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

Abstract

From 2014 to 2021 the panzootic H5N8 clade 2.3.4.4 AIV spread on an unprecedented scale to wild and domestic birds worldwide. Compared to the parental clade 2.3.4.4A viruses (designated  H5N8-A) in 2013/2015, viruses in clade 2.3.4.4B (designated H5N8-B) from 2015 to 2021 caused high mortality in wild birds and Pekin ducks and were detected recently in mammals. In this comprehensive study, we analyzed pathogenicity and virulence markers of H5N8-A and H5N8-B in chickens, ducks and mice. Recombinant H5N8-A, H5N8-B and H5N8-A viruses carrying gene segment(s) from H5N8-B were successfully constructed using reverse genetics. In vitro, compared to H5N8-A, H5N8-B virus exhibited increased binding affinity to avian-sialic acid receptors and higher NA activity. Both H5N8-A and H5N8-B blocked IFN-β induction efficiently despite the striking preferential differences in the NS1 C-terminus domain (CTE). In chickens, both viruses were highly virulent, while H5N8-B was more virulent in Pekin ducks and mice than H5N8-A. In ducks, HA, NA and NS played a role in H5N8-B virulence and transmission. Moreover, we found that mutations in the NS1 CTE of H5N8-B was crucia for virus adaptation in mammals.

Talk at the Junior Scientist Symposium 2022, Greifswald, Germany (14 - 16 November, 2022)

Thijs Kuiken

Talk at the JNCC-BTO UK Workshop on HPAI and wild birds, online (10 November, 2022)

Thijs Kuiken

Abstract

Highly pathogenic avian influenza (HPAI) typically emerges in commercial poultry farms by conversion of an innocuous wild-type virus—aptly called low pathogenic avian influenza virus—into one that causes high mortality in poultry. The current H5N1 HPAI virus—clade 2.3.4.4b of the A/Goose/Guangdong/1/96 lineage—originated in a commercial goose farm in China in 1996 and spread across the rapidly growing poultry populations in Asia, substantially spilling over into wild birds in 2005. The virus caused numerous wild bird outbreaks, typically during autumn and winter, but has persisted year-round in wild birds in Europe since 2021. This year, it has spread quickly to breeding seabirds in Europe, North America, and Africa. For many of these long-lived species, already threatened by loss of habitat and climate change, the resulting mortality will have a large impact on their populations. Short-term recommendations include accurate monitoring of the virus and associated mortality in wild birds, and, where appropriate, coordinated removal of infected wild bird carcasses to limit virus spread. Long-term recommendations include enhanced protection of seabird and waterbird sites, vaccination of poultry against HPAI, reduction of poultry farm size and density, and avoidance of waterbird-rich areas as a location for poultry farms. The ongoing global spread of HPAI H5 viruses stresses the importance of international cooperation to better understand the global epidemiology of avian influenza, and is a call to re-assess the poultry sector in a way that embraces the One Health perspective: to sustainably balance and optimize the health of people, animals and ecosystems.

Talk at the BWDS DSWH joint conference, Utrecht, The Netherlands (13 October, 2022)

Scolamacchia F., Mulatti P., Fornasiero D., Dorotea T., Manca G., Mannelli A.

Poster at the ECVPH AGM & Annual Scientific Conference 2022, Athens, Greece (28-30 September, 2022)

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

Abstract

Avian influenza viruses (AIV), members of the RNA family Orthomyxoviridae, infect a wide range of wild and terrestrial birds. In wild waterfowl, the natural reservoir, AIV are thought to be in a static evolution; however, the transmission of AIV to poultry is accompanied by mutations in different viral proteins including the neuraminidase (NA). NA is a surface glycoprotein and varies into 9 subtypes (N1-N9). It is formed of stalk and head domains and mediates virus release from infected cells. Deletions in the NA stalk domain (NAdel) particularly in H5N1 viruses have been observed and linked to increased virus adaptation and/or virulence in chickens. Here, we investigated the impact of NA deletion or extension on biological fitness of two other NA subtypes: high pathogenic H5N8 (2016), with naturally full NA stalk and low pathogenic H7N7 (2011) with a natural NA stalk deletion. Using reverse genetics, we generated an H5N8 with a deletion in the stalk domain and another H7N7 with NA stalk extension. The replication of these four viruses in experimentally inoculated chickens and ducks was studied. In vitro data revealed no significant impact on virus replication on avian or mammalian cells. In contrast, reduction in the NA activity after elongation of the H7N7 NA stalk could be shown. In vivo, viruses carrying full NA stalk domain exhibited higher virulence and more efficient replication in chickens and ducks. These results indicate that the NA stalk deletion affects virus fitness in vivo and in vitro.

Talk at the 2nd Summer School "Infection Biology" 2022, Greifswald, Germany (26 - 28 September, 2022)

Scheibner, David; Ulrich, Reiner G.; Mettenleiter, Thomas C.; Abdelwhab, El-Sayed M.

Abstract

Highly pathogenic (HP) avian influenza viruses (AIV) cause severe mortality in chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), although turkeys are more sensitive than chickens. Little is known about the virulence determinants in both bird species, beyond the polybasic cleavage site (CS) in the hemagglutinin (HA). In 2015, HPAIV H7N7 and a putative low pathogenic (LP) ancestor were isolated from the same chicken farm. In addition to the polybasic HA CS, mutations in all gene segments were observed. The aim of this study was to investigate the impact of mutations in different segments in addition to the polybasic CS on virulence, transmission, replication and tissue tropism in chickens and turkeys. Using reverse genetics different virus reassortants with an LP backbone carrying the polybasic CS and single or multiple HP segments were generated. Interestingly, viruses carrying the HP NS or M gene segments or NS segment were as virulent and transmissible as the HPAIV in turkeys and chickens, respectively. All viruses were detected in the brain, although the endotheliotropism was exclusively found in chickens, where the HACS and to lesser extent NA are major determinants for the endotheliotropism. In vitro, HP exhibited lower NA and higher polymerase activities compared to LP. Taken together, this study showed two major differences between chickens and turkeys: more genetic constellations to confer high virulence and transmission in turkeys than in chickens and neurotropism in turkeys vs. endothelial tropism in chickens.

Poster at the 2nd Summer School "Infection Biology" 2022, Greifswald, Germany (26 - 28 September, 2022)

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

Abstract

From 2014 to 2021 the panzootic H5N8 clade 2.3.4.4 AIV spread on an unprecedented scale to wild and domestic birds worldwide. Compared to the parental clade 2.3.4.4A viruses (designated H5N8-A) in 2013/2015, viruses in clade 2.3.4.4B (designated H5N8-B) from 2015 to 2021 caused high mortality in wild birds and Pekin ducks, and were detected recently in mammals including humans, foxes and dead seals. In this comprehensive study, we analyzed pathogenicity and virulence markers of H5N8-A and H5N8-B in chickens, ducks and mice and determined the genetic markers for efficient transmission of H5N8 viruses in Pekin ducks and chickens. Recombinant H5N8-A, H5N8-B and H5N8-A viruses carrying gene segment(s) from H5N8-B were successfully constructed using reverse genetics. In vitro, compared to H5N8-A, H5N8-B virus exhibited increased binding affinity to avian-sialic acid receptors and higher NA activity. Both H5N8-A and H5N8-B blocked IFN-β induction efficiently despite the striking preferential differences in the NS1 C-terminus domain (CTE). In chickens, both viruses were highly virulent, while H5N8-B was more virulent in Pekin ducks and mice than H5N8-A. In ducks, HA, NA and NS played a role in H5N8-B virulence and transmission. Mutations in the NS1 CTE of H5N8-B (i) reduced virus shedding, transmission and/or endothelioptropism in chickens and ducks, (ii) reduced virulence and replication in mice and (iii) contributed to the high efficiency of H5N8-B for blocking IFN and apoptosis inductions in human lung cells indicating a role in virus adaptation in mammals.

Poster at the 2nd Summer School "Infection Biology" 2022, Greifswald, Germany (26 - 28 September, 2022)

Gianpiero Zamperin, Alice Bianco, Jacqueline Smith, Alessio Bortolami, Lonneke Vervelde, Alessia Schivo, Andrea Fortin, Sabrina Marciano, Valentina Maria Panzarin, Eva Mazzetto, Adelaide Milani, Yohannes Berhane, Paul Digard, Francesco Bonfante, Isabella Monne

Abstract

Background
Influenza A viruses (AIVs) are important human and veterinary health pathogens, with avian species representing their natural hosts. There are many subtypes of AIVs, each characterized by a specific pair of surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). AIVs infecting poultry can be divided into two groups, according to the severity of the disease they cause. So far, only H5 and H7 subtypes have showed the capability to mutate from low to highly pathogenic avian influenza (LPAI, HPAI). Once this happens, the mutation results in severe epizootics with up to 100% mortality. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored.

Methods
We selected two pairs of H7 low-pathogenic viruses with a distinctive ability to evolve from LPAI to HPAI under natural conditions. One pair of LPAI virus precursor of HPAI strains (precHP) included the H7N1 A/chicken/Italy/1279/1999 and the H7N3 A/chicken/BC/CN006/2004. The other pair included two viruses that have not evolved into highly pathogenic strains (nevoLP), even after mid- to long-term circulation in Galliformes: the H7N3 A/turkey/Italy/2962/03 and H7N2 A/chicken/Italy/1670/15. We did an in vivo infection of six-week-old female SPF white leghorn chickens. RNA samples were isolated from tracheas at 24, 36, 48 at 72 hours post-infection and subjected to library production and sequencing. High quality data were aligned to host genome to perform differential expression analysis. Differentially expressed genes (FDR < 0.05, |log2FC| ≥ 1) were used for GO enrichment analysis (FDR < 0.05) and pathway analysis with IPA.

Results
NevoLP enriched terms included chromosome segregation, cell division, cell cycle, biosynthetic process, cellular component biogenesis, ATP metabolic process, cellular metabolic process, suggesting that infected cells were busy multiplying and producing proteins, ATP and other components. PrecHP enriched terms included immune response, immune effector process, response to external stimulus, response to biotic stimulus and actin filament-based process, indicating a host immune response as the main reaction to the viral infection.

Conclusion
Our challenge study demonstrated that the number and type of DEGs can profoundly vary between viruses and even within the same HA subtype, pathotype, species and tissue. It is interesting to highlight that both the H7 HPAI precursors showed a very high number of general enriched terms related to immune response. These findings suggest that an exacerbated innate immune response may represent a selective factor shaping AIV pathogenicity evolution.

Poster at the OPTIONS XI for the Control of INFLUENZA Conference, Belfast, UK (26-29 September, 2022)

Lalidia Bruno Ouoba, Habibata Lamouni Zerbo Ouermi, Bianca Zecchin, Giacomo Barbierato, Sandaogo Hamidou Ouandaogo, Elisa Palumbo, Edoardo Giussani, Alessio Bortolami, Calogero
Terregino, Mariétou Guitti Kindo, Dominique Guigma, Nicolas Barro, Alice Fusaro , Isabella Monne

Abstract

Background
Since 2006, the West African poultry population has been seriously hit by different waves of highly pathogenic avian influenza (HPAI) H5Nx viruses, brought by migratory birds, with the last introduction of the H5N1 subtype reported in late 2020. In January 2017, the H9N2 subtype of the G1 lineage was identified for the first time in Burkina Faso and since then it has been reported in several West African countries. The co-circulation of these two zoonotic subtypes in this region is a cause for concern not only for animal health and economic damage, but also for the possible emergence of reassortant viruses with unknown biological properties and public health implications.

In December 2021, three HPAI H5N1 outbreaks were reported in poultry farms from three distinct regions of Burkina Faso. Here we describe the complete genome characterization of the first H5N1/H9N2 reassortant virus in West Africa.

Methods
By using an Illumina MiSeq platform, we generated the whole genome sequences of three H5N1 viruses from Burkina Faso. Maximum likelihood phylogenetic trees were obtained for all the eight gene segments using IQTree v1.6.6 and the time to the most recent common ancestor (tMRCA) was estimated for the hemagglutinin (HA) gene using the BEAST v.1.10.4 package.

Results
Phylogenetic analysis of the HA gene shows that the HPAI H5N1 viruses from the three distinct outbreaks in Burkina Faso cluster together (similarity of 99%-99.2%) within clade 2.3.4.4B, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in 2021-2022. The phylogenetic trees of the other gene segments confirm this clustering, except for the PA gene where the three H5N1 viruses from Burkina Faso cluster with H9N2 viruses collected in West Africa between 2017 and 2020. The tMRCA of the three H5N1 under study was June 2021 (95% HPD March-August 2021).

These reassortant viruses possess several mutations that may be associated to an increased zoonotic potential, including the HA-S137A (H3 numbering) mutation, which has been demonstrated to increase virus binding to human receptors, and the PA-367K mutation, which has been associated to a more efficient H5N1 virus replication in primary human bronchial epithelial (NHBE) cells, in A549 and Calu-3 cells.

Conclusion
Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of an H5N1/H9N2 reassortant virus in West Africa raises concerns about its possible impact on animal and human health. Further studies to evaluate the biological significance of this emerging genotype and to assess its spread are urgently required.

Poster at the OPTIONS XI for the Control of INFLUENZA Conference, Belfast, UK (26-29 September, 2022)

Mathis Funk, Monique I. Spronken, Anja C. de Bruin, Aartjan J.W. te Velthuis, Alexander P. Gultyaev, Ron A.M. Fouchier, Mathilde R. Richard

Poster at the OPTIONS XI for the Control of INFLUENZA Conference, Belfast, UK (26-29 September, 2022)

Bianca Zecchin, Alice Fusaro , Giacomo Barbierato, Edoardo Giussani, Diletta Fornasiero, Francesca Scolamacchia, Paolo Mulatti, Annalisa Salviato, Alessia Sc hivo, Elisa Palumbo, Maria Varotto, Federica Gobbo, Isabella Monne, Calogero Terregino

Abstract

The last two epidemic waves caused by the H5Nx Highly Pathogenic Avian Influenza (HPAI) viruses of clade 2.3.4.4b occurring in Europe in 2020-2021 and 2021-2022 had devastating economic impacts on the poultry industry of several countries. Between October 2021 and April 2022, Italy reported 317 HPAI H5N1 outbreaks in poultry, which caused the death of 13'700'000 domestic birds. Most of the outbreaks occurred in the highly density poultry populated areas of northern Italy. To shed light on the number of introductions and diffusion dynamics of the H5N1 in Italy, evolutionary analyses (BEAST v1.10.4, Network10, IQtree v1.6.6) were performed on the complete genomes of 342 viruses identified in poultry (N=320) and wild birds (N=22), generated using an Illumina MiSeq platform.

Our analysis revealed at least twelve distinct introductions (seven in domestic birds) which had been caused by six distinct genotypes originating from different reassortment events. We observed a clear clustering by province, which suggested the occurrence of several secondary outbreaks. The Bayesian phylogeographic analysis suggested that the provinces of Verona and Mantua (north eastern Italy) had been the main source of the virus for the other provinces. Furthermore, the Bayesian analyses showing the species contribution to the viral spread revealed that, among the infected species, turkeys acted as the most likely source of the virus for the other domestic birds. Some mutations associated with an increased binding to human-type receptors were detected in the HA protein of all (S137A, H3 numbering) or few (S159N/T160A, H3 numbering) viruses. Besides, two mutations associated to an increased polymerase activity in mammalian cells (NP-N319K and PB2-K482R) were detected in a H5N1 virus from a wild bird.

Our genetic investigation of the HPAI H5N1 viruses responsible of the 2021-2022 Italian epidemic revealed the incursions of multiple viral genotypes. Nevertheless, a single genotype accounted for most of the outbreaks in poultry. Mutations associated with mammalian adaptation were only sporadically identified in the analysed viruses from avian hosts. However, the persistent circulation and continuous emergence of new reassortant viruses in Italy and Europe raise concern for animal and public health.

Poster at the 12th ESVV International Congress, Ghent, Belgium (20-23 September, 2022)

Thijs Kuiken

Abstract

The A/Goose/Guangdong/1/96 (GsGd) lineage of highly pathogenic avian influenza (HPAI) H5 virus first emerged in poultry in Southeast Asia more than 25 years ago. For the first time in 2005, the virus spread from Asia to Russia, western Europe, Africa and the Middle East, causing high mortality in wild birds and poultry. This spread was a result of unprecedented long-distance transport of HPAI virus, in which wild migratory ducks, geese and swans were implicated. Since then, HPAI GsGd H5 outbreaks have frequently occurred in Europe. 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 unusually high presence of the viruses in European wild bird populations in late winter and spring 2021, as well as the greater involvement of barnacle and greylag geese in the epidemiology of HPAI in Europe since October 2020, may explain why spread to Newfoundland happened this winter (2021/2022), and not in the previous winters. To prevent and mitigate the risk of viral spread, it will be vital to further increase surveillance of wild birds in North America and South America, as well as at migration stop-over stations in Iceland and Greenland. The ongoing global spread of HPAI H5 viruses stresses the importance of close international cooperation and data exchange to better understand the global epidemiology of avian influenza, and is a call to re-assess the poultry sector in a way that embraces the One Health perspective: to sustainably balance and optimize the health of people, animals and ecosystems.

Talk at the 12th ESVV International Congress, Ghent, Belgium (20-23 September, 2022)

Jonas Waldenström

Talk at the 6TH PAN-EUROPEAN DUCK SYMPOSIUM, Coimbra, Portugal (19-23 September, 2022)

Mathilde Richard

Lecture at the EpiViral summer school, Alveiro, Portugal (09 September, 2022)

Angele Breithaupt, Claudia Blaurock, Florian Pfaff, Thomas C. Mettenleiter and Elsayed M. Abdelwhab

Abstract

In gallinaceous birds, chickens and turkeys, the transition of low pathogenic (LP) avian influenza virus (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). For unknown reasons, turkeys are succumbed to higher morbidity and mortality to HPAIV-infection than chickens. Here, we determined the pathogenesis of H7N1 virus using recombinant Lp, Hp and Lp H7N1 carrying pCS (Lp_poly) in turkeys and chickens, and explored the host responses in this species compared to chickens. We found that recombinant Lp_poly was avirulent in chickens, but exhibited high virulence in turkeys indicating that virulence determinants vary in these two galliform species. Strikingly, the virulence of Hp in chickens and turkeys was associated with extensive tropism to the endothelium, pCS conferred endotheliotropism only in turkeys. Transcriptome analysis indicated that turkeys mount a different host response (e.g. RSAD2, USP18) than chickens (e.g. Mx1) particularly from genes involved in RNA metabolism and immune response. Together, these findings indicate variable pathogenesis, virulence determinants and host response in two closely related galliformes, turkeys and chickens, after the infection with HPAIV H7N1 and may explain the high vulnerability of turkeys to HPAIV.

Poster at the 8th International Influenza Meeting 2022, Muenster, Germany (2-4 September, 2022)

Thijs Kuiken

Talk at the 15th International Seabird Group Conference, Cork, Ireland (22 August, 2022)

Diletta Fornasiero, Tiziano Dorotea, Bianca Zecchin, Alice Fusaro, Matteo Mazzucato, Paolo Mulatti

Abstract

Objective(s)
From October 2021, Italy has been involved in a severe highly pathogenic avian influenza (HPAI) epidemic sustained by a H5N1 virus. As of February 2021, 309 outbreaks in poultry were  notified mainly in the north-eastern Regions. Despite enhanced biosecurity and surveillance measures, the epidemic spread unprecedentedly fast, with massive economic impacts. We present a preliminary assessment of the potential reasons leading to such rapid spread, accounting for both epidemiological evidences and phylogenetic support.

Materials and methods Epidemiological investigations mainly aimed at identifying risk factors and contacts between infected and non-infected farms occurring during a temporal risk window. Investigated contacts included: movements of vehicles/personnel, sharing of equipment, and/or belonging to the same owners.
These data where then integrated with genetic information of the complete genome of 298 viruses, and geographical distances between outbreaks, to assess possible scenarios of the epidemic evolution, hence granting insights in the disease dynamics.

Results
Preliminary phylogenetic analyses indicated at least 7 new introductions from wild to domestic birds, while contact-tracing activities showed 37 at-risk contacts related to vehicles/personnel movements, and 65 outbreaks belonging to the same owners. The close proximity of farms in such densely populated poultry areas, and at-risk contacts appear to be the main factors driving the rapid spread of the disease, leading to several secondary outbreaks. The phylogenetic and network analyses based on complete genome pinpointed that other potential unregistered and uncontrolled contacts between outbreaks occurred.

Conclusion
The integration of different analyses permits to monitor the epidemic dynamic, allowing real-time interventions to re-modulate the implementation of control measures. The recent epidemic demonstrates the need to further strengthen the biosecurity measures, considering the limited possibility of reducing the farms density, and plausibly the reassessment of the poultry sector organization to contain the risk of future epidemics.

Talk at the 16th International Symposium of Veterinary Epidemiology and Economics, Halifax, Canada (7-12 August, 2022)

Matteo Mazzucato, Paola Bonato, Monica Lorenzetto, Matteo Trolese, Grazia Manca, Nicola Ferrè

Abstract

Objective(s)
From October 2021, North-eastern Italy was involved in a severe H5N1 High Pathogenic Avian Influenza epidemic. In less than three months, more than 300 outbreaks were notified with massive repercussions on the poultry production. The high number of daily cases hampered the capacity to counter the disease spread, prompting for the development of a tool to keep track of the evolving epidemiological situation and control measures applied. Therefore, a Support Decision System was developed, exploiting well-established data interoperability standards, data warehouse framework, and web-based GIS technologies. This led to an integrated system for visualising, querying and analysing data, ultimately allowing the interaction between different operators/experts, such as Epidemiologists, GIS and Data manager, and decision makers.

Materials and methods 
A dedicated Data Warehouse (DWH) infrastructure was developed using RDBMS Oracle®11g to harmonise data from different sources (e.g. spatial data, animal registry, production). The available data and epidemiological information were collected and visualised via a set of web applications, developed using HTML5, JavaScript, PHP/Java technologies, on RDBMS PostGreSQL®. All the official documents, on each outbreak and control measures, were stored using a web-based document and activity flows software (ARXivarNEXT).

Results
The developed SDS provides a useful tool to explore data under different perspectives. Information can be visualised both spatially (webGIS) and in non-spatial ways (tables, charts) through dedicated web applications, thanks to the implementation of the DWH, integrating different types of data from multiple databases. In addition,ArxivarNEXT allows to quickly find and retrieve all the formal documents.

Conclusion
The proposed architecture overcomes the weakness of a closely coupled design, enabling dynamic data integration from different providers, allowing interoperability between data and application. The SDS for the management of the HPAI epidemic has proved its helpfulness in decision making,
showing the real time progression of the disease spread.

Talk at the 16th International Symposium of Veterinary Epidemiology and Economics, Halifax, Canada (7-12 August, 2022)

M.I. Spronken, M. Funk, A.P. Gultyaev, A.C.M. de Bruin, R.A.M. Fouchier, M.R. Richard

Abstract

Upon introduction of H5 or H7 low pathogenic avian influenza viruses (LPAIV) into poultry, highly pathogenic viruses (HPAIV) may emerge, resulting in severe disease with mortality rates of up to 100%. The LPAIV to HPAIV transition is the result of the acquisition of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA). HAs with an MBCS are cleaved by ubiquitous furin-like proteases, supporting systemic viral dissemination in poultry. The exact molecular mechanism underlying this genetic change and why MBCS acquisitions have been exclusively observed in H5 and H7 subtypes remain unknown. We developed a novel, high-throughput and sensitive system with which insertions at the HA cleavage site are detected. Single nucleotides are deleted in the cleavage site of HA. Upon use of these templates in reverse genetics, viruses with HAs presenting insertions or deletions (indels) to repair the HA frame are under strong positive selection. Predicted RNA stem-loop structures of H5 and H6 HA cleavage site regions were the starting point in understanding the features of H5 HA (RNA structure/sequence) that promote insertions. H6 HA was selected as no H6 viruses have acquired an MBCS in nature so far. Indels were detected in a representative LPAIV H5 cleavage site at a low frequency. Indel frequency was increased when the number of adenines (A) in the loop was increased, even upon only one single nucleotide substitution. In contrast to H5, multiple substitutions to increase the number of As and resemble the H5 loop sequence were required in H6 for indels to be detected, yet at lower frequency than in H5. These data shows that sequence is a key determinant driving insertions at the HA cleavage site HA, potentially explaining why HPAIVs have emerged from H5 but not from H6 LPAIVs.

Talk at the Negative Strand Virus meeting, Braga, Portugal (17 June, 2022)

Anja de Bruin, Monique Spronken, Adinda Kok, Miruna Rosu, Dennis de Meulder, Stefan van Nieuwkoop, Pascal Lexmond, Theo Bestebroer, Sander Herfst, Ron Fouchier, and Mathilde Richard

Poster at the Negative Strand Virus meeting, Braga, Portugal (17 June, 2022)

Thijs Kuiken

Presentation for EU Parliament Intergroup on the welfare and conservation of animals, Brussel, Belgium (24 March, 2022)

Thijs Kuiken

Talk at the EPIC conference, Edinburgh, UK (01 March, 2022)

David Scheibner, Angele Breithaupt, Christine Luttermann, Diana Palme, Maryna Kuryshko, Claudia Blaurock, Thomas C. Mettenleiter and Elsayed M. Abdelwhab

Abstract

Background
Avian influenza viruses (AIV) are a common threat to poultry and human health and cause periodically huge socioeconomic losses worldwide. Depending on the two major surface glycoproteins, 16 hemagglutinin (HA) and 9 neuraminidase (NA) distinct subtypes can be distinguished. All HA/NA subtypes are circulating in wild waterfowl, the natural reservoirs. In domestic birds, AIV are either low pathogenic (LPAIV) causing mild or no clinical signs or highly pathogenic (HP) causing up to 100% mortality in few days. In chickens, LPAIV H5 and H7 viruses can evolve to HPAIV due to acquisition of polybasic amino acids in the HA cleavage site (HACS). Conversely, ducks rarely succumb to morbidity and mortality after HPAIV infections and the HACS alone is not the main virulence determinant. Since 2014, H5N8 clade 2.3.4.4 caused high mortality in domestic and wild birds worldwide in three major waves in 2014, 2016 and 2020/2021. Although all H5N8 viruses possessed polybasic HACS and were highly virulent in chickens, viruses in 2016-2021 only were high pathogenic in Pekin ducks. In this study, the virulence determinants leading to differences in virus phenotypes between German H5N8 clade 2.3.4.4 viruses in 2014 and 2016 were analyzed.

Methods
Sequence analysis of H5N8 viruses from 2014 and 2016 revealed several mutations in all eight gene segments. Using reverse genetics, single, double and multiple H5N8-2016 segments were swapped with those from H5N8-2014 and recombinant viruses were rescued and characterized. The minimal genetic constellation for the exhibition of high virulence in Pekin ducks was determined.

Results
In vivo, the H5N8-2016 HA alone did not increase the virulence of H5N8-2014 in Pekin ducks. We further tested double reassortants of H5N8-2014 carrying HA in addition to single segments from H5N8-2016. Virulence of H5N8-2014 increased dramatically after reassortment of H5N8-2016 HA in combination with NP, NS and/or NA. The high mortality caused by these viruses was associated with the degree of endothelial tropism and systemic spread in different tissues. In vitro, the replication of the H5N8-2014 virus in primary duck cells was increased by swapping the HA and NP from H5N8-2016. The latter exhibited increased HA receptor binding affinity and NA activity compared to the H5N8-2014 virus.

Conclusion
Taken together, in this study we determined the multifactorial genetic constellation and underlying mechanism for the high virulence of H5N8 clade 2.3.4.4 viruses in Pekin ducks, which improves our current understanding for the evolution of HPAIV in different avian species.

Talk at the 8th ESWI Influenza Conference, virtual (4 - 7 December 2021)

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

Abstract

Background
Since 1997, avian influenza viruses (AIV) of H5Nx goose/Guangdong lineage continue to cause severe losses in poultry and wild birds and pose a serious pandemic risk. This H5Nx lineage exhibited high flexibility for mutations resulting in tens of clades/subclades and undergone several reassortment events. From 2014 to 2021, the panzootic H5N8 clade 2.3.4.4 AIV spread on an unprecedented scale to wild and domestic birds worldwide. Compared to the parent clade 2.3.4.4A viruses (designated H5N8-A) in 2013/2015, viruses in clade 2.3.4.4B (designated H5N8-B) from 2015 to 2021 caused high mortality in wild birds and Pekin ducks and was isolated recently from humans and dead seals. Therefore, there is an urgent need to keep vigilance to assess periodically the virulence and transmissibility of H5N8-B in different animals and understand the molecular mechanisms underlying potential virus adaptation. In this comprehensive study, we determined the pathogenicity and virulence markers of H5N8-A and H5N8-B in chickens, ducks and mice and determined the genetic markers for the efficient transmission of H5N8 viruses in Pekin ducks and chickens.

Methods
Recombinant H5N8-A, H5N8-B and H5N8-A viruses carrying gene segment(s) from H5N8-B were  successfully constructed using reverse genetics. Replication efficiency in avian and mammalian cells, receptor-binding affinity, sialidase and polymerase activities, interferon (IFN)-antagonism and apoptosis induction were assessed in vitro. In vivo, chickens and ducks (n= 10 each) were inoculated oculonasal with recombinant viruses and one-day post-inoculation five birds were added to assess chicken-to-chicken or duck-to-duck transmission, respectively. Morbidity, mortality, virus excretion, tissue distribution and histopathological alterations were described. Furthermore, in mice, we assessed the impact of selected recombinant H5N8-B viruses on bodyweight gain, mortality, virus replication in the lungs and interferon induction.

Results
In vitro, compared to H5N8-A,H5N8-B virus exhibited increased binding affinity to avian-sialic acid receptors and higher NA activity. In duck cells, but not in chicken or human cells, H5N8-B replicated at higher levels than H5N8-A. Both H5N8-A and H5N8-B blocked IFN-β induction efficiently despite of the striking preferential differences in the NS1 C-terminus domain (CTE). In chickens, both viruses were highly virulent, while H5N8-B was more virulent in Pekin ducks and mice than H5N8-A. In ducks, HA, NA and NS played a role in H5N8-B virulence and transmission. Mutations in the NS1 CTE of H5N8-B (i) reduced virus shedding, transmission and/or endothelioptropism in chickens and ducks, (ii) reduced virulence and replication in mice and (iii) contributed to the high efficiency of H5N8-B for blocking IFN and apoptosis inductions in human lung cells indicating a role in virus adaptation in mammals.

Conclusion
The recent H5N8-B virus exhibited high virulence in chickens, Pekin ducks and mice compared to the predecessor H5N8-A virus. NS segment contributes to H5N8-B fitness in birds and mammals.

Talk at the 8th ESWI Influenza Conference, virtual (4 - 7 December 2021)

Susanne Koethe, Lorenz Ulrich, Angele Breithaupt, Christian Grund, Anne Pohlmann, Timm Harder and Martin Beer

Abstract

Migratory waterfowl are potential vectors for the long-range transmission of avian influenza viruses (AIV) globally. In 2013, low pathogenic avian influenza viruses (LPAIV) of subtype H7N9 emerged in China and evolved into highly pathogenic avian influenza virus (HPAIV) variants that were first detected in the beginning of 2017. Extensive co-circulation of both pathotypes was particularly restricted to galliform poultry but caused several thousand severe human infections with a high fatality ratio in China. The systematic surveillance revealed constant evolution of AIV H7N9 and reassortment events with the Eurasian wild bird gene pool. Spillover and adaptation of either LPAIV or HPAIV H7N9 from galliform to anseriform poultry into migratory waterfowl would justify severe concerns about a possible transcontinental spread of these highly zoonotic viruses.
The potential of AI H7N9 viruses to adapt to wild waterfowl was determined using an LPAIV (A/duck/Japan/AQ-HE28-3/2016) and two HPAIV H7N9 isolates, (A/duck/Japan/AQ-HE29-22/2017 (HE29-22), and (A/duck/Japan/AQ-HE29–52/2017) (HE29-52)). Replication competence and transmissibility, respectively, were characterized in embryonated duck eggs, in one-week old ducklings and in four-week old ducks.
The LPAIV strainrevealed no pathogenicity and transmissibility in ducks. In contrast to the LPAIV, the two HP variants induced systemic infection in duck embryos. When applied intra-muscularly to one-week old ducklings, the HPAIV HE29-22 and HE29-52 isolates caused mortality, indicating increased virulence. In four-week old ducks, oronasal inoculation of both respective HPAIV H7N9 isolates lead to virus shedding and efficient transmission to contact ducks without onset of disease.
The data indicate a significant adaptive potential of HPAIV, but not LPAIV, H7N9 isolates to ducks. Asymptomatic but productive infection in four-week old ducks justifies fears of undetected regional and possibly global virus spread with ducks and migratory anseriform birds, respectively.

Talk at the 8th ESWI Influenza Conference, virtual (4 - 7 December 2021)

Anja C.M. de Bruin, Monique I.J. Spronken, Theo M. Bestebroer, Pascal Lexmond, Zhen Wei Marcus Tong, Michelle Baker,Kirsty R. Short Ron A.M. Fouchier and Mathilde J. Richard

Abstract

Introduction
Highly pathogenic avian influenza viruses (HPAIV) of the subtypes H5 and H7 emerge from low pathogenic precursors upon transmission from wild waterfowl, their reservoir, to poultry. In poultry, HPAIV are responsible for systemic disease with marked endotheliotropism resulting in outbreaks with mortality rates as high as 100%. The HPAIV phenotype is the result of the acquisition of multiple basic amino acids at the cleavage site of the hemagglutinin (HA) protein. This allows for HA activation by ubiquitously expressed proteases which leads to systemic virus dissemination. HPAIV infections in wild birds are, in general, of milder pathogenicity and are not associated with endothelial cell tropism. The involvement of endothelium in immunopathogenesis is well established in mammalian influenza infections, but data from avian species is lacking. This study investigates virus replication, in relation to protease expression, and immune responses in chicken and duck endothelial cells which could contribute to observed differences in HPAIV pathogenesis.

Methods
Primary endothelial cell cultures of both duck and chicken origin were established, in which viral growth kinetics and host responses were compared. Expression of HA-activating proteases was analyzed by PCR followed by functional characterization in an overexpression system. A transcriptomics approach was applied to HPAIV-infected cells to study host responses and the production of inflammatory cytokines was studied during infection and after Toll-like receptor stimulation.

Results
Surprisingly, the amount of produced infectious virions upon HPAIV infection was comparable between primary chicken and duck endothelial cells, showing only a trend towards lower initial infection percentages in duck cells than in chicken cells. Messenger RNA of the HPAIV-activating proteases furin and pc5/6 were expressed in duck endothelial cells and duck furin cleaved HPAIV HA when both proteins were overexpressed in a furin-deficient cell line. Transcriptomic analysis of chicken endothelial cells hinted towards a more pro-inflammatory state, which was supported by high IL-6 and IL-8 mRNA induction upon Poly(I:C) treatment. Duck endothelial cells overexpress genes within pathways related to immune regulation and the type I interferon response.

Conclusion
Primary duck endothelial cells sustained multi-cycle growth of HPAIV which was supported by the expression of adequate HPAIV-activating proteases. Despite comparable levels of virus replication, host responses upon infection differed between chicken and duck endothelial cells. Chicken cells mounted a pro-inflammatory response whereas duck cells increased their antiviral defenses. The lack of endotheliotropism of HPAIV in ducks in vivo is not fully recapitulated within our monoculture experiments. Therefore, we are currently developing an epithelial-endothelial coculture setting to study viral dissemination, barrier integrity, and host responses.

Talk at the 8th ESWI Influenza Conference, virtual, (4 - 7 December 2021)

Thijs Kuiken

Talk at the Dutch National Zoonoses Symposium (25 November 2021)

Gianpiero Zamperin, Alice Bianco, Jacqueline Smith, Alessio Bortolami, Lonneke Vervelde, Alessia Schivo, Andrea Fortin, Sabrina Marciano, Valentina Panzarin, Eva Mazzetto, Adelaide Milani, Yohannes Berhane, Paul Digard, Francesco Bonfante, Isabella Monne

Abstract

Influenza A viruses (AIVs) are important veterinary and human health pathogens and avian species represent their natural hosts. Many subtypes of AIVs exist, each characterized by a specific pair of surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). AIVs infecting poultry can be divided into two distinct groups on the basis of the severity of the disease they cause. Once low pathogenic avian influenza (LPAI) viruses of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating to highly pathogenic avian influenza (HPAI). This mutation results in severe epizootics with up to 100% mortality, with major economic impact on poultry production. It follows that LPAI infections with viruses of the H5 and H7 subtypes are of great concern. It is still unclear why the H5 and H7 subtypes are more prone to evolve into HPAI forms than any other AIV HA subtypes. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In order to close such gap, we studied the early stages of transcriptome response in the tracheal tissue of chickens infected with two pairs of H7 strains which demonstrated a distinct ability to evolve their pathotype in the field. Our results show that H7 precursors of HP viruses trigger the host immune response, while in chickens infected with the H7 strains that do not mutate to the HP phenotype, the majority of differentially expressed genes were involved in metabolic and cellular division processes, with very low involvement of immunity genes. These findings suggest that, in the fight between host and virus, the immune response may represent a selective factor favoring the mutation of influenza viruses themselves.

Poster at the MEEGID XV - 15th International Conference on Molecular Epidemiology and Evolutionary Genetics of Infectious Diseases (2 - 5 November 2021)

Thijs Kuiken

Presentation in the zoo "Ouwehands", Rhenen (23 October 2021)

Mathilde Richard

Virology course (4 - 8 October 2021)

David Scheibner, Reiner Ulrich, Olanrewaju I. Fatola, Marcel Gischke, Ahmed Salaheldin, Jutta Veits, Ulrike Blohm, Thomas C. Mettenleiter and Elsayed M. Abdelwhab

Abstract

Highly pathogenic (HP) avian influenza viruses (AIV) cause severe mortality in chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), although turkeys are more sensitive than chickens. Little is known about the virulence determinants in both bird species, beyond the polybasic cleavage site (CS) in the hemagglutinin (HA). In 2015, HPAIV H7N7 and a putative low pathogenic (LP) ancestor were isolated from the same chicken farm. In addition to the polybasic HA CS, mutations in all gene segments were observed. The aim of this study was to investigate the impact of mutations in different segments in addition to the polybasic CS on virulence, transmission, replication and tissue tropism in chickens and turkeys. Using reverse genetics different virus reassortants with an LP backbone carrying the polybasic CS and single or multiple HP segments were generated. Interestingly, viruses carrying the HP NS or M gene segments or NS segment were as virulent and transmissible as the HPAIV in turkeys and chickens, respectively. All viruses were detected in the brain, although the endothelial tropism was exclusively found in chickens where the HACS and to lesser extent NA are major determinants for the endotheliotropism. In vitro characterization revealed comparable replication kinetics of all viruses but lower NA and higher polymerase activity of HP compared to LP. Taken together, this study showed two major differences between chickens and turkeys: more genetic constellations to confer high virulence and transmission in turkeys than in chickens and neurotropism in turkeys vs. endothelial tropism in chickens. M and NS genes were required for widespread distribution and severe inflammation particularly in the brain and lymphatic organs.

Poster at the 30th Annual Meeting of the Society for Virology (24-26 March, 2021)

Thijs Kuiken

Webinar NCOH (28 January 2021)

Anja de Bruin

Talk at the 13th CEIRS meeting, virtual, (11-15 January 2021)

Anja de Bruin

Poster at the 23rd Moledular Medicine Day, Rotterdam, (14 March 2019)