Workshop on Research Gap Analysis in Animal Influenza 8 and 9 January 2015, Parma 1, 2

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1 EFSA supporting publication 2015:EN-0787 EVENT REPORT Workshop on Research Gap Analysis in Animal Influenza 8 and 9 January 2015, Parma 1, 2 European Food Safety Authority and the European Commission s Directorates General for Agriculture and Rural Development and for Research and Innovation 3 European Food Safety Authority (EFSA), Parma, Italy and the European Commission s Directorates General for Agriculture and Rural Development and for Research and Innovation, Brussels, Belgium ABSTRACT Fighting animal influenza is a major challenge for animal and human health, requiring continuous knowledge building and development of appropriate control tools. Over the last ten years, the European Union (EU) has funded a series of projects in the field of animal influenza mainly in the context of the Research Framework Programmes. While these and other international initiatives have built on the knowledge of animal influenza, there is a need for continuous priority research gap analysis. Starting from the outcomes from recent research gap analysis exercises in the field of animal influenza, the workshop aimed at identifying priority research areas based on expert opinion. An exercise was structured around four central themes: host-pathogen interaction, diagnosis, surveillance and prevention and control. Ten different research priority subjects were identified: (1) identify virus and host determinants of virus replication to understand host range restriction and to identify mechanisms by which viruses adapt to new host species, (2) integrated and multiplexed rapid molecular tests, (3) improve serological tests, (4) improve virus recovery methods, (5) develop integrated risk assessment tools, (6) interface studies of different host species, (7) analyse risk of introduction into EU (risk factors and mechanisms/ preventive measures) and early detection, (8) develop an efficacious emergency vaccination program for animal influenza viruses with pandemic and/or epizootic potential, (9) validate biosecurity measures to avoid introduction & spread of avian influenza, and (10) research on the development of routine vaccination. Any further ranking is challenging, being this highly dependent on the specific research needs and objectives, and whether the focus is on basic or applied research. Recommendations were made in order to sustain availability of data generated during research studies, and to encourage coordinated EU and international funding in research so to efficiently achieve best progress and outcomes possible. 1 Question No EFSA-Q Disclaimer: The views or positions expressed in this publication do not necessarily represent in legal terms the official position of the European Food Safety Authority. The European Food Safety Authority assumes no responsibility or liability for any errors or inaccuracies that may appear. 3 Acknowledgement: EFSA and EC wishes to thank the experts Jabbar Ahmed, Francesca Ambrosini, Anette Bøtner, Ian Brown, Michel Bublot, Giovanni Cattoli, Thomas Chambers, Johannes Charlier, Gwenaelle Dauphin, Ron Fouchier, Cyril Gay, Bruno Goddeeris, Timm Harder, Guus Koch, Mikael Leijon, Alex Morrow, Gounalan Pavade, Ben Peeters, Dirk Pfeiffer, Thomas Vahlenkamp, Saskia Van De Zande, Wim Van Der Poel, Kristien Van Reeth; Mike Sharp, Petra Muellner; Pasi Penttinen from the European Centre for Disease Prevention and Control (ECDC); Jean-Charles Cavitte, Laszlo Kuster, Maria Pittman, Luis Vivas-Alegre from the European Commission; Marianne Carson, Andrea Gervelmeyer, Per Have and Frank Verdonck from EFSA for the preparation and participation in the workshop and support provided to this scientific output. Any enquiries related to this output should be addressed to ALPHA@efsa.europa.eu Suggested citation: European Food Safety Authority, Workshop on animal influenza. EFSA supporting publication 2015:EN-0787, 40 pp. Available online: European Food Safety Authority, 2015

2 European Food Safety Authority and the European Commission s Directorates General for Agriculture and Rural Development and for Research and Innovation, 2015 KEY WORDS Animal influenza viruses, gap analysis, knowledge gap, One Health, pandemic, priority research, expert opinion, zoonotic EFSA supporting publication 2015:EN

3 SUMMARY Over the last ten years, the European Union (EU) has funded in the context of the Research Framework Programmes a series of projects in the field of animal influenza 4. EFSA has funded the FLURISK project that developed a risk assessment methodological framework for potential pandemic influenza strains. All these projects have improved our scientific understanding of the virus and facilitated collaboration between scientists, risk assessors/managers and policy makers. However, gap analyses recently performed by the FLURISK consortium 5, AHVLA 6, the OFFLU and Star-IDAZ initiative 7 and the USDA Animal Influenza Viruses Gap Analysis workshop 8 show that there is still a need to improve our knowledge about this important transboundary zoonosis. To feed the reflection on the future research agenda on animal influenza, the European Commission's (EC) Directorate General for Agriculture and Rural Development and the European Food Safety Agency (EFSA), in collaboration with EC DG Research and Innovation, organised a priority research gap analysis workshop on 8-9 January 2015 at EFSA premises in Parma. Fighting animal influenza is a major challenge for animal and human health that requires continuous knowledge building and development of appropriate control tools. Influenza has been identified as one of three top priorities in the tripartite collaboration among the International Organisations (OIE, FAO, WHO). This event aimed at mapping a number of research projects supported by the EU since 2002 in the area of animal influenza, whether specialised or with a component on animal influenza. The discussions focussed mostly on the state of play, the knowledge gaps and priorities for research subjects on animal influenza. In doing so, participants took into account the European and International framework on animal influenza and related policy needs. Twenty-four scientists, including representatives of EU funded projects, delegates of European and International organisations (FAO and OIE) as well as the United States Agricultural Research Service (USDA-ARS) participated in the workshop together with a dozen of representatives from European Commission's Agricultural, Research and Innovation and Health and Food Safety Directorate-Generals, European Centre for Disease Control, European Food Safety Authority and the EU Reference Laboratory for Avian Influenza. After an introductory speech on the epidemiological update and EU policy overview, the presentations and discussions were organised around four themes: host-pathogen interactions, diagnostics, surveillance and risk assessment and prevention and control. For each of these themes, an overview of current knowledge as well as knowledge gaps identified were first presented and discussed, based on pre-workshop preparatory work carried out and building on research gaps identified by previous initiatives. Following this, breakout sessions were used to carry out an exercise to gather expert opinion on research gap priorities. A list of predefined questions (i.e. criteria and sub-criteria) was used in order to support and justify the research priority subjects identified.. While undoubtedly the EU funded projects have contributed to the progress made in the knowledge about this epidemic, transboundary disease affecting a number of animal species and humans, major gaps still exist in host-pathogen interaction, surveillance and risk assessment, prevention and control and, to a lesser extent, diagnostics. Ten research priority subjects were identified and described among four main research domains: hostpathogen interaction, diagnosis, surveillance and prevention and control. The identified subjects were: (1) Identify virus and host determinants of virus replication to understand host range restriction and to IDAZ_Executive_Summary_final.pdf 8 EFSA supporting publication 2015:EN

4 identify mechanisms by which viruses adapt to new host species, (2) Integrated and multiplexed rapid molecular tests, (3) Improve serological tests, (4) Improve virus recovery methods, (5) Develop integrated risk assessment tools, (6) Interface studies of different host species, (7) Analyse risk of introduction into EU (risk factors and mechanisms/ preventive measures) and early detection, (8) Develop an efficacious emergency vaccination program for animal influenza viruses with pandemic and/or epizootic potential, (9) Validate biosecurity measures to avoid introduction & spread of avian influenza, and (10) Research on the development of routine vaccination. Any further ranking of these research priority areas would depend on the specific objective under consideration, and whether the focus is based on basic or applied research. It would therefore be important to examine which research priorities provide maximum scientific, policy and socioeconomic impact depending on the scope sought. It was generally acknowledged that integration of diagnostic tools, linking of databases and networks as well as international cooperation are critical. Besides technical improvements, there is a need to involve scientists from the social sciences in order to achieve greater impact in animal influenza diagnosis, surveillance and control. A key recommendation of the workshop was the need to consider maintenance issues of the databases and networks after the end of projects in order to maximise the use of the data generated. There is also a need to encourage the EU and international funding and research organisations to efficiently coordinate upcoming initiatives in the field of animal influenza research in order to achieve best progress and outcomes possible. EFSA supporting publication 2015:EN

5 TABLE OF CONTENTS Abstract... 1 Summary... 3 Table of contents... 5 Background as provided by the European Commission and EFSA... 6 Terms of reference as provided by the European Commission and EFSA... 6 Workshop Introduction Pre-workshop preparatory work Expert workshop on research gap analysis on Animal Influenza General overview of knowledge and data gaps Host-pathogen interactions Diagnosis Surveillance and Risk Assessment Prevention and control Expert opinion on research gap priorities Host-pathogen interactions Diagnosis Surveillance and risk assessment Prevention and control Challenges to priority ranking of research needs across the four areas Conclusions and recommendations References Appendices Appendix A. Knowledge and data gaps identified by previous exercises Appendix B. Research subjects identified by previous exercises Abbreviations EFSA supporting publication 2015:EN

6 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION AND EFSA Workshop on animal influenza Influenza infections are a major threat to animal and public health. A swine-origin influenza virus has become the most recent human pandemic virus and avian influenza viruses of different subtypes have caused enormous damage to the poultry industry and in some cases human health issues. Over the last ten years, the European Union (EU) has funded in the context of the Research Framework Programmes a series of projects in the field of animal influenza 9. EFSA has funded the FLURISK project that developed a risk assessment methodological framework for potential pandemic influenza strains. All these projects have improved our scientific understanding of the virus and facilitated collaboration between scientists, risk assessors/managers and policy makers. However, gap analyses recently performed by the FLURISK consortium 10, AHVLA 11, the OFFLU and Star-IDAZ initiative 12 and the USDA Animal Influenza Viruses Gap Analysis workshop 13 show that there is still a need to improve our knowledge. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION AND EFSA "EFSA is requested to provide support in the organisation of a review exercise of EU the achievements and limitations of EU funded projects in the field of animal influenza. This review should be expert-driven and result in the preparation of workshop where research gaps and needs would be discussed and prioritised, taking into account the European and International framework including the policy needs perspective." IDAZ_Executive_Summary_final.pdf 13 EFSA supporting publication 2015:EN

7 WORKSHOP 1. Introduction The EC Directorate-General for Agriculture and Rural Development (DG-AGRI), opened the Workshop by providing a background for the Workshop and outlining its objectives: Through a review of achievements and limitations of EU projects of the last 10 years and other research initiatives, discuss gaps and priorities for research needs on animal influenza, taking into account the European and International framework and the policy needs perspective. Animal influenza has a severe impact not only for animal health through multiple pandemics, but also severe effects on industry, economic impacts and through its zoonotic potential. The preparatory work for the Workshop looked at animal influenza projects in the EU, their achievements and limitations (see chapter 3). The preparatory work and the workshop itself were also intended to improve understanding between different project coordinators by sharing information on EU-based animal influenza initiatives. Discussions during the workshop aimed to define knowledge and data gaps and to define, ideally also prioritise, possible research subjects to the animal influenza situation, primarily in Europe. The list of prioritised research subjects could be taken on board by funding organisations and schemes to stimulate research on animal influenza in an efficient and coordinated manner within Europe. To that end, those priorities can also be taken into account within Horizon 2020, the EU s biggest research and innovation framework programme to date. The EC Directorate-General for Health and Food Safety (SANTE) gave an overview of EU Policy on animal health and an epidemiological update on the current animal influenza situation in the EU. The EU strategy for animal health is based on the belief that Prevention is better than cure, which aims to take a preventative rather than a reactive approach to health. The action plan for the animal health strategy is centred around four pillars of activities: (1) prioritisation of EU intervention, (2) a modern EU animal health framework, (3) improve prevention, crisis preparedness and (4) science, innovation and research. The underlying principles for the 4 pillars are partnership and communication. Key actions within each respective pillar are: (1) study on categorisation of animal diseases, (2) proposal for a EU Regulation on animal health, (3) evaluation of EU rapid response capacity and development of EU vaccine banks and (4) research. In 2014 a new framework regulation for animal health was proposed by the EC, which included renewed features such as animal health responsibilities (including those of farmers), biosecurity issues, disease categorisation and prioritisation, and disease surveillance. Terrestrial, aquatic and other animals and the movements of these would fall under this framework. The framework would provide for delegated and implementing acts targeting disease control, movement of animals and products and the entry into the EU of animals and products. It is expected that this framework will be adopted by the Council and European Parliament and published in 2015, with delegated and implementing acts to be fine-tuned and in place by However, the regulation on animal health will not lead to major changes to the existing animal influenza policy. It is expected that the new regulation will be easier and faster to update when new scientific information becomes available. Several outbreaks of influenza occurred in Europe during the last decade. The most recent animal influenza outbreaks were caused by highly pathogenic avian influenza (HPAI) H5N8 in poultry and wild birds involving four Member States (MSs) in the autumn of 2014: Germany, the Netherlands, the UK and Italy. Only indoor poultry farms have been affected in this current outbreak, involving 343,000 birds as of December Lessons learned from these events are that the response to avian influenza (AI) outbreaks works well, with robust emergency procedures and contingency plans in place. Furthermore, AI disease control is well accepted by stakeholders and there is good balance between regulatory prescription and flexibility for MSs. However, challenges remain for biosecurity, surveillance and AI vaccination. EFSA supporting publication 2015:EN

8 Biosecurity measures are the first line of defence against avian influenza virus (AIV) introduction in poultry flocks, but solid scientific evidence about the real efficacy of these measures is often lacking or has not been quantified. Different poultry systems such as free range poultry provide challenges for biosecurity, and there is a need for protection against direct and indirect virus introduction. Even though the latest EU outbreak only involved indoor poultry farms this observation might be biased as for instance migratory routes in the Netherlands are mainly situated in the western part of the country where there are no outdoor poultry farms. The question remains how to better protect free-range farms and to determine the contribution of poultry trade versus migratory birds to AI epidemiology. The current EU AI surveillance strategy has been in place since 2003 and is based on a risk-based approach. It is fit for purpose and intended to be affordable for MS. Effective surveillance requires clearly defined objectives and should help inform risk managers in order to trigger veterinary action. It should further be appropriate to the type of species and poultry production systems involved. EFSA recommended both active and passive surveillance of wild birds (EFSA, 2014). EU legislation allowing for both emergency and preventive vaccination has been in place since The decision to vaccinate lies with the individual MSs. Vaccination must be accompanied by appropriate surveillance of vaccinated birds and control of their movements. However, currently there is very little use of vaccination in poultry and zoo birds in MSs. Many MSs do not consider having advantages in using emergency vaccination with the currently available vaccines mainly due to too the late onset of protection. Although an internationally recognised measure, vaccination does have implications for trade. In addition, within the animal/human influenza interface, in the event of outbreaks on holdings keeping pigs and poultry legislation requires testing of pigs and other mammals as appropriate. Here, the EU Reference Laboratory for AI (EURL-AI) has an important role by reviewing the zoonotic aspects of animal influenza, monitoring the suitability of current protocols and the supply of antigen. It is fortunate that the EURL is also the OIE Reference Laboratory for swine influenza. When dealing with zoonotic influenza, cooperation between the European Centre for Disease Prevention and Control (ECDC) and EFSA is especially important ensuring that a One Health approach is taken for this zoonotic disease. 2. Pre-workshop preparatory work Pre-workshop preparatory work focused on mapping animal influenza EU-funded projects conducted in the last 10 years. Information from documents provided by the project coordinators was extracted by a contractor. This information was then summarised by four reviewing experts with expertise but who have not been involved in AI during the last 10 years. Particular attention was given to four strategic areas of animal influenza research: (a) host-pathogen interaction, (b) diagnosis, (c) surveillance and (d) prevention and control. In addition, outputs from previous workshops organized in 2014 by the United States Department of Agriculture (USDA, 2014) and by OFFLU-STAR-IDAZ (2014) were summarised. The findings were used to create an overview of the current understanding of animal influenza and elaborate a preliminary list of knowledge gaps and possible research subjects for animal influenza within the EU. All the collected information was shared with the workshop participants before the workshop itself and briefly presented during the workshop. Group discussions and breakout sessions took place to identify and analyse the most knowledge gaps and possible research subjects on animal influenza within the European context. EFSA supporting publication 2015:EN

9 Human Pig Poultry Other species Host-pathogen interaction Diagnostics Surveillance and risk assessment Prevention and control Workshop on animal influenza Twenty-two EU-funded projects with at least one component on animal influenza were selected. Information was obtained for 20 of these projects (Table 1). Table 1: Mapping of species and areas covered by projects 14 with at least one component on animal influenza and funded by the EU in the last 10 years, in alphabetic order. Project Species covered by the project Area covered by the project ANIHWA No No Yes No High High Not Not AIV VACC No No Yes No Not High Not High DIAGNOSIS AVIFLU No No Yes No High High Not High ConFluTech No Yes Yes Yes Not Medium Medium Medium EPIZONE Yes Yes Yes Yes Not Low Low Low ESNIP2 No Yes No No Medium High High Not ESNIP3 No Yes No No Not High High Not FLUAID No Yes Yes No High High Not High FLU-LAB-NET No No Yes Yes Not High High Not FLUPATH No Yes Yes Yes High Not Not Not FLUPIG No Yes No No High Not Not Medium FLURESIST No No Yes No Medium Not Medium Medium FLURISK Yes Yes Yes Yes Not Not High Not FLUTEST No No Yes Yes Not High Medium Not FLUTRAIN No Yes Yes Yes Not High Not Not HEALTHY No information submitted for this exercise POULTRY INN-FLU No No No Yes High Low Not Not 14 For background information on these or other EU-funded projects please visit the Community Research and Development Information Service (CORDIS) at: EFSA supporting publication 2015:EN

10 Human Pig Poultry Other species Host-pathogen interaction Diagnostics Surveillance and risk assessment Prevention and control Workshop on animal influenza Project Species covered by the project Area covered by the project LAB-ON-SITE Yes Yes Yes Yes Not High Not Not NEW-FLUBIRD No No No Yes Medium Medium High Not NOVADUCK No No No Yes Not Not Not High RISKSUR Yes Yes Yes Yes Not Not High Not RIVERS No information submitted for this exercise The documents available for mapping were primarily reports, scientific articles and workshop outcomes provided by the project coordinators. For some projects, only high level summary information was provided whereas very detailed information was provided for other projects. Twelve projects encompassed pigs in their research. Poultry were the species targeted by most of the analysed projects since 18 projects focussed on chickens and/or ducks, and/or turkeys. Nine projects covered more than one species, five of which including humans. Research areas covered by the projects were: 12 projects involved diagnostics, nine involved surveillance and risk assessment, seven involved prevention and control and three involved hostpathogen interactions. A scoring system (high, medium, low or not ) was used to present the relevance of each project to the four strategic areas. Cross-cutting activities, embracing more than one strategic area of research, were carried out by seven projects while the remainder focussed on one area of relevance. Figure 1 shows the mapped projects according to: a) the species covered by the research activities on animals and/or humans, and b) the project timeframe. In addition DISCONTOOLS and STAR-IDAZ are coordination actions focussed respectively on disease prioritization and gap analyses from a mostly animal health industry perspective, and for coordination of research funding at global level. DISCONTOOLS performed an evaluation on avian and swine influenza and STAR-IDAZ coorganised with OIE the OFFLU workshop in Spring A few projects and specific topics were selected for presentation during the workshop (see presentations 15 ) last accessed 16 March 2015 EFSA supporting publication 2015:EN

11 Figure 1: Project mapping in time and coverage of animal (presented as blue a ) or human aspects (presented as red h ) of influenza. Several knowledge and data gaps (Appendix A) as well as potential future research subjects (Appendix B) for animal influenza identified in previous workshops and some EU-funded projects, were listed within the four main headings. From these, short lists were extracted by four reviewing experts and presented during the workshop (see presentations). The recurrent issue identified from the projects was the need for better integration; for example, through the integration of animal influenza activities such as surveillance and risk assessment, together with consideration of the different aspects of the transmission continuum and integration across animal species. There is a critical need for research programs to take a holistic or systems approach, integrating for example molecular epidemiology with basic research in predictive biology. Taking a holistic One Health approach to animal influenza is therefore vital. 3. Expert workshop on research gap analysis on Animal Influenza 3.1. General overview of knowledge and data gaps The presentations of selected EU projects and topics were used as a basis for discussions on the current understanding of animal influenza within the four central themes: host-pathogen interaction, diagnosis, surveillance and prevention and control. The short lists of knowledge and data gaps (see below) were presented and reviewed according to the opinion of the participating experts. EFSA supporting publication 2015:EN

12 Host-pathogen interactions Workshop on animal influenza Specific knowledge about animal influenza viruses and their interaction with the host contributes to the understanding of disease pathogenesis in the individual host and virus transmission in the population. The physical and biological properties of a pathogen are one of three fundamental elements of the epidemiological triad, which also includes the host and the environment. Understanding these processes is critical for the design of diagnostic and intervention tools aimed at infection control and eradication. Gaps exist in the knowledge of many areas of influenza virus pathogenesis. For instance, although the role of the hemagglutinin (HA) protein in the pathogenesis of HPAI viruses is well characterized (Mair et al., 2014), the role of other determinant(s) contributing to virulence is not fully understood (e.g. INN-FLU). Endocytosis and efficiency of membrane fusion and viral replication were mentioned to be influenced also by many cell-specific factors and hence affect the host-pathogen interaction. Moreover, the proteome of influenza A viruses turns out to be much more complex than thought since influenza A viruses have evolved different mechanisms of non-canonical translation (e.g. leaky ribosomal scanning and alternative open reading frames) (Vasin et al., 2014). The function of the novel, probably accessory, proteins and their contributions to the pathogenesis of influenza are still yet to be determined. There is a great need for basic understanding of the molecular pathogenesis of the virus in different animal species, including differences in binding motifs of hemagglutinin genes and optimization of polymerase complex for adaptation to individual species or families of animals, and the changes needed to move across different animal species or families. Data have been produced describing the interaction of influenza viruses with host cells (e.g. FLUAID). Further identifying the molecular determinants of tissue tropism is required as well as relating these to infectivity and pathophysiologic changes. Beyond the characterization of receptor specificity for different moieties of sialic acid, which is not completely definitive, there is little known about the determinants of tissue specificity within a host. Experimental infections of key bird species has allowed the identification of species that could act as spreaders (e.g. NEW FLUBIRD, INN-FLU). However, the capacity of an AIV to infect pigs under experimental conditions may not be sufficient to allow transmission to other pigs or ferrets (an animal model for human influenza infections). The transmission mechanisms between hosts are poorly understood and remain a fundamental knowledge gaps. Although lot of studies have generated data on transmission in bird populations (e.g. AVIFLU) and transmission from wild birds to domestic poultry (e.g. INN-FLU) or from avian to pigs and subsequently to humans (e.g. ESNIP2, FLUPIG and FLUPATH), pathways of interactions between species still need further investigations. The monitoring of contact frequencies between wild and captive birds showed that the numbers of contacts are limited and occur at certain times of the year. Tracking of shelducks (INN-FLU) revealed common flyways but very different behavior in places visited at geographical and habitat typology level. It illustrates how difficult it is to generalize migration strategies of wild birds and to speculate on places visited. Analysis of virus survival in the environment, fresh poultry commodities and litter is very difficult to undertake (e.g. FLURESIST) but is required to generate data that are very for risk assessment, risk management and understanding influenza virus ecology. The role of pigs in the generation of pandemic viruses has been investigated (e.g. FLUPIG) and genetic markers characterizing the pandemic potential of an influenza virus have been analysed. Nevertheless the factors that drive the emergence and the presence of these genetic markers are not EFSA supporting publication 2015:EN

13 well known. Also the identification of genetic markers that determine the pathogenicity of HPAI for ducks requires further characterization. The mechanism of immunological protection against influenza viruses, either through innate, adaptive or vaccine-induced responses, emphasizes the need for gaining a thorough understanding of the host immune response in order to advance more effective control strategies. Progress has been made in the analysis of both innate and acquired immune responses against influenza infections in different species (e.g. FLUAID; FLUPATH; Crisci et al., 2013), but there remain major gaps, for instance in understanding what constitutes an optimal acquired immune response for effective suppression of shedding to interrupt transmission. Differences in interferon production have been observed between ducks and chickens in different organs (e.g. FLUPATH, INN-FLU), but it is unknown if these responses are correlated with difference in clinical outcome. Adaptive immune responses are relatively late to limit virus replication and prevent severe pathology. It was therefore questioned whether prophylactic stimulation of the innate immune response could be an effective way of controlling virus replication. It is also not clear what the optimal balance is for cellular versus humoral, or systemic versus mucosal responses for an effective immune response. More knowledge is also required on the contribution of the immune response to pathogenesis and why some birds and mammals are resistant to HPAI, and others are highly susceptible. Inherent differences between field and laboratory settings have compromised the translation of scientific achievements from the laboratory into the field. Most research so far has been performed in vitro, and it has been found that in vivo and in vitro experimental results may not be consistent. This problem needs to be addressed in several areas of animal influenza research. Many questions remain across a broad range of aspects of influenza virology. Crucial are issues of pathogenesis, tissue tropism and transmission both among hosts of a particular species and between hosts of different species. In reviewing the state of scientific knowledge of animal influenza viruses, the international community noted that the fundamental understanding of drivers of virulence, hostrange and adaptation process to new host species, and transmission between animals of same or divergent species, and importantly between animals and humans, remains rudimentary (USDA, 2014). Priority data gaps identified by previous initiatives on host-pathogen interaction have been listed (Appendix A). As an output of the discussions, the participating experts agreed on the following general knowledge and data gaps in the area of host-pathogen interaction: a. Knowledge regarding virus-host interactions, and on factors that affect disease pathogenesis, to allow more effective infection control, such as: Genetic variations and phenotypic traits of the virus that determine virulence, host specificity and zoonotic potential. Mechanisms of virus adaptation to different host species. Factors that determine tissue tropism of the virus. Mechanisms of gene re-assortment between virus subtypes. Mechanisms of resistance in different host species. Existence of carrier states in certain species or immune compromised animals. Differences in pathogen interaction between field and laboratory studies (e.g. role of age component, co-infections) EFSA supporting publication 2015:EN

14 b. Understanding of virus-host interactions and factors that impact on transmissibility to allow more effective infection control, such as: Role of aerosol or neighbourhood transmission. Role of different host species in the genesis of new geno/phenotypes, persistence and spread of the virus Factors controlling inward and outward spread Pathways of interactions between different host species. Survival of influenza virus in different environments and their role in transmission. Reservoirs and sources of transmission of virus capable of infecting humans remain unknown. c. Understanding of sequence difference of virus-host interactions and factors that impact on disease (including immune responses) to allow more effective infection control, such as: Insufficient knowledge of many areas of influenza immunology, such as importance of mucosal versus systemic immunity, cellular versus humoral immunity in different species, or other/novel mechanisms. Duration of carriage of virus in immunosuppressed or partially immune animals or certain species, particularly during migration. Contribution of immune response to pathogenesis (vaccines) Diagnosis The EU AI Diagnostic Manual (Decision 2006/437/EC) describes the diagnostic methods for influenza in poultry, covers a wide range of scenarios from suspicion to freedom testing and closely relates to the OIE Terrestrial Manual for Diagnostic Tests. Because of the highly variable nature of influenza viruses, continued monitoring of tests for their ability to detect variants is necessary. The use of both a screening test and a confirmatory test is recommended in most cases. The diagnostic steps for confirmation of AI can follow the classical pathway (virus isolation in embryonated fowl eggs, serological characterization (HI) and pathotyping by animal experiment (IVPI)), the molecular pathway (M-gene PCR, HA-gene PCR and pathotyping by sequencing the HA-cleavage site [H5 and H7 subtypes only]) or a combination of both. Similar diagnostic methods are available for influenza in swine (except for pathotyping). A lot of projects developed and validated diagnostic tools, mainly for avian species but also pigs (e.g. AIV VACC DIAGNOSIS, AVIFLU, LAB-ON-SITE, FLUAID and FLUTEST). Progress has been made to harmonize protocols amongst MS and beyond (e.g. ESNIP projects, FLUTRAIN, OFFLU- OIE manual). However, test methods such as HI are difficult to standardize and variation between laboratories can still be high. Proficiency tests are organized annually for AI NRLs by the EURL but there are no statutory activities ongoing for swine or equine influenza due to absence of legal requirements. Current tests for virus detection, usually PCRs, are generally sensitive, accurate and reliable, although new technology can always improve sensitivity and specificity. New methodologies became available in the recent years such as microarray applications, next-generation sequencing, alternative substrates to eggs, a range of ELISA s and the ability to define antigenic properties with cartography. Therefore, the EU AI Diagnostic Manual is reviewed, if needed. Integration of diagnostic methods, linking diagnostics with analysis e.g. for surveillance purposes, and further development of pen-side tests need to be stimulated to deliver test results in a timely and cost- EFSA supporting publication 2015:EN

15 effective manner. The characteristics of currently available penside tests are inferior to other diagnostic methods available in Europe at the moment, but these tests are already useful in areas with a less developed lab infrastructure. Another key animal influenza diagnostic gap is in subtype identification of virus and in the identification of the subtype specificity of sera. Better serological tests are needed, both to determine the subtype specificity of antibody (i.e., what subtypes has an animal been infected with) and to characterize the antigenic differences among animal influenza isolates. Characterization of the antibody response and antigenic differences among animal influenza isolates are critical for updating vaccines and evaluating vaccine-induced protection. Hemagglutination inhibition (HI) assay is the current standard for identifying the subtype specificity of sera and to characterize antigenic differences. This is a cumbersome test that lacks precision. Next generation sequencing provides faster and more detailed sequence information. Datasets become increasingly large and much more information becomes available. The phylogenetic analysis becomes more complex and increases difficult computational demands and bioinformatic skills. Furthermore, these molecular techniques do not provide accurate info on the antigenic characteristics of an influenza virus. Characterization of viruses requires isolation of the virus, as time consuming egg inoculation techniques are used. The availability of this technique is restricted to some laboratories and requires availability of a source of embryonated chicken SPF eggs. This is the reason for the need to generate highly permissive cell lines to cultivate the virus. Data sharing and data analysis has considerably improved over the last decade via the implementation of communication networks via internet (e.g. FluLabNet), sequence deposition in GISAID, rapid virus sharing and collaboration with EU institutions (e.g. ECDC and EFSA). Technological developments in mobile telecommunication should be exploited to facilitate real-time data transfer in the near future. OFFLU initiatives actively coordinate knowledge sharing at a global level (e.g. subgroup range of technical and review activities, interaction with public health via VCM, H5 evolution working group). However, coordinated structures are mainly in place for AI and less for swine and equine influenza. Another issue raised by the participants of the workshop is that databases and networks generated by projects are often not maintained when the project ends. It was felt that there is a need to integrate existing databases and maintain them, instead of developing new ones. Some obstacles in enhancing diagnostics are process based, for example sample collection in a medium that does not require the cold chain would be beneficial for virus isolation, which is frequently used to confirm the results of molecular tests (which do not require viable virus). In addition and importantly, influenza virus isolates are necessary to characterize the biology and genetics of new isolates. A need to analyse virus isolates in different species has been highlighted. Further, it was highlighted that current pathotyping tests are for galliformes but not necessarily for anseriformes. The participants of the workshop agreed that besides some technological improvements on diagnostic methods, there is a strong need to reduce the time between first detection of clinical signs at a farm and delivery of samples to a lab. Farmers and veterinarians attitudes towards reporting AI suspect cases contribute to this issue. Involvement of experts from social sciences will be required to make progress on this aspect of influenza detection. As an output of the discussions, the participating experts agreed on the following general knowledge and data gaps in the area of diagnostics: Integrated and multiplexed rapid molecular tests (both pen-side and high-throughput lab tests) to detect and characterize all influenza viruses timely and cost effectively. Better serological tests to determine the subtype specificity of antibodies (i.e. to identify what subtype an animal has been infected with) and to determine antigenic characteristics of EFSA supporting publication 2015:EN

16 influenza viruses for improved evaluation of cross reaction/protection between viruses/vaccines. Improve virus recovery methods relating to sample sources, quality and virus isolation Integration of diagnostic methodologies with surveillance (real time data exchange) Integration of diagnostics with bioinformatic approaches to assist decision making Surveillance and Risk Assessment The ability to identify and characterize Influenza A viruses circulating in animals is a prerequisite for outbreak preparedness. Understanding the key determinants of influenza infection and transmission dynamics would help develop robust surveillance and control measures. Surveillance in animals, which per definition should aim to provide information for action, is, however, mostly driven by the objective of safeguarding livestock health and international trade. Surveillance activities therefore currently mainly target known viruses notifiable to the World Organisation for Animal Health (OIE), such as highly pathogenic influenza viruses and low pathogenic AIV of the H5 and H7 subtypes in poultry and equine influenza (EI), or H1N1pdm09, H1N2 or H3N2 viruses in swine. Molecular epidemiology has been used in the field of animal influenza (e.g. Bataille et al., 2011; Van Borm et al., 2014) and is nowadays bringing together phylogenetic data with information on space, time and host species (Alkhamis et al., 2013) to improve our understanding of outbreaks. Also mathematical models are increasingly regarded as an important tool for instance in evaluating the effectiveness of control measures or for the design of a surveillance program (e.g. FLUTEST; Daly et al., 2013). However, models are only as good as the assumptions made in constructing them. The better the data available to feed into models, the more reliable inferences will be. An early warning system for the threat posed to animal and human health by AIV in migratory birds has been established (e.g. NEW FLUBIRD). Since influenza virus strains can circulate in animals without causing clinical signals, surveillance that only focuses on clinically ill animals will likely not be sufficient. Three risk assessment frameworks were developed and the RISKSUR project just started with the aim to develop a design and evaluation framework for risk based surveillance systems. The FLURISK prototype influenza risk assessment framework (IRAF) model ranks animal viruses according to their potential to infect humans. Opportunity maps are generated to highlight high risk regions. This work shows the global need for increasing surveillance that targets potentially zoonotic influenza viruses in animal species. Due to the gaps in scientific knowledge and data availability, the model focused solely on AIV. The US Centres for Disease Control and Prevention (CDC) developed an influenza risk assessment tool (IRAT) to assess the potential pandemic risk posed by influenza A viruses that currently circulate in animals but not in humans, based on two different scenarios: emergence and public health impact. The real-time risk model emerging pandemic threats plus (EPT+) has been developed by FAO/USAID and identifies drivers of influenza emergence at livestock-wildlife-human interface. Integration of the IRAF, IRAT and EPT+ models is required as well as linking them with existing disease information systems (e.g. EMPRES-i). The establishment of scientific networks on epizootic viral diseases including influenza and capacity building disease monitoring and molecular epidemiology amongst MS and with international cooperation targeted countries further facilitated data collection and exchange (e.g. EPIZONE, ESNIP projects, ConFluTech). Gaps in the availability of comprehensive surveillance systems for animal influenza viruses exist, in addition to the limited understanding of virus evolution and population dynamics. Current surveillance of equine, swine and avian populations for influenza viruses lacks capability to provide a clear understanding of circulating viruses. There is a need for systematic analysis of field viruses, particularly those that display higher than usual levels of variation in their genetic and phenotypic traits. Furthermore the value of targeted (or risk-based) surveillance activities has been demonstrated EFSA supporting publication 2015:EN

17 for instance in backyard and free range systems or those with low biosecurity, husbandry production systems intermingled with human living spaces and locations where poorly implemented vaccination campaigns may encourage the evolution of escape mutants. In a survey of national animal influenza surveillance programmes, the capacity to detect influenza viruses in animals with pandemic and zoonotic potential has been assessed (Von Dobschuetz et al., 2014). It was found that most regions prioritized surveillance in domestic poultry over wild birds, with the exception of Europe and Oceania where both domestic and wild birds are given similar importance. Less than 1% of all reported surveillance components (in Europe, the Americas and Oceania) were specifically designed to identify influenza viruses in domestic pigs with a possible pandemic impact. Whereas AI is a notifiable disease, swine influenza is not. Pigs are considered reservoirs for older human (and avian) virus genes. Swine-adapted viruses only occasionally infect people: the incidence is unknown but infections in humans are mostly dead-end. The 2009 pandemic H1N1 virus is an exception. This virus was able to transmit from human to human although the underlying mechanism is largely unknown. There are distinct swine influenza lineages in North America, Europe and Asia, which also have regional differences. Surveillance of swine influenza is important to gain insights into the public health risk of influenza in swine. There are data about the sero-prevalence of various swine influenza viruses in Belgium, France, Italy, Spain and the United Kingdom, but there are no coordinated activities ongoing. Surveillance results were being reported at regular intervals in the majority of surveillance system components, ranging from quarterly to annual reporting intervals. In instances where animals tested positive, the public health sector was alerted for about half of the components targeting avian, swine, and pandemic influenza (61%, 51%, and 50%, respectively) (Von Dobschuetz et al., 2014). This indicates a need to further work towards a One Health approach for AI. Ongoing projects, such as PREDEMICS and ANTIGONE (both funded by the FP7 health programme) could make an important contribution to this. Sequencing activities were found to take place in about 25% of the surveillance components analysed and in 20% of the surveillance components (128/587) sequences would be submitted to one of the public genetic databases (GenBank (48/128), GISAID (11/128), IRD (4/128) or another public access database (65/128)). This illustrates that a significant improvement in data sharing has taken place in the last decade but further efforts are required. Epidemiological data in public sequence databases should be available together with information on temporal, demographic and geographic processes of viral spread and knowledge on the intra-host and outbreak dynamics of mutation occurrence to enhance the surveillance of the virus. Further improvement in data collection and data sharing will require involvement of social scientists to ensure and increase engagement of all stakeholders. The participants indicated an overall need for: Efficient sampling methods; Rapid detection of emerging new strains; Identifying highly pathogenic influenza virus in animals and influenza viruses with zoonotic infection and/or pandemic potential, Assure support from stakeholders for monitoring or surveillance activities (social science), Infrastructure funding potentially necessary, Open data access, Scope for research projects beyond EU, global approach needed for research projects, Virus sample banks linked with databases must be maintained. As an output of the discussions, the participating experts agreed on the following general knowledge and data gaps in the area of surveillance and risk assessment : EFSA supporting publication 2015:EN