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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.



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.



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.



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.