Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021.

Highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage (GsGd), which threaten the health of poultry, wildlife and humans, are spreading across Asia, Europe, Africa and North America but are currently absent from South America and Oceania. In December 2021, H5N1 HPAI viruses were detected in poultry and a free-living gull in St. John's, Newfoundland and Labrador, Canada. Our phylogenetic analysis showed that these viruses were most closely related to HPAI GsGd viruses circulating in northwestern Europe in spring 2021. Our analysis of wild bird migration suggested that these viruses may have been carried across the Atlantic via Iceland, Greenland/Arctic or pelagic routes. The here documented incursion of HPAI GsGd viruses into North America raises concern for further virus spread across the Americas by wild bird migration.

The role of national and international veterinary laboratories.

In the field of diagnostic test validation, World Organisation for Animal Health (OIE) Reference Laboratories (RLs) have a pivotal role and provide the international community with impartial advice and support in the selection, development and validation of diagnostic tests, which can be applied to the specialist diseases for which they are designated. National RLs provide an invaluable function in supporting the introduction, ongoing validation and application of validated diagnostic tests in line with international standards. Experienced staff with extensive knowledge of such systems and access to specialist facilities for conducting work are available to monitor changes or advancements in technology. They consider their relevance and value to evolving diagnostic test requirements. Reference Laboratories often have a broad mandate of activity linking research or development programmes and surveillance activities to benefit the continual assessment and, if necessary, improvement of diagnostic tools. Reference Laboratories maintain or have access to unique biological archives (known positive and negative sample populations) and produce international reference standards, both of which are vital in establishing the necessary and detailed validation of any diagnostic test. Reference Laboratories act either singularly or in collaborative partnerships with other RLs or science institutes, but also, when required, and with impartiality, with the commercial sector, to ensure new tests are validated according to OIE standards. They promote and apply formal programmes of quality assurance (including proficiency testing programmes) for newly validated tests, ensuring ongoing monitoring and compliance with standards, or as required set out any limitations or uncertainties. Reference Laboratories publish information on test validation in the scientific literature and on relevant websites, as well as disseminating information at workshops and international conferences. Furthermore, they can offer training in the processes and systems underpinning test validation.

Dans le domaine de la validation des tests de diagnostic, les Laboratoires de référence de l'Organisation mondiale de la santé animale (OIE) jouent un rôle central et fournissent à la communauté internationale des conseils impartiaux ainsi qu'un soutien pour la sélection, la mise au point et la validation des tests de diagnostic utilisés pour la détection des maladies correspondant à leur domaine de spécialisation. Les Laboratoires de référence nationaux remplissent une fonction inestimable en facilitant l'introduction, la validation continue et l'application de tests de diagnostic validés conformément aux normes internationales. Ces laboratoires sont dotés de personnels expérimentés possédant une connaissance approfondie de ces systèmes et qui ont accès à des installations spécialisées pour mener à bien leurs opérations et suivre de près les changements ou les avancées technologiques. Ils peuvent ainsi examiner leur pertinence et intérêt au regard de l'évolution des exigences relatives aux tests de diagnostic. Le mandat des Laboratoires de référence recouvre souvent un large éventail d'activités reliant les programmes de recherche ou développement et les activités de surveillance, ce qui permet de réaliser une évaluation continue des outils diagnostiques et, si besoin, de procéder à leur amélioration. Les Laboratoires de référence entretiennent ou ont accès à des banques de matériels biologiques uniques (panels d'échantillons positifs et négatifs connus) et produisent des réactifs de référence internationale, deux catégories de matériels essentielles pour procéder à la validation point par point d'un test diagnostique suivant les critères requis. Les Laboratoires de référence interviennent individuellement ou en partenariat avec d'autres Laboratoires de référence ou instituts scientifiques, mais aussi, lorsque c'est nécessaire et dans le respect des règles d'impartialité, avec le secteur privé, afin de s'assurer que les nouveaux tests sont validés conformément aux normes de l'OIE. Ils soutiennent et appliquent des programmes officiels d'assurance de la qualité (y compris en participant à des programmes d'essais d'aptitude inter-laboratoires) pour les tests nouvellement validés et garantissent leur suivi continu ainsi que leur conformité avec les normes, ou, suivant les cas, définissent les limites ou le niveau d'incertitude à prendre en considération. Les Laboratoires de référence publient les données relatives à la validation des tests dans des journaux scientifique et sur les sites Web pertinents et diffusent également des informations sur le sujet lors d'ateliers et de conférences internationales. En outre, ils peuvent proposer des formations sur les procédures et les systèmes qui sous-tendent la validation des tests.

En el terreno de la validación de pruebas de diagnóstico, los Laboratorios de Referencia de la Organización Mundial de Sanidad Animal (OIE) cumplen una función central y proporcionan a la comunidad internacional servicios de apoyo y asesoramiento imparcial para la selección, el desarrollo y la validación de pruebas de diagnóstico, que pueden aplicarse a la enfermedad para la que cada laboratorio esté designado. Los laboratorios de referencia nacionales cumplen una inestimable función de apoyo a la implantación, la continua validación y la utilización de pruebas de diagnóstico validadas con arreglo a las normas internacionales. Disponen de personal experimentado y muy buen conocedor de estos sistemas y de acceso a instalaciones especializadas de trabajo, lo que les permite seguir de cerca los cambios o adelantos tecnológicos y estudiar su utilidad o interés en relación con la evolución de los requisitos de las pruebas de diagnóstico. Los Laboratorios de Referencia suelen tener un mandato amplio, que a los programas de investigación y desarrollo aúna actividades de vigilancia, en aras de la continua evaluación y, en caso necesario, mejora de las herramientas de diagnóstico. Estos laboratorios poseen (o tienen acceso a) archivos biológicos únicos (conjuntos de muestras probadamente positivas y negativas) y elaboran patrones de referencia internacional, elementos ambos indispensables para llevar a buen fin la necesaria validación detallada de toda prueba de diagnóstico. Los Laboratorios de Referencia pueden trabajar en solitario o en colaboración con otros Laboratorios de Referencia, con institutos científicos e incluso, cuando hace falta, y procediendo con imparcialidad, con entidades del sector privado, a fin de garantizar que toda nueva prueba sea validada con arreglo a las normas de la OIE. También promueven y llevan adelante programas oficiales de garantía de la calidad de pruebas recién validadas (incluidos programas de pruebas de competencia), lo que asegura un seguimiento continuo y el cumplimiento de la normativa en todo momento, o fijan, cuando es necesario, limitaciones o niveles de incertidumbre. Asimismo, estos laboratorios publican datos sobre la validación de pruebas en revistas científicas y sitios web conexos y difunden información al respecto en talleres y conferencias internacionales. Además, pueden impartir formación sobre los procesos y sistemas que fundamentan la validación de pruebas de diagnóstico.

Highly pathogenic avian influenza virus H5N6 (clade 2.3.4.4b) has a preferable host tropism for waterfowl reflected in its inefficient transmission to terrestrial poultry.

Highly-pathogenic avian influenza virus (HPAIV) H5N6 (clade 2.3.4.4b) incurred into Europe in late 2017 and was predominantly detected in wild birds, with very few terrestrial poultry cases. Pekin ducks directly-infected with a UK virus (H5N6-2017) were donors of infection to investigate contact transmission to three recipient species: Ducks, chickens and turkeys. H5N6-2017 transmission to ducks was 100% efficient, but transmission to in-contact galliforme species was infrequent and unpredictable, thereby reflecting the European 2017-2018 H5N6 epidemiology. Although only two of 28 (7%) infected ducks died, the six turkeys and one chicken which became infected all died and displayed systemic H5N6-2017 dissemination, while pathogenesis in ducks was generally milder. Analysis of H5N6-2017 progeny in the contacts revealed no emergent polymorphisms in an infected duck, but the galliforme species included changes in the polymerase (PB2 A199T, PA D347A), matrix (M1 T218A) and neuraminidase genes (T88I). H5N6-2017 environmental contamination was associated with duck shedding.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008.

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006-2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Evaluation of ELISA and haemagglutination inhibition as screening tests in serosurveillance for H5/H7 avian influenza in commercial chicken flocks.

Avian influenza virus (AIV) subtypes H5 and H7 can infect poultry causing low pathogenicity (LP) AI, but these LPAIVs may mutate to highly pathogenic AIV in chickens or turkeys causing high mortality, hence H5/H7 subtypes demand statutory intervention. Serological surveillance in the European Union provides evidence of H5/H7 AIV exposure in apparently healthy poultry. To identify the most sensitive screening method as the first step in an algorithm to provide evidence of H5/H7 AIV infection, the standard approach of H5/H7 antibody testing by haemagglutination inhibition (HI) was compared with an ELISA, which detects antibodies to all subtypes. Sera (n = 1055) from 74 commercial chicken flocks were tested by both methods. A Bayesian approach served to estimate diagnostic test sensitivities and specificities, without assuming any 'gold standard'. Sensitivity and specificity of the ELISA was 97% and 99.8%, and for H5/H7 HI 43% and 99.8%, respectively, although H5/H7 HI sensitivity varied considerably between infected flocks. ELISA therefore provides superior sensitivity for the screening of chicken flocks as part of an algorithm, which subsequently utilises H5/H7 HI to identify infection by these two subtypes. With the calculated sensitivity and specificity, testing nine sera per flock is sufficient to detect a flock seroprevalence of 30% with 95% probability.

Companion Animals as a Source of Viruses for Human Beings and Food Production Animals.

Companion animals comprise a wide variety of species, including dogs, cats, horses, ferrets, guinea pigs, reptiles, birds and ornamental fish, as well as food production animal species, such as domestic pigs, kept as companion animals. Despite their prominent place in human society, little is known about the role of companion animals as sources of viruses for people and food production animals. Therefore, we reviewed the literature for accounts of infections of companion animals by zoonotic viruses and viruses of food production animals, and prioritized these viruses in terms of human health and economic importance. In total, 138 virus species reportedly capable of infecting companion animals were of concern for human and food production animal health: 59 of these viruses were infectious for human beings, 135 were infectious for food production mammals and birds, and 22 were infectious for food production fishes. Viruses of highest concern for human health included hantaviruses, Tahyna virus, rabies virus, West Nile virus, tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus, Aichi virus, European bat lyssavirus, hepatitis E virus, cowpox virus, G5 rotavirus, influenza A virus and lymphocytic choriomeningitis virus. Viruses of highest concern for food production mammals and birds included bluetongue virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, Rift Valley fever virus, porcine circovirus, classical swine fever virus, equine herpesvirus 9, peste des petits ruminants virus and equine infectious anaemia virus. Viruses of highest concern for food production fishes included cyprinid herpesvirus 3 (koi herpesvirus), viral haemorrhagic septicaemia virus and infectious pancreatic necrosis virus. Of particular concern as sources of zoonotic or food production animal viruses were domestic carnivores, rodents and food production animals kept as companion animals. The current list of viruses provides an objective basis for more in-depth analysis of the risk of companion animals as sources of viruses for human and food production animal health.

Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Virus: Equivocal Pathogenicity and Implications for Surveillance Following Natural Infection in Breeder Ducks in the United Kingdom.

Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7-day period. Post-mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low-to-moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter-current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine.

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian-like H1N1 and two reassortant H1N2 and H3N2 viruses with human-origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross-reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.

Cytokine Expression at Different Stages of Influenza A(H1N1)pdm09 Virus Infection in the Porcine Lung, Using Laser Capture Microdissection.

Pandemic influenza A(H1N1)pdm09 virus has retained its ability to infect swine whilst developing the ability to transmit effectively between humans, thus making the pig a valuable model for studying disease pathogenesis in both species. Lung lesions in pigs caused by infection with influenza A viruses vary in both their severity and distribution with individual lung lobes exhibiting lesions at different stages of infection pathogenic development and disease resolution. Consequently, investigating interactions between the virus and host and their implications for disease pathogenesis can be complicated. Studies were undertaken to investigate the discrete expression of pro- and anti-inflammatory mediators during lung lesion formation in pigs during infection with influenza A(H1N1)pdm09 (A/Hamburg/05/09) virus. Laser capture microdissection was used to identify and select lung lobules containing lesions at different stages of development. Dissected samples were analysed using quantitative RT-PCR to assess pro- and anti-inflammatory cytokine mRNA transcripts. Differential expression of the immune mediators IL-8, IL-10 and IFN-γ was observed depending upon the lesion stage assessed. Upregulation of IFN-γ, IL-8 and IL-10 mRNA was observed in stage 2 lesions, whereas decreased mRNA expression was observed in stage 3 lesions, with IL-8 actively downregulated when compared with controls in both stage 3 and stage 4 lesions. This study highlighted the value of using laser capture microdissection to isolate specific tissue regions and investigate subtle differences in cytokine mRNA expression during lesion development in pigs infected with influenza A(H1N1)pdm09.

The evolution of pigeon paramyxovirus type 1 (PPMV-1) in Great Britain: a molecular epidemiological study.

Newcastle disease (ND), caused by virulent strains of avian paramyxovirus type 1 (APMV-1), is considered throughout the world as one of the most important animal diseases. For over three decades now, there has been a continuing panzootic caused by a variant virulent APMV-1 strain, so-called pigeon paramyxovirus type 1 (PPMV-1), primarily in racing pigeons, which has also spread to wild birds and poultry. PPMV-1 isolations have been made in Great Britain every year since 1983. In this study, we have completed a comparative phylogenetic analysis based on a 374 nucleotide section of the fusion protein gene of 63 isolates of PPMV-1 that were isolated over a 26-year period; 43 of these were sequenced for this study. Phylogenetic analysis of these sequences revealed that all were closely related and placed in the genetic sublineage 4b (VIb), subdivision 4biif.

Antigenic and genetic analyses of isolate APMV/wigeon/Italy/3920-1/2005 indicate that it represents a new avian paramyxovirus (APMV-12).

Isolate wigeon/Italy/3920-1/2005 (3920-1) was obtained during surveillance of wild birds in November 2005 in the Rovigo province of Northern Italy and shown to be a paramyxovirus. Analysis of cross-haemagglutination-inhibition tests between 3920-1 and representative avian paramyxoviruses showed only a low-level relationship to APMV-1. Phylogenetic analysis of the whole genome and each of the six genes indicated that while 3920-1 grouped with APMV-1 and APMV-9 viruses, it was quite distinct from these two. In the whole-genome analysis, 3920-1 had 52.1 % nucleotide sequence identity to the closest APMV-1 virus, 50.1 % identity to the APMV-9 genome, and less than 42 % identity to representatives of the other avian paramyxovirus groups. We propose isolate wigeon/Italy/3920-1/2005 as the prototype strain of a further APMV group, APMV-12.

The evolutionary dynamics of influenza A virus adaptation to mammalian hosts.

Few questions on infectious disease are more important than understanding how and why avian influenza A viruses successfully emerge in mammalian populations, yet little is known about the rate and nature of the virus' genetic adaptation in new hosts. Here, we measure, for the first time, the genomic rate of adaptive evolution of swine influenza viruses (SwIV) that originated in birds. By using a curated dataset of more than 24 000 human and swine influenza gene sequences, including 41 newly characterized genomes, we reconstructed the adaptive dynamics of three major SwIV lineages (Eurasian, EA; classical swine, CS; triple reassortant, TR). We found that, following the transfer of the EA lineage from birds to swine in the late 1970s, EA virus genes have undergone substantially faster adaptive evolution than those of the CS lineage, which had circulated among swine for decades. Further, the adaptation rates of the EA lineage antigenic haemagglutinin and neuraminidase genes were unexpectedly high and similar to those observed in human influenza A. We show that the successful establishment of avian influenza viruses in swine is associated with raised adaptive evolution across the entire genome for many years after zoonosis, reflecting the contribution of multiple mutations to the coordinated optimization of viral fitness in a new environment. This dynamics is replicated independently in the polymerase genes of the TR lineage, which established in swine following separate transmission from non-swine hosts.

Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys.

The purpose of this study was to determine whether pooling avian influenza (AI)-positive swabs with negative swabs has a detrimental effect on the sensitivity of AI real-time reverse transcription-polymerase chain reactions (rRT-PCRs). Cloacal and buccal swabs were sampled daily from 12 turkeys infected with A/goose/England/07(H2N2). For half the turkeys, each swab was mixed with four swabs from known AI-negative turkeys, and for the other half the swabs were tested individually. Bayesian modelling was used to (i) determine whether pooling the positive swabs compromised the cycle threshold (C(t)) value obtained from the rRT-PCRs, and (ii) estimate the likelihood of detection of an H2N2 infected turkey flock via rRT-PCR for pooled and individually tested swabs (cloacal and buccal) vs. the number of days post-infection of the flock. Results indicated that there was no significant effect of compromising AI rRT-PCR sensitivity by pooling a weak positive swab with negative swabs on the Ct values which were obtained. Pooled sampling was able to widen the detection window compared to individual sampling, for the same number of rRT-PCR tests. This indicates that pooled sampling would be an effective method of reducing the number of tests to be performed to determine flock status during an AI outbreak and for surveillance.

Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests.

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

First reported detection of a low pathogenicity avian influenza virus subtype H9 infection in domestic fowl in England.

In December 2010, infection with a H9N1 low pathogenicity avian influenza (LPAI) virus was detected in a broiler breeder flock in East Anglia. Disease suspicion was based on acute drops in egg production in two of four sheds on the premises, poor egg shell quality and evidence of diarrhoea. H9N1 LPAI virus infection was confirmed by real-time reverse transcription PCR. Sequencing revealed high nucleotide identity of 93.6 per cent and 97.9 per cent with contemporary North American H9 and Eurasian N1 genes, respectively. Attempted virus isolation in embryonated specific pathogen free (SPF) fowls' eggs was unsuccessful. Epidemiological investigations were conducted to identify the source of infection and any onward spread. These concluded that infection was restricted to the affected premises, and no contacts or movements of poultry, people or fomites could be attributed as the source of infection. However, the infection followed a period of extremely cold weather and snow which impacted on the biosecurity protocols on site, and also led to increased wild bird activity locally, including waterfowl and game birds around the farm buildings. Analysis of the N1 gene sequence suggested direct introduction from wild birds. Although H9 infection in poultry is not notifiable, H9N2 LPAI viruses have been associated with production and mortality episodes in poultry in many parts of Asia and the Middle East. In the present H9N1 outbreak, clinical signs were relatively mild in the poultry with no mortality, transient impact on egg production and no indication of zoonotic spread. However, this first reported detection of H9 LPAI virus in chickens in England was also the first H9 UK poultry case for 40 years, and vindicates the need for continued vigilance and surveillance of avian influenza viruses in poultry populations.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

Descriptive clinical and epidemiological characteristics of influenza A H1N1 2009 virus infections in pigs in England.

Infection of pigs with influenza A H1N1 2009 virus (A(H1N1)pdm09) was first detected in England in November 2009 following global spread of the virus in the human population. This paper describes clinical and epidemiological findings in the first English pig farms in which A(H1N1)pdm09 influenza virus was detected. These farms showed differences in disease presentation, spread and duration of infection. The factors likely to influence these features are described and relate to whether pigs were housed or outdoors, the age of the pigs, inter-current disease and the management system of the unit. Infection could be mild or clinically inapparent in breeding pigs with more typical respiratory disease being identified later in their progeny. Mortality was low where disease was uncomplicated by environmental stresses or concurrent infections. Where deaths occurred in pigs infected with A(H1N1)pdm09 influenza, they were mainly due to other infections, including streptococcal disease due to Streptococcus suis infection. This paper demonstrates the ease with which A(H1N1)pdm09 virus was transmitted horizontally and maintained in a pig population.

The ecology and age structure of a highly pathogenic avian influenza virus outbreak in wild mute swans.

The first UK epizootic of highly pathogenic (HP) H5N1 influenza in wild birds occurred in 2008, in a population of mute swans that had been the subject of ornithological study for decades. Here we use an innovative combination of ornithological, phylogenetic and immunological approaches to investigate the ecology and age structure of HP H5N1 in nature. We screened samples from swans and waterbirds using PCR and sequenced HP H5N1-positive samples. The outbreak's origin was investigated by linking bird count data with a molecular clock analysis of sampled virus sequences. We used ringing records to reconstruct the age-structure of outbreak mortality, and we estimated the age distribution of prior exposure to avian influenza. Outbreak mortality was low and all HP H5N1-positive mute swans in the affected population were <3 years old. Only the youngest age classes contained an appreciable number of individuals with no detectable antibody responses to viral nucleoprotein. Phylogenetic analysis indicated that the outbreak strain circulated locally for ~1 month before detection and arrived when the immigration rate of migrant waterbirds was highest. Our data are consistent with the hypothesis that HP H5N1 epizootics in wild swans exhibit limited mortality due to immune protection arising from previous exposure. Our study population may represent a valuable resource for investigating the natural ecology and epidemiology of avian influenza.

Virological surveillance and preliminary antigenic characterization of influenza viruses in pigs in five European countries from 2006 to 2008.

This study presents the results of the virological surveillance for swine influenza viruses (SIVs) in Belgium, UK, Italy, France and Spain from 2006 to 2008. Our major aims were to clarify the occurrence of the three SIV subtypes - H1N1, H3N2 and H1N2 - at regional levels, to identify novel reassortant viruses and to antigenically compare SIVs with human H1N1 and H3N2 influenza viruses. Lung tissue and/or nasal swabs from outbreaks of acute respiratory disease in pigs were investigated by virus isolation. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined using standard methods. Of the total 169 viruses, 81 were classified as 'avian-like' H1N1, 36 as human-like H3N2 and 47 as human-like H1N2. Only five novel reassortant viruses were identified: two H1N1 viruses had a human-like HA and three H1N2 viruses an avian-like HA. All three SIV subtypes were detected in Belgium, Italy and Spain, while only H1N1 and H1N2 viruses were found in UK and Northwestern France. Cross-hemagglutination inhibition (HI) tests with hyperimmune sera against selected older and recent human influenza viruses showed a strong antigenic relationship between human H1N1 and H3N2 viruses from the 1980s and H1N2 and H3N2 human-like SIVs, confirming their common origin. However, antisera against human viruses isolated during the last decade did not react with currently circulating H1 or H3 SIVs, suggesting that especially young people may be, to some degree, susceptible to SIV infections.