Consequences and global risks of highly pathogenic avian influenza outbreaks in poultry in the United Kingdom

Wild birds in the Orders Anseriformes and Charadriiformes are the natural reservoirs for avian influenza (AI) viruses. Although they are often infected with multiple AI viruses, the significance and extent of acquired immunity in these populations is not understood. Pre-existing immunity to AI virus has been shown to modulate the outcome of a highly pathogenic avian influenza (HPAI) virus infection in multiple domestic avian species, but few studies have addressed this effect in wild birds. In this study, the effect of pre-exposure to homosubtypic (homologous hemagglutinin) and heterosubtypic (heterologous hemagglutinin) low pathogenic avian influenza (LPAI) viruses on the outcome of a H5N1 HPAI virus infection in wood ducks (Aix sponsa) was evaluated. Pre-exposure of wood ducks to different LPAI viruses did not prevent infection with H5N1 HPAI virus, but did increase survival associated with H5N1 HPAI virus infection. The magnitude of this effect on the outcome of the H5N1 HPAI virus infection varied between different LPAI viruses, and was associated both with efficiency of LPAI viral replication in wood ducks and the development of a detectable humoral immune response. These observations suggest that in naturally occurring outbreaks of H5N1 HPAI, birds with pre-existing immunity to homologous hemagglutinin or neuraminidase subtypes of AI virus may either survive H5N1 HPAI virus infection or live longer than naïve birds and, consequently, could pose a greater risk for contributing to viral transmission and dissemination. The mechanisms responsible for this protection and/or the duration of this immunity remain unknown. The results of this study are important for surveillance efforts and help clarify epidemiological data from outbreaks of H5N1 HPAI virus in wild bird populations.

Outbreak of highly pathogenic avian influenza caused by Asian lineage h5n1 virus in turkeys in Great Britain in January 2007

H7 influenza viruses circulate in wild birds in the United States, but when the virus emerges in domestic poultry populations, the frequency of human exposure and the potential for human infections increases. An H7N8 highly pathogenic avian influenza (HPAI) virus and an H7N8 low-pathogenic avian influenza (LPAI) virus were recently isolated from commercial turkey farms in Indiana. To determine the risk that these influenza viruses pose to humans, we assessed their pathogenesis and transmission in vitro and in mammalian models. We found that the H7N8 HPAI virus exhibited enhanced virulence, and although transmission was only observed with the H7N8 LPAI virus, the ability of this H7 virus to transmit in a mammalian host and quickly evolve to a more virulent strain is cause for concern. Our findings offer important insight into the potential for emerging H7 avian influenza viruses to acquire the ability to cause disease and transmit among mammals.

Characterization of the low-pathogenic H5N1 avian influenza virus in South Korea

Avian Influenza Virus Infections in Humans

The prevalence of infection with avian influenza (AI) virus varies significantly between taxonomic Orders and even between species within the same Order. The current understanding of AI infection and virus shedding parameters in wild birds is limited and largely based on trials conducted in mallards (Anas platyrhynchos). The objective of the present study was to provide experimental data to examine species-related differences in susceptibility and viral shedding associated with wild bird-origin low-pathogenicity avian influenza (LPAI) viruses in multiple duck species and gulls. Thus mallards, redheads (Aythya americana), wood ducks (Aix sponsa), and laughing gulls (Leucophaeus atricilla) were inoculated experimentally with three wild mallard-origin LPAI viruses representing multiple subtypes. Variation in susceptibility and patterns of viral shedding associated with LPAI virus infection was evident between the duck and gull species. Consistent with the literature, mallards excreted virus predominantly via the gastrointestinal tract. In wood ducks, redheads, and laughing gulls, AI virus was detected more often in oropharyngeal swabs than cloacal swabs. The results of this study suggest that LPAI shedding varies between taxonomically related avian species. Such differences may be important for understanding the potential role of individual species in the transmission and maintenance of LPAI viruses and may have implications for improving sampling strategies for LPAI detection. Additional comparative studies, which include LPAI viruses originating from non-mallard species, are necessary to further characterize these infections in wild avian species other than mallards and provide a mechanism to explain these differences in viral excretion.

The mutation of low pathogenic avian influenza (LPAI) virus to highly pathogenic avian influenza (HPAI) virus is a major factor in the disease burden from avian influenza in poultry flocks. In order to update the understanding on this mutation, an extensive literature review on mechanisms and risk factors for a LPAI to HPAI mutation was performed. Information about documented HPAI outbreaks since 1959 were collected from the literature and from the World Organisation of Animal Health (OIE) website. For outbreaks where a LPAI precursor had been identified, the genetic sequences of both HPAI and LPAI viruses were downloaded from Genbank or GISAID, if available. Experimental studies on the LPAI to HPAI mutation were obtained from the literature. Where possible, data on host-specific and environment-specific elements related to the LPAI to HPAI mutation were collected. In total, 42 HPAI outbreaks occurring between 1959 and 2016 were reviewed. The involvement of wild birds in these outbreaks most often was unknown. In 6 outbreaks, closely related LPAI viruses were isolated from wild birds, and in 10 outbreaks, a LPAI precursor was detected before emergence of HPAI. Poultry species in which LPAI to HPAI mutagenesis was confirmed were chicken, turkey and ostrich. Both in nature and experimentally, only H5 and H7 viruses evolved towards the HPAI phenotype. Experimentally, passaging in chicken embryo cells, in embryonated chicken eggs or in chickens allowed either selection of minority variants in the field isolate or acquisition of a multi-basic cleavage site of HPAI hemagglutinin (HA). The acquisition of multiple basic amino acids at the HA cleavage site probably occurred via recombination, insertion or mutation. Neither the size of the index farm, nor the type of farm management, nor the age of the affected poultry seemed to play a role in HPAI emergence.

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Analysis of low pathogenic avian influenza (LPAI) viruses circulating in the Netherlands in a previous study revealed associations of specific hemagglutinin (HA) and neuraminidase (NA) subtypes with wild bird or poultry hosts. In this study, we identified putative host associations in LPAI virus internal proteins. We show that LPAI viruses isolated from poultry more frequently carried the allele A variant of the nonstructural protein (NS) gene, compared to wild bird viruses. We determined the susceptibility of chickens to wild bird–associated subtypes H3N8 and H4N6 and poultry-associated subtypes H8N4 and H9N2, carrying either NS allele A or B, in an infection experiment. We observed variations in virus shedding and replication patterns, however, these did not correlate with the predicted wild bird- or poultry-associations of the viruses. The experiment demonstrated that LPAI viruses of wild bird-associated subtypes can replicate in chickens after experimental infection, despite their infrequent detection in poultry. Although the NS1 protein is known to play a role in immune modulation, no differences were detected in the limited innate immune response to LPAI virus infection. This study contributes to a better understanding of the infection dynamics of LPAI viruses in chickens.

ABSTRACTAvian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks.RESEARCH HIGHLIGHTSCanaries are susceptible to infection with H5/H7 LPAI viruses.Canaries inoculated with LPAI viruses excrete large amounts of viral RNA.Buccal swabs may be appropriate specimens for AI virus detection in canaries.The control of canaries for LPAI virus detection should not be overlooked.

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Low-pathogenicity avian influenza (LPAI) viruses of subtypes H5 and H7 have the ability to spontaneously mutate to highly pathogenic (HPAI) virus variants, causing high mortality in poultry. The highly pathogenic phenotype is caused by mutation of the hemagglutinin (HA) cleavage site, but additional mutations may play a role. Evidence from the field for the switch to high pathogenicity remains scarce. This study provides direct evidence for LPAI-to-HPAI virus mutation during H7N3 infection of a turkey farm in the Netherlands. No severe clinical symptoms were reported at the farm, but deep sequencing of isolates from the infected turkeys revealed a minority of HPAI virus sequences (0.06%) in the virus population. The HPAI virus contained a 12-nucleotide insertion in the HA cleavage site that was likely introduced by a single event as no intermediates with shorter inserts were identified. This suggests nonhomologous recombination as the mechanism of insertion. Analysis of different organs of the infected turkeys showed the largest amount of HPAI virus in the lung (4.4%). The HPAI virus was rapidly selected in experimentally infected chickens after both intravenous and intranasal/intratracheal inoculation with a mixed virus preparation. Full-genome sequencing revealed that both pathotypes contained a deletion in the stalk region of the neuraminidase protein. We identified additional mutations in HA and polymerase basic protein 1 (PB1) in the HPAI virus, which were already present as minority variants in the LPAI virus population. Our findings provide more insight into the molecular changes and mechanisms involved in the emergence and selection of HPAI viruses.IMPORTANCE Low-pathogenicity avian influenza (LPAI) viruses circulate in wild birds and can be transmitted to poultry. LPAI viruses can mutate to become highly pathogenic avian influenza (HPAI) viruses causing severe disease and death in poultry. Little is known about this switch to high pathogenicity. We isolated an LPAI H7N3 virus from an infected turkey farm and showed that this contains small amounts of HPAI virus. The HPAI virus rapidly outcompeted the LPAI virus in chickens that were experimentally infected with this mixture of viruses. We analyzed the genome sequences of the LPAI and HPAI viruses and identified several changes that may be important for a virus to become highly pathogenic. This knowledge may be used for timely identification of LPAI viruses that pose a risk of becoming highly pathogenic in the field.

Dendritic cells (DCs) are professional antigen-presenting cells, which are key components of the immune system and involved in early immune responses. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with avian influenza virus (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death. For this study, chicken bone marrow-derived DC (ckBM-DC)s were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphologies, and evaluate virus replication. A strong proinflammatory response was observed at 8 hours post infection, via upregulation of chicken interleukin-1β and stimulation of the interferon response pathway. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The increase in activation of infected DCs may be indicative of a dysregulated immune response typically seen with HPAI infections.

Influenza A viruses infect a wide range of animals including poultry, wild birds, pigs, horses, dogs, and marine mammals. Influenza in animals threatens animal health and welfare, agricultural productivity, public health, food security, and the livelihoods of farmers across the globe. The recent H1N1 pandemic of 2009, continuous reporting of zoonotic infections with highly pathogenic avian influenza (HPAI) H5N1 and other avian and swine influenza viruses, such as H3N2v, raise ongoing concerns regarding the emergence of zoonotic viruses with pandemic potential.1 Different strains of influenza A virus show host specificity and are often defined by the species in which they are initially found to be circulating. However, over time the situation becomes more complicated as the viruses continuously evolve, through mutation and reassortment, and in some cases are transmitted from species to species. The next major pandemic is likely to be caused by a strain of influenza virus that is new to that generation; a virus to which the human population has little or no immunity. Almost certainly such a strain would contain genes from influenza viruses that have been circulating in animals. A better understanding of the mechanisms responsible for interspecies transmission, and information on host adaptation and pathogenicity are needed to allow more informed assessment as to when and where the next pandemic may arise. Timely identification of viruses with pandemic potential could ultimately reduce the impact of a new pandemic. With current levels of knowledge and surveillance, it is not possible to accurately assess geographic location of all animal influenza viruses, the production systems in which they circulate, and which of these viruses may be transmitted to and adapt in the human population. This makes predictions about which strains of influenza virus to select for vaccines for pandemic preparedness a challenging task. With mortality rates reaching up to 100% in affected populations, HPAI viruses continue to have a devastating impact in poultry populations. Low-pathogenic avian influenza (LPAI) viruses also have a significant and variable impact on poultry production, depending on the strain and health status of the birds, and may evolve into HPAI viruses, for example, the recent HPAI H7N3 outbreak in Jalisco, Mexico.2, 8 These impacts together with consequential cost of control and trade measures, aimed at preventing further spread, lead to huge economic costs. There is often a greater impact on countries with a lower Gross Domestic Product (GDP), who rely on agriculture for economic development and for sustenance. Early detection of HPAI and H5 and H7 LPAI virus infections in poultry is essential for an effective response which relies on a combination of classic control measures (culling infected flocks and high risk contacts, disinfection, biosecurity, and trade measures) and, where appropriate, vaccination.3 Delays in detection lead to spiraling costs to keep epidemics under control and make the disease more difficult to eradicate. In the case of H5N1 HPAI, effective control in the animal source is needed to reduce the public health risk. Developing countries do not always have the resources to maintain the infrastructure and technical capacities needed for rapid and accurate diagnostic testing and characterization of viral strains. These countries rely on international reference laboratories to test their specimens and characterize the viruses. Research is needed to develop accurate, cheap, and robust diagnostic tests to ensure that the disease is detected early, with sufficient confidence, to allow timely initiation of effective response measures. For initial disease confirmation, both sensitivity and specificity of tests are essential because the implications of false-negative and false-positive results can be considerable. Animal influenza is not only a constraint for agriculture and food production. Equine influenza is an ongoing problem for companion and competition horses and has a huge impact on the horse racing industry. The 2007 equine influenza outbreak in Australia is estimated to have cost the horse racing and gambling industries 3·6 billion Australian dollars in lost revenue.4 Research, accompanied by increased global surveillance, is needed to ensure that the equine sector is able to access effective up-to-date vaccines so that many can continue to benefit from the enjoyment and financial gains that horse sports offer. Some suggest that historic accounts showing a temporal relationship between respiratory disease in horses and humans may implicate influenza viruses.5 However, these accounts date back to a time before influenza viruses had been isolated, and a clear link to influenza as we know it would be difficult to prove. Influenza viruses of subtype H3N8 currently circulating in the horse population have also crossed the species barrier and become established in dogs. However, this subtype and other strains of equine influenza viruses do not appear to be a significant zoonosis despite intense exposure of owners to their horses and dogs, and vice versa. An understanding of the underlying reasons for this may help to explain why other influenza viruses are zoonotic. Effective and cost-effective control require targeting resources for optimal impact. Research is needed to gain an understanding of how control measures can be better targeted. More rapid control in the animal population will limit impacts on animal health and public health when the influenza virus is zoonotic and will minimize costs in terms of production losses and access to international markets. Currently, vaccination does not always prevent infection of birds nor does it prevent infected vaccinated birds from shedding virus. If vaccines are not adequately matched antigenically to circulating field viruses and at least 60–80% of the susceptible populations are immunized, vaccination as a program will not be effective. Further research is needed to improve the effectiveness of the control measures themselves, such as vaccination, and to provide improved access to resources needed for control. Despite unprecedented levels of international investment to support avian influenza surveillance between 2004 and 2009, global surveillance for influenza viruses in animals is woefully inadequate, with too little being undertaken without adequate coordination. Improvements in surveillance are required to provide early warning for effective control and to inform much needed research.6 As well as surveillance in domestic animals (poultry, horses and pigs), surveillance in wildlife is important; it is now evident that wild birds also play a role in the primary introduction of avian influenza in previously disease-free areas.7 Today, we are not able to fully manage the threats and impacts from animal influenza. In one form or another, influenza A viruses are circulating in every country on the planet. Our understanding of the mechanisms responsible for interspecies transmission, adaptation, and pathogenicity is incomplete, and the methods for risk assessment and disease control are rudimentary. Challenges to reduce threats from animal influenzas are considerable and will only be improved through extensive research and innovation. Continued reports of notifiable avian influenza8 and animal influenza associated human infections highlight the need to monitor influenza viruses in all animal species to better understand their role in causing pandemics and severe zoonotic infections, and in reducing agricultural productivity. OFFLU is the World Organisation for Animal Health (OIE) – Food and Agricultural Organization of the United Nations (FAO) global network of expertise on animal influenza, established in 2005 to address the animal and public health threats from H5N1 HPAI. Since then, its mandate has been extended to cover all animal influenza viruses. OFFLU is unique in that its participation comprises a global representation of leading experts in animal influenza including researchers, diagnosticians, policy makers, economists, and epidemiologists. One of OFFLU's core objectives is to advocate for more research, to highlight specific influenza research objectives, promote their development, and to ensure coordination. OFFLU works closely with WHO on all influenza issues at the human–animal interface, including identifying commonly agreed research priorities. Following the OFFLU annual technical meeting in 2010 attended by avian, swine, equine, and public health experts, it was decided that there was an urgent need to develop a Research Agenda to highlight and coordinate research priorities for the animal influenza sector. The Research Agenda highlighted needs in different animal species and at the human–animal interface. It is designed to help policy makers, researchers, and donors ensure that their efforts and resources are targeted to areas where there is an identified need. The agenda should also be used to leverage funds for animal influenza research. In today's world where there is a huge volume of information of variable quality, the OFFLU Research Agenda has been designed to be concise and digestible; it comprises only 11 pages. The animal and human influenza networks share the common goal of reducing public health threats from animal influenza viruses. OFFLU has been working closely with its parent organizations, the OIE and the FAO, and its partner the World Health Organization (WHO) to ensure that its efforts are complementary and well coordinated. In 2008 and 2010, the OIE-WHO-FAO tripartite held joint technical consultations on avian influenza at the human–animal interface in Verona, Italy and discussed other technical interface topics of common interest. The experts identified that more research is needed on modes of transmission, behaviors associated with increased risks of transmission, virologic and ecologic aspects, and viral persistence in the environment to address the human exposure risks to H5N1 infection.9 Importantly in practical terms, OFFLU contributes animal influenza data to the biannual WHO influenza vaccine composition meetings. This information is critical in allowing selection of the most appropriate strains of virus for vaccines to protect against potential zoonotic pandemic influenza, including H5N1 and H9N2 avian influenza.10 Where zoonotic strains of influenza are undergoing antigenic drift in animal populations, the situation is being monitored in real time to allow selection of relevant influenza virus seed strains and antigens for vaccines for public health preparedness. OFFLU and WHO experts are working together to better understand which animal influenza viruses may pose a risk to human health. This ongoing risk assessment is supported by OFFLU's drive to improve and better collate data from avian, swine, and equine influenza surveillance programs world-wide. Animal influenza research is suffering from donor fatigue, and it is a continuing challenge to ensure that sufficient resources can be secured to address the priorities that have been identified, ultimately to improve health and economies. It is considered possible to prevent a human influenza pandemic by identifying influenza viruses with pandemic potential in animal species; this will only be achieved through further influenza research studies in animals. A large body of animal influenza data has been generated in recent years, presenting a real opportunity to increase our understanding of how to identify risk and better control the adverse effects of influenza in animals and at the human–animal interface. Many questions still remain to be answered. Structured and coordinated research toward prioritized goals and objectives will greatly facilitate this. The 'OFFLU Research Agenda' is a first for the animal health sector and will help to steer animal influenza research toward the identified objectives, providing maximum benefits for public and animal health. The influenza research priorities focus on several topics including control and education, diagnostics, epidemiology, immunology and immune responses, pathogenesis, transmission, vaccines and vaccination and virus characteristics and evolution in poultry, wild birds, swine and equine. The full OFFLU research agenda can be viewed at http://www.offlu.net/fileadmin/home/en/publications/pdf/OFFLU_Research_Priorities_photo.pdf. The authors would like to thank David Swayne, Ian Brown, Kristien Van Reeth and Ann Cullinane. The authors have no potential conflicts of interest to declare.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.