OIE Reference Laboratory Reports Activities in 2010

Name of disease (or topic) for Avian influenza which you are a designated OIE Reference Laboratory:

Address of laboratory: Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), Viale dell’Università, 10 – 35020 Legnaro (Padua), Italy

Tel.: (+39-049) 808.43.79

Fax: (+39-049) 808.43.60

e-mail address: [email protected]

website:

Name of Head of Laboratory (Responsible Official):

Name of OIE Reference Expert: Ilaria Capua

Name of writer of this report (if different from above):

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 1 Avian influenza

Part I: Summary of general activities related to the disease

1. Test(s) in use/or available for the specified disease/topic at your laboratory

Test For Specificity Total ELISA Antibody Group 559 DIVA test (iIFA) Antibody Subtype 161 Haemagglutination inhibition test (HI) Antibody Subtype 40373 Haemagglutination inhibition test (HI) Viral typing Subtype 1685 Neuraminidase inhibition test (NI) Viral typing Subtype 90 ELISA Viral antigen Group 757 Inoculation of SPF embryonated chicken eggs Virus isolation Group 2987 rRT-PCR Virus Group 24847 (reverse transcriptase- polymerase chain reaction) Sequencing Virus Pathotype 96

2. Production and distribution of diagnostic reagents

In 2010, 26,595 ml of reference antigens and 3,375 ml of reference sera were produced. The list of reagents produced is shown in detail below: Avian influenza antigens (H1-H16) Infectious bronchitis antigens Newcastle disease antigen Infectious bursal disease antigen Avian Paramyxovirus antigens (APMV-1, APMV-9) Infectious laryngotracheitis antigen Reovirus antigen Negative serum Positive sera for avian influenza virus (H1-H16) Positive serum for Newcastle disease virus Positive Avian Paramyxovirus serum (APMV-1, APMV-9) Infectious bursal disease serum Infectious laryngotracheitis serum Reovirus serum Hev Hepatitis E serum

 A total of 2,314 ml of antigens were distributed, of these 1,139 ml nationally and 1,175 ml internationally.  A total of 1,492 ml of sera were supplied, of these 660 ml nationally and 832 ml internationally.

Part II: Activities specifically related to the mandate of OIE Reference Laboratories

3. International harmonisation and standardisation of methods for diagnostic testing or the production and testing of vaccines

Ring trials in which the OIE Italian laboratory participated during 2010 EU serological and virological identification ring trial.

2 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 Avian influenza

Ring trials organized by the OIE Italian laboratory during 2010 National Ring-test In 2010 a serological and virological ring test was organised by the Italian National Reference Centre to assess the capability of Italian public laboratories to identify selected antigens and to detect antibodies in avian sera to avian influenza viruses and Newcastle disease and to standardize the diagnostic procedures used by the different laboratories.

All the laboratories were able to detect H5 and H7 subtypes viruses by virological, serological and molecular methods. Furthermore, most laboratories were able to detect the H1N1 pandemic virus, although without specific reagents to detect this viral subtype. The number of reagents will be increased in order to enable different laboratories to enlarge the diagnostic efforts to identify influenza viruses in general.

International Ring-test In addition to the National Ring-Test, the Reference Centre has organized a Ring-test for avian influenza and Newcastle disease for African reference laboratories, and for laboratories in the Balkan region and in the Middle East. This initiative was funded by FAO, and by FAO ECTAD of Mali.

All the laboratories were able to detect antibodies against H7 and H5 subtypes by HI test and to detect H5 viral RNA by molecular methods (RT-PCR and RRT-PCR). Areas of improvement have been identified in the serological assay (AGID) and in molecular methods (e.g. RT-PCR and RRT-PCR) to identify H7 subtype viruses. Corrective actions are being implemented to improve the accuracy of these tests in specific laboratories. This proficiency test was essential in providing the necessary feedback to FAO, donors and reference laboratories to better design future training courses and provide technical support of the network

4. Preparation and supply of international reference standards for diagnostic tests or vaccines

See point 2 above.

5. Research and development of new procedures for diagnosis and control

Development of an antigen capture ELISA for the rapid detection of avian influenza virus in the feathers of poultry

It has been recently reported that feathers, together with faeces and respiratory secretions, could represent a potential source of infection by avian influenza viruses (AIV). In this study, we have developed a double antibody sandwich enzymed-linked immunosorbent assay (DAS-ELISA), to detect AIV in feathers of experimentally infected poultry. Briefly, two separate monoclonal antibodies against the nucleoprotein (NP) of AIV were used to develop an ELISA assay, one as a capture antibody and the other as a detection antibody. The test has been applied to feathers collected from experimentally infected ducks and chickens. The statistical analysis, to assess the specificity and sensitivity of the test, is currently ongoing.

6. Collection, analysis and dissemination of epizootiological data relevant to international disease control

The results of all diagnostic investigations related to Avian influenza (AI) have been notified to the OIE, according to its guidelines. This diagnostic effort contributed to improve knowledge on the global spread of avian influenza viruses. IZSVe was also actively involved in epidemiological and molecular studies of avian influenza viruses of H5N1 and H9N2 subtypes.

In particular, IZSVe investigated the impact of vaccination on the genetic evolution of H5N1 viruses in Egypt. Since 2006 H5N1 HPAI viruses belonging to clade 2.2.1 have been extensively circulating in the poultry population in Egypt. In order to control the disease in poultry and reduce the risk for human health, stamping out and vaccination have been applied on a large-scale. Despite this, the virus became endemic in Egypt. The results obtained, suggest that vaccines and vaccination practices may impact the evolutionary rate and the occurrence of mutations in H5N1 viruses circulating in poultry. This should be taken into account when considering vaccination as a tool for controlling this infection. Proper vaccine strain selection, the use of good quality vaccines, implementation of rigorous vaccination practices and monitoring of field vaccination coverage and vaccine efficacy must all be seriously considered to reduce the risk of vaccine failures and major antigenic drift.

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 3 Avian influenza

IZSVe also performed a molecular epidemiological study of the HA gene of H9N2 viruses isolated in the Middle East, from 2004-2009. Genetic data were generated in order to elucidate the molecular characteristics and the phylogenetic relationships of 21 H9N2 viruses identified in 4 distinct Middle Eastern Countries, namely Jordan, Iraq, Saudi Arabia and the United Arab Emirates (UAE between 2004 and 2009). Analysis of deduced amino acid sequences of the HA glycoprotein revealed that 6 viruses from Saudi Arabia, two viruses from Dubai and all the viruses isolated in Kurdistan-Iraq contained the substitution Q226L (H3 numbering) at the receptor binding site. This mutation is associated with a preferential receptor binding specificity for sialic acid 2,6-linked galactose and displays human virus-like cell tropisms. Molecular analysis allowed us also to identify a Saudi Arabian strain, exhibiting 3 out of 4 amino acid residues at the receptor binding site previously associated with respiratory transmission in ferrets (Sorrel et al., 2009). The genetic variability identified between the H9N2 viruses used in the present study combined with the identification of specific substitutions which are believed to modify the efficiency of the replication in non-avian species, highlight the need to constantly monitor the evolution of this potentially pandemic strain in the Middle East

7. Provision of consultant expertise to OIE or to OIE Members

OIE Twinning project between the Italian OIE/FAO reference laboratory for Avian Influenza and Newcastle Disease and National Centre for Animal and Plant Health (Censa) of Cuba (final workshop, November 2010). This twinning had the aim of implementing capacity building of the Cuban laboratory in order to bring it closer to OIE reference laboratory status by improving standards in specific selected areas.

Collaboration Agreement between IZSVe e IVO (Iranian Veterinary Organization) with the aim of giving scientific and technical support on Avian influenza and Newcastle disease to the Iranian veterinary service.

8. Provision of scientific and technical training to personnel from other OIE Members

During 2010 IZSVe continued and increased the training activities on avian influenza (AI) and Newcastle disease (ND). In particular, it managed training courses within the framework of European funded projects (EPIZONE, FLUTRAIN), organizing training courses on avian influenza diagnosis and epidemiology, for veterinarians and technicians. The list of visitors is reported in Table 2.

During 2010, IZSVe’s staff members carried out assessment missions on laboratories which applied for such activity. The list of the missions is reported below:

 20-23 March 2010 (Qatar), OIE meeting for a possible twinning project between IZSVe and National veterinary laboratory  March 2010 Cairo (Egypt), Meeting with IFT Corporation to create a collaboration for the management of the H5N1 crisis  October 2010 Cairo (Egypt), Workshop on the surveillance activity for Avian influenza in Egypt

Table 2: List of people attending courses at IZSVe during 2010

Name Laboratory Country Training subject Period

Central Veterinary Laboratory, Rosa-Stella Mbulu Ministry of Agriculture, Water Namibia Diagnosis of AI and NDV 15/02/2010 26/02/2010 and Forestry

Department of Veterinary Canaan Tinashe Technical Services, Ministry of Zimbabwe Diagnosis of AI and NDV 15/02/2010 26/02/2010 Agriculture

Central Veterinary Research Wegdan Hassan Ali Abdel Laboratories, Department of Sudan Diagnosis of AI and NDV 15/02/2010 26/02/2010 El Mageed Virology

Name Laboratory Country Training subject Period

4 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 Avian influenza

Istituto Zooprofilattico Diagnosis of major avian Maria Lucia Mandola Sperimentale del Piemonte, Italy diseases-Laboratory quality 07/04/2010 08/04/2010 Liguria e Valle d’Aosta assurance

National Veterinary Research Tony Joannis Nigeria Research studies 01/09/2009 30/05/2010 Institute

Virginia-Maryland Regional United Jeffrey Christ Diagnosis of AI and NDV 01/06/2010 08/08/2010 College of Veterinary Medicine States

Cairo Poultry Company (CPC) Rabab Amin Ahmed Khalifa Egypt Molecular diagnosis of AI 07/06/2010 11/06/2010 Laboratory

Cairo Poultry Company (CPC) Dina Moustafa Ali Hamed Egypt Diagnosis of AI 07/06/2010 11/06/2010 Laboratory

Medwey School of Pharmacy Pseudoparticles Eleonora Molesti University of Greenwich and Italy 01/10/2010 22/10/2010 neutralization assay for AIV Kent a Medway

Centre de Recerca en Sanitat Kateri Bertran Dols Spain Diagnosis of AI 10/10/2010 31/01/2011 Animal (CReSA)

Department of Clinical Sciences, Diagnosis of AI and major Hesamodin Emadi Chashmi Iran 18/10/2010 18/04/2011 Faculty of Veterinary Medicine avian diseases

Universidade Luterana Do Diagnosis of AI and major Laìs Pancotto Brasil 25/10/2010 24/12/2010 Brasil avian diseases

Mamoona Chaudhry University of Edimburg (UK) Pakistan Molecular techniques for AI 8/11/2010 17/11/2010 diagnosis and sequencing

9. Provision of diagnostic testing facilities to other OIE Members

IZSVe has supported the Veterinary Services of foreign countries by providing official confirmatory diagnosis of routine cases identified during surveillance activities in domestic and wild birds.

Table 3 indicates the number of samples from foreign countries submitted for Avian Influenza diagnosis in 2010.

Table 3: Samples from foreign Countries submitted for Avian Influenza diagnosis at IZSVe

Country Serological tests Virological tests Molecular tests Total

BANGLADESH 0 17 56 73 BULGARIA 0 0 1 1 BURKINA FASO 12 0 50 62 EGYPT 74 212 712 898 JORDAN 0 7 14 21 IRAN 0 10 149 159 KYRGYZSTAN 2 0 0 2 MYANMAR 80 23 468 571 PAKISTAN 0 30 368 398

Country Serological tests Virological tests Molecular tests Total

TURKEY 0 239 2 241

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 5 Avian influenza

ZIMBAWE 0 1 425 426 LITHUANIA 0 0 308 308 UK 12 112 0 124 Total 180 651 2,832 3,866

10. Organisation of international scientific meetings on behalf of OIE or other international bodies

In 2010 IZSVe organized the following conference:

27-29 April, 2010 Verona (Italy): “2nd OIE/FAO/WHO Scientific consultation on influenza and other emerging infectious diseases at the human-animal interface”. The meeting gathered a group of experts in Verona, Italy, to focus on emerging zoonoses including influenza and identify commonalities at the human-animal interface. The main purpose of the meeting was to provide a technical basis for developing or modifying policies and strategies to more effectively prepare for and respond to the next emerging event.

11. Participation in international scientific collaborative studies

Working groups  Dr. Ilaria Capua and Dr. Isabella Monne – Members of EFSA working group on “scientific opinion on the monitoring for emergence of possible new pandemic strains of influenza in animals”, Panel on Animal Health and Welfare (AHAW) European Food Safety Authority (EFSA)”.

Research projects  CARIPLO-“Novel Strategies of vaccine design to prevent emerging and pandemic influenza virus infections NoFlu”  NEW FLUBIRD – “Network for early warning of influenza viruses in migratory birds in Europe”  FLUPATH-“Avian Influenza: impact of virus-host interactions on pathogenesis and ecology”  FLUTRAIN – “Training and technology transfer of Avian Influenza diagnostics and disease management skills”  FLURESIST-“Studies on Avian Influenza virus survival in poultry commodities, poultry manure and the environment”  FLU-LAB-NET-“Development and enhancement of laboratory networks for avian influenza”  FLUPIG-“Pathogenesis and transmission of influenza in pigs”  EPIZONE-“Network of Excellence for Epizootic Disease Diagnosis and Control”

12. Publication and dissemination of information relevant to the work of OIE (including list of scientific publications, internet publishing activities, presentations at international conferences)

1. Anderson T.C., De Benedictis P., Perez A., Viale E., Veggiato C., Tiozzo Caenazzo S., Crawford C., Capua I. (2010)."A Diagnostic Algorithm for Detection of Antibodies to Influenza A in Dogs"; Proceding of The International Symposium on Neglected Influenza Viruses; Amelia Island (Florida) 3-5 February. 2. Anderson T., Capua I., Dauphin G., Donis R., Fouchier R., Mumford E., Peiris M., Swayne D., Thiermann A. (2010).“FAO-OIE-WHO Joint Technical Consultation on Avian Influenza at the Human-Animal Interface” Influenza Other Respi Viruses, 4 Suppl 1:1-29. 3. Beato MS., Monne I., Mancin M., Bertoli E., Capua I. (2010). “A proof-of-principle study to identify suitable vaccine seed candidates to combat introductions of Eurasian lineage H5 and H7 subtype avian influenza viruses. Avian Pathol., 39(5):375-82. 4. Capua I., Alexander D. (2010). “Perspectives on the global threat: the challenge of avian influenza viruses for the world's veterinary community” Avian Dis., 54(1 Suppl):176-8. 5. Capua I. (2010). "Patogeni emergenti nell'era globalizzata"; Proceedings 44th Congresso Nazionale SIRM - Verona 11-15 Giugno 2010. 6. Capua I., Cattoli G. (2010). “One flu for One Health” Emerg Infect Dis., 16(4):719. 7. Cecchinato M., Ceolin C., Busani L., Dalla Pozza M.,Terregino C., Moreno A., Bonfanti L., Marangon S. (2010). "Low Pathogenicity Avian Influenza in Italy During 2007 and 2008: Epidemiology and Control"; Avian Diseases 54(1):323-328.

6 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 Avian influenza

8. Cecchinato M., Comin A., Bonfanti L. , Terregino C., Monne I, Lorenzetto M., Marangon S. (2010). “Epidemiology and control of Low Pathogenicity Avian Influenza infections in rural poultry in Italy. Avian Diseases in press. 9. Cilloni,F., Toffan,A.; Giannecchini,S.; Clausi,V.; Azzi A.; Capua I.; Terregino C. (2010). "Increased pathogenicity and shedding in chickens of a wild bird-origin LPAI virus of the H7N3 subtype following multiple in vivo passages in quail and turkey"; Avian Diseases 54:555-557. 10. Dauphin G., Hamilton K., Kim L.M., Choudhury B., Capua I., Edwards S. (2010). “Main achievements of the World Organisation for Animal Health/United Nations Food and Agriculture Organization network on animal influenza” Avian Dis., 54(1 Suppl):380-3. 9. 11. Dauphin G., Mumford L., Thiermann A., Capua I. (2010) “Opening letter. Avian influenza at the human animal interface” Influenza Other Respi. Viruses, 4 Suppl 1:i. 12. De Benedictis P., Anderson T. C., Perez A., Viale E., Veggiato C., Tiozzo Caenazzo S., Crawford P. C., Capua I. (2010). “A diagnostic algorithm for detection of antibodies to influenza A viruses in dogs in Italy (2006–2008)” J Vet Diagn Invest., 22: 914-920. 13. Di Lullo G.; Soprana E.; Panigada M.; Palini A.; Agresti A.; Comunian C.; Milani A.; Capua I.; Erfle V.; Siccardi G.A. (2010). "The combination of marker gene swapping and fluorescence-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use"; Journal of Virological Methods, 163(2):195-204. 14. Dundon W.G., De Benedictis P., Veggiato C., Capua I. (2010). "Serological evidence of H1N1 infection in dogs in Italy"; Proceding of The International Symposium on Neglected Influenza Viruses; Amelia Island (Florida) 3-5 February 2010. 15. Dundon W.G., De Benedictis P., Viale E., Capua I.(2010). "Serological evidence of H1N1 infection in dogs in Italy"; Emerging Infectious Diseases, 16(12):2019-21. 16. Fereidouni S.R., Werner O., Starick E., Beer M., Harder T.C., Aghakhan M., ModirroustaH., AminiH., Moghaddam M.K., Bozorghmehrifard M.H., Akhavizadegan M.A., Gaidet N., Newman S.H., Hammoumi S., Cattoli G., Globig A., Hoffmann B., Sehati M.E., Masoodi S., Dodman T., Hagemeijer W., Mousakhani S., Mettenleiter T.C. (2010). "Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007; Virol J. 19;7:43. 17. Fusaro A., Monne I., Cattoli G., De Nardi R., Salviato A., Moreno Martin A., Capua I., and Terregino C. (2010) “Gene segment reassortment between Eurasian and American clades of avian influenza virus in Italy”. Arch Virol., 155(1):77-81. 18. Fusaro A., Nelson M.I., Joannis T., Bertolotti L., Monne I., Salviato A., Olaleye O., Shittu I., Sulaiman L., Lombin L.H., Capua I., Holmes E.C., Cattoli G. (2010). “Evolutionary dynamics of multiple sublineages of H5N1 influenza viruses in Nigeria from 2006 to 2008”. J Virol., 84(7):3239-47. 19. Giannecchini S., Clausi V., Di Trani L., Falcone E., Terregino C., Toffan A., Cilloni F. Matrosovich M., Gambaryan A.S., Bovin N.V., Delogu M., Capua I., Donatelli I., Azzi A.(2010). “Molecular adaptation o an H7N3 wild duck influenza virus following experimental multiple passages in quail and turkey”. Virology, 408:167-173. 20. Keiner B., Maenz B., Wagner R., Cattoli G., Capua I., Klenk HD. (2010). "Intracellular distribution of NS1 correlates with the infectivity and interferon antagonism of an Avian Influenza virus (H7N1). Journal of Virology, 84(22), 11858-11865. 21. Rigoni, M. , Toffan, A., Viale, E. , Mancin, M. , Cilloni, F. , Bertoli, E., Salomoni, A., Marciano, S. , Milani, A., Zecchin, B. , Capua, I., Cattoli G., (2010). “The mouse model is suitable for the study of viral factors governing transmission and pathogenesis of highly pathogenic avian influenza (HPAI) viruses in mammals”, Veterinary Research, 41:66. 22. Smietanka K., Fusaro A., Domanska-Blicharz K., Salviato A., Monne I., Dundon W. G., Cattoli G., Minta Z. (2010). “Full-length Genome Sequencing of the Polish 1 HPAI H5N1 Viruses Suggests Separate Introductions in 2006 and 2007”. Avian Dis., 54(1 Suppl):335-9. 23. Terregino C., Toffan A., Cilloni F., Monne I., Bertoli E., Castellanos L., Amarin N., Mancin M., Capua I.; (2010). "Evaluation of the protection induced by avian influenza vaccines containing Mexican H5N2 LPAI seed strain against a 2008 Egyptian H5N1 virus belonging to clade 2.2.1 by means of serological and in vivo tests"; Avian Pathology, vol 39(3), 215-222. 24. Toffan A.; Olivier A.; Mancin M.; Tuttoilmondo V.; Facco D.; Capua I.; Terregino C. (2010). "Evaluation of different serological tests for the detection of antibodies against highly pathogenic avian influenza in experimentally infected ostriches (Struthio camelus)"; Avian Pathology, 39 (1), 11-15. 25. Uttenthal A., Parida S., Rasmussen T.B., Paton D.J., Haas B., Dundon W.G. “Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza” Expert Rev. Vaccines. 9(1):73-87. 26. Zecchin, B., Munir, M., Zohari, S., Cattoli, G. Berg, M., Capua, I., Dundon W.G. (2010). Analysis of the interferon abrogation ability of the non-structural 1 protein from influenza A. Fourth Annual EPIZONE meeting June 7-10, St Malo, France.

Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010 7 Avian influenza

27. Zecchin, B, Munir, M., Zohari, S., Cattoli, G., Berg, M., Capua, I., Dundon W.G. (2010). The interferon abrogation of the Non-structural I proetin from influenza A is not effected by carboxy terminal length variations. 4th European congress of Virology, 7-11, April, Cernobbio, Italy.

13. Inscription of diagnostic kits on the OIE Register

i) Did you participate in expert panels for the validation of candidate kits for inscription on the OIE Register? If yes, for which kits?

ii) Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for which kits?

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8 Annual reports of OIE Reference Laboratories and Collaborating Centres, 2010