BACKGROUND DOCUMENT STREAM GLOBAL INFLUENZA PROGRAMME

WHO 1 PUBLIC HEALTH RESEARCH

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s d AGENDA FOR d v a e n INFLUENZA e c n e h s lt c a ie e nc c h e li to a pub ddress unmet

REDUCING RISK Reducing the risk of emergence

2017 UPDATE of a pandemic influenza WHO/WHE/IHM/GIP/2017.4 © World Health Organization 2017

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WHO Public Health Research Agenda for Influenza 2017 Update Contents Abbreviations 1.0 INTRODUCTION 1 Substream 1.1 FACTORS ASSOCIATED WITH THE EMERGENCE OF INFLUENZA VIRUSES WITH ZOONOTIC OR PANDEMIC POTENTIAL 3 RESEARCH RECOMMENDATION 1.1.1 Major progress and observations 1.1.1 Unmet public health needs and remaining knowledge gaps 1.1.1 RESEARCH RECOMMENDATION 1.1.2 Major progress and observations 1.1.2 Unmet public health needs and remaining knowledge gaps 1.1.2 RESEARCH RECOMMENDATION 1.1.3 Major progress 1.1.3 Unmet public health needs and remaining knowledge 1.1.3

Substream 1.2 FACTORS ASSOCIATED WITH HUMAN INFECTION AT THE HUMAN–ANIMAL INTERFACE 7

RESEARCH RECOMMENDATION 1.2.1 Major progress and observations 1.2.1 Unmet public health needs and remaining knowledge gaps 1.2.1 RESEARCH RECOMMENDATION 1.2.2 Major progress and observations 1.2.2 Unmet public health needs and remaining knowledge gaps 1.2.2 RESEARCH RECOMMENDATION 1.2.3 Major progress 1.2.3 Unmet public health needs and remaining knowledge 1.2.3

Substream 1.3 SURVEILLANCE AT THE HUMAN–ANIMAL INTERFACE 10 RESEARCH RECOMMENDATION 1.3.1 Major progress 1.3.1 Unmet public health needs and remaining knowledge gaps 1.3.1 RESEARCH RECOMMENDATION 1.3.2 Major progress 1.3.2 Unmet public health needs and remaining knowledge gaps 1.3.2 RESEARCH RECOMMENDATION 1.3.3 Major progress 1.3.3 Unmet public health needs and remaining knowledge 1.3.3 RESEARCH RECOMMENDATION 1.3.4 Major progress – Research recommendation 1.3.4 Unmet public health needs and remaining knowledge 1.3.4

Substream 1.4 PREVENTIVE MEASURES TO REDUCE THE RISK OF EMERGENCE OF ZOONOTIC AND PANDEMIC INFLUENZA VIRUSES 14

RESEARCH RECOMMENDATION 1.4.1 Major progress 1.4.1 Unmet public health needs and remaining knowledge gaps 1.4.1 RESEARCH RECOMMENDATION 1.4.2 Major progress 1.4.2 Unmet public health needs and remaining knowledge gaps 1.4.2 RESEARCH RECOMMENDATION 1.4.3 Major progress 1.4.3 Unmet public health needs and remaining knowledge 1.4.3 RESEARCH RECOMMENDATION 1.4.4 Major progress 1.4.4 Unmet public health needs and remaining knowledge 1.4.4 REFERENCES 19 Abbrevations AI Avian Influenza AA Amino Acids CDC Centers for Disease Control and Prevention CEIRS Centers of Excellence for Influenza Research and Surveillance EPT Emerging Pandemic Threats FAO Food and Agriculture Organization of the United Nations HA Haemagglutinin IAVs Influenza A Viruses IFITM3 interferon-inducible transmembrane 3 IRC International Research Consortium BACKGROUND DOCUMENT LPMs Live Poultry Markets NA Neuraminidase STREAM NGS Next-Generation Sequencing REDUCING THE RISK OF EMERGENCE OF A PANDEMIC INFLUENZA 1 NS Non-Structural OIE World Organization for Animal Health PA Polymerase Acidic PB2 Polymerase Basic 2 POC Point of Care rRT-PCR Real-Time Reverse Transcription Polymerase Chain Reaction SRA Strategic Research Agenda USAID United States Agency for International Development USDA United States Department of Agriculture WHO World Health Organization STREAM 1. Introduction 1

The current review of the WHO Research Agenda Stream 1 relating to the risk of emergence of a pandemic influenza from an animal reservoir was conducted against a background of international activities that helped to inform the review. There is consistency among the priorities identified here and internationally.

The Global Strategic Alliances for the Coordination of Research on the Major Infectious Diseases of Animals and Zoonoses (STAR-IDAZ) has developed a strategic research agenda (SRA) (STAR-IDAZ, 2015) for the major infectious diseases of production animals including zoonoses. The SRA covers three main areas: structural and political aspects, technological developments, and specific topics or diseases. STAR-IDAZ funded the development of influenza-specific recommendations (OFFLU, 2014) that address surveillance and risk assessment, diagnostics development, prevention and control, vaccine development, host–pathogen interaction, and socioeconomics and policy.

The United States Department of Agriculture (USDA) Agricultural Research Service developed an animal influenza viruses gap analysis (USDA, 2014). The analysis identified the following priority areas: • understanding of viral evolution in animal populations; • viral pathogenesis; • understanding of transmission and epidemiology; and • development of improved countermeasures such as vaccines and diagnostics.

A similar European initiative is DISCONTOOLS (2016), which publishes gap assessments for both avian and swine influenza.

A new initiative is the STAR-IDAZ International Research Consortium (STAR-IDAZ, 2016), a consortium of research funders and programme owners that aims to maximize funding for coordinated animal health research. The deliverables include candidate vaccines, diagnostics, therapeutics and other animal health products, procedures, and key scientific information and tools to support risk analysis and disease control.

A bibliometrics study for the period 2006–2013 (Ducrot et al., 2015) analysed the research subjects and international collaborations in infectious diseases of production animals. For virology, 19% of publications were on the Orthomyxoviridae, increasing at the rate of 13% per annum – a growth of 170% over the study period. Although the study was too broadly based to identify work on influenza A viruses (IAVs) specifically, it indicated a surge in publications regarding the Orthomyxoviridae in farm animals over the period up to and including 2013.

Important research has been conducted into the molecular biology of IAV infections, especially how the virus–host interactions of zoonotic infections differ from those of seasonal influenza. Work is directed to understanding the molecular processes that result in some IAVs being usually confined to a single host type (avian or mammalian), and the changes in virus and the corresponding biology of the host that allow cross-species transmission. Research involves virus–host receptor interactions; virus replication within

1 host-cell cytoplasm and nucleus; and innate and adaptive host immunity to the virus, and the mechanisms that allow some viral strains to modulate this immunity.

Exposure to poultry is still the major source of human infection with avian influenza viruses, with many factors contributing to the risk. Similarly, close contact between pigs and people is required for human infection with IAVs of porcine origin, a situation that is more complex epidemiologically because infections can pass in each direction. Reassortant IAVs with genes from human seasonal influenza strains are frequently isolated from pigs.

IAVs of the H5 subtype are becoming more genetically complex, through both antigenic drift and reassortants. Some novel viruses (e.g. H5N6) have infected humans and caused severe disease, a disturbing new development. Poultry H5 viruses have infected wild birds, probably from a source in domestic water birds. Migratory birds have spread infections internationally and between continents. An H5N8 IAV detected in China in 2010 caused disease in poultry in South Korea in 2014, and was subsequently detected in Europe and North America (albeit of different lineages). An H5N8 virus is currently being spread across Europe in a second wave of infections. The H5 involved, which is of highly pathogenic avian influenza (HPAI) Clade 2.3.4.4, has sometimes subsequently reassorted with an N gene from an IAV circulating in wild birds in the destination countries. These complex new developments increase the risk of emergence of dangerous zoonotic viruses.

A major new pandemic influenza risk arose with the reporting of human infections with a new avian influenza A (H7N9) virus in China in March 2013. Again, the risk for human infection lies in close contact with infected poultry. Live poultry markets (LPMs) are frequently implicated as sources of human infection, but risks in raising village, rooftop or backyard poultry have also been identified.

There has been an increase in surveillance for IAVs in animals – especially in wild birds, poultry and pigs – facilitated by agencies funding work internationally and also expedited through negotiations with pig farmer organizations. Recent work highlighting the antigenic diversity of global swine influenza viruses stresses the urgent need to monitor these viruses closely. Further, as examples of reverse zoonoses from humans to swine continue to occur, so does the genetic diversity of circulating viruses. To keep pace, classification systems and database tools have been created. However, surveillance is still somewhat opportunistic, and provides patchy coverage rather than comprehensive global monitoring. IAV surveillance is not a routine feature of livestock farming. The animal health sector, through OFFLU – the research organization of the World Organisation for Animal Health/Food and Agriculture Organization of the United Nations (OIE/FAO) – contributes to the WHO vaccine antigen strain selection process. However, the value of this process is restricted by a reluctance of some countries to submit viruses internationally for analysis.

Some progress has been made in containing disease caused by H5 HPAI. However, current control practices in areas where zoonotic influenza viruses are endemic can only reduce, not eliminate, human exposures. There are no specific, coordinated measures in place for some viruses (e.g. H9N2 avian influenza or swine influenza). Zoonotic infections will continue unless different approaches are adopted.

In more affluent countries, disease control in poultry by stamping out (i.e. by culling) can result in an influenza-free poultry sector. However, such measures are disruptive, with potential animal welfare concerns, environmental issues and impacts on the food supply. Stamping out is

2 considered by some to be an outdated method of control for a disease with no carrier state (even if it is still effective). In less affluent countries, measures based on stamping out for avian influenza (AI) will only result in “case harvesting”, regardless of the incentives paid to farmers.

Vaccines help to reduce levels of disease and shedding of HPAI in poultry. Immunity in vaccinated populations is often suboptimal, and antigenic drift complicates vaccination. Farmers in high-risk areas want vaccine, but vaccines still require individual animal administration – a major drawback. Movement management remains weak in places where the virus is endemic. These issues are unlikely to be resolved in the foreseeable future. Some poultry farming systems (e.g. extensive duck production) facilitate transmission of influenza viruses. Well-managed chicken farms can remain IAV-free, except in high-risk areas, where even usually sound biosecurity measures can fail. IAV in pigs is a problem for many large-scale farms, with human–animal and animal–human transmission increasing the challenge of establishing effective biosecurity.

LPM in well-resourced countries or regions can be managed to limit infection with IAV through the use of measures based on strict source control, as is done for Hong Kong Special Administrative Region (SAR). Other supplementary measures (e.g. rest days) help but are secondary. Although a shift towards centralized slaughtering is occurring, popular demand for sale of live birds persists.

Behavioural change of all the people and agencies involved in animal product value chains has been difficult to achieve through the methods used so far. Research should focus on technically feasible solutions that are also economically profitable, socially acceptable and politically expedient. Where individual or farm-level profitability is problematic, research is required on measures that can be supported through market interventions and incentives and, where possible, government support of infrastructure through credits, grants and the provision of information. These interventions need to be assessed using scientific methods such as randomized control trials.

Substream 1.1 Factors associated with the emergence of influenza viruses with zoonotic or pandemic potential

Research recommendation 1.1.1 Investigate virus-specific factors associated with zoonotic and pandemic potential (e.g. infectivity, transmissibility and pathogenicity).

Major progress/observations 1.1.1 Since 2011, new zoonotic viruses have emerged (H7N9, H10N8 and H5N6) (Belser et al., 2016; Joseph et al., 2016; Neumann & Kawaoka, 2015; Richard & Fouchier, 2016; Sun & Liu, 2015; Zhang et al., 2014). Phylogenetic analyses show a natural history associated with enzootic viruses of poultry in Asia, particularly reassortment with H9N2 viruses; for example: • there is increased evidence that transmission and pathogenicity do not necessarily share the same loci (Imai et al., 2012; Linster et al., 2014; Obadan et al., 2015); • zoonotic viruses (H5, H7 and H9 IAV) show a preference for α2,6 sialic acid receptors; and • some of these zoonotic viruses (H5 and H9) appear to require little or no adaptation for airborne transmission in mammals (Gao et al., 2009; Imai et al., 2013; Kimble et al., 2011; Sorrell et al., 2009; Wan et al., 2008). 3 Two independent studies showed that airborne transmission in ferrets of two different H5N1 viruses is linked to improved α2,6 sialic acid binding (Q226L or G228S) and loss of a glycosylation site at the 158–160 sequon (Herfst et al., 2012; Imai et al., 2012). Additional mutations in haemagglutinin (HA) were identified – H110Y (Linster et al., 2014) and T318I (Imai et al., 2012) – that decrease the threshold pH at which membrane fusion is achieved by HA, correlated with increased thermostability. Zhang et al. (2013b) showed that several H5N1 reassortants (with at least the polymerase acidic [PA] and non-structural [NS] segments from a pH1N1 virus) were transmitted via the airborne route between guinea pigs, without the need for mammalian adaptation substitutions. Chen et al. (2012) showed that an H5N1 reassortant virus containing HA with substitutions Q196R, Q226S and G228S, and the neuraminidase (NA) gene of a human H3N2 virus, was transmitted via the airborne route between ferrets. • In general, H7 subtype avian influenza viruses, regardless of pathotype and including the Asian H7N9, can be efficiently transmitted by contact in the ferret model, but they are not readily transmissible via the airborne route (Belser et al., 2016; Belser et al., 2013; Gabbard et al., 2014; Richard et al., 2013; Zhu et al., 2013a). A switch in receptor specificity was not required for airborne transmission of an HPAI virus H7N1, although the virus did not transmit as well as human influenza viruses do in this model (Sutton et al., 2014). • Changes in polymerase basic 2 (PB2) at positions 271, 594, 627 and 701 have been observed, associated with mammalian adaptation (Sun & Liu, 2015). The E627K is the best-characterized mutation and is the one that shows a clear effect on modulating airborne transmission in mammals. • The matrix segment of pH1N1 favours airborne transmission of pH1N1 and IAV in pigs, with the triple reassortant gene cassette (Lakdawala et al., 2011). • IAV have emerged in dogs (H3N2, H3N8) and seals (H3N8); they may have zoonotic potential based on receptor binding and transmission in ferrets (Karlsson et al., 2014; Kim et al., 2013; Lyoo et al., 2015; Lyoo et al., 2016). • New type A influenza viruses in fruit bats have been identified that are unique (Mehle, 2014). Some level of compatibility between the internal gene constellation of bat flu-A and other flu-A viruses has been observed, as well as anti-interferon (anti-IFN) attributes in the NS1 protein of the bat flu-A that are similar to what has been described for other flu-A viruses (Ma, García-Sastre & Schwemmle, 2015; Mehle, 2014; Turkington et al., 2015; Zhao et al., 2016b)

Unmet public health needs and remaining knowledge gaps 1.1.1 • Studies to establish the flexibility of amino acids in the receptor-binding site and how sialic acid binding is affected by those amino acids will allow more precise risk assessments and will increase the “predictive value” regarding viruses identified through surveillance. • Further understanding of genetic changes associated with zoonotic or pandemic potential is needed in order to better refine risk assessments. Studies should not be limited to amino acid changes in the virus, but should also investigate virus–host interactions at the protein and nucleic acid levels, and regulation of virus replication. • In the United States of America (USA), new regulations on the so-called “gain-of-function research of concern”1 may impede research into the mechanisms controlling airborne transmission of zoonotic influenza viruses. Alternative systems are needed to address the impact of mammalian-adaptive changes in avian influenza viruses, and their impact on transmission and pathogenesis.

1 Gain-of-function studies, or research that improves the ability of a pathogen to cause disease, help to define the fundamental nature of human–pathogen interactions, making it possible to assess the pandemic potential of emerging infectious agents, informing public health and preparedness efforts, and furthering medical countermeasure development (US Department of Health and Human Services, 2014). 4 Assessment of research recommendation 1.1.1 The recommendation should be retained.

Research recommendation 1.1.2 Assess the animal host-specific factors associated with zoonotic and pandemic potential (e.g. infectivity, transmissibility and pathogenicity).

Major progress and observations 1.1.2 The respiratory tissues of ferrets express both a2–3- and a2–6-linked sialic acids; they also express the Sda epitope (NeuAcalpha2–3(GalNAcbeta1–4)Galbeta1–4GlcNAc) and sialylated N,N’-diacetyllactosamine (NeuAcalpha2–6GalNAcbeta1–4GlcNAc), which have not been observed in human respiratory surface epithelium. The Sda epitope reduces potential binding sites for avian viruses and thus may have implications for the usefulness of the ferret in IAV studies (Jia et al., 2014).

Information between 2009 and 2011 NS1 controls RIG-I activation by binding to TRIM25 and thus inhibiting IFN-beta activation (Gack et al., 2009; Rajsbaum et al., 2012). Some human and avian influenza viruses control the expression of induced genes in infected cells, including IFN-beta, by binding to the host-cell factor CPSF30 (cleavage and polyadenylation specific factor 4, 30 kDa subunit), and inhibiting the processing and nuclear export of host mRNAs (Ayllon et al., 2014; Engel, 2013; Ramos et al., 2013).

New information A species-specific difference in host protein ANP32A accounts for the suboptimal function of AI polymerase in mammalian cells. Avian ANP32A (also known as pp32) has an extra 33 amino acids between the leucine-rich repeats and the C-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of AI polymerase, returning it to levels similar to those of the mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, but its insertion into human ANP32A or ANP32B supported AI polymerase function (Long et al., 2016).

Host factors pp32 and APRIL interact with a free form of the viral polymerase complex contributing to primer-independent synthesis of cRNA from the vRNA template (Sugiyama et al., 2015).

Knockdown of DDX17 in human cells inhibited a human-adapted (627K) PB2 polymerase but increased the avian-adapted PB2 627E counterpart. The chicken DDX17 homologue was required for efficient avian (627E) and human (627K) virus infection in chicken cells (Bortz et al., 2011).

A proteomic analysis of host proteins interacting with polymerase subunits identified more than 300 human proteins that bound to PA alone, which might explain why a complex combination of amino acid signatures modulates PA activity in human cells (compared with avian cells). A second viral protein generated from mRNA of segment 3 was discovered; termed PA-X, it comprises the first 191 amino acids containing the endonuclease domain of PA, but then through a frameshift gains a novel C terminus with a unique sequence of 61 amino acids. PA-X may control host gene expression by destroying host mRNAs, but its role in zoonotic viruses is not well characterized (Bortz et al., 2011).

5 PB1-F2 localizes to the mitochondria and interacts with mitochondrial antiviral signalling protein (MAVS) to inhibit IFN induction. A single amino acid substitution (N66S) in PB1-F2 increases the virulence of an H5N1 virus as well as the 1918 pandemic virus in mice (Varga et al., 2012; Varga et al., 2011). PB1-F2 is required for prolonged shedding of virus in ducks but has little effect on virulence in avian species (Schmolke et al., 2011). In pigs, PB1-F2 effects appear to be strain specific, and this is probably the case with respect to other hosts (Pena et al., 2012a; Pena et al., 2012b).

The NP N319K mutation has been shown to increase avian viral replication in mammalian cells by enhancing the interaction with importin-alpha isoforms (Gabriel, Herwig & Klenk, 2008). Structural components (P-complex) of IAV have been shown to interact with RIG-I (Li et al., 2014b).

Unmet public health needs and remaining knowledge gaps 1.1.2 • Incomplete understanding of host–virus interactions and how host factors modulate the transmission of influenza viruses in birds and mammals. • Zoonotic IAVs have emerged, mostly in poultry and pigs, but host–virus interactions in each remain largely unexplored, particularly in pig cells.

Assessment of research recommendation 1.1.2 The recommendation should be retained

Research recommendation 1.1.3 Study the environmental and animal management or husbandry-specific factors associated with zoonotic and pandemic potential (e.g. infectivity, transmissibility).

Major progress 1.1.3 Most epidemiological studies identified risk factors for the transmission of animal IAVs among host animals, including spread to other farms or premises and the environment, but not specifically to humans. Zoonotic influenza transmission was most frequently associated with direct contact with infected poultry or swine, but also with contaminated environments, and untreated or improperly disposed waste such as offal and manure.

Risk factors for zoonotic influenza transmission included: • failure of hand washing; • not wearing masks; • extensive pig–human contacts; • suboptimal biosecurity practices; • feeding and cleaning practices that exposed children to contact with infected poultry; • contamination of the close environment due to slaughtering poultry within homes; • lack of personal protective barriers; • poor waste management that exposed the communities to slaughter by-products and dead chickens; and • extensive and intimate human–animal interactions and exposure to slaughter of avian influenza infected poultry in LPMs.

6 Detection of airborne virus in LPMs and experimental transmission of H5N1 to ferrets by slaughter of poultry in LPMs suggest respiratory transmission from poultry to humans.

Risk factors for influenza transmission within agricultural systems include commingling of pigs and chickens, suboptimal biosecurity practices, using manure as field fertilizer, hiring boars from outside, trading activities, lack of education to small farmers on biosecurity practices, and improper disposal of dead poultry and faeces.

Unmet public health needs and remaining knowledge 1.1.3 • Few studies have examined the impact of individual biosecurity interventions on prevention of animal-to-human transmission in relation to environmental or husbandry practices. • There is a disconnect between public and animal health sectors in working in a One Health way to identify environmental and husbandry factors associated with zoonotic IAV transmission. Much work is public health centric, without adequate agricultural expertise on field and laboratory studies.

Assessment of research recommendation 1.1.3 There is overlap between the material reviewed for this recommendation and Recommendations 1.2.1 and 1.2.2, and the material reviewed under Substream 1.4. Consolidation of recommendations should be considered.

Substream 1.2 Factors associated with human infection at the human–animal interface

Research recommendation 1.2.1 Investigate potential modes of transmission for human infection with animal viruses.

Major progress 1.2.1 Studies have been conducted on avian H5, H7 and H9, and swine H1 and H3 virus subtypes, to investigate the incidence of zoonotic infections from occupational exposure (e.g. at farms, markets and slaughterhouses), among the general public (e.g. visitors to LPMs and fairs), and upon exposure to infected family members (contact-tracing activities).

Serological studies indicate that zoonotic infections may occur more frequently than the laboratory-confirmed cases reported to WHO imply (Khan et al., 2015; Wang et al., 2014; Yang et al., 2016). Interpretation of and comparison among serological studies has been problematic because of variability in assay methods and their standardization; issues include specificity, arbitrary cut-offs, cross-reactivity with human influenza and confirmatory testing (Khan et al., 2015; Sikkema et al., 2016).

Direct exposure to poultry and pigs remains a high risk factor for zoonotic IAV infection. Improperly managed LPMs are important, and interventions (e.g. closure, rest days and overnight cleaning) are proposed to reduce virus burden (Peiris et al., 2016; Zhu et al., 2016). The FAO has produced a market biosecurity guide (FAO, 2015).

7 Contact tracing of people with laboratory-confirmed infection with either H7N9 or H5N1 reveals plausible human-to-human transmission, but no evidence for sustained transmission (Aditama et al., 2011; Aditama et al., 2012). The studies suggested that familial or genetic factors contributed to the human-to-human virus transmission in Indonesia.

Concurrent infections with human and zoonotic IAVs have been reported (Li et al., 2014a; Zhang et al., 2015; Zhu et al., 2013b), including nosocomial transmission of a mix of human and avian viruses (Chen et al., 2016). However, the potential for reassortment and for increased pandemic risk remains unclear.

Although the exact routes of human infection with zoonotic IAVs remain unproven, both avian (Jonges et al., 2015; Zhou et al., 2016) and swine IAVs (Choi et al., 2015; Corzo et al., 2013) have been detected in air samples. Techniques used in human studies (Milton et al., 2013) may be applicable to zoonotic settings.

Unmet public health needs and remaining knowledge gaps 1.2.1 • Continued characterization of IAVs of poultry and pigs, which continue to evolve and emerge (e.g. H5Nx, H6N1, H10N8 and swine reassortants). • More precise identification of the main routes, risk factors and behaviours leading to zoonotic infections with avian or swine IAVs – standardized, quantitative tests are needed to measure infectious IAV in air. • Further study of reassortment between human and animal IAVs upon coinfection in humans or animals, and the associated pandemic risk. • Improved practical measures to reduce zoonoses at the human–animal interface (e.g. on farms, and at live poultry markets, slaughtering places and fairs). • Better definition of the at-risk human population, and development of fit-for-purpose mitigation strategies to reduce zoonotic infections. • Harmonization of serological tests among diagnostic and research laboratories in public health and animal health sectors.

Assessment of research recommendation 1.2.1 There is overlap between the material reviewed for this recommendation and Recommendations 1.1.3 and 1.2.2, and the material reviewed under Substream 1.4. Consolidation of recommendations should be considered.

Research recommendation 1.2.2 Study the role of human behavioural factors associated with infection by animal viruses.

Major progress 1.2.2 Human infection with zoonotic AI is related to poultry exposure, especially at LPMs, with limited human-to-human transmission. Closure of LPMs was reported to reduce the risk of H7N9 human infections by 97–99% (range 68–100%, across four cities). Similarly, occupational exposure to pigs has been suggested as a risk factor for human infection with pandemic H1N1.

A meta-analysis of studies of seroprevalence of influenza A (H9N2) in humans indicated that only a small proportion of exposed people have antibodies. Comparison among studies was hampered by lack of standardization of serological tests, including their positive–negative cut-off.

8 In Africa, risk factors for human infections with H5N1 included: • lack of: o compartmentalization; o hygiene; o training or education on basic preventive behaviours; o incentives to prevent contamination or disease dissemination; • dual use of the distribution system; • incentives to spread diseases by selling infected chickens; and • poor biosecurity and veterinary services, which increase the risk of contamination in the whole poultry products process and food chain.

Unmet public health needs and remaining knowledge gaps 1.2.2 • Specific risk factors, activities and behaviours that caused human infections with animal influenza viruses have not yet been fully elucidated, and the most challenging scientific questions (e.g. the actual modes of transmission from animals or the environments) remain unsolved. • Existing knowledge is not comprehensively applied in some outbreaks; translational research is required. • Better animal (or tissue or other) biological models for zoonotic influenza transmissibility would permit more detailed research to support management of the risk of cross-species transmission.

Assessment of research recommendation 1.2.2 There is overlap between the material reviewed for this recommendation and Recommendations 1.1.3 and 1.2.1, and the material reviewed under Substream 1.4. Consolidation of recommendations should be considered.

Research recommendation 1.2.3 Determine the genetic or other factors related to human susceptibility to infection with animal viruses.

Major progress 1.2.3 Most genetics studies have tried to associate human single nucleotide polymorphisms (SNPs) with disease severity, not necessarily with susceptibility. Nevertheless, the advances in this area are detailed below.

SNPs within interferon-induced transmembrane protein-3 (IFITM3) were shown to influence severity of influenza: • SNPs within IFITM3 were originally detected as a phenotype in knockout mice; • a specific SNP was validated in a cohort of severe human influenza cases – this minor IFITM3 allele (SNP rs12252-C), which alters a splice acceptor site, was enriched in hospitalized individuals; the minor CC genotype IFITM3 has reduced influenza virus restriction in vitro; • the minor allele is important in Han Chinese, being found in 69% of Chinese patients with severe pandemic influenza A H1N1/09 virus disease compared with 25% of those with mild infection; • subsequent unpublished results suggest that rs12252-C may not be the SNP responsible – another SNP in the promoter region may be the causative mutation; and

9 • in summary, support for rs12252 is largely restricted to Han Chinese, with significant evidence that rs12252 does not identify cases at risk of severe influenza illness in European populations.

Other genetic association studies have been done, but none have been validated to any degree. Genes associated include FCGR2A, RPAIN, C1QBP, CCR5, TNF, LTA, LGALS1, SFTPA2, SFTPB, TMPRSS2 and ST3GAL1.

A rare null mutation in IRF7 in a child was shown to be responsible for severe influenza. The child was unable to produce an interferon response.

Researchers created an algorithm to predict the strength of human leukocyte antigen (HLA) alleles to bind conserved viral sequences, and consequently the magnitude of the CD8-T cell response. In a small human cohort, the findings were validated; that is, there was a correlation between the magnitude of the CD8 response and the HLA allele.

Further refinement of the mode of action of the well-described restriction factor Mx1 identified critical residues and showed that the impact of Mx1 depends on the genetic context. Other genetic loci associated with susceptibility to influenza have been explored in various inbred mouse lines (e.g. BxD and collaborative cross).

Unmet public health needs and remaining knowledge gaps 1.2.3 • There are few studies in humans to address SNPs associated with susceptibility – more are needed. Limitations include cost, cohorts and sensitivities to genetic studies. The availability of full genomes (human) may accelerate these studies. • More relevant animal models are needed; murine models have not provided as much information as hoped. • Additional research is needed to address the levels of age-specific human population immunity to avian and swine influenza viruses, to better assess serological correlates of infection.

Assessment of research recommendation 1.2.3 The recommendation should be retained.

Substream 1.3 Surveillance at the human–animal interface

Research recommendation 1.3.1 Develop research strategies for joint animal and human health surveillance systems to monitor influenza viruses with zoonotic or pandemic potential in countries with varying capacity and resources.

Major progress 1.3.1 OFFLU continues to contribute animal health data (on epidemiological, antigenic and genetic characteristics of zoonotic influenza viruses) to the WHO process to identify candidate vaccine viruses for potential use in human vaccines (WHO, 2016). The Centers of Excellence for Influenza Research and Surveillance (CEIRS) network has also made significant contributions to global surveillance studies; these studies have provided insights into the natural history of IAVs in wild birds, virus ecology and viral hosts (CEIRS, 2016). 10 The virology and epidemiology of influenza infections in pigs has been extensively described (Nelson & Vincent, 2015; Nelson et al., 2016). Four-way linkage programmes between laboratory and field agencies from both public and animal health sectors have been initiated in Africa and South- East Asia.

Unmet public health needs and remaining knowledge gaps 1.3.1 • A more structured, comprehensive and geographically representative surveillance of farmed animal populations is needed.

Assessment of research recommendation 1.3.1 There is overlap in the material reviewed for this recommendation and Recommendation 1.3.3. Consolidation of recommendations should be considered.

Research recommendation 1.3.2 Develop diagnostic assays to support joint animal and human health surveillance systems to monitor influenza viruses with zoonotic or pandemic potential in countries with varying capaci- ty and resources.

Major progress 1.3.2 Development of diagnostic tools across virus subtypes and lineages has been advanced by national veterinary and public health laboratories, academia, OFFLU reference laboratories and WHO collaborating centres for influenza surveillance and research. These organizations continue to develop assays to improve sensitivity, specificity, speed and ease of use.

OFFLU laboratories continue to support the optimization and validation of real-time reverse transcription polymerase chain reaction (rRT-PCR) assays and other diagnostic tools and approaches for improved virus detection in wild birds and poultry (OFFLU, 2016). Subtype-specific advances were made in molecular assays to detect A(H7N9) virus in poultry, and the spread of A(H5N8) and A(H5N2) in Asia, the Americas and Europe (Bao et al., 2015a; Bevins et al., 2016; Heine et al., 2015; Hu et al., 2015). Advances were also made in the development of assays to detect novel or emerging threats, such as influenza D virus in swine and cattle (Chiapponi et al., 2016; Ferguson et al., 2015), and new lineages of swine influenza viruses (Lung et al., 2017).

There has also been development of multiplex, real-time TaqMan assays, microarrays and loop- mediated isothermal amplification (LAMP) assays for detection of avian, equine and swine influenza viruses (Bao et al., 2015b; Fereidouni & Globig, 2013; Lung et al., 2017; Okamatsu et al., 2016; Zhang et al., 2013a). Development of tests to detect emerging subtypes or lineages allowed rapid detection of and response to new threats. Detection of emerging viruses, such as influenza D or new lineages of A(H3N2) variant viruses, remains important for development of diagnostics (Bose et al., 2014; Monavari, Mollaie & Fazlalipour, 2014; White et al., 2016).

Next-generation sequencing (NGS) is increasingly being used as a universal tool for detection and surveillance of influenza virus in both human and animal specimens. A growing number of laboratories worldwide are exploring NGS for primary diagnosis owing to its sensitivity for samples with low viral RNA concentrations (Wang et al., 2015; Zhao et al., 2016a). NGS approaches have added capacity for metagenomics sequencing, allowing for characterization of multiple pathogens from a single specimen (Lysholm et al., 2012).

11 Point-of-care (POC) assays and their validation are growing areas of development and utility (Douthwaite et al., 2016; Song et al., 2012; Wang et al., 2015). Portability and use in field or clinical settings are important, but POC diagnostics have not been implemented in some critical settings; for example, in many hospitals and clinics, during field investigations and in developing countries (Abd El Wahed, Weidmann & Hufert, 2015; Tuttle et al., 2015).

Unmet public health needs and remaining knowledge gaps 1.3.2 • Further development of POC diagnostics with improved specificity is needed. Public health clinics and hospitals should compare more of their test results to laboratory-based (molecular) methods, to validate sensitivity and specificity. Animal health laboratories should use and validate more POC assays so that field and outbreak specimens can be more rapidly analysed. Field testing of rapid diagnostic or novel assays that use “field-ready” instrumentation is needed. • Additional work towards development and validation of NGS platforms for diagnostic applications is critical. • Additional multiplex assays that can detect all subtypes and major lineages are needed to improve surveillance capacity and rapid response.

Assessment of research recommendation 1.3.2 The recommendation should be retained.

Research recommendation 1.3.3 Perform operational research to evaluate and improve joint animal and human health surveillance systems to monitor influenza viruses with zoonotic or pandemic potential in countries with varying capacity and resources.

Major progress 1.3.3 OFFLU continues to develop systems for monitoring influenza viruses in animals and in people at the animal–human interface, with a focus on regional hotspots and host-specific surveillance (OFFLU, 2013; 2014). The CEIRS laboratories continue to advance surveillance in animal populations and in humans at high risk of exposure (CEIRS, 2016). The United States Agency for International Development (USAID) Emerging Pandemic Threats (EPT) programme has strengthened capacity to detect emergence in wildlife populations within geographical hotspots, and has improved diagnostic and laboratory capacity across human and domestic animal and livestock sectors (Dixon, Dar & Heymann, 2014). Additional risk assessment studies are ongoing to better understand the antigenic distance between human seasonal influenza viruses and circulating swine influenza strains, in order to assess potential pre-existing immunity in the population (Lewis et al., 2016).

Interagency collaboration and funding within national systems require systematic partnership, communication and benefit sharing to maximize outcomes. For example, the USDA and the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, have improved swine influenza surveillance in the USA through recognition of potential exposure risks, at-risk populations and emerging zoonotic viruses (USDA, 2010). WHO, in collaboration with FAO and OIE, continued to address the need for improved joint surveillance systems to monitor animal and human populations for zoonotic influenza (Forcella et al., 2015; Yang et al., 2016).

12 Four-way linking projects have promoted national frameworks for data sharing, risk assessment and risk communication among public and animal health laboratories and administrators. Such projects strengthen the capacity to detect, report and assess risks from emerging influenza viruses for both animal and human health sectors.

Development of host-specific strategies to monitor animals and at-risk human populations for influenza viruses (OFFLU, 2013) is critical. Strategic, active surveillance will provide baseline data about risks with certain animal–human interfaces. Targeted surveillance in animal hosts and populations to detect IAVs with pandemic and zoonotic potential has improved in some regions. A focus on these specific populations and interfaces will expand our capacity to detect and respond to emerging viruses (Nelson et al., 2016; Von Dobschuetz et al., 2015).

Unmet public health needs and remaining knowledge gaps 1.3.3 • Wildlife disease hotspots are an important knowledge gap. Surveillance that monitors the impact of IAVs on wild species, environmental transmission and seasonality due to migration are needed. • Global transmission of IAVs from humans to swine (i.e. reverse zoonosis) is being detected more frequently (Nelson & Vincent, 2015). Introductions and evolution of human-origin seasonal IAVs in pig populations will continue. Strategies to mitigate associated risks are needed; for example, by reviewing husbandry practices and personal protective equipment, monitoring for illness among workers and vaccinating workers. • Long-term support of joint animal–human health surveillance systems is needed to demonstrate their effectiveness in controlling disease in both sectors. Acceptance of One Health needs evidence of the socioeconomic value that the approach can provide (Vandersmissen & Welburn, 2014).

Assessment of research recommendation 1.3.3 There is overlap between the material reviewed for this recommendation and Recommendation 1.3.1. Consolidation of recommendations should be considered.

Research recommendation 1.3.4 Develop research to establish social, political, economic and legal strategies for wider (broader) animal influenza outbreak reporting.

Major progress – Research recommendation 1.3.4 • A report has demonstrated the strength of combining epidemiological and economic model analysis to gain insight into a range of consequences, and thus serving as a decision support tool outbreak response, albeit in resource-rich settings (Backer et al., 2015). • The OIE regularly reviews the OIE Terrestrial Animal Health Code that sets the standards to be followed regarding avian influenza in the conduct of international trade (OIE, 2016). • The OFFLU website maintains a record of the amino acid motifs observed in reported HPAI viruses. • Some OIE Member Countries have updated their national plans with specific policies on avian influenza (e.g. Australia and the USA). • In the USA, there has been a review of the statutes, regulations and online reportable animal disease lists from the 50 states and the District of Columbia, to describe the legal landscape of animal disease reporting (Allen, 2015).

13 • There is growing awareness among animal health providers of the importance of culturally sensitive dialogue to develop acceptable AI prevention and control options in developing countries (Daniels et al., 2014). • Questions have been raised about what is an appropriate level of surveillance in the intensive animal industries, how to fund such surveillance and how to respond to findings appropriately and cost-effectively (Daniels, 2014).

Unmet public health needs and remaining knowledge gaps 1.3.4 • Financial feasibility and social acceptability of control strategies need to be reviewed. • There is a need for further analysis of short- and long-term implications for export and aftermath costs of control strategies. • There is a need to establish fit-for-purpose criteria to better evaluate or test animals, to improve national and international import and export regulations. • Few studies have been undertaken in developing countries. The socioeconomic impacts of the ongoing AI outbreak in West Africa have yet to be reported or assessed. • Animal and zoonotic disease detection and reporting has been slow and ineffective, despite One Health initiatives and international obligations in less industrialized countries, and with respect to influenza in pigs. • There is a need for more research on coping strategies to reduce panic from animal disease threats.

Assessment of research recommendation 1.3.4 The outcomes expected from this recommendation should be retained. Remaining knowledge gaps include the appropriate tactics for social, political, economic and legal strategies. Such tactics will be needed if these strategies are to keep up with technology applications and the challenges inherent in implementing the One Health approach.

Substream 1.4 Preventive measures to reduce the risk of emergence of zoonotic and pandemic influenza viruses

Research recommendation 1.4.1 Investigate and develop animal intervention strategies (e.g. culling, vaccination and biosecurity) under different epidemiological and field conditions that can reduce risk of zoonotic infection.

Major progress 1.4.1 Vaccination Progress in vaccination includes: • reviews of existing and novel vaccines (Rahn et al., 2015; Spackman & Pantin-Jackwood, 2014); • assessments of vaccination in specific places: Viet Nam (Cuong et al., 2016) and Nepal (Karki et al., 2015); • papers on vector vaccines, subunit vaccines and novel vaccines for pigs (Borggren et al., 2016); • live virus vaccines (Gambaryan et al., 2016); • improved adjuvants, such as Toll-like receptor agonists and universal vaccines (Dabaghian et al., 2014);

14 • better understanding of why vaccination programmes “fail” (Swayne et al., 2015); • a prototype vaccine decision support tool (Castellan et al., 2014); and • a review of vaccination (Sims et al., 2016).

Stamping out Progress in stamping out influenza includes: • issues identified through field experiences (e.g. from the USA in 2014–2015, with 50.4 million head), such as the difficulty of rapid stamping out on large farms; • biotherapeutics for use in animals such as farmed poultry, such as defective interfering RNA (Smith et al., 2016) – there are examples from foot and mouth disease that might be applicable (Diaz-San Segundo et al., 2016); • genetic resistance in poultry – there has been limited progress in attempts to genetically enhance poultry (Blohm et al., 2016); • farm biosecurity – failures in USA outbreaks have been assessed, as has transmission by fomite and airborne spread (less important) (Torremorell et al., 2016); and • identification of the need for enhanced air treatment and faecal handling (Leibler et al., 2017), and the adoption of air treatment systems in some pig farms.

Unmet public health needs and remaining knowledge gaps 1.4.1 Stamping out of infections in poultry (culling and on-farm biocontainment) In relation to stamping out of infections in poultry, there is a need for: • alternatives to stamping out (e.g. live virus or vector vaccines, innate immune system stimulators and engineering methods to enhance biocontainment); • alternatives to the use of foam for killing of birds in cold conditions; • alternatives to shutting down ventilation for use on large farms, where the capacity for CO2 or foam has been exceeded or is impractical (e.g. for caged layers); and • ways to effectively contain or eliminate virus in resource-poor countries, including methods for humane killing of poultry in low-resource settings, and partial culls coupled with quarantine.

Vaccination of animals In relation to vaccination of animals, there is a need for: • vaccines for mass application to poultry (air, droplet, water); • better vaccines for ducks; • better vaccines for pigs, to avoid vaccine-enhanced respiratory disease; • vaccines that minimize or cope with antigenic variation (e.g. ancestral strain, multivalent, conserved dual arm, higher antigen payload and better adjuvants); and • optimization of approaches for testing of vaccine efficacy using experimental models and field studies; and identification of molecular markers of antigenicity and sequence-based approaches to optimize vaccine strain selection and antigenic characterization.

Farm biosecurity (bioexclusion) In relation to farm biosecurity, there is a need for: • practical, low-cost ways to improve bioexclusion on high-risk farms; • biosecurity practices that are practical and behaviourally acceptable in more traditional production systems, and an understanding of why recommended measures may not be adopted (i.e. the social acceptability of biosecurity measures); and • enhanced air treatment and handling of faeces.

15 Assessment of research recommendation 1.4.1 Research is still needed on improved interventions for control and prevention of zoonotic IAVs in animals.

Research recommendation 1.4.2 Develop human intervention strategies related to the animal–human interface (e.g. behaviour, legal approaches and biosecurity procedures) in different social and cultural contexts.

Major progress 1.4.2 • Various assessments have been made of biosecurity in developing countries. • A biosecurity guide to markets has been published (FAO, 2015). • Market upgrades have been attempted in various countries; for example, facilities have been improved, and work practices have sometimes been improved (in Indonesia and Viet Nam); however, reversion to earlier practices has sometimes been observed. • Healthy Livestock, Healthy Village programmes have been developed – for example, in Cambodia and Indonesia (Patrick et al., 2014) – and there have been various studies of market and value chains, with FAO publishing a guide to market chain studies (FAO, 2012).

Unmet public health needs and remaining knowledge gaps 1.4.2 If significant progress is to be made in preventing exposure to zoonotic influenza, the incremental approach used over the past 10 years will not be sufficient.All of the following social, economic and political aspects need to be included in research (with research on the management of the commons, as appropriate).

Stamping out of infections in poultry (culling and on-farm biocontainment) In relation to stamping out of infections in poultry, there is a need for: • ways to effectively contain or eliminate virus in resource-poor countries; • financing strategies that cover the value of the animals and compensate for the revenues lost where stamping out is used; and • ways to overcome international resistance to measures other than stamping out that may have equivalent effects in managing risks of onward viral transmission; there is also a need for associated tools for assessment and comparison of alternative methods. Vaccination of animals (and humans) In relation to vaccination, there is a need for: • efforts to better mitigate reverse zoonosis, to prevent establishment of seasonal influenza viruses in animal populations; • universal vaccines for humans in contact with swine and poultry; • systems that facilitate vaccination of target populations at village or commune level, as required; and • better understanding of the resistance to the use of vaccine in situations where H5 and H7 viruses are endemic and are unlikely to be eradicated. Market biosecurity and hygiene (for remaining markets) In relation to market by security and hygiene, there is a need for: • exploration of ways for orderly closure of improperly managed large LPMs (i.e. those with no source control and other supplementary measures) and traders’ yards; • better coordination between the civic agencies responsible for markets, slaughter and processing points, and veterinary authorities; and 16 • better methods to prevent airborne IAV infection of people in markets, including safe methods for processing of poultry in low-resource settings.

Movement management of livestock (cross border or farm to market) In relation to movement management of livestock, there is a need for: • better understanding of successful cooperatives that market products as “healthy and safe” (e.g. how do they ensure traceability, and what are the replicable factors for success?); • studies of social and economic networks of traders and transporters, their practices of selecting and managing animals, and their management of animals during outbreak periods – this information is needed to develop appropriate changes; • enhanced traceability of food production animals (large consignments); and • systems for cost-neutral legalizing illegal cross-border trade.

Assessment of research recommendation 1.4.2 Research is still needed on improved interventions for control and prevention of zoonotic influenza in animals and viral spillover to humans.

Research recommendation 1.4.3 Conduct operational research to integrate animal and human health strategies for prevention.

Major progress 1.4.3 • A market closure study in China reported a reduction in human H7N9 cases. • A value chain study in Indonesia showed consumer support for poultry products from farms that implemented appropriate biosecurity activities. • An ethnographic study in Bangladesh villages showed that knowledge dissemination failed to improve behaviour for safe poultry slaughtering. • Poultry in the Netherlands (and other countries) were kept indoors once H5N8 was detected in Europe (in November 2016). • Vaccine antigens for poultry in China, Indonesia and Hong Kong SAR have been updated – different systems were used, but all were based on identification of antigenic variants and appropriate changes to vaccine antigens. • Testing of poultry destined for Hong Kong SAR identified H7N9 positive consignments before the consignments could enter retail markets. • Information has been collected to justify targeting of vaccination (e.g. a greater focus on duck vaccination, and designation of not-to-be-vaccinated areas in China). • There has been increased enforcement against smuggling of poultry into Viet Nam, to prevent H7N9 virus from gaining entry to the country.

Unmet public health needs and remaining knowledge gaps 1.4.3 The unmet public health needs and remaining knowledge gaps for Recommendation 1.4.3 are the same as those for Recommendation 1.4.2 above.

Assessment of research recommendation 1.4.3 Research is still needed on improved interventions for control and prevention of zoonotic influenza in animals, and viral spillover to humans.

17 Research recommendation 1.4.4 Assess and evaluate the public health, social and other impacts of intervention strategies under different epidemiological and field conditions to optimize their effectiveness.

Major progress 1.4.4 • The main public health impact of existing control and preventive measures is that virus continues to circulate and evolve, albeit at levels that are not resulting in many cases of disease in humans. • Any measure that reduces exposure of humans to zoonotic influenza viruses is expected to have a positive effect on public health; however, given the relatively low number of cases, the effect has been difficult to quantitate. • The World Bank prepared documents that discuss the cost of emerging diseases, especially pandemic influenza. • Work at the human–animal interface through the USAID EPT-2 programme includes IAVs, but few assessments have been made of the benefits of early warning. 2 • Social impacts of intervention strategies: there have been some studies on the social impacts of measures such as the shift to larger intensive farms, poultry production clusters, changes to marketing practices such as consumers’ desire for live poultry sales, and acceptance by traders of measures such as rest days.

Unmet public health needs and remaining knowledge gaps 1.4.4 • Vaccination cost studies covering all aspects of delivery and also cost–effectiveness studies, recognizing separately the benefit to the farmer and the benefit to society through reduced risk of zoonotic influenza. • Recognition that vaccination will rarely be done well in less affluent value chain settings, and hence that alternative interventions are needed. • Research is needed on the acceptability, cost and barriers to uptake of any proposed improved intervention for control of zoonotic influenza.

Assessment of research recommendation 1.4.4 Research is still needed on improved interventions for control and prevention of zoonotic influenza in animals, and viral spillover to humans.

2 An example would be early warning of H5N8 by FAO in September 2016 – to what extent did this early warning reduce risk? It did not have any effect in India, but might have had some effect in Europe, making it possible to respond rapidly when the virus arrived. However, no specific action was taken by most countries until the virus was detected.

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