[Show abstract][Hide abstract] ABSTRACT: Genetic sequences of a highly pathogenic avian influenza (H5N8) virus in England have high homology to those detected in mainland Europe and Asia during 2014. Genetic characterization suggests this virus is an avian-adapted virus without specific affinity for zoonoses. Spatio-temporal detections of H5N8 imply a role for wild birds in virus spread.
[Show abstract][Hide abstract] ABSTRACT: Swine influenza causes concern for global veterinary and public health officials. In continuing two previous networks that initiated the surveillance of swine influenza viruses (SIVs) circulating in European pigs between 2001 and 2008, a third European Surveillance Network for Influenza in Pigs (ESNIP3, 2010-2013) aimed to expand widely the knowledge of the epidemiology of European SIVs. ESNIP3 stimulated programs of harmonized SIV surveillance in European countries and supported the coordination of appropriate diagnostic tools and subtyping methods. Thus, an extensive virological monitoring, mainly conducted through passive surveillance programs, resulted in the examination of more than 9 000 herds in 17 countries. Influenza A viruses were detected in 31% of herds examined from which 1887 viruses were preliminary characterized. The dominating subtypes were the three European enzootic SIVs: avian-like swine H1N1 (53.6%), human-like reassortant swine H1N2 (13%) and human-like reassortant swine H3N2 (9.1%), as well as pandemic A/H1N1 2009 (H1N1pdm) virus (10.3%). Viruses from these four lineages co-circulated in several countries but with very different relative levels of incidence. For instance, the H3N2 subtype was not detected at all in some geographic areas whereas it was still prevalent in other parts of Europe. Interestingly, H3N2-free areas were those that exhibited highest frequencies of circulating H1N2 viruses. H1N1pdm viruses were isolated at an increasing incidence in some countries from 2010 to 2013, indicating that this subtype has become established in the European pig population. Finally, 13.9% of the viruses represented reassortants between these four lineages, especially between previous enzootic SIVs and H1N1pdm. These novel viruses were detected at the same time in several countries, with increasing prevalence. Some of them might become established in pig herds, causing implications for zoonotic infections.
PLoS ONE 12/2014; 9(12):e115815. DOI:10.1371/journal.pone.0115815 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Skeletal muscle, at 30 to 40% of body mass, is the most abundant soft tissue in the body. Besides its primary function in movement and posture, skeletal muscle is a significant innate immune organ with the capacity to produce cytokines and chemokines and respond to proinflammatory cytokines. Little is known about the role of skeletal muscle during systemic influenza A virus infection in any host and particularly avian species. Here we used primary chicken and duck multinucleated myotubes to examine their susceptibility and innate immune response to influenza virus infections. Both chicken and duck myotubes expressed avian and human sialic acid receptors and were readily susceptible to low-pathogenicity (H2N3 A/mallard duck/England/7277/06) and high-pathogenicity (H5N1 A/turkey/England/50-92/91 and H5N1 A/turkey/Turkey/1/05) avian and human H1N1 (A/USSR/77) influenza viruses. Both avian host species produced comparable levels of progeny H5N1 A/turkey/Turkey/1/05 virus. Notably, the rapid accumulation of viral nucleoprotein and matrix (M) gene RNA in chicken and duck myotubes was accompanied by extensive cytopathic damage with marked myotube apoptosis (widespread microscopic blebs, caspase 3/7 activation, and annexin V binding at the plasma membrane). Infected chicken myotubes produced significantly higher levels of proinflammatory cytokines than did the corresponding duck cells. Additionally, in chicken myotubes infected with H5N1 viruses, the induction of interferon beta (IFN-β) and IFN-inducible genes, including the melanoma differentiation-associated protein 5 (MDA-5) gene, was relatively weak compared to infection with the corresponding H2N3 virus. Our findings highlight that avian skeletal muscle fibers are capable of productive influenza virus replication and are a potential tissue source of infection.
IMPORTANCE Infection with high-pathogenicity H5N1 viruses in ducks is often asymptomatic, and skeletal muscle from such birds could be a source of infection of humans and animals. Little is known about the ability of influenza A viruses to replicate in avian skeletal muscle fibers. We show here that cultured chicken and duck myotubes were highly susceptible to infection with both low- and high-pathogenicity avian influenza viruses. Infected myotubes of both avian species displayed rapid virus accumulation, apoptosis, and extensive cellular damage. Our results indicate that avian skeletal muscle fibers of chicken and duck could be significant contributors to progeny production of highly pathogenic H5N1 viruses.
Journal of Virology 12/2014; 89(5). DOI:10.1128/JVI.03421-14 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-014-0118-3) contains supplementary material, which is available to authorized users.
Veterinary Research 12/2014; 45(1):118. DOI:10.1186/s13567-014-0118-3 · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Transboundary and Emerging Diseases 06/2014; DOI:10.1111/tbed.12232 · 2.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.
PLoS ONE 01/2014; 9(1):e87076. DOI:10.1371/journal.pone.0087076 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The divergence of the hemagglutinin gene of A/goose/Guangdong/1/1996-lineage H5N1 viruses during 2011 and 2012 (807 new sequences collected through December 31, 2012) was analyzed by phylogenetic and p-distance methods to define new clades using the pre-established nomenclature system. Eight new clade designations were recommended based on division of clade 1.1 (Mekong River Delta), 22.214.171.124 (Indonesia), 2.2.2 (India/Bangladesh), 126.96.36.199 (Egypt/Israel), and 188.8.131.52 (Asia). A simplification to the previously defined criteria, which adds a letter rather than number to the right-most digit of fifth-order clades, was proposed to facilitate this and future updates.
Influenza and Other Respiratory Viruses 01/2014; DOI:10.1111/irv.12230 · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm(2), which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.
[Show abstract][Hide abstract] ABSTRACT: Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-γ. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-γ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-γ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin.
Virus Research 09/2013; 178(2). DOI:10.1016/j.virusres.2013.09.015 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular characterization studies of a diverse collection of avian influenza viruses (AIVs) have demonstrated that AIVs' greatest genetic variability lies in the HA, NA, and NS genes. The objective here was to quantify the association between geographical locations, periods of time, and host species and pairwise nucleotide variation in the HA, NA, and NS genes of 70 isolates of H5N1 highly pathogenic avian influenza virus (HPAIV) collected from October 2005 to December 2007 from birds in Romania. A mixed-binomial Bayesian regression model was used to quantify the probability of nucleotide variation between isolates and its association with space, time, and host species. As expected for the three target genes, a higher probability of nucleotide differences (odds ratios [ORs] > 1) was found between viruses sampled from places at greater geographical distances from each other, viruses sampled over greater periods of time, and viruses derived from different species. The modeling approach in the present study maybe useful in further understanding the molecular epidemiology of H5N1 HPAI virus in bird populations. The methodology presented here will be useful in predicting the most likely genetic distance for any of the three gene segments of viruses that have not yet been isolated or sequenced based on space, time, and host species during the course of an epidemic.
[Show abstract][Hide abstract] ABSTRACT: 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.
Archives of Virology 05/2013; 158(11). DOI:10.1007/s00705-013-1735-2 · 2.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RESUMEN Modelo de la asociación entre espacio, tiempo y especies hospedadoras con variaciones de los genes HA, NA y NS de virus de la influenza aviar H5N1 altamente patógenos aislados de aves en Rumania entre los años 2005 al 2007. Los estudios de caracterización molecular de una colección diversa de virus de la influenza aviar (AIV) han demostrado que la mayor variabilidad genética de estos virus se encuentra en los genes HA, NA, y NS. El objetivo fue cuantificar la asociación entre la localización geográfica, los períodos de tiempo, y las especies hospedadoras con la variación en los pares de nucleótidos dentro de los genes HA, NA y NS de 70 aislamientos de virus de la influenza aviar altamente patógenos H5N1 (HPAIV) recolectados entre octubre de 2005 a diciembre del 2007 de aves en Rumania. Se utilizó un modelo de regresión bayesiana mixto binomial para cuantificar la probabilidad de variación de nucleótidos entre los aislamientos y su relación con el espacio, el tiempo, y las especies hospedadoras. Como se esperaba, para los tres genes estudiados, una mayor probabilidad de diferencias de nucleótidos (odds ratio [OR] > 1) se encontró entre los virus recolectados de lugares con grandes distancias geográficas entre sí, los virus detectados a lo largo de grandes períodos de tiempo, y los virus derivados de diferentes especies. El enfoque de este modelo en el presente estudio puede resultar útil para comprender aún más la epidemiología molecular del virus de la influenza aviar H5N1 altamente patógeno en poblaciones de aves. La metodología presentada, puede ser útil para predecir durante el curso de una epidemia, la distancia genética más probable para cualquiera de los tres segmentos de genes de virus que aún no han sido aislados, o secuenciados con base en el espacio, en el tiempo y en las especies hospedadoras.
[Show abstract][Hide abstract] ABSTRACT: Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.
Zoonoses and Public Health 04/2013; 61(1). DOI:10.1111/zph.12049 · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Philosophical Transactions of The Royal Society B Biological Sciences 02/2013; 368(1614):20120382. DOI:10.1098/rstb.2012.0382 · 7.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In January 2012, influenza virus researchers from around the world announced a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. Now that the aims of the voluntary moratorium have been met in some countries and are close to being met in others, we declare an end to the voluntary moratorium on avian flu transmission studies.
[Show abstract][Hide abstract] ABSTRACT: To simulate a field situation in which pigs are in close contact with poultry and thus provide a potential mixing vessel for avian, swine and human influenza viruses, uninfected pigs were placed in contact with Pekin ducks or chickens infected with a H5N1 highly pathogenic avian influenza (HPAI) virus. To sustain prolonged exposure, newly inoculated birds were added at regular intervals. Although influenza virus was detected in birds and environmental samples, 14 days exposure to infected birds failed to produce evidence of infection in the pigs. The ability of pigs to generate reassortant viruses with these particular virus variants (H5N1 clade 2.2.1) may therefore be limited.