAAntigenicity difference R-7 vaccine protects only 60%High mortality in geese

H5N5 virus (A/duck/Guangdong/wy24/2008) is the first detected reassortant subtype within this clade25

[de Varis, 2015]

Clade 2.3.4.4 is widely spread in the world

Verhagen, 201526

Outline Avian influenza virus Evolution of H5N1 AIVVirus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region27

AI outbreaks in poultry farms in Taiwan1. H6N1 since 1972

2. H5N2 LPAI outbreaks in chicken farms in Dec. 2003 - Mar. 2004. H5N2 HPAI appeared in chicken farms

3. The appearance of novel H5 HPAI in Taiwan in 2015

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Phylogenetic tree of H6N1 in Taiwan

29[Wang, exp]

PB2 E627K is critical for virus growth and plaque size in MDCK cells

E627K: Mutation of the 627 of PB2 from E to K30

[Wang, exp]

40994093PLAQUE

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Discovery Studio shows the HA structure of Taiwan H6N131

Check the location of amino acid change32

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34HA1 changes more than HA2[Wang, 2011]

H5N2 avian influenza outbreaks - in Dec. 2003 to Mar. 2004

Low pathogenic avian influenza (LPAI) H5N2 virus caused outbreaks in 21 chicken farms in December 2003 to March 2004.No relationship with wild bird H5N2.Sequence is similar to Mexican vaccine strains35

2016/7/22

Epi curve of H5N2 LPAI in 2004-2012

2004 2008 2010 2011 2012YearNo. of cases36

362016/7/22

37

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(American lineage) (Eurasian lineage) 2012/12/13Phylogenetic tree of HA geneCK/TW/2004CK/Tw/2010-2012CK/Tw/2008-2012CK/TW

DK/TW/H5N2Smuggled bird/H5N1WB/Tw/H5

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Genetic analysis of the eight segments of Tw/H5N2 AIVs

American lineage strain(s)-Mexican-like virusesEurasian lineage strain(s)-H6N1/TW-like viruses Taiwan H5N2 AIV

Tw/H5N2 AIV was a reassortant whose HA and NA gene segments belonged to the American lineage and the internal genes to the Eurasian lineage.39

2016/7/22

Novel H5 outbreaks in Taiwan in 201540

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HA phylogenetic tree of AIVs in 2015Two cladesNovel H5Classical H5

Transportation of geese spread the virus

H5 viruses from different countiesshow the same sequenceH5 spreads from one county to another viatransportation

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HPAI outbreak in Taiwan in 2015

Totally confirmed 965 farmsnovel H5N8210Novel H5N319Novel H5N2413nH5N8 & nH5N266nH5N2 & nH5N32Slaughterhouse22nH5N2, nH5N3, H5N8131cH5N2 land fowl57nH5N2, cH5N2 land fowl1H5 water fowl25H5 land fowl21

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4444Multi-basic amino acid in cleavage sites of Novel H5 HPAI viruses Cleavage siteCritical basic aaPhenotypeA/Goose/Taiwan/0103 (H5N8)PLRERRRKR/GLF5HPA/Goose/Taiwan/0104 (H5N2)PLRERRRKR/GLF5HPA/Goose/Taiwan/01042 (H5N3)PLRERRRKR/GLF5HP

Clinical signs in different poultry in novel H5 HPAI in Taiwan 201545

Geese46

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Ducks

H5N8 HPAIV is poorly chicken adaptedhigh chicken infectious dose50 = 105.7 EID50 vs 102.4 EID50 of A/h\Hong Kong/486/1997 (H5N1)A limit blood vessel endothelial cell replicationPoorly field transmissible (less than transmission cutoff 104.7 EID50)48[Bertran, 2016:190]

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Chickens

Outline Avian influenza virus Evolution of H5N1 AIVRisk factors for virus persistence and pathogenicityThe appearance of H5N8Avian influenza outbreaks in TaiwanTransboundary cooperation in the Asian Pacific region50

Wild birds in duck and chicken farms51

Chinese bulbuls get AIV H5N3 infection in Taiwan in Jan 2015

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Night heron gets AIV H5N2 infection in Taiwan in Jan 201553

Black spoon bill gets AIV nH5 infection in Taiwan54

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Sampling by Taipei Wild Bird Society

Bird fecal sample collectionssince 1998 till now

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56Active surveillance in wild birds in TaiwanYearDucksShorebirdsGullsEgrettasOthersTotal19981,457003001,48719991,418750001,49320001,825440001,86920012,516453002,56420022,060652720982,88220031,83155359002,44320041,9021,14919041063,35120052,8061,357112381944,50720062,3521,4131614361794,541200720082009201020112,6452,7682,7552,7912,3011,1479208357541,0682080577940317389417504326211089118264,1504,2654,1554,2463,761Sum31,42710,0348732,46184145,714isolates34014261363Prevalence(%)1.080.140.240.230.240.80

Detected subtypesH1N1, H1N2, H1N3, H2N3, H2N7, H2N9, H3N2, H3N6, H3N7, H3N8, H3N9, H4N2, H4N3, H4N5,H4N6, H4N7, H4N8,H5N2, H5N6, H6N1, H6N2, H6N5, H6N9, H7N1, H7N2, H7N3, H7N5, H7N6, H7N7, H7N9, H8N3, H8N4, H9N6, H9N9, H10N1, H10N2, H10N3, H10N4, H10N6, H10N7, H10N8, H10N9, H11N3, H11N9, H12N2, H14N7

All isolates are LPAIVSubtypeCleavage site of HA proteinH7PEIPKGR*GLFH5PQRETR*GLF

561.08%0.8%

Five million birds use the East Asia-Australasian Flyway (blue)

[U.S. Fish and Wildlife Service/Alaska/Wikimedia][OneOcean.org]57

Some migrating birds appear in NTU campus only in winter each year58

a heron

Extensive surveillance for HPAIV in wild birds is essential through transboundary cooperation in the Asian Pacific region countries 59

http://www.oneocean.org/

The distribution of Chinese Crested-tern (Sterna bernsteini)

http://www.birdlife-asia.org/eng/chinese_crested_tern/

ConclusionThe unprecedented epidemic of HPAI is not yet under controlThe novel H5 AIV has different antigenicity and different pathogenicityThe novel H5 HA is very aggressive to re-assort with other genes, forms H5N8, H5N3 and H5N2Migrating birds are to blame for spreadingCooperation among countries61

Thanks for your attention62

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