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Infectivity and transmissibility of an avian H3N1 infuenza virus in pigs

Wojciech Stadejek, Koen Chiers and Kristien Van Reeth


In 2019 a low pathogenic H3N1 avian infuenza virus (AIV) caused an outbreak in Belgian poultry farms, characterized by an unusually high mortality in chickens. Infuenza A viruses of the H1 and H3 subtype can infect pigs and become established in swine populations. Therefore, the H3N1 epizootic raised concern about AIV transmission to pigs and from pigs to humans. Here, we assessed the replication efciency of this virus in explants of the porcine respiratory tract and in pigs, using virus titration and/or RT-qPCR. We also examined transmission from directly, intranasally inoculated pigs to contact pigs. The H3N1 AIV replicated to moderate titers in explants of the bronchioles and lungs, but not in the nasal mucosa or trachea. In the pig infection study, infectious virus was only detected in a few lung samples collected between 1 and 3 days post-inoculation. Virus titers were between 1.7 and 4.8 log10 TCID50. In line with the ex vivo experiment, no virus was isolated from the upper respiratory tract of pigs. In the transmission experiment, we could not detect virus transmission from directly inoculated to contact pigs. An increase in serum antibody titers was observed only in the inoculated pigs. We conclude that the porcine respiratory tract tissue explants can be a useful tool to assess the replication efciency of AIVs in pigs. The H3N1 AIV examined here is unlikely to pose a risk to swine populations. However, continuous risk assessment studies of emerging AIVs in pigs are necessary, since diferent virus strains will have diferent genotypic and phenotypic traits.


Genetic variation in chicken interferon signalling pathway genes in research lines showing differential viral resistance

Joshua Mountford, Almas Gheyas, Lonneke Vervelde, Jacqueline Smith


Avian viruses of economic interest are a significant burden on the poultry industry, affecting production traits and resulting in mortality. Furthermore, the zoonosis of avian viruses risks pandemics developing in humans. Vaccination is the most common method of controlling viruses; however current vaccines often lack cross-protection against multiple strains of each virus. The mutagenicity of these viruses has also led to virulent strains emerging that can overcome the protection offered by vaccines. Breeding chickens with a more robust innate immune response may help in tackling current and emerging viruses. Understanding the genetic evolution of different lines will thus provide a useful tool in helping the host in the fight against pathogens. This study focuses on the interferon genes and their receptors in different chicken lines that are known to be more resistant or susceptible to particular avian viruses. Comparing genotypic differences in these core immune genes between the chicken lines may explain the phenotypic differences observed and aid the identification of causative variations. The relative resistance/susceptibility of each line to viruses of interest (Marek’s disease virus, infectious bursal disease, infectious bronchitis virus and avian influenza virus) has previously been determined. Here we identify single nucleotide polymorphisms in interferons and downstream genes. Functional prediction tools were used to identify variants that may be affecting protein structure, mRNA secondary structure or transcription factor and micro-RNA binding sites. These variants were then considered in the context of the research lines and their distribution between phenotypes. We highlight 60 variants of interest in the interferon pathway genes that may account for susceptibility/resistance to viral pathogens.



Connect to Protect: Dynamics and Genetic Connections of Highly Pathogenic Avian Influenza Outbreaks in Poultry from 2016 to 2021 in Germany

Jacqueline King, Christoph Staubach, Christiane Lüder, Susanne Koethe, Anne Günther, Lina Stacker, Dennis Rubbenstroth, Klaas Dietze, Christian Grund, Franz J. Conraths, Timm Harder, Martin Beer and Anne Pohlmann


During autumn/winter in 2016–2017 and 2020–2021, highly pathogenic avian influenza viruses (HPAIV) caused severe outbreaks in Germany and Europe. Multiple clade H5 HPAI subtypes were responsible for increased mortality in wild birds and high mortality and massive losses in the poultry sector. To clarify putative entry sources and delineate interconnections between outbreaks in poultry holdings and wild birds, we applied whole-genome sequencing and phylodynamic analyses combined with the results of epidemiological outbreak investigations. Varying outbreak dynamics of the distinct reassortants allowed for the identification of individual, putatively wild bird-mediated entries into backyard holdings, several clusters comprising poultry holdings, local virus circulation for several weeks, direct farm-to-farm transmission and potential reassortment within a turkey holding with subsequent spill-over of the novel reassorted virus into the wild bird population. Whole-genome sequencing allowed for a unique high-resolution molecular epidemiology analysis of HPAIV H5Nx outbreaks and is recommended to be used as a standard tool. The presented detailed account of the genetic, temporal, and geographical characteristics of the recent German HPAI H5Nx situation emphasizes the role of poultry holdings as an important source of novel genetic variants and reassortants.



Evidence for Different Virulence Determinants and Host Response after Infection of Turkeys and Chickens with Highly Pathogenic H7N1 Avian Influenza Virus

Claudia Blaurock, Florian Pfaff, David Scheibner, Bernd Hoffmann, Alice Fusaro, Isabella Monne, Thomas C. Mettenleiter, Angele Breithaupt, Elsayed M. Abdelwhab


Wild birds are the reservoir for all avian influenza viruses (AIV). In poultry, the transition from low pathogenic (LP) AIV of H5 and H7 subtypes to highly pathogenic (HP) AIV is accompanied mainly by changing the hemagglutinin (HA) monobasic cleavage site (CS) to a polybasic motif (pCS). Galliformes, including turkeys and chickens, succumb with high morbidity and mortality to HPAIV infections, although turkeys appear more vulnerable than chickens. Surprisingly, the genetic determinants for virulence and pathogenesis of HPAIV in turkeys are largely unknown. Here, we determined the genetic markers for virulence and transmission of HPAIV H7N1 in turkeys, and we explored the host responses in this species compared to those of chickens. We found that recombinant LPAIV H7N1 carrying pCS was avirulent in chickens but exhibited high virulence in turkeys, indicating that virulence determinants vary in these two galliform species. A
transcriptome analysis indicated that turkeys mount a different host response than do chickens, particularly from genes involved in RNA metabolism and the immune response. Furthermore, we found that the HA glycosylation at residue 123, acquired by LP viruses shortly after transmission from wild birds and preceding the transition from LP to HP, had a role in virus fitness and virulence in chickens, though it was not a prerequisite for high virulence in turkeys. Together, these findings indicate variable virulence determinants and host responses in two closely related galliformes, turkeys and chickens, after infection with HPAIV H7N1. These results could explain the higher vulnerability to HPAIV of turkeys compared to chickens.


Hemagglutinin Subtype Specificity and Mechanisms of Highly Pathogenic Avian Influenza Virus Genesis

Anja C. M. de Bruin, Mathis Funk, Monique I. Spronken, Alexander P. Gultyaev, Ron A. M. Fouchier and Mathilde Richard


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.


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

V. Caliendo, N. S. Lewis, A. Pohlmann, S. R. Baillie, A. C. Banyard, M. Beer, I. H. Brown, R. A. M. Fouchier, R. D. E. Hansen, T. K. Lameris, A. S. Lang, S. Laurendeau, O. Lung, G. Robertson, H. van der Jeugd, T. N. Alkie, K. Thorup, M. L. van Toor, J. Waldenström, C. Yason, T. Kuiken, & Y. Berhane


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.


In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis

Mathis Funk, Anja C. M. de Bruin, Monique I. Spronken, Alexander P. Gultyaev and Mathilde Richard


A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.


Has Epizootic Become Enzootic? Evidence for a Fundamental Change in the Infection Dynamics of Highly Pathogenic Avian Influenza in Europe, 2021

Anne Pohlmann, Jacqueline King, Alice Fusaro, Bianca Zecchin, Ashley C. Banyard, Ian H. Brown, Alexander M. P. Byrne, Nancy Beerens, Yuan Liang, Rene Heutink, Frank Harders, Joe James, Scott M. Reid, Rowena D. E. Hansen, Nicola S. Lewis, Charlotte Hjulsager, Lars E. Larsen, Siamak Zohari, Kristofer Anderson, Caroline Bröjer, Alexander Nagy, Vladimir Savič, Steven van Borm, Mieke Steensels, Francois-Xavier Briand, Edyta Swieton, Krzysztof Smietanka, Christian Grund, Martin Beer, Timm Harder


Phylogenetic evidence from the recent resurgence of high-pathogenicity avian influenza (HPAI) virus subtype H5N1, clade, observed in European wild birds and poultry since October 2021, suggests at least two different and distinct reservoirs. We propose contrasting hypotheses for this emergence: (i) resident viruses have been maintained, presumably in wild birds, in northern Europe throughout the summer of 2021 to cause some of the outbreaks that are part of the most recent autumn/winter 2021 epizootic, or (ii) further virus variants were reintroduced by migratory birds, and these two sources of reintroduction have driven the HPAI resurgence. Viruses from these two principal sources can be distinguished by their hemagglutinin genes, which segregate into two distinct sublineages (termed B1 and B2) within clade, as well as their different internal gene compositions. The evidence of enzootic HPAI virus circulation during the summer of 2021 indicates a possible paradigm SHIFT in the epidemiology of HPAI in Europe.


Genetic Determinants for Virulence and Transmission of the Panzootic Avian Influenza Virus H5N8 Clade in Pekin Ducks

David Scheibner, Angele Breithaupt, Christine Luttermann, Claudia Blaurock, Thomas C. Mettenleiter, Elsayed M. Abdelwhab


Waterfowl is the natural reservoir for avian influenza viruses (AIV), where the infection is mostly asymptomatic. In 2016, the panzootic high pathogenicity (HP) AIV H5N8 of clade (designated H5N8-B) caused significant mortality in wild and domestic ducks, in stark contrast to the predecessor virus from 2014 (designated H5N8-A). Here, we studied the genetic determinants for virulence and transmission of H5N8 clade in Pekin ducks. While ducks inoculated with recombinant H5N8-A did not develop any clinical signs, H5N8-B-inoculated and cohoused ducks died after showing neurological signs. Swapping of the HA gene segments did not increase virulence of H5N8-A but abolished virulence and reduced systemic replication of H5N8-B. Only H5N8-A carrying H5N8-B HA, NP, and NS with or without NA exhibited high virulence in inoculated and contact ducks, similar to H5N8-B. Compared to H5N8-A, HA, NA, NS, and NP proteins of H5N8-B possess peculiar differences, which conferred increased receptor binding affinity, neuraminidase activity, efficiency to inhibit interferon-alpha induction, and replication in vitro, respectively. Taken together, this comprehensive study showed that HA is not the only virulence determinant of the panzootic H5N8-B in Pekin ducks, but NP, NS, and to a lesser extent NA were also necessary for the exhibition of high virulence in vivo. These proteins acted synergistically to increase receptor binding affinity, sialidase activity, interferon antagonism, and replication. This is the first ad-hoc study to investigate the mechanism underlying the high virulence of HPAIV in Pekin ducks.


Highly pathogenic avian influenza virus incursions of subtype H5N8, H5N5, H5N1, H5N4, and H5N3 in Germany during 2020-21

Jacqueline King, Timm Harder, Anja Globig, Lina Stacker, Anne Günther, Christian Grund, Martin Beer, and Anne Pohlmann


From October 2020 to July 2021, five different subtypes (H5N8, H5N5, H5N1, H5N4, and H5N3) and seven genotypes of highly pathogenic avian influenza viruses (HPAIV) belonging to clade were detected in a broad array of avian hosts in Germany. Initial incursion by wild birds with an unprecedented involvement of charadriiforme species at the Wadden Sea coast only carrying subtype H5N3, lateral spread between poultry with detection of novel reassortants and mixed infections in poultry holdings, suspected spillback of HPAIV from poultry to wild birds, and detection of HPAIV-infected wild birds during the following summer in 2021 were hallmarks of this epizootic. Local reassortment events with low pathogenic AIV strains were detected by phylogenetic analyses, with a dominating HP H5N8 and later HP H5N1 strain responsible for most cases. In addition, the first-ever described HPAIV strain of subtype H5N4 could be genetically characterized.


Hotspots in the grid: Avian sensitivity and vulnerability to collision risk from energy infrastructure interactions in Europe and North Africa

Jethro G. Gauld, João P. Silva, Philip W. Atkinson, Paul Record, Marta Acácio, Volen Arkumarev, Julio Blas, Willem Bouten, Niall Burton, Inês Catry, Jocelyn Champagnon, Gary D. Clewley, Mindaugas Dagys, Olivier Duriez, Klaus-Michael Exo, Wolfgang Fiedler, Andrea Flack, Guilad Friedemann, Johannes Fritz, Clara Garcia-Ripolles, Stefan Garthe, Dimitri Giunchi, Atanas Grozdanov, Roi Harel, Elizabeth M. Humphreys, René Janssen, Andrea Kölzsch, Olga Kulikova, Thomas K. Lameris, Pascual López-López, Elizabeth A. Masden, Flavio Monti, Ran Nathan, Stoyan Nikolov, Steffen Oppel, Hristo Peshev, Louis Phipps, Ivan Pokrovsky, Viola H. Ross-Smith, Victoria Saravia, Emily S. Scragg, Andrea Sforzi, Emilian Stoynov, Chris Thaxter, Wouter Van Steelant, Mariëlle van Toor, Bernd Vorneweg, Jonas Waldenström, Martin Wikelski, Ramūnas Žydelis, Aldina M. A. Franco


  1. Wind turbines and power lines can cause bird mortality due to collision or electrocution. The biodiversity impacts of energy infrastructure (EI) can be minimised through effective landscape-scale planning and mitigation. The identification of high-vulnerability areas is urgently needed to assess potential cumulative impacts of EI while supporting the transition to zero carbon energy.
  2. We collected GPS location data from 1,454 birds from 27 species susceptible to collision within Europe and North Africa and identified areas where tracked birds are most at risk of colliding with existing EI. Sensitivity to EI development was estimated for wind turbines and power lines by calculating the proportion of GPS flight locations at heights where birds were at risk of collision and accounting for species' specific susceptibility to collision. We mapped the maximum collision sensitivity value obtained across all species, in each 5 × 5 km grid cell, across Europe and North Africa. Vulnerability to collision was obtained by overlaying the sensitivity surfaces with density of wind turbines and transmission power lines.
  3. Results: Exposure to risk varied across the 27 species, with some species flying consistently at heights where they risk collision. For areas with sufficient tracking data within Europe and North Africa, 13.6% of the area was classified as high sensitivity to wind turbines and 9.4% was classified as high sensitivity to transmission power lines. Sensitive areas were concentrated within important migratory corridors and along coastlines. Hotspots of vulnerability to collision with wind turbines and transmission power lines (2018 data) were scattered across the study region with highest concentrations occurring in central Europe, near the strait of Gibraltar and the Bosporus in Turkey.
  4. Synthesis and applications. We identify the areas of Europe and North Africa that are most sensitive for the specific populations of birds for which sufficient GPS tracking data at high spatial resolution were available. We also map vulnerability hotspots where mitigation at existing EI should be prioritised to reduce collision risks. As tracking data availability improves our method could be applied to more species and areas to help reduce bird-EI conflicts.



Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses

Anja C. M. de Bruin, Monique I. Spronken, Theo M. Bestebroer, Ron A. M. Fouchier and Mathilde Richard


Highly pathogenic avian influenza viruses (HPAIVs) cause fatal systemic infections in chickens, which are associated with endotheliotropism. HPAIV infections in wild birds are generally milder and not endotheliotropic. Here, we aimed to elucidate the species-specific endotheliotropism of HPAIVs using primary chicken and duck aortic endothelial cells (chAEC and dAEC respectively). Viral replication kinetics and host responses were assessed in chAEC and dAEC upon inoculation with HPAIV H5N1 and compared to embryonic fibroblasts. Although dAEC were susceptible to HPAIV upon inoculation at high multiplicity of infection, HPAIV replicated to lower levels in dAEC than chAEC during multi-cycle replication. The susceptibility of duck embryonic endothelial cells to HPAIV was confirmed in embryos. Innate immune responses upon HPAIV inoculation differed between chAEC, dAEC, and embryonic fibroblasts. Expression of the pro-inflammatory cytokine IL8 increased in chicken cells but decreased in dAEC. Contrastingly, the induction of antiviral responses was stronger in dAEC than in chAEC, and chicken and duck fibroblasts. Taken together, these data demonstrate that although duck endothelial cells are permissive to HPAIV infection, they display markedly different innate immune responses than chAEC and embryonic fibroblasts. These differences may contribute to the species-dependent differences in endotheliotropism and consequently HPAIV pathogenesis.


Migration distance affects how closely Eurasian wigeons follow spring phenology during migration

Mariëlle L. van Toor, Sergey Kharitonov, Saulius Švažas, Mindaugas Dagys, Erik Kleyheeg, Gerard Müskens, Ulf Ottosson, Ramunas Žydelis and Jonas Waldenström


Background: The timing of migration for herbivorous migratory birds is thought to coincide with spring phenology as emerging vegetation supplies them with the resources to fuel migration, and, in species with a capital breeding strategy also provides individuals with energy for use on the breeding grounds. Individuals with very long migration distances might however have to trade of between utilising optimal conditions en route and reaching the breeding grounds early, potentially leading to them overtaking spring on the way. Here, we investigate whether migration distance afects how closely individually tracked Eurasian wigeons follow spring phenology during spring migration.
Methods: We captured wigeons in the Netherlands and Lithuania and tracked them throughout spring migration to identify staging sites and timing of arrival. Using temperature-derived indicators of spring phenology, we investigated how maximum longitude reached and migration distance afected how closely wigeons followed spring. We further estimated the impact of tagging on wigeon migration by comparing spring migratory timing between tracked individuals and ring recovery data sets.
Results: Wigeons migrated to locations between 300 and 4000 km from the capture site, and migrated up to 1000 km in a single day. We found that wigeons migrating to more north-easterly locations followed spring phenology more closely, and increasingly so the greater distance they had covered during migration. Yet we also found that despite tags equalling only around 2% of individual’s body mass, individuals were on average 11–12 days slower than ring-marked individuals from the same general population.
Discussion: Overall, our results suggest that migratory strategy can vary dependent on migration distance within species, and even within the same migratory corridor. Individual decisions thus depend not only on environmental cues, but potentially also trade-ofs made during later life-history stages.


Encephalitis and Death in Wild Mammals at a Rehabilitation Center after Infection with Highly Pathogenic Avian Influenza A(H5N8) Virus, United Kingdom

Tobias Floyd, Ashley C Banyard, Fabian Z X Lean, Alexander M P Byrne, Edward Fullick, Elliot Whittard, Benjamin C Mollett, Steve Bexton, Vanessa Swinson, Michele Macrelli, Nicola S Lewis, Scott M Reid, Alejandro Núñez, J Paul Duff, Rowena Hansen, Ian H Brown


We report a disease and mortality event involving swans, seals, and a fox at a wildlife rehabilitation center in the United Kingdom during late 2020. Five swans had onset of highly pathogenic avian influenza virus infection while in captivity. Subsequently, 5 seals and a fox died (or were euthanized) after onset of clinical disease. Avian-origin influenza A virus subtype H5N8 was retrospectively determined as the cause of disease. Infection in the seals manifested as seizures, and immunohistochemical and molecular testing on postmortem samples detected a neurologic distribution of viral products. The fox died overnight after sudden onset of inappetence, and postmortem tissues revealed neurologic and respiratory distribution of viral products. Live virus was isolated from the swans, seals, and the fox, and a single genetic change was detected as a potential adaptive mutation in the mammalian-derived viral sequences. No human influenza-like illness was reported in the weeks after the event.



Active Surveillance for Highly Pathogenic Avian Influenza Viruses in Wintering Waterbirds in Northeast Italy, 2020–2021

Federica Gobbo, Diletta Fornasiero, Maria Alessandra De Marco, Bianca Zecchin, Paolo Mulatti, Mauro Delogu and Calogero Terregino


The increasing involvement of wild waterfowl in H5 Highly Pathogenic Avian Influenza Virus (HPAIV) circulation continues to pose a threat to animal and public health worldwide. In winter 2020–2021, two field surveillance activities were carried out on a weekly basis, through virological and serological analyses, in 823 hunted and 521 trapped migratory aquatic birds in northeast Italy. Sixty Eurasian teals were recaptured several times, which allowed us to follow the progression of the HPAI H5 infection in naturally infected wild waterfowl. Oropharyngeal, cloacal, and feather swabs (OS, CS and FS) were collected from each duck and tested by real time rRT-PCR Type A influenza. The identified viruses were characterized and pathotyped by sequencing. Several viruses belonging to three different HPAI H5 subtypes were detected: H5N8, H5N5, and H5N1. High prevalence of infection with HPAI H5 clade during November–December 2020 (up to 27.1%) was observed in captured Eurasian teals, while infection rates in hunted dabbling ducks, mainly Eurasian wigeons, showed the highest prevalence of infection in November 2020 (8.9%) and January 2021 (10.2%). All HPAI positive birds were also clinically healthy when recaptured weeks apart. The OS and FS showed the highest detection efficiency of HPAIV. Our results highlight that HPAI passive surveillance should be complemented by a targeted active surveillance to more efficiently detect novel HPAI viruses.



Limited onward transmission potential of reassortment genotypes from chickens co-infected with H9N2 and H7N9 avian influenza viruses

Wen Su, Sin Fun Sia, Ka-Tim Choy, Yue Ji, Dongdong Chen, Eric Ho Yin Lau, Guanghua Fu, Yu Huang, Jinhua Liu, Malik Peiris, Juan Pu, and Hui-Ling Yen

The segmented genome of influenza A virus has conferred significant evolutionary advantages to this virus through genetic reassortment, a mechanism that facilitates the rapid expansion of viral genetic diversity upon influenza co-infections. Therefore, co-infection of genetically diverse avian influenza viruses in poultry may pose a significant public health risk in generating novel reassortants with increased zoonotic potential. This study investigated the reassortment patterns of a Pearl River Delta-lineage avian influenza A(H7N9) virus and four genetically divergent avian influenza A(H9N2) viruses upon co-infection in embryonated chicken eggs and chickens. To characterize “within-host” and “between-host” genetic diversity, we further monitored the viral genotypes that were subsequently transmitted to contact chickens in serial transmission experiments. We observed that co-infection with A(H7N9) and A(H9N2) viruses may lead to the emergence of novel reassortant viruses in ovo and in chickens, albeit with different reassortment patterns. Novel reassortants detected in donor chickens co-infected with different combinations of the same A(H7N9) virus and different A(H9N2) viruses showed distinct onward transmission potential to contact chickens. Sequential transmission of novel reassortant viruses was only observed in one out of four co-infection combinations. Our results demonstrated different patterns by which influenza viruses may acquire genetic diversity through co-infection in ovoin vivo, and under sequential transmission conditions.



Compositional biases in RNA viruses: Causes, consequencesand applications

Eleanor R. Gaunt and Paul Digard

If each of the four nucleotides were represented equally in the genomes of viruses and the hosts they infect, each base would occur at a frequency of 25%. However, this is not observed in nature. Similarly, the order of nucleotides is not random (e.g., in the human genome, guanine follows cytosine at a frequency of ~0.0125, or a quarter the number of times predicted by random representation). Codon usage and codon order are also nonrandom. Furthermore, nucleotide and codon biases vary between species. Such biases have various drivers, including cellular proteins that recognize specific patterns in nucleic acids, that once triggered, induce mutations or invoke intrinsic or innate immune responses. In this review we examine the types of compositional biases identified in viral genomes and current understanding of the evolutionary mechanisms underpinning these trends. Finally, we consider the potential for large scale synonymous recoding strategies to engineer RNA virus vaccines, including those with pandemic potential, such as influenza A virus and Severe Acute Respiratory Syndrome Coronavirus Virus 2.


Genetic and antigenic evolution of H1 swine influenza A viruses isolated in Belgium and the Netherlands from 2014 through 2019

Sharon Chepkwony, Anna Parys, Elien Vandoorn, Wojciech Stadejek, Jiexiong Xie, Jacqueline King, Annika Graaf, Anne Pohlmann, Martin Beer, Timm Harder & Kristien Van Reeth

Surveillance of swine influenza A viruses (swIAV) allows timely detection and identification of new variants with potential zoonotic risks. In this study, we aimed to identify swIAV subtypes that circulated in pigs in Belgium and the Netherlands between 2014 and 2019, and characterize their genetic and antigenic evolution. We subtyped all isolates and analyzed hemagglutinin sequences and hemagglutination inhibition assay data for H1 swIAV, which were the dominant HA subtype. We also analyzed whole genome sequences (WGS) of selected isolates. Out of 200 samples, 89 tested positive for swIAV. swIAV of H1N1, H1N2 and H3N2 subtypes were detected. Analysis of WGS of 18 H1 swIAV isolates revealed three newly emerged genotypes. The European avian-like H1 swIAV (lineage 1C) were predominant and accounted for 47.2% of the total isolates. They were shown to evolve faster than the European human-like H1 (1B lineage) swIAV, which represented 27% of the isolates. The 2009 pandemic H1 swIAV (lineage 1A) accounted for only 5.6% of the isolates and showed divergence from their precursor virus. These results point to the increasing divergence of swIAV and stress the need for continuous surveillance of swIAV.


Preferential Selection and Contribution of Non-Structural Protein 1 (NS1) to the Efficient Transmission of Panzootic Avian Influenza H5N8 Virus Clades and B in Chickens and Ducks

Claudia Blaurock, Angele Breithaupt, David Scheibner, Ola Bagato, Axel Karger, Thomas C. Mettenleiter, and Elsayed M. Abdelwhab

Highly pathogenic avian influenza virus H5N8 clade caused outbreaks in poultry at an unprecedented global scale. The virus was spread by wild birds in Asia in two waves: clade in 2014/2015 and clade from 2016 up to today. Both clades were highly virulent in chickens, but only clade B viruses exhibited high virulence in ducks. Viral factors which contribute to virulence and transmission of these panzootic H5N8 viruses are largely unknown. The NS1 protein, typically composed of 230 amino acids (aa), is a multifunctional protein which is also a pathogenicity factor. Here, we studied the evolutionary trajectory of H5N8 NS1 proteins from 2013 to 2019 and their role in the fitness of H5N8 viruses in chickens and ducks. Sequence analysis and in vitro experiments indicated that clade and clade viruses have a preference for NS1 of 237 aa and 217 aa, respectively, over NS1 of 230 aa. NS217 was exclusively seen in domestic and wild birds in Europe. The extension of the NS1 C terminus (CTE) of clade B virus reduced virus transmission and replication in chickens and ducks and partially impaired the systemic tropism to the endothelium in ducks. Conversely, lower impact on fitness of clade A virus was observed. Remarkably, the NS1 of clade A and clade B, regardless of length, was efficient in blocking interferon (IFN) induction in infected chickens, and changes in the NS1 C terminus reduced the efficiency for interferon antagonism. Together, the NS1 C terminus contributes to the efficient transmission and high fitness of H5N8 viruses in chickens and ducks.



The C-Terminus of Non-Structural Protein 1 (NS1) in H5N8 Clade Avian Influenza Virus Affects Virus Fitness in Human Cells and Virulence in Mice

Claudia Blaurock, Ulrike Blohm, Christine Luttermann, Julia Holzerland, David Scheibner, Alexander Schäfer, Allison Groseth, Thomas C. Mettenleiter & Elsayed M. Abdelwhab

Avian influenza viruses (AIV) H5N8 clade pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade (H5N8-A) from 2013-2015 and clade (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.



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

A.H. Seekings, C.J. Warren, S.S. Thomas, S. Mahmood, J. James, A.M.P. Byrne, S. Watson, C. Bianco, A. Nunez, I.H. Brown, S.M. Brookes, M.J. Slomka

Highly-pathogenic avian influenza virus (HPAIV) H5N6 (clade 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.



Human Infection with Eurasian Avian-Like Swine Influenza A(H1N1) Virus, the Netherlands, September 2019

Anna Parys, Elien Vandoorn, Jacqueline King, Annika Graaf, Anne Pohlmann, Martin Beer, Timm Harder, and Kristien Van Reeth

We report a zoonotic infection of a pig farmer in the Netherlands with a Eurasian avian-like swine influenza A(H1N1) virus that was also detected in the farmed pigs. Both viruses were antigenically and genetically characterized. Continued surveillance of swine influenza A viruses is needed for risk assessment in humans and swine.


Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites

Alexander P Gultyaev, Monique I Spronken, Mathis Funk, Ron A M Fouchier, Mathilde Richard

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype ha. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.



Non‑basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys

Claudia Blaurock, David Scheibner, Maria Landmann, Melina Vallbracht, Reiner Ulrich, Eva Böttcher‑Friebertshäuser, Thomas C. Mettenleiter & Elsayed M. Abdelwhab

H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cellto-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.



Enterotropism of highly pathogenic avian influenza virus H5N8 from the 2016/2017 epidemic in some wild bird species

Valentina Caliendo, Lonneke Leijten, Lineke Begeman, Marjolein J. Poen, Ron A. M. Fouchier, Nancy Beerens & Thijs Kuiken


In 2016/2017, H5N8 highly pathogenic avian infuenza (HPAI) virus of the Goose/Guangdong lineage spread from Asia to Europe, causing the biggest and most widespread HPAI epidemic on record in wild and domestic birds in Europe. We hypothesized that the wide dissemination of the 2016 H5N8 virus resulted at least partly from a change in tissue tropism from the respiratory tract, as in older HPAIV viruses, to the intestinal tract, as in low pathogenic avian infuenza (LPAI) viruses, allowing more efcient faecal-oral transmission. Therefore, we determined the tissue tropism and associated lesions in wild birds found dead during the 2016 H5N8 epidemic, as well as the pattern of attachment of 2016 H5N8 virus to respiratory and intestinal tissues of four key wild duck species. We found that, out of 39 H5N8-infected wild birds of 12 species, four species expressed virus antigen in both respiratory and intestinal epithelium, one species only in respiratory epithelium, and one species only in intestinal epithelium. Virus antigen expression was association with infammation and necrosis in multiple tissues. The level of attachment to wild duck intestinal epithelia of 2016 H5N8 virus was comparable to that of LPAI H4N5 virus, and higher than that of 2005 H5N1 virus for two of the four duck species and chicken tested. Overall, these results indicate that 2016 H5N8 may have acquired a similar enterotropism to LPAI viruses, without having lost the respirotropism of older HPAI viruses of the Goose/Guangdong lineage. The increased enterotropism of 2016 H5N8 implies that this virus had an increased chance to persist long term in the wild waterbird reservoir.


Genesis and spread of multiple reassortants during the 2016/2017 H5 avian influenza epidemic in Eurasia

Samantha J. Lycett, Anne Pohlmann, Christoph Staubach, Valentina Caliendo, Mark Woolhouse, Martin Beer, Thijs Kuiken, and Global Consortium for H5N8 and Related Influenza Viruses


Highly pathogenic avian influenza (HPAI) viruses of the H5 A/goose/ Guangdong/1/96 lineage can cause severe disease in poultry and wild birds, and occasionally in humans. In recent years, H5 HPAI viruses of this lineage infecting poultry in Asia have spilled over into wild birds and spread via bird migration to countries in Europe, Africa, and North America. In 2016/2017, this spillover resulted in the largest HPAI epidemic on record in Europe and was associated with an unusually high frequency of reassortments between H5 HPAI viruses and cocirculating low-pathogenic avian influenza viruses. Here, we show that the seven main H5 reassortant viruses had various combinations of gene segments 1, 2, 3, 5, and 6. Using detailed time-resolved phylogenetic analysis, most of these gene segments likely originated from wild birds and at dates and locations that corresponded to their hosts’ migratory cycles. However, some gene segments in two reassortant viruses likely originated from domestic anseriforms, either in spring 2016 in east China or in autumn 2016 in central Europe. Our results demonstrate that, in addition to domestic anseriforms in Asia, both migratory wild birds and domestic anseriforms in Europe are relevant sources of gene segments for recent reassortant H5 HPAI viruses. The ease with which these H5 HPAI viruses reassort, in combination with repeated spillovers of H5 HPAI viruses into wild birds, increases the risk of emergence of a reassortant virus that persists in wild bird populations yet remains highly pathogenic for poultry.


Different environmental gradients associated to the spatio-temporal and genetic pattern of the H5N8 highly pathogenic avian influenza outbreaks in poultry in Italy

Francesca Scolamacchia, Paolo Mulatti, Matteo Mazzucato, Marco Barbujani, William T. Harvey, Alice Fusaro, Isabella Monne, Stefano Marangon

Comprehensive understanding of the patterns and drivers of avian influenza outbreaks is pivotal to inform surveillance systems and heighten nations’ ability to quickly detect and respond to the emergence of novel viruses. Starting in early 2017, the Italian poultry sector has been involved in the massive H5N8 highly pathogenic avian influenza epidemic that spread in the majority of the European countries in 2016/2017. Eighty-three outbreaks were recorded in north-eastern Italy, where a densely populated poultry area stretches along the Lombardy, Emilia-Romagna and Veneto regions. The confirmed cases, affecting both the rural and industrial sectors, depicted two distinct epidemic waves. We adopted a combination of multivariate statistics techniques and multi-model regression selection and inference, to investigate how environmental factors relate to the pattern of outbreaks diversity with respect to their spatiotemporal and genetic diversity. Results showed that a combination of eco-climatic and host density predictors were associated with the outbreaks pattern, and variation along gradients was noticeable among genetically and geographically distinct groups of avian influenza cases. These regional contrasts may be indicative of a different mechanism driving the introduction and spreading routes of the influenza virus in the domestic poultry population. This methodological approach may be extended to different spatiotemporal scale to foster site-specific, ecologically informed risk mitigating strategies.



Novel HPAIV H5N8 Reassortant (Clade Detected in Germany

Jacqueline King, Christoph Schulze, Andreas Engelhardt, Andreas Hlinak, Sara-Lisa Lennermann, Kerstin Rigbers, Jasmin Skuballa, Christoph Staubach, Thomas C. Mettenleiter, Timm Harder, Martin Beer, and Anne Pohlmann

A novel H5N8 highly pathogenic avian influenza virus (HPAIV) was detected in a greater white-fronted goose in January 2020 in Brandenburg, Germany, and, in February 2020, in domestic chickens belonging to a smallholding in Baden-Wuerttemberg, Germany. Full-genome sequencing was conducted on the MinION platform, enabling further phylogenetic analyses. The virus of clade holds six segments from a Eurasian/Asian/African HPAIV H5N8 reassortant and two segments from low pathogenic avian influenza H3N8 subtype viruses recently detected in wild birds in Central Russia. These new entries continue to show the reassortment potential of the clade H5Nx viruses, underlining the necessity for full-genome sequencing and continuous surveillance.


Precision cut lung slices: a novel versatile tool to examine host–pathogen interaction in the chicken lung

Karen Jane Bryson, Damien Garrido, Marco Esposito, Gerry McLachlan, Paul Digard, Catherine Schouler, Rodrigo Guabiraba, Sascha Trapp and Lonneke Vervelde

The avian respiratory tract is a common entry route for many pathogens and an important delivery route for vaccination in the poultry industry. Immune responses in the avian lung have mostly been studied in vivo due to the lack of robust, relevant in vitro and ex vivo models mimicking the microenvironment. Precision-cut lung slices (PCLS) have the major advantages of maintaining the 3-dimensional architecture of the lung and includes heterogeneous cell populations. PCLS have been obtained from a number of mammalian species and from chicken embryos. However, as the embryonic lung is physiologically undifferentiated and immunologically immature, it is less suitable to examine complex host–pathogen interactions including antimicrobial responses. Here we prepared PCLS from immunologically mature chicken lungs, tested different culture conditions, and found that serum supplementation has a detrimental effect on the quality of PCLS. Viable cells in PCLS remained present for ≥ 40 days, as determined by viability assays and sustained motility of fluorescent mononuclear phagocytic cells. The PCLS were responsive to lipopolysaccharide stimulation, which induced the release of nitric oxide, IL-1β, type I interferons and IL-10. Mononuclear phagocytes within the tissue maintained phagocytic activity, with live cell imaging capturing interactions with latex beads and an avian pathogenic Escherichia coli strain. Finally, the PCLS were also shown to be permissive to infection with low pathogenic avian influenza viruses. Taken together, immunologically mature chicken PCLS provide a suitable model to simulate live organ responsiveness and cell dynamics, which can be readily exploited to examine host–pathogen interactions and inflammatory responses.


The Pathogenesis of H7 Highly Pathogenic Avian Influenza Viruses in Lesser Scaup (Aythya affinis).

Christopher B. Stephens, Diann J. Prosser, Mary J. Pantin-Jackwood, Alicia M. Berlin, Erica Spackman 

Waterfowl are the natural hosts of avian influenza virus (AIV), and through migration spread the virus worldwide. Most AIVs carried by wild waterfowl are low pathogenic strains; however, Goose/Guangdong/1996 lineage clade H5 highly pathogenic (HP) AIV now appears to be endemic in wild birds in much of the Eastern Hemisphere. Most research efforts studying AIV pathogenicity in waterfowl thus far have been directed toward dabbling ducks. In order to better understand the role of diving ducks in AIV ecology, we previously characterized the pathogenesis of clade H5 HPAIV in lesser scaup (Aythya affinis). In an effort to further elucidate AIV infection in diving ducks, the relative susceptibility and pathogenesis of two North American lineage H7 HPAIV isolates from the most recent outbreaks in the United States was investigated. Lesser scaup were inoculated with either A/turkey/IN/1403-1/2016 H7N8 or A/chicken/TN/17-007147-2/2017 H7N9 HPAIV by the intranasal route. The approximate 50% bird infectious dose (BID50) of the H7N8 isolate was determined to be 103 50% egg infectious doses (EID50), and the BID50 of the H7N9 isolate was determined to be <102 EID50, indicating some variation in adaptation between the two isolates. No mortality or clinical disease was observed in either group except for elevated body temperatures at 2 and 4 days postinoculation (DPI). Virus shedding was detected up to 14 DPI from both groups, and there was a trend for shedding to have a longer duration and at higher titer levels from the cloacal route. These results demonstrate that lesser scaup are susceptible to both H7 lineages of HPAIV, and similar to dabbling duck species, they shed virus for long periods relative to gallinaceous birds and don't present with clinical disease.


Spatiotemporal reconstruction and transmission dynamics during the 2016–17 H5N8 highly pathogenic avian influenza epidemic in Italy

William T. Harvey, Paolo Mulatti, Alice Fusaro, Francesca Scolamacchia, Bianca Zecchin, Isabella Monne, Stefano Marangon

Effective control of avian diseases in domestic populations requires understanding of the transmission dynamics facilitating viral emergence and spread. In 2016–17, Italy experienced a significant avian influenza epidemic caused by a highly pathogenic A(H5N8) virus, which affected domestic premises housing around 2.7 million birds, primarily in the north-eastern regions with the highest density of poultry farms (Lombardy, Emilia-Romagna and Veneto). We perform integrated analyses of genetic, spatiotemporal and host data within a Bayesian phylogenetic framework. Using continuous and discrete phylogeography, we estimate the locations of movements responsible for the spread and persistence of the epidemic. The information derived from these analyses on rates of transmission between regions through time can be used to assess the success of control measures. Using an approach based on phylogenetic–temporal distances between domestic cases, we infer the presence of cryptic wild bird-mediated transmission, information that can be used to complement existing epidemiological methods for distinguishing transmission within the domestic population from incursions across the wildlife–domestic interface, a common challenge in veterinary epidemiology. Spatiotemporal reconstruction of the epidemic reveals a highly skewed distribution of virus movements with a high proportion of shorter distance local movements interspersed with occasional long-distance dispersal events associated with wild birds. We also show how such inference be used to identify possible instances of human-mediated movements where distances between phylogenetically linked domestic cases are unusually high.


Disentangling the role of Africa in the global spread of H5 highly pathogenic avian influenza

Alice Fusaro, Bianca Zecchin, Bram Vrancken, Celia Abolnik, Rose Ademun, Abdou Alassane, Abdelsatar Arafa, Joseph Adongo Awuni, Emmanuel Couacy-Hymann, M.’ Bétiégué Coulibaly, Nicolas Gaidet, Emilie Go-Maro, Tony Joannis, Simon Dickmu Jumbo, Germaine Minoungou, Clement Meseko, Maman Moutari Souley, Deo Birungi Ndumu, Ismaila Shittu, Augustin Twabela, Abel Wade, Lidewij Wiersma, Yao P. Akpeli, Gianpiero Zamperin, Adelaide Milani, Philippe Lemey & Isabella Monne

The role of Africa in the dynamics of the global spread of a zoonotic and economically-important virus, such as the highly pathogenic avian influenza (HPAI) H5Nx of the Gs/GD lineage, remains unexplored. Here we characterise the spatiotemporal patterns of virus diffusion during three HPAI H5Nx intercontinental epidemic waves and demonstrate that Africa mainly acted as an ecological sink of the HPAI H5Nx viruses. A joint analysis of host dynamics and continuous spatial diffusion indicates that poultry trade as well as wild bird migrations have contributed to the virus spreading into Africa, with West Africa acting as a crucial hotspot for virus introduction and dissemination into the continent. We demonstrate varying paths of avian influenza incursions into Africa as well as virus spread within Africa over time, which reveal that virus expansion is a complex phenomenon, shaped by an intricate interplay between avian host ecology, virus characteristics and environmental variables.


Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017–2018

Anne Pohlmann, Donata Hoffmann, Christian Grund, Susanne Koethe, Daniela Hüssy, Simone M. Meier, Jacqueline King, Jan Schinköthe, Reiner Ulrich, Timm Harder, and Martin Beer

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade isolates found in Germany in 2017–2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.


Role of Endothelial Cells in the Pathogenesis of Influenza in Humans

Kirsty R ShortThijs KuikenDebby Van Riel

Not available


Intercontinental spread of Asian-origin H7 avian influenza viruses by captive bird trade in 1990`s. Infection, Genetics and Evolution

Dong-Hun Lee, Mary Lea Killian, Mia K. Torchetti, Ian Brown, Nicola Lewis, Yohannes Berhane, David E. Swayne

Wild bird migration and illegal trade of infected poultry, eggs, and poultry products have been associated with the spread of avian influenza viruses (AIV). During 1992–1996, H7N1 and H7N8 low pathogenic AIV (LPAIV) were identified from captive wild birds; such as Pekin robin (Leiothrix lutea), magpie robin (Copsychus saularis), flycatcher sp. (genus Empidonax), a species of softbill and parakeet, sun conure (Aratinga solstitialis), painted conure (Pyrrhura picta), fairy bluebird (Irena puella), and common iora (Aegithina tiphia), kept in aviaries or quarantine stations in England, The Netherlands, Singapore and the United States (U.S.). In this study, we sequenced these H7 viruses isolated from quarantine facilities and aviaries using next-generation sequencing and conducted a comparative phylogenetic analysis of complete genome sequences to elucidate spread patterns. The complete genome sequencing and phylogenetic analysis suggested that H7 viruses originated from a common source, even though they were obtained from birds in distant geographical regions. All H7N1 and H7N8 viruses were LPAIV, except a H7N1 highly pathogenic AIV (HPAIV), A/Pekin robin/California/30412/1994(H7N1) virus. Our results support the continued need for regulation of the captive wild bird trade to reduce the risk of introduction and dissemination of both LPAIV and HPAIV throughout the world.


Variable impact of the hemagglutinin polybasic cleavage site on virulence and pathogenesis of avian influenza H7N7 virus in chickens, turkeys and ducks

David Scheibner, Reiner Ulrich, Olanrewaju I . Fatola, Annika Graaf, Marcel Gischke, Ahmed H. Salaheldin, Timm C . Harder, Jutta Veits, Thomas C . Mettenleiter & Elsayed M. Abdelwhab

Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1-H16) and 9 neuraminidase (NA; N1-N9) subtypes. All AIV are low pathogenic (LP) in birds, but subtypes H5 and H7 AIV can evolve into highly pathogenic (HP) forms. In the last two decades evolution of HPAIV H7 from LPAIV has been frequently reported. However, little is known about the pathogenesis and evolution of HP H7 from LP ancestors particularly, in non-chicken hosts. In 2015, both LP and HP H7N7 AIV were isolated from chickens in two neighbouring farms in Germany. Here, the virulence of these isogenic H7N7 LP, HP and LP virus carrying a polybasic HA cleavage site (HACS) from HP (designated LP-Poly) was studied in chickens, turkeys and different duck breeds. The LP precursor was avirulent in all birds. In contrast, all inoculated and contact chickens and turkeys died after infection with HP. HP infected Pekin and Mallard ducks remained clinically healthy, while Muscovy ducks exhibited moderate depression and excreted viruses at significantly higher amounts. The polybasic HACS increased virulence in a species-specific manner with intravenous pathogenicity indices of 3.0, 1.9 and 0.2 in chickens, turkeys and Muscovy ducks, respectively. Infection of endothelial cells was only observed in chickens. In summary, Pekin and Mallard were more resistant to HPAIV H7N7 than chickens, turkeys and Muscovy ducks. The polybasic HACS was the main determinant for virulence and endotheliotropism of HPAIV H7N7 in chickens, whereas other viral and/or host factors play an essential role in virulence and pathogenesis in turkeys and ducks.


Loss of fitness in mallards of Mexican H7N3 highly pathogenic avian influenza virus after circulating in chickens.

Sung-Su Youk, Dong-Hun Lee, Christina M. Leyson, Diane Smith, Miria Ferreira Criado, Eric DeJesus, David E. Swayne, Mary J. Pantin-Jackwood

Outbreaks of highly pathogenic avian influenza (HPAI) virus subtype H7N3 have been occurring in commercial chickens in Mexico since its first introduction in 2012. In order to determine changes in virus pathogenicity and adaptation in avian species, three H7N3 HPAI viruses from 2012, 2015, and 2016 were evaluated in chickens and mallards. All three viruses caused high mortality in chickens when given at medium to high doses and replicated similarly. No mortality or clinical signs and similar infectivity were observed in mallards inoculated with the 2012 and 2016 viruses. However, the 2012 H7N3 HPAI virus replicated well in mallards and transmitted to contacts, whereas the 2016 virus replicated poorly and did not transmit to contacts, which indicates that the 2016 virus is less adapted to mallards. In vitro, the 2016 virus grew slower and to lower titers than did the 2012 virus in duck fibroblast cells. Full-genome sequencing showed 115 amino acid differences between the 2012 and the 2016 viruses, with some of these changes previously associated with changes in replication in avian species, including hemagglutinin (HA) A125T, nucleoprotein (NP) M105V, and NP S377N. In conclusion, as the Mexican H7N3 HPAI virus has passaged through large populations of chickens in a span of several years and has retained its high pathogenicity for chickens, it has decreased in fitness in mallards, which could limit the potential spread of this HPAI virus by waterfowl.


Virulence of three European highly pathogenic H7N1 and H7N7 avian influenza viruses in Pekin and Muscovy ducks

David Scheibner, Claudia Blaurock, Thomas C. Mettenleiter and Elsayed M. Abdelwhab

Background: There is paucity of data on the virulence of highly pathogenic (HP) avian influenza viruses (AIV) H7 in ducks compared to HPAIV H5. Here, the virulence of HPAIV H7N1 (designated H7N1-FPV34 and H7N1-It99) and H7N7 (designated H7N7-FPV27) was assessed in Pekin and/or Muscovy ducklings after intrachoanal (IC) or intramuscular (IM) infection.
Results: The morbidity rate ranged from 60 to 100% and mortality rate from 20 to 80% depending on the duck species, virus strain and/or challenge route. All Muscovy ducklings inoculated IC with H7N7-FPV27 or H7N1-FPV34 exhibited mild to severe clinical signs resulting in the death of 2/10 and 8/10 ducklings, respectively. Also, 2/10 and 6/9 of inoculated Muscovy ducklings died after IC or IM infection with H7N1-It99, respectively. Moreover, 5/10 Pekin ducklings inoculated IC or IM with H7N1-It99 died. The level of virus detected in the oropharyngeal swabs was higher than in the cloacal swabs.
Conclusion: Taken together, HPAIV H7 cause mortality and morbidity in Muscovy and Pekin ducklings. The severity of disease in Muscovy ducklings depended on the virus strain and/or route of infection. Preferential replication of the virus in the respiratory tract compared to the gut merits further investigation.


A brief history of bird flu

Samantha J. Lycett, Florian Duchatel and Paul Digard

In 1918, a strain of influenzaAvirus caused a human pandemic resulting in the deaths of 50 million people. A century later, with the advent of sequencing technology and corresponding phylogenetic methods, we know much more about the origins, evolution and epidemiology of influenza epidemics. Here we review the history of avian influenza viruses through the lens of their genetic makeup: from their relationship to human pandemic viruses, starting with the 1918 H1N1 strain, through to the highly pathogenic epidemics in birds and zoonoses up to 2018. We describe the genesis of novel influenza A virus strains by reassortment and evolution in wild and domestic bird populations, as well as the role of wild bird migration in their long-range spread. The emergence of highly pathogenic avian influenza viruses, and the zoonotic incursions of avian H5 and H7 viruses into humans over the last couple of decades are also described. The threat of a new avian influenza virus causing a human pandemic is still present today, although control in domestic avian populations can minimize the risk to human health.


A Comprehensive Model for the Quantitative Estimation of Seed Dispersal by Migratory Mallards

Erik Kleyheeg, Wolfgang Fiedler, Kamran Safi, Jonas Waldenström, Martin Wikelski and Mariëlle Liduine van Toorn

Long-distance seed dispersal is an important ecosystem service provided by migratory animals. Plants inhabiting discrete habitats, like lakes and wetlands, experience dispersal limitation, and rely heavily on zoochory for their spatial population dynamics. Granivorous waterbirds may disperse viable seeds of wetland plants over long distances during migration. The limited knowledge of waterbird migration has long hampered the evaluation of the importance of waterbirds in seed dispersal, requiring key metrics such as realistic dispersal distances. Using recent GPS tracking of mallards during spring migration, we built a mechanistic seed dispersal model to estimate realistic dispersal distances. Mallards are abundant, partially migratory ducks known to consume seeds of >300 European plant species. Based on the tracking data, we informed a mallard migration simulator to obtain a probabilistic spring migration model for the mallard population wintering at Lake Constance in Southern Germany. We combined the spring migration model with seed retention curves to develop seed dispersal kernels. We also assessed the effects of pre-migratory fasting and the availability of suitable deposition habitats for aquatic and wetland plants. Our results show that mallards at Lake Constance can disperse seeds in the northeastern direction over median distances of 293 and 413 km for seeds with short and long retention times, respectively, assuming a departure immediately after foraging. Pre-migratory fasting strongly affected the dispersal potential, with only 1–7% of ingested seeds left for dispersal after fasting for 12 h. Availability of a suitable deposition habitat was generally <5% along the migratory flyway. The high probability of seed deposition in a freshwater habitat during the first stopover, after the mallards completed the first migratory flight, makes successful dispersal most likely to happen at 204–322 km from Lake Constance. We concluded that the directed long-distance dispersal of plant seeds, realized by mallards on spring migration, may contribute significantly to large scale spatial plant population dynamics, including range expansion in response to shifting temperature and rainfall patterns under global warming. Our dispersal model is the first to incorporate detailed behavior of migratory waterbirds and can be readily adjusted to include other vector species when tracking data are available.


Clade H5 North American Highly Pathogenic Avian Influenza Viruses Infect, but Do Not Cause Clinical Signs in, American Black Ducks (Anas rubripes).

Erica Spackman, Diann J. Prosser, Mary Pantin-Jackwood, Christopher B. Stephens, Alicia M. Berlin 

Highly pathogenic avian influenza virus (HPAIV) from the goose/Guangdong/1996 clade H5 lineage spread from Asia into North America in 2014, most likely by wild bird migrations. Although several variants of the virus were detected, H5N8 and H5N2 were the most widespread in North American wild birds and domestic poultry. In early 2015, the H5N2 virus spread through commercial poultry in the Midwest, and >50 million chickens and turkeys died or had to be culled. Related H5 HPAIVs are still endemic in much of the Eastern Hemisphere. The wild bird species that were involved with dissemination of the virus in North America are not known. Dabbling ducks, especially mallards (Anas platyrhynchos), typically have the highest detection rates for avian influenza viruses. To better characterize the wild avian species that could spread the virus, American black ducks (Anas rubripes), which are closely related to mallards, were challenged with the North American H5N2 and H5N8 index HPAIV isolates: A/Northern Pintail/WA/40964/2014 H5N2 and A/Gyrfalcon/WA/41088/2014 H5N8. Although the American black ducks could be infected with low doses of both isolates (≤102 50% egg infective doses), ducks shed the H5N2 longer than the H5N8 (10 VS. 7 days) and the titers of virus shed were higher. Although there were too few ducks available on which to draw definitive conclusions, this suggests that American black ducks could serve as a more efficient reservoir for the H5N2 virus than the H5N8 virus.


Integration of genetic and epidemiological data to infer H5N8 HPAI virus transmission dynamics during the 2016-2017 epidemic in Italy

P. Mulatti, A. Fusaro, F. Scolamacchia, B. Zecchin, A. Azzolini, G. Zamperin, C. Terregino, G. Cunial, I. Monne & S. Marangon

Between October 2016 and December 2017, several European Countries had been involved in a massive Highly Pathogenic Avian Influenza (HPAI) epidemic sustained by H5N8 subtype virus. Starting on December 2016, also Italy was affected by H5N8 HPAI virus, with cases occurring in two epidemic waves: the first between December 2016 and May 2017, and the second in July-December 2017. Eighty-three outbreaks were recorded in poultry, 67 of which (80.72%) occurring in the second wave. A total of 14 cases were reported in wild birds. Epidemiological information and genetic analyses were conjointly used to get insight on the spread dynamics. Analyses indicated multiple introductions from wild birds to the poultry sector in the first epidemic wave, and noteworthy lateral spread from October 2017 in a limited geographical area with high poultry densities. Turkeys, layers and backyards were the mainly affected types of poultry production. Two genetic sub-groups were detected in the second wave in non-overlapping geographical areas, leading to speculate on the involvement of different wild bird populations. The integration of epidemiological data and genetic analyses allowed to unravel the transmission dynamics of H5N8 virus in Italy, and could be exploited to timely support in implementing tailored control measures.


As the Duck Flies—Estimating the Dispersal of Low-Pathogenic Avian Influenza Viruses by Migrating Mallards

Mariëlle L. van Toor, Alexis Avril, Guohui Wu, Scott H. Holan and Jonas Waldenström

Many pathogens rely on the mobility of their hosts for dispersal. In order to understand and predict how a disease can rapidly sweep across entire continents, illuminating the contributions of host movements to disease spread is pivotal. While elegant proposals have been made to elucidate the spread of human infectious diseases, the direct observation of long-distance dispersal events of animal pathogens is challenging. Pathogens like avian influenza A viruses, causing only short disease in their animal hosts, have proven exceptionally hard to study. Here, we integrate comprehensive data on population and disease dynamics for low-pathogenic avian influenza viruses in one of their main hosts, the mallard, with a novel movement model trained from empirical, high-resolution tracks ofmallardmigrations. This allowed us to simulate individualmallard migrations from a key stopover site in the Baltic Sea for the entire population and link these movements to infection simulations. Using this novel approach, we were able to estimate the dispersal of low-pathogenic avian influenza viruses by migrating mallards throughout several autumn migratory seasons and predicted areas that are at risk of importing these viruses. We found that mallards are competent vectors and on average dispersed viruses over distances of 160 km in just 3 h. Surprisingly, our simulations suggest that such dispersal events are rare even throughout the entire autumn migratory season. Our approach directly combines simulated population-level movements with local infection dynamics and offers a potential converging point for movement and disease ecology.

A viral race for primacy: co-infection of a natural pair of low and highly pathogenic H7N7 avian influenza viruses in chickens and embryonated chicken eggs

Annika Graaf, Reiner Ulrich, Pavlo Maksimov, David Scheibner, Susanne Koethe, Elsayed M. Abdelwhab,
Thomas C. Mettenleiter, Martin Beer and Timm Harder

Highly pathogenic avian influenza virus (HPAIV) infection in poultry caused devastating mortality and economic losses.
HPAIV of subtypes H5 and H7 emerge from precursor viruses of low pathogenicity (LP) by spontaneous mutation
associated with a SHIFT in the susceptibility of the endoproteolytic cleavage site of the viral hemagglutinin protein from
trypsin- to furin-like proteases. A recently described natural pair of LP/HP H7N7 viruses derived from two spatiotemporally
linked outbreaks in layer chickens was used to study how a minority of mutated HP virions after de novo
generation in a single host might gain primacy. Co-infection experiments in embryonated eggs and in chickens were
conducted to investigate amplification, spread and transmissionof HPAIV within a poultry population that experiences
concurrent infection by an antigenically identical LP precursor virus. Simultaneous LPAIV co-infection (inoculum dose
of 106 egg-infectious dose 50% endpoint (EID50)/0.5 mL) withincreasing titers of HPAIV from 101 to 105.7 EID50/0.5 mL)
had a significant impeding impact on HP H7 replication, viral excretion kinetics, clinical signs and histopathological
lesions (in vivo) and on embryo mortality (in ovo). LP/HP co-infected chickens required a hundredfold higher virus
dose (HPAIV inoculum of 105 EID50) compared to HPAIV mono-infection (HPAIV inoculum of 103 EID50) to develop
overt clinical signs, mortality and virus spread to uninfected sentinels. Escape and spread of HP phenotypes after de
novo generation in an index host may therefore be highly precarious due to significant competition with cocirculating
LP precursor virus.

The culture of primary duck endothelial cells for the study of avian influenza

Raissa L. Davis, Geunho Choi, Thijs Kuiken, Pascale Quéré, Sascha Trapp, Kirsty R. Short and Mathilde Richard

Background:  Endothelial cells play a major role in highly pathogenic avian influenza (HPAI) virus pathogenesis in
gallinaceous poultry species (e.g. chicken, turkey and quail). Upon infection of gallinaceous poultry with HPAI viruses,
endothelial cells throughout the body become rapidly infected, leading to systemic dissemination of the virus,
disseminated intravascular coagulation, oedema and haemorrhaging. In contrast, the pathogenesis of HPAI
viruses in most wild bird species (e.g. duck, goose and gull species) is not associated with endothelial tropism.
Indeed, viral antigen is not found in the endothelial cells of most wild bird species following infection with HPAI
viruses. This differential endothelial cell tropism in avian species is poorly understood, mainly due to the absence of
appropriate cell culture systems.
Results:  Here, we describe the isolation and purification of primary duck endothelial cells from the aorta or bone
marrow of Pekin duck embryos. Cells were differentiated in the presence of vascular endothelial growth factor and, if
needed, enriched via fluorescent-activated cell sorting based on the uptake of acetylated low-density lipoprotein. The
expression of von Willebrand factor, a key marker of endothelial cells, was confirmed by polymerase chain reaction.
Monocultures of duck endothelial cells, either derived from the aorta or the bone marrow, were susceptible to infection
with an H5N1 HPAI virus but to a much lesser extent than chicken endothelial cells.
Conclusions:  The methods described herein to isolate and purify duck endothelial cells from the aorta or bone marrow
could also be applied to obtain microvascular endothelial cells from other tissues and organs, such as the lung or the
intestine, and represent a valuable tool to study the pathogenesis of avian viruses.

The post-2009 influenza pandemic era: time to revisit antibody immunodominance

Kristien Van Reeth

The current inactivated influenza vaccines rely on the induction of neutralizing antibodies against the head domain of the viral hemagglutinin (HA). The HA head contains five immunodominant antigenic sites, all of which are subject to antigenic drift, thereby limiting vaccine efficacy. Bypassing the immune system’s tendency to focus on the most variable regions of the HA may be a step toward more broadly protective influenza vaccines.
However, this requires a better understanding of the biological meaning of immunodominance, and of the hierarchy between different antigenic sites. In this issue of the JCI, Liu et al. determined the immunodominance of the five antigenic sites of the HA head in experimentally infected mice, guinea pigs, and ferrets. All three species exhibited different preferences for the five sites of the 2009 pandemic H1N1 strain. Moreover, human subjects exhibited yet a different pattern of immunodominance following immunization with the standard inactivated influenza vaccine. Together, these results have important implications for influenza vaccine design and interpretation of animal models.

White-Tailed Sea Eagle (Haliaeetus albicilla) Die-Off Due to Infection with Highly Pathogenic Avian Influenza Virus, Subtype H5N8, in Germany

Oliver Krone, Anja Globig, Reiner Ulrich, Timm Harder, Jan Schinköthe, Christof Herrmann, Sascha Gerst, Franz J. Conraths, and Martin Beer

In contrast to previous incursions of highly pathogenic avian influenza (HPAIV) H5 viruses, H5N8 clade viruses caused numerous cases of lethal infections in white-tailed sea eagles (Haliaeetus albicilla) affecting mainly young eagles (younger than five years of age) in Germany during winter 2016/2017. Until April 2017, 17 HPAIV H5N8-positive white-tailed sea eagles had been detected (three found alive and 14 carcasses) by real-time RT-PCR and partial nucleotide sequence analyses. Severe neurological clinical signs were noticed which were corroborated by immunohistopathology revealing mild to moderate, oligo- to multifocal necrotizing virus-induced polioencephalitis. Lethal lead (Pb) concentrations, a main factor of mortality in sea eagles in previous years, could be ruled out by atomic absorption spectrometry. HPAIV H5 clade reportedly is the first highly pathogenic influenza virus known to induce fatal disease in European white-tailed see eagles. This virus strain may become a new health threat to a highly protected species across its distribution range in Eurasia. Positive cloacal swabs suggest that eagles can spread the virus with their faeces.

A novel European H5N8 influenza A virus has increased virulence in ducks but low zoonotic potential

Christian Grund, Donata Hoffmann, Reiner Ulrich, Mahmoud Naguib, Jan Schinköthe, Bernd Hoffmann, Timm Harder, Sandra Saenger, Katja Zscheppang, Mario Tönnies, Stefan Hippenstiel, Andreas Hocke, Thorsten Wolff, Martin Beer

We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future work flow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.

Integrating animal movement with habitat suitability for estimating dynamic migratory connectivity

Marielle L. van Toor, Bart Kranstauber, Scott H. Newman, Diann J. Prosser, John Y. Takekawa, Georgios Technitis, Robert Weibel, Martin Wikelski, Kamran Safi

Context High-resolution animal movement data are becoming increasingly available, yet having a multitude of empirical trajectories alone does not allow us to easily predict animal movement. To answer ecological and evolutionary questions at a population level, quantitative estimates of a species’ potential to link patches or populations are of importance.
Objectives We introduce an approach that combines movement-informed simulated trajectories with an environment-informed estimate of the trajectories’ plausibility to derive connectivity. Using the example of bar-headed geese we estimated migratory connectivity at a landscape level throughout the annual cycle in their native range.
Methods We used tracking data of bar-headed geese to develop a multi-state movement model and to estimate temporally explicit habitat suitability within the species’ range. We simulated migratory movements between range fragments, and calculated a
measure we called route viability. The results are compared to expectations derived from published literature.
Results Simulated migrations matched empirical trajectories in key characteristics such as stopover duration. The viability of the simulated trajectories was similar to that of the empirical trajectories. We found that, overall, the migratory connectivity was higher within the breeding than in wintering areas, corroborating previous findings for this species.
Conclusions We show how empirical tracking data and environmental information can be fused for meaningful predictions of animal movements throughout the year and even outside the spatial range of the available data. Beyond predicting migratory connectivity, our framework will prove useful for modelling ecological processes facilitated by animal movement, such as seed dispersal or disease ecology.