Rapid and Accuracy Diagnosis of Highly Pathogenic Avian Influenza (H5N8) Virus used for the Control of the Outbreak in the Republic of Korea

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1 Rapid and Accuracy Diagnosis of Highly Pathogenic Avian Influenza (H5N8) Virus used for the Control of the Outbreak in the Republic of Korea Third Global Conference of OIE Reference Centres Incheon(Seoul), Korea, October 2014 Lee, Youn-Jeong Avian Influenza Lab. Animal and Plant Quarantine Agency Republic of Korea

2 Organization of Veterinary Authorities Ministry of Agriculture Food and Rural Affairs (MAFRA) Livestock Policy Bureau Animal and Plant Quarantine Agency (QIA) Provincial Government (16) General Division of Animal Health Animal Health Management Division Regional Offices of QIA (6) Provincial Veterinary Services (44) Central Provincial Government Government

3 Organization of QIA

4 Control Measures for HPAI Stamping-out policy (culling) Prompt depopulation : infected farm, farms within 500m radius Pre-emptive culling : within 3 km radius, dangerous contact Zoning and movement restriction 500 m radius : contaminated zone 500m 3 km : protection zone 3 km 10km : surveillance zone 3km 10km Vaccination prohibited Movement restriction were lifted 30 days after the last culling

5 DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC MAR JUN SEP DEC H5 HPAI Epidemics in Korea 03/04 H5N1 06/07 H5N H5N1 10/11 H5N H5N farms - 8 Dk farms - 9 Ck farms 7 farms - 2 Dk farms - 4 Ck farms - 1 Qa farm 33 farms - 6 Dk farms - 21 Ck farms - 6 Dk/Ck mixed 53 farms - 33 Dk farms - 18 Ck farms - 1 Qa, 1 Ph 31 farms - 19 Dk farms - 11 Ck farms - 1 Gs

6 Outbreak of H5N8 HPAI in Korea Poultry Date: Region: Gochang in Jeonbuk(JB) province Farm: breeder duck farm week-old breeder ducks 16,000 birds Reporting: farmer regional veterinary office QIA Clinical signs: Decrease of egg production (above 50%) Increase of death (1-2 bird 7 birds) Depression Wild birds Date: Region: Donglim reservoir near the index case farm Reporting: QIA team for epidemiological investigation hundreds of dead Baikal Teal in Donglim reservoir Data: Epidemiological Division of QIA, 2014

7 Isolation of H5N8 Viruses in Poultry & Wild Birds H5N8 outbreaks: 31 confirmed cases/37 suspect cases in poultry H5N8 virus: 278 viruses No. of cases No. of virus isolation Poultry Wild bird Environment Negative Total 1, , *170,153 samples Weeks number, 2014 Poultry Wild birds

8 breeder duck broiler duck breeder chicken broiler chicken layer Korean native chicken Ostrich Goose Mallard Quail Mixed Baikal Teal Bean Goose White-fronted Goose Mallard Spot-billed duck Common Teal Swan Coot little Grebe Great Egret feces Affected species in poultry and wild birds H5N8 viruses from poultry (231 cases) Duck (174): 75% Chicken (48): 21% Others (9): 4% H5N8 viruses from wild birds (38 cases) Dead bird (20) Captured live bird (9): Mallard(3), spot-billed duck(2), common teal(4) Feces (9) Duck Chicken Others Domestic poultry Wild birds

9 H5 Gene 91 Baikal Teal/KoreH66/2014(H5N8) White-fronted Goose/KoreH231/2014(H5N8) Baikal Teal/KoreH84/2014(H5N8) Bean Goose/KoreH328/2014(H5N8) Baikal Teal/KoreH68/2014(H5N8) Mallard/KoreH207/2014(H5N8) breeder chicken/koreh122/2014(h5n8) breeder duck/koreh249/2014(h5n8) Baikal Teal/KoreH41/2014(H5N8) Mallard/KoreH297/2014(H5N8) Bean Goose/KoreH53/2014(H5N8) breeder chicken/koreh250/2014(h5n8) breeder duck/koreh128/2014(h5n8) Bean Goose/KoreH40/2014(H5N8) broiler duck/koreh48/2014(h5n8) broiler duck/koreh49/2014(h5n8) broiler duck/koreh65/2014(h5n8) broiler duck/koreh145/2014(h5n8) Baikal Teal/KoreH96/2014(H5N8) Tundra Swan/KoreH411/2014(H5N8) Common Teal/KoreH455-30/2014(H5N8) Spot-billed Duck/KoreH455-42/2014(H5N8) broiler duck/korebuan2/2014(h5n8) broiler duck/koreh133/2014(h5n8) broiler duck/koreh29/2014(h5n8) broiler duck/koreh31/2014(h5n8) broiler duck/koreh47/2014(h5n8) Korean native chicken/koreh257/2014(h5n8) Baikal Teal/KoreH80/2014(H5N8) 100 Coot/KoreH81/2014(H5N8) Baikal Teal/KoreH62/2014(H5N8) broiler duck/koreh32/2014(h5n8) breeder duck/koreh200/2014(h5n8) Baikal Teal/KoreDonglim3/2014(H5N8) breeder duck/koreh158/2014(h5n8) duck/jiangsu/m234/2012(h5n2) 99 wild duck/shandong/628/2011(h5n1) duck/jiangsu/k1203/2010(h5n8) breeder duck/koregochang1/2014(h5n8) 100 Baikal Teal/KoreH52/2014(H5N8) 100 chicken/chinah/2012(h5n1) 99 goose/eastern Chin1112/2011(H5N2) duck/eastern Chin1111/2011(H5N2) 100 duck/guangdong/wy11/2008(h5n5) 82 duck/guangdong/wy19/2008(h5n5) duck/eastern Chin008/2008(H5N5) 100 quail/jiangsu/k0104/2010(h5n5) chicken/vietnam/ncvd-404/2010(h5n1) Hunan/2/2009(H5N1) chicken/sichuan/81/2005(h5n1) Anhui/1/2005(H5N1) duck/anhui/1/2006(h5n1) Japanese white-eye/hk/1038/2006(h5n1) Jiangsu/1/2007(H5N1) chicken/vietnam/ncvd-281/2009(h5n1) environment/guizhou/2/2009(h5n1) chicken/vietnam/ncvd-279/2009(h5n1) Vietnam/HN31323/2007(H5N1) chicken/fujian/1/2007(h5n1) chicken/hong Kong/AP156/2009(H5N1) chicken/jiangsu/xz/2010(h5n1) 100 peregrine falcon/hong Kong/810/2009(H5N1) chicken/guiyang/3055/2005(h5n1) goose/guiyang/3422/2005(h5n1) chicken/egypt/1029/2010(h5n1) turkey/turkey/1/2005(h5n1) bar-headed goose/qinghai/12005(h5n1) 2.2 chicken/bangladesh/ /2010(h5n1) chicken/koreis/2006(h5n1) chicken/korees/2003(h5n1) crow/kyoto/53/2004(h5n1) 2.5 chicken/deli/bppvri 238 2/2008(H5N1) Indonesi625/2006(H5N1) chicken/indonesibl/2003(h5n1) chicken/central JavUT3091/2005(H5N1) chicken/yunnan/447/2005(h5n1) duck/hunan/70/2004(h5n1) pigeon/zhejiang/17/2005(h5n1) duck/yunnan/1126/2006(h5n1) goose/guangxi/345/2005(h5n1) Guangxi/1/2009(H5N1) chicken/koregimje/2008(h5n1) whooper swan/akit1/2008(h5n1) 77 chicken/vietnam/ncvd-399/2010(h5n1) Hubei/1/2010(H5N1) Hong Kong/6841/2010(H5N1) tiger/jiangsu/01/2013(h5n1) 90 whooper swan/mongoli1/2010(h5n1) Eurasian eagle owl/kore23/2010(h5n1) 97 whooper swan/hokkaido/3/2011(h5n1) chicken/koreiksan/2010(h5n1) mandarin duck/korepsc24-24/2010(h5n1) chicken/thailand/cu-354/2008(h5n1) 1 99 CambodiU /2010(H5N1) chicken/vietnam/ncvd-380/2009(h5n1) 1.1 goose/guangdong/1/1996(h5n1) 100 Hong Kong/156/1997(H5N1) 0 97 Hong Kong/483/1997(H5N1) 0 wild duck/korepsc18-15/10(h5n8) 100 wild duck/korepsc18-20/10(h5n8) duck/ireland/113/1983(h5n8) 100 turkey/ireland/1378/1983(h5n8) ruddy turnstone/new Jersey/828227/2001(H5N8) 100 avian/colorado/456648/2006(h5n8) 89 mallard/californi2559p/2011(h5n8) mallard/minnesot355811/2000(h5n5) 100 mallard/mn/105/2000(h5n5) A B H5 Gs/Gd lineage Korea LPAI H5N8 Ireland HPAI H5N8 American LPAI H5N8 0.05

10 H5N2 H5N8 H11N9 A/duck/Estern China/1111/2011 A/duck/Jiangsu/k1203/2010 A/environment/Jiangxi/28/2009 Avian Influenza Viruses in Eastern China during Layer Chicken farm, Kumamoto prefecture in Japan(4.13) Group A Wild birds: 27 cases Poultry: 18 cases Group B Wild birds: 1 case Poultry: 1 case Duck in LBM, Zhejiang of China 2013 EID online(5.6) HPAI H5N8 virus of Korea, 2014 Total 47 viruses were analyzed in all 8 segments

11 Procedures for HPAI Suspected Cases Recognize clinical signs Farmer Report to Provincial Veterinary Service History taking and sampling Provincial Veterinary Service Screening test using antigen detection kit (Type A) Transportation samples to QIA Direct RNA detection by RT-PCR, real-time RT-PCR Virus isolation by egg inoculation (HA, RT-PCR & Sequencing) QIA Assessment of pathogenicity ELISA, HI test

12 The Application of Antigen Detection Kit It was developed in 2004 during first H5N1 HPAI outbreak Detection Limit: HA unit, EID 50 /0.1ml Sample collection Extract Reaction Results

13 The Application of Antigen Detection Kit Suspected cases Species Virus isolation Type A Suspected Species Virus Type A cases isolation Field Lab Field Lab 1st Duck + ND + 16 th Chicken + ND + 2 nd Duck + ND + 17 th Duck + ND + 3th Duck + ND + 18 th Chicken th Duck + ND + 19 th Duck + ND + 5 th Chicken th Chicken - ND - 6 th Duck + ND + 21th Chicken + +(4/4) + 7 th Duck + ND + 22th Chicken + +(5/10) + 8 th Duck + ND + 23th Duck + +(6/6) + 9 th Duck - ND - 24 th Chicken + +(4/5) + 10 th Duck + ND + 25 th Chicken + +(2/5) + 11 th Duck + ND + 26 th Duck + +(2/2) + 12 th Chicken - ND - 27 th Duck + ND + 13 th Duck + ND + 28 th Chicken + +(4/10) + 14 th Chicken - ND - 15 th Chicken + +(4/12) + Total 23/28 8/9 23/28 ND: Not done or no information from regional Veterinary Service Field: Samples were oropharyngeal and cloacal swab at the farm Lab: Samples were tissues of dead birds

14 Laboratory Test for AIV Diagnosis Direct RNA Detection Virus Isolation & Identification Serological Test Standard RT-PCR & Sequencing analysis Egg Inoculation, HA AGID Real time RT-PCR RT-PCR & Sequencing Analysis C-ELISA Pathogenicity test in Animal HI test

15 Direct RNA Detection by RT-PCR Materials & Methods AIV multi RT-PCR kit(in-house; Intron Co.): premix with specific Primer sets Traditional RT-PCR: H typing and N typing M, NP gene Negative M, NP and H5 + N8 positive H5 Product of H5 cleavage site N8 Pathotyping (sequencing) AIV- H5 or H7 AIV

16 Comparison of RT-PCR & real time RT-PCR H5N8 Virus* Real time RT-PCR** Traditional RT-PCR (Virus dilution) M gene (Ct) H5 AIV multi kit (in-house; Intron Co.) M H5-HA H5-cleavage1 H5N8 (10-1 ) O O O H5N8 (10-2 ) O O N/A H5N8 (10-3 ) O O N/A H5N8 (10-4 ) N/A N/A N/A N/A H5N8 (10-5 ) N/A N/A N/A N/A H5N8 (10-6 ) N/A N/A N/A N/A N/A H5N8 (10-7 ) N/A N/A N/A N/A N/A * H5N8 virus: A/duck/Korea/Buan2/2014 (H5N8) ** real time RT-PCR: QIA use the same primer and probe set with OIE reference lab(vla)

17 Setting of real time RT-PCR for pathotyping Forward primer Reverse primer Cleavage site Conserve region Primer Name Sequence of primers and probes Nucleotide position References rh5_960f GGGGAATGCCCCAAATATGT Hofmann et al., rh5_1195r TTTTGTCAATGATTGAGTTGACCTTATTGG QIA rh5_conserve_hex_1061 HEX-TTGGAGCTATAGCAGGTTTTATAGAGG-BHQ Hofmann et al., Probe rh5_cs_fam_1029_a* FAM-AGAGARAGAAGAAGAAAAAGAGGACT-TAMRA QIA rh5_cs_fam_1029_b** FAM-AGAGAAAGAAGAAGAAAAAGAGACTGTTT-TAMRA QIA rh5_cs_fam_1029_c*** FAM-AGAGAGAAGAAGAAAAAAGAGAGGACTA-TAMRA Hofmann et al. *Detection for cleavage site of 2008/2010/2014(clade 2,3,2,1, clade <Buan2>) H5 HPAI virus of Korea **Detection for cleavage site of 2014(clade ,Gochang1>) H5 HPAI virus of Korea *** Detection for cleavage site of 2003/2006 H5 (clade 2.5, clade 2.2) HPAI virus of Korea

18 Efficacy test of real time RT-PCR for pathotyping H5N8 Virus* Real time RT-PCR** Real time RT-PCR for pathotyping Traditional RT-PCR (Virus dilution) M gene (Ct) H5 H5 H5- cleavage AIV multi kit (in-house; Intron Co.) H5- M H5 cleavage H5N8 (10-1 ) O O O H5N8 (10-2 ) O O N/A H5N8 (10-3 ) O O N/A H5N8 (10-4 ) N/A N/A N/A N/A N/A N/A H5N8 (10-5 ) N/A N/A N/A N/A N/A N/A H5N8 (10-6 ) N/A N/A N/A N/A N/A N/A N/A H5N8 (10-7 ) N/A N/A N/A N/A N/A N/A N/A * H5N8 virus: A/duck/Korea/Buan2/2014 (H5N8) ** real time RT-PCR: QIA use the same primer and probe set with OIE reference lab(vla)

19 Virus Isolation by Egg Inoculation Virus Isolation Advantage Gold Standard Virus available for further characterization Sensitive for avian viruses Disadvantage SPF eggs expensive Can be time consuming Safety issues with handling infectious material

20 Antigenic Subtyping Advantages Used to identify isolates No non-specific inhibitors Disadvantages Time consuming Complex Requires experience to interpret Requires well-characterized reagents

21 Antibody Detection Methods Agar gel immunodiffusion (AGID) 6 Group-specific test for influenza A virus Enzyme-linked immunoassay (ELISA) Alternative method to the AGID Competitive ELISA was developed and applied Haemagglutination inhibition test (HI) H type specific test Antibodies are subtyped as H5 and H7

22 Antibody detection in poultry & wild birds Results Chickens are all negative for H5 Ab Some of ducks and goose are positive for H5 Ab H5 antibody positive rate is unusually high in wild bird of this year Species No. of tested farm No. of tested birds No. of Ab positive farms No. of H5 Ab positive Chicken , Ducks , Goose Others (mixed) Wild birds

23 Diagnostic Procedures Clinical signs and Gross Lesions Sample: Tissue of dead bird, OP CL swab, serum Clinical signs: egg production, depression, death Gross lesions: hemorrhage in ovary, necrosis in pancreas Virus isolation Virus isolation by egg inoculation Gene detection Direct RT-PCR & real time RT-PCR Egg inoculation HA test RT-PCR Sequence analysis Sero-typing

24 Acknowledgements (QIA) Emergency Preparedness and Response Center Department of Animal Disease Control & Quarantine Animal Disease Control Division Veterinary Epidemiology Division Department of Animal & Plant Health Research Avian Disease Division (MAFRA) Regional Office for Animal Disease Control Veterinary School