http://repub.eur.nl/res/aut/868/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/39081/ 2012-06-27T00:00:00Z Avian influenza virus (AIV) surveillance studies in wild birds are usually conducted in rural areas and nature reserves. Less is known of avian influenza virus prevalence in wild birds located in densely populated urban areas, while these birds are more likely to be in close contact with humans. Influenza virus prevalence was investigated in 6059 wild birds sampled in cities in the Netherlands between 2006 and 2009, and compared with parallel AIV surveillance data from low urbanized areas in the Netherlands. Viral prevalence varied with the level of urbanization, with highest prevalence in low urbanized areas. Within cities virus was detected in 0.5% of birds, while seroprevalence exceeded 50%. Ring recoveries of urban wild birds sampled for virus detection demonstrated that most birds were sighted within the same city, while few were sighted in other cities or migrated up to 2659 km away from the sample location in the Netherlands. Here we show that urban birds were infected with AIVs and that urban birds were not separated completely from populations of long-distance migrants. The latter suggests that wild birds in cities may play a role in the introduction of AIVs into cities. Thus, urban bird populations should not be excluded as a human-animal interface for influenza viruses. http://repub.eur.nl/res/pub/39085/ 2012-06-22T00:00:00Z Highly pathogenic avian influenza A/H5N1 virus can cause morbidity and mortality in humans but thus far has not acquired the ability to be transmitted by aerosol or respiratory droplet ("airborne transmission") between humans. To address the concern that the virus could acquire this ability under natural conditions, we genetically modified A/H5N1 virus by site-directed mutagenesis and subsequent serial passage in ferrets. The genetically modified A/H5N1 virus acquired mutations during passage in ferrets, ultimately becoming airborne transmissible in ferrets. None of the recipient ferrets died after airborne infection with the mutant A/H5N1 viruses. Four amino acid substitutions in the host receptor-binding protein hemagglutinin, and one in the polymerase complex protein basic polymerase 2, were consistently present in airborne-transmitted viruses. The transmissible viruses were sensitive to the antiviral drug oseltamivir and reacted well with antisera raised against H5 influenza vaccine strains. Thus, avian A/H5N1 influenza viruses can acquire the capacity for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic influenza. http://repub.eur.nl/res/pub/39115/ 2012-04-01T00:00:00Z Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4 (6-O-HSO3)GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO3)GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry. http://repub.eur.nl/res/pub/34656/ 2011-09-01T00:00:00Z The ability to attach to host cells is one of the main determinants of the host range of influenza A viruses. By using virus histochemistry, we investigate the pattern of virus attachment of both a human and an avian influenza virus in colon and trachea sections from 12 wild bird species. We show that significant variations exist, even between closely related avian species, which suggests that the ability of wild birds to serve as hosts for influenza viruses strongly varies among species. These results will prove valuable to assess the possibilities of interspecies transmission of influenza viruses in natural environments and better understand the ecology of influenza. http://repub.eur.nl/res/pub/33415/ 2011-06-01T00:00:00Z A multibasic cleavage site (MBCS) in the haemagglutinin (HA) protein of influenza A virus is a key determinant of pathogenicity in chickens, and distinguishes highly pathogenic avian influenza (HPAI) viruses from low pathogenic avian influenza viruses (LPAI). An MBCS has only been detected in viruses of the H5 and H7 subtypes. Here we investigated the phenotype of a human H3N2 virus with an MBCS in HA. Insertion of an MBCS in the H3N2 virus resulted in cleavage of HA and efficient replication in Madin-Darby canine kidney cells in the absence of exogenous trypsin in vitro, similar to HPAI H5N1 virus. However, studies in ferrets demonstrated that insertion of the MBCS into HA did not result in increased virus shedding, cellular host range, systemic replication or pathogenicity, as compared with wild-type virus. This study indicates that acquisition of an MBCS alone is insufficient to increase pathogenicity of a prototypical seasonal human H3N2 virus. http://repub.eur.nl/res/pub/33458/ 2011-05-01T00:00:00Z Migratory animals may play an important role in connecting disparate ecosystems, including the introduction of various pathogens. The incidence of these pathogens may vary over time and space, such that events along the entire migratory flyway are likely to be important in the interaction between pathogens and their migratory hosts. On this premise, the annual cycle of a naturally occurring host-pathogen system was reconstructed by examining infection with and antibodies to avian influenza virus along the flyway of a long-distance Arctic migrant, the Svalbard-breeding pink-footed goose Anser brachyrhynchus. A highly-localized transmission period was identified in winter, in contrast to the north-south decline expected from dabbling ducks, indicating the dynamics of infection may differ among host species. In spring, 63% (95% CI: 57.1, 68.9) of adults had detectable antibodies to the nucleoprotein of avian influenza virus, compared to just 15% (95% CI: 8.7, 23.4) of juveniles, suggesting inter-annual antibody maintenance. Nevertheless, adult seroprevalence declined by approximately 30% from spring to late summer, indicating significant seroreversion in the population. Integrating these findings in an epidemiological model, detectable antibodies to nucleoprotein were estimated to persist for just 343 days (95% CI: 221, 607); considerably shorter than for other wildlife diseases in long-lived bird species. The investigation of wildlife diseases in migratory populations is an inherently complex task, yet, by integrating disease incidence and seroprevalence along a migratory flyway, our findings suggest that the ecological interactions and life history of the host, as well as the life-history of the pathogen, can influence the dynamics of infection and host immune response. http://repub.eur.nl/res/pub/21931/ 2010-12-01T00:00:00Z Recent demand for increased understanding of avian influenza virus in its natural hosts, together with the development of high-throughput diagnostics, has heralded a new era in wildlife disease surveillance. However, survey design, sampling, and interpretation in the context of host populations still present major challenges. We critically reviewed current surveillance to distill a series of considerations pertinent to avian influenza virus surveillance in wild birds, including consideration of what, when, where, and how many to sample in the context of survey objectives. Recognizing that wildlife disease surveillance is logistically and financially constrained, we discuss pragmatic alternatives for achieving probability-based sampling schemes that capture this host-pathogen system. We recommend hypothesis-driven surveillance through standardized, local surveys that are, in turn, strategically compiled over broad geographic areas. Rethinking the use of existing surveillance infrastructure can thereby greatly enhance our global understanding of avian influenza and other zoonotic diseases. http://repub.eur.nl/res/pub/27928/ 2010-11-01T00:00:00Z The pathogenesis of lower respiratory tract disease from the pandemic 2009 H1N1 (H1N1v) influenza A virus is poorly understood. Therefore, either H1N1v virus or a seasonal human H1N1 influenza A virus was inoculated into cynomolgus macaques as a nonhuman primate model of influenza pneumonia, and virological, pathological, and microarray analyses were performed. Macaques in the H1N1v group had virus-associated diffuse alveolar damage involving both type I and type II alveolar epithelial cells and affecting an average of 16% of the lung area. In comparison, macaques in the seasonal H1N1 group had milder pulmonary lesions. H1N1v virus tended to be reisolated from more locations in the respiratory tract and at higher titers than seasonal H1N1 virus. In contrast, differential expression of messenger RNA transcripts between H1N1v and seasonal H1N1 groups did not show significant differences. The most upregulated genes in H1N1v lung samples with lesions belonged to the innate immune response and proinflammatory pathways and correlated with histopathological results. Our results demonstrate that the H1N1v virus infects alveolar epithelial cells and causes diffuse alveolar damage in a nonhuman primate model. Its higher pathogenicity compared with a seasonal H1N1 virus may be explained in part by higher replication in the lower respiratory tract. http://repub.eur.nl/res/pub/21058/ 2010-08-01T00:00:00Z Reverse genetics can be used to produce recombinant influenza A viruses containing virtually every desired combination of hemagglutinin (HA) and neuraminidase (NA) genes using the virus backbone of choice. Here, a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes, H1-H16 and N1-N9, was established. HA and NA genes were selected based on sequence analyses of influenza virus genes available from public databases. Prototype Eurasian HA and NA genes were cloned in bidirectional reverse genetics plasmids. Recombinant viruses based on the virus backbone of A/PR/8/34, and containing a variety of HA and NA genes were produced in 293T cells. Virus stocks were produced in MDCK cells and embryonated chicken eggs. These plasmids and viruses may be useful for numerous purposes, including influenza virus research projects, vaccination studies, and to serve as reference reagents in diagnostic settings. http://repub.eur.nl/res/pub/27601/ 2010-08-01T00:00:00Z The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA0(the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtypespecific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype. Copyright http://repub.eur.nl/res/pub/28195/ 2010-07-07T00:00:00Z Recent outbreaks of highly pathogenic avian influenza (HPAI) in poultry have raised interest in the interplay between avian influenza (AI) viruses and their wild hosts. Studies linking virus ecology to host ecology are still scarce, particularly for non-duck species. Here, we link capture-resighting data of greater white-fronted geese Anser albifrons albifrons with the AI virus infection data collected during capture in The Netherlands in four consecutive winters. We ask what factors are related to AI virus prevalence and whether there are ecological consequences associated with AI virus infection in staging white-fronted geese. Mean seasonal (low pathogenic) AI virus prevalence ranged between 2.5 and 10.7 per cent, among the highest reported values for non-duck species, and occurred in distinct peaks with near-zero prevalence before and after. Throat samples had a 2.4 times higher detection frequency than cloacal samples. AI virus infection was significantly related to age and body mass in some but not other winters. AI virus infection was not related to resighting probability, nor to maximum distance travelled, which was at least 191 km during the short infectious lifespan of an AI virus. Our results suggest that transmission via the respiratory route could be an important transmission route of AI virus in this species. Nearzero prevalence upon arrival on their wintering grounds, in combination with the epidemic nature of AI virus infections in white-fronted geese, suggests that white-fronted geese are not likely to disperse Asian AI viruses from their Siberian breeding grounds to their European wintering areas. http://repub.eur.nl/res/pub/27428/ 2010-07-01T00:00:00Z The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to α2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with α2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished. Copyright http://repub.eur.nl/res/pub/27386/ 2010-04-01T00:00:00Z In the first 6 months of the H1N1 swine-origin influenza virus (S-OIV) pandemic, the vast majority of infections were relatively mild. It has been postulated that mutations in the viral genome could result in more virulent viruses, leading to a more severe pandemic. Mutations E627K and D701N in the PB2 protein have previously been identified as determinants of avian and pandemic influenza virus virulence in mammals. These mutations were absent in S-OIVs detected early in the 2009 pandemic. Here, using reverse genetics, mutations E627K, D701N, and E677G were introduced into the prototype S-OIV A/Netherlands/602/2009, and their effects on virus replication, virulence, and transmission were investigated. Mutations E627K and D701N caused increased reporter gene expression driven by the S-OIV polymerase complex. None of the three mutations affected virus replication in vitro. The mutations had no major impact on virus replication in the respiratory tracts of mice and ferrets or on pathogenesis. All three mutant viruses were transmitted via aerosols or respiratory droplets in ferrets. Thus, the impact of key known virulence markers in PB2 in the context of current S-OIVs was surprisingly small. This study does not exclude the possibility of emergence of S-OIVs with other virulence-associated mutations in the future. We conclude that surveillance studies aimed at detecting S-OIVs with increased virulence or transmission should not rely solely on virulence markers identified in the past but should include detailed characterization of virus phenotypes, guided by genetic signatures of viruses detected in severe cases of disease in humans. Copyright http://repub.eur.nl/res/pub/27603/ 2010-04-01T00:00:00Z Background. The newly emerged influenza A(H1N1) virus (new H1N1 virus) is causing the first influenza pandemic of this century. Three influenza pandemics of the previous century caused variable mortality, which largely depended on the development of severe pneumonia. However, the ability of the new H1N1 virus to cause pneumonia is poorly understood. Methods. The new H1N1 virus was inoculated intratracheally into ferrets. Its ability to cause pneumonia was compared with that of seasonal influenza H1N1 virus and highly pathogenic avian influenza (HPAI) H5N1 virus by using clinical, virological, and pathological analyses. Results. Our results showed that the new H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by HPAI H5N1 virus. The new H1N1 virus replicated well throughout the lower respiratory tract and more extensively than did both seasonal H1N1 virus (which replicated mainly in the bronchi) and HPAI H5N1 virus (which replicated mainly in the alveoli). High loads of new H1N1 virus in lung tissue were associated with diffuse alveolar damage and mortality. Conclusions. The new H1N1 virus may be intrinsically more pathogenic for humans than is seasonal H1N1 virus. http://repub.eur.nl/res/pub/27652/ 2010-02-01T00:00:00Z Two viruses isolated during the highly pathogenic avian influenza (HPAI) H7N7 virus outbreak in The Netherlands in 2003, one isolated from a person with conjunctivitis and one from a person who died as the result of infection, were used for an in vitro study of influenza A virus pathogenicity factors. The two HPAI H7N7 viruses differed in 15 amino acid positions in five gene segments. Assays were designed to investigate the role of each of these substitutions in attachment and entry, transcription and genome replication, and virus production and release as determined by hemagglutinin (HA), polymerase proteins, nonstructural protein 1 (NS1), and neuraminidase (NA). These in vitro studies confirmed the roles of the E627K substitution in basic polymerase 2 (PB2) and the A143T substitution in HA in pathogenicity observed in a mouse model previously. However, the in vitro studies identified a contribution of acidic polymerase (PA) and NA to the efficient replication in human cells of the fatal case virus, despite the fact that these are rarely marked as determinants of pathogenicity in in vivo studies. With the exception of PB2 E627K, all substitutions contributing to enhanced replication of the fatal case virus in vitro were present in poultry viruses prior to transmission to the human fatal case, indicating that viruses with enhanced replication efficiency in the mammalian host can be generated in poultry. Thus, detailed in vitro analyses of mutations facilitating replication of avian influenza viruses in mammalian cells are important to assess the zoonotic risk posed by these viruses and, in addition, highlight the value of in vitro studies to complement animal models. Copyright http://repub.eur.nl/res/pub/27044/ 2009-10-23T00:00:00Z The recent introductions of highly pathogenic avian influenza (HPAI) H5N1 virus in wild birds and its subsequent spread throughout Asia, the Middle East, Africa and Europe has put a focus on the role of wild birds in the geographical spread of HPAI H5N1 virus. Large-scale surveillance programs are ongoing to determine a potential role of wild birds in H5N1 virus spread and to serve as sentinel systems for introductions into new geographical regions. The unprecedented scale and coverage of these surveillance programs offer a unique opportunity to expand our current knowledge on the ecology of LPAI in wild migratory birds. We provide an update on the current knowledge on the relation between host and virus ecology. http://repub.eur.nl/res/pub/19334/ 2009-09-01T00:00:00Z The largest recorded outbreak of highly pathogenic avian influenza virus of the subtype H7N7 occurred in the Netherlands in 2003. We describe the immunohistochemical and histopathologic findings of 3 chickens naturally infected during this outbreak. Influenza virus antigen occurred in endothelial cells and mononuclear cells of all tissues examined and occurred in parenchymal cells of heart, lung, kidney, pancreas, and trachea, often associated with multifocal inflammation and necrosis. These findings are consistent with the acute stage of highly pathogenic avian influenza from other subtypes. In the severely edematous wattle skin, most endothelial cells contained virus antigen, while in all other tissues virus antigen was only detected in a few endothelial cells. Virus histochemistry showed that this H7N7 virus attached to more endothelial cells in wattle skin than in other vascular beds. This might explain, at least partly, the tropism of the virus and the associated severity of lesions in this tissue. http://repub.eur.nl/res/pub/25229/ 2009-07-24T00:00:00Z The swine-origin A(H1N1) influenza virus that has emerged in humans in early 2009 has raised concerns about pandemic developments. In a ferret pathogenesis and transmission model, the 2009 A(H1N1) influenza virus was found to be more pathogenic than a seasonal A(H1N1) virus, with more extensive virus replication occurring in the respiratory tract. Replication of seasonal A(H1N1) virus was confined to the nasal cavity of ferrets, but the 2009 A(H1N1) influenza virus also replicated in the trachea, bronchi, and bronchioles. Virus shedding was more abundant from the upper respiratory tract for 2009 A(H1N1) influenza virus as compared with seasonal virus, and transmission via aerosol or respiratory droplets was equally efficient. These data suggest that the 2009 A(H1N1) influenza virus has the ability to persist in the human population, potentially with more severe clinical consequences. http://repub.eur.nl/res/pub/27162/ 2009-07-07T00:00:00Z http://repub.eur.nl/res/pub/25213/ 2009-03-22T00:00:00Z The natural reservoir of influenza A virus is waterfowl, particularly dabbling ducks (genus Anas). Although it has long been assumed that waterfowl are asymptomatic carriers of the virus, a recent study found that low-pathogenic avian influenza (LPAI) infection in Bewick's swans (Cygnus columbianus bewickii) negatively affected stopover time, body mass and feeding behaviour. In the present study, we investigated whether LPAI infection incurred ecological or physiological costs to migratory mallards (Anas platyrhynchos) in terms of body mass loss and staging time, and whether such costs could influence the likelihood for long-distance dispersal of the avian influenza virus by individual ducks. During the autumn migrations of 2002-2007, we collected faecal samples (n=10918) and biometric data from mallards captured and banded at Ottenby, a major staging site in a flyway connecting breeding and wintering areas of European waterfowl. Body mass was significantly lower in infected ducks than in uninfected ducks (mean difference almost 20g over all groups), and the amount of virus shed by infected juveniles was negatively correlated with body mass. There was no general effect of infection on staging time, except for juveniles in September, in which birds that shed fewer viruses stayed shorter than birds that shed more viruses. LPAI infection did not affect speed or distance of subsequent migration. The data from recaptured individuals showed that the maximum duration of infection was on average 8.3 days (s.e. 0.5), with a mean minimum duration of virus shedding of only 3.1 days (s.e. 0.1). Shedding time decreased during the season, suggesting that mallards acquire transient immunity for LPAI infection. In conclusion, deteriorated body mass following infection was detected, but it remains to be seen whether this has more long-term fitness effects. The short virus shedding time suggests that individual mallards are less likely to spread the virus at continental or intercontinental scales. http://repub.eur.nl/res/pub/25242/ 2009-03-01T00:00:00Z Influenza A virus surveillance studies of wild bird populations are essential to improving our understanding of the role of wild birds in the ecology of low-pathogenic avian influenza viruses and their potential contribution to the spread of H5N1 highly pathogenic avian influenza viruses. Whereas the primary results of such surveillance programs have been communicated extensively, practical considerations and technical implementation options generally receive little attention. In the present study, the data obtained from 39,490 samples were used to compare the impacts of variables such as the sampling procedure, storage and transport conditions, and the choice of molecular and classical diagnostic tests on the outcome of the results. Molecular diagnostic tests allowed estimation of the virus load in samples, which has implications for the ability to isolate virus. Virus isolation in embryonated eggs was more sensitive than virus isolation in cell cultures. Storage and transport conditions had less of an impact on diagnostics by the use of molecular tests than by the use of classical approaches. These findings indicate that molecular diagnostic tests are more sensitive and more reliable than classical tests. In addition, molecular diagnostic tests facilitated analyses in real time and allowed the discrimination of H5 influenza viruses with low and high pathogenicities without the need for virus isolation. Critical assessment of the methods used in large surveillance studies like this will facilitate comparison of the results between studies. Moreover, the lessons learned from current large-scale influenza A virus surveillance activities could be valuable for other pathogen surveillance programs in the future. Copyright http://repub.eur.nl/res/pub/15050/ 2007-07-01T00:00:00Z During the highly pathogenic avian influenza (HPAI) H7N7 virus outbreak in The Netherlands in 2003, 88 infected persons suffered from mild illnesses, and 1 died of pneumonia. Here, we studied which of the 14 amino acid substitutions observed between the fatal case (FC) virus and a conjunctivitis case (CC) virus determined the differences in virus pathogenicity. In virus-attachment experiments, the CC and FC viruses revealed marked differences in binding to the lower respiratory tract of humans. In a mouse model, the hemagglutinin (HA) gene of the FC virus was a determinant of virus tissue distribution. The lysine at position 627 of basic polymerase 2 (PB2) of the FC virus was the major determinant of pathogenicity and tissue distribution. Thus, remarkable similarities were revealed between recent HPAI H5N1 and H7N7 viruses. We conclude that the influenza virus HA and PB2 genes should be the prime targets for molecular surveillance during outbreaks of zoonotic HPAI viruses. http://repub.eur.nl/res/pub/15057/ 2007-05-01T00:00:00Z Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus-host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds. http://repub.eur.nl/res/pub/8202/ 2006-12-22T00:00:00Z Influenza A virus behoort tot de familie van Orthomyxoviridae. Infl uenza A virussen zijn onregelmatig gevormde virussen van ongeveer 120 nm groot. Het genoom van influenza A virussen is gesegmenteerd en bestaat uit negatief-strengs RNA. De acht gensegmenten coderen voor 11 verschillende eiwitten. Infl uenza A virussen worden onderverdeeld op basis van de oppervlakte eiwitten; hemagglutinine (HA, een eiwit dat zorg draagt voor de binding van het virus aan en binnendringen van de gastheercel) en neuraminidase (NA, een eiwit dat zorgt voor de efficiënte verspreiding na vermenigvuldiging van het virus). Er waren tot voor kort 15 verschillende subtypen van HA geïdentificeerd en 9 verschillende subtypen van NA. Dit leidt tot 135 potentiële subtype combinaties zoals H1N1, H3N2, H5N1 en H7N7. Aviaire influenza virussen zijn verder in te delen op grond van biologische eigenschappen: laag pathogene aviaire influenza virussen (LPAI), die geen tot milde ziekteverschijnselen veroorzaken en hoog pathogene aviare influenza virussen (HPAI), die massale sterfte onder pluimvee kunnen veroorzaken (ook bekend als vogelpest). Influenza A virus is voornamelijk bekend als veroorzaker van de drie pandemieën (wereldwijde uitbraken) van de afgelopen eeuw: de H1N1 Spaanse griep in 1918, de H2N2 Aziatische griep in 1957 en de H3N2 Hong Kong griep in 1968. Daarnaast is het ook bekend van de jaarlijks terugkerende griepepidemieën (lokale uitbraken) en van uitbraken van vogelpest. Hoewel het bekend is dat alle infl uenza A virussen hun oorsprong hebben in wilde vogels, is er weinig bekend over het voorkomen van aviaire influenza virussen in wilde vogels in Europa. Het onderzoek beschreven in dit proefschrift richtte zich in eerste instantie op het in kaart brengen van de complexe ecologie van aviaire influenza virussen in hun natuurlijke gastheer, de wilde vogels, en vervolgens op de veranderingen die ten grondslag liggen aan het vermogen van deze virussen om overgedragen te kunnen worden naar de mens. Hoofdstuk 1 geeft een algemene inleiding en een overzicht van de huidige kennis over het voorkomen van infl uenza A virussen in wilde vogels. Hierbij wordt ingegaan op de rol die verschillende vogelfamilies spelen in de ecologie van infl uenza A virussen zoals de Anseriformes (eenden, ganzen en zwanen) en Charadriformes (meeuwen, alken en steltlopers). http://repub.eur.nl/res/pub/15052/ 2005-12-01T00:00:00Z In 2003 an outbreak of highly pathogenic avian influenza virus (H7N7) struck poultry in The Netherlands. A European Commission directive made vaccination of valuable species in zoo collections possible under strict conditions. We determined pre- and post-vaccination antibody titres in 211 birds by haemagglutination inhibition test as a measure of vaccine efficacy. After booster vaccination, 81.5% of vaccinated birds developed a titre of > or =40, while overall geometric mean titre (GMT) was 190 (95% CI: 144-251). Birds of the orders Anseriformes, Galliformes and Phoenicopteriformes showed higher GMT, and larger percentages developed titres > or =40 than those of the other orders. Antibody response decreased with increasing mean body weight in birds > or =1.5 kg body weight. In the vicinity of the outbreak, H7N7 was detected by RT-PCR in wild species (mallards and mute swans) kept in captivity together with infected poultry, illustrating the potential threat of transmission from poultry into other avian species, and the importance of protecting valuable avian species by means of vaccination. http://repub.eur.nl/res/pub/13914/ 2005-10-01T00:00:00Z In 2003, an outbreak of highly pathogenic avian influenza occurred in The Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, approximately 1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used. http://repub.eur.nl/res/pub/13697/ 2005-03-01T00:00:00Z In wild aquatic birds and poultry around the world, influenza A viruses carrying 15 antigenic subtypes of hemagglutinin (HA) and 9 antigenic subtypes of neuraminidase (NA) have been described. Here we describe a previously unidentified antigenic subtype of HA (H16), detected in viruses circulating in black-headed gulls in Sweden. In agreement with established criteria for the definition of antigenic subtypes, hemagglutination inhibition assays and immunodiffusion assays failed to detect specific reactivity between H16 and the previously described subtypes H1 to H15. Genetically, H16 HA was found to be distantly related to H13 HA, a subtype also detected exclusively in shorebirds, and the amino acid composition of the putative receptor-binding site of H13 and H16 HAs was found to be distinct from that in HA subtypes circulating in ducks and geese. The H16 viruses contained NA genes that were similar to those of other Eurasian shorebirds but genetically distinct from N3 genes detected in other birds and geographical locations. The European gull viruses were further distinguishable from other influenza A viruses based on their PB2, NP, and NS genes. Gaining information on the full spectrum of avian influenza A viruses and creating reagents for their detection and identification will remain an important task for influenza surveillance, outbreak control, and animal and public health. We propose that sequence analyses of HA and NA genes of influenza A viruses be used for the rapid identification of existing and novel HA and NA subtypes. http://repub.eur.nl/res/pub/13290/ 2004-02-03T00:00:00Z Highly pathogenic avian influenza A viruses of subtypes H5 and H7 are the causative agents of fowl plague in poultry. Influenza A viruses of subtype H5N1 also caused severe respiratory disease in humans in Hong Kong in 1997 and 2003, including at least seven fatal cases, posing a serious human pandemic threat. Between the end of February and the end of May 2003, a fowl plague outbreak occurred in The Netherlands. A highly pathogenic avian influenza A virus of subtype H7N7, closely related to low pathogenic virus isolates obtained from wild ducks, was isolated from chickens. The same virus was detected subsequently in 86 humans who handled affected poultry and in three of their family members. Of these 89 patients, 78 presented with conjunctivitis, 5 presented with conjunctivitis and influenza-like illness, 2 presented with influenza-like illness, and 4 did not fit the case definitions. Influenza-like illnesses were generally mild, but a fatal case of pneumonia in combination with acute respiratory distress syndrome occurred also. Most virus isolates obtained from humans, including probable secondary cases, had not accumulated significant mutations. However, the virus isolated from the fatal case displayed 14 amino acid substitutions, some of which may be associated with enhanced disease in this case. Because H7N7 viruses have caused disease in mammals, including horses, seals, and humans, on several occasions in the past, they may be unusual in their zoonotic potential and, thus, form a pandemic threat to humans.