US-UK BBSRC-NIFA Collab: Evolution of the high pathogenicity phenotype in avian influenza virus

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute


Influenza A virus can infect a wide range of hosts, but its main reservoir is wild aquatic waterfowl. From these hosts, the virus can infect domestic birds and is of particular concern to the poultry industry, where infection can cause high mortality. This in turn can jeopardize the food supply chain, while outbreaks also have the potential to affect human health.

Outbreaks of avian influenza (AI) in domestic poultry show a broad range of disease severity, from mild symptoms to rapid fatality. Of particular concern are outbreaks of High Pathogenicity Avian Influenza (HPAI). To date, only the H5 and H7 HA subtypes of AIV have caused HPAI outbreaks. HPAI can be introduced directly from wild birds, but is more commonly associated with the development of increased disease severity from a progenitor Low Pathogenicity Avian Influenza (LPAI). Acquisition of a high pathogenicity phenotype is associated with introduction of a polybasic cleavage site (PBCS) in HA. HA must be cleaved to be activated, and the presence of a PBCS means the protein can be activated by a broader range of host cell proteases, thus increasing the tissues affected by the virus, and leading to systemic spread and death. However acquisition of a PBCS is insufficient to increase the pathogenicity of the virus in all cases, and some H5/H7 viruses with a PBCS display a low pathogenicity phenotype in birds. Therefore there is an urgent need to understand other factors affecting acquisition of a HPAI phenotype in order to more accurately assess the risk associated with emerging AIV infections. We believe that the viral ion channel protein M2, and a recently identified variant of this called M42, play roles in the LPAI to HPAI transition through a number of mechanisms, and this proposal will explore this hypothesis.

We recently identified an M2 variant called M42, which differs only slightly from M2 in its extracellular domain. This minor difference was enough to cause a shift in sub cellular localisation of M42 relative to M2, to the Golgi apparatus. We will test the hypothesis that the altered subcellular localisation of M42 is important for regulating HA activity, and M42 expression levels may affect the acquisition of a high pathogenicity phenotype.

M2 has been proposed to form the basis of a universal vaccine for use in human and poultry which takes advantage of the fact that the extracellular domain of M2 is conserved amongst virus subtypes. Since the extracellular domain of M42 is different from M2, expression of M42 has also been identified as a mechanism by which influenza virus can escape from inhibition by antibodies targeted against M2 in cell culture studies. To date, no studies examining whether expression of M42 could provide a mechanism for AIV to overcome this vaccination strategy have been performed in birds.

Overall, in this proposal we will investigate (i) if the M2/M42 proteins of avian influenza virus are involved in the change from low pathogenic to highly pathogenic virus and (ii) if the change from M2 to M42 is of importance to escape vaccine responses.

Technical Summary

We have two hypotheses to test: (i) expression of M42 is important to H5 HPAI viruses and (ii) the ability of IAV strains to switch between M2 and M42 expression is an important consideration for vaccines directed against the M2 ectodomain.
We have selected A/chicken/Pennsylvania/1/1983 as a LPAI and A/chicken/Pennsylvania/1370/1983 (H5N2) as its HPAI derivative. Viruses will be rescued from synthetic cDNA clones and tagged versions of M2 and M42 proteins will be subcloned to investigate the role these proteins play in regulating HA during the LPAI to HPAI transition.
We will use fusion proteins in combination with mutagenesis to identify amino acids in the extracellular domain of M2/M42 that determine localization and take advantage of the ability to mutagenize M42 without altering the M1 coding sequence in a parallel approach using virus infection. Virus fitness will be assessed using plaque assays.
Other in vitro experiments using rescued viruses will examine the effect of M2/ M42 on HA synthesis, maturation and glycosylation. This will be assessed using HA transfection and M2/42 co-transfection or virus superinfection contexts. HA synthesis and trafficking will be assessed by western blotting, metabolic labeling, red cell binding assays, immunofluorescence and FACS. HA glycosylation will be tested by assessing EndoH sensitivity and lectin staining.
In vivo experiments will be focused on understanding the role of M42/M2 expression on the LPAI to HPAI transition using the rescued viruses. Panels of viruses with different levels of M2/M42 will be used inoculated chickens and domestic ducks (naïve birds will be cohoused to examine transmission). Birds will be monitored for clinical signs of disease and necropsied for virus titration and evaluation of virus tissue tropism by immunohistochemistry. The role of M42 expression in providing an escape route from the universal vaccine directed against the M2 extracellular domain will be tested in vitro and in vivo.

Planned Impact

The work proposed has direct relevance to the strategic priorities of the BBSRC - Animal Health. Avian influenza virus continues to pose a threat to the poultry industry; not only do influenza outbreaks cause devastating losses to the poultry industry and thereby threaten food security, but they also pose risks to human health. A better understanding of the molecular events involved in the evolution of low-pathogenic AI (LPAI) and highly pathogenic AI (HPAI) strains and how the virus might respond to the application of vaccine-driven selective immunological pressure will inform control measures against this important pathogen. Our research fits within the Animal Health priority area, as it is research on a viral emerging disease that is also a disease of agriculturally relevant animals of high economic consequence in BOTH the US and UK. It is relevant to pathogen emergence, transmission, infectivity and pathogenesis and to next generation vaccines with particular emphasis on approaches using new techniques and methodologies.

The following stakeholders have been identified as beneficiaries of this work:

The poultry production industry
Influenza outbreaks cost the UK and US poultry industry millions of pounds and resulted in the destruction of millions of birds. Understanding the evolution of the high pathogenicity phenotype in avian influenza virus will benefit the development of a universal influenza vaccine, ensuring that poultry farming remains not only a secure food source but also increases the economic competitiveness of the UK and the USA. Our increasing reliance on industrial level poultry farming coupled with an increasing threat from HPAI represents a vulnerability that must be addressed.

The poultry breeding industry
The consequences of improved vaccines and disease resistance may provide a panel of phenotypic biomarkers which could be developed as affordable tools to inform breeding strategy. We have established collaborations with major poultry breeding companies that will ensure any commercially useful results can be translated into practice.

The animal health industry
The RI has established collaborations, including direct support, with several vaccine companies that have resulted in ongoing assessment of potential vaccine candidates and immunomodulatory products. The data generated during this project will provide important considerations for the development of universal vaccine strategies.

Animal welfare
The reduction of disease as a result of improved vaccine strategies supports the Five Freedoms implicit to animal welfare as set out by the Farm Animal Welfare Council.

General public and the environment
The consequences of understanding the evolution of avian influenza into a devastating highly pathogenic form are of importance to both animal and human health. The data generated during this project will provide important considerations for the development of universal vaccine strategies which are not only applicable for the poultry industry but a similar M2 based vaccine is posed and tested for the use in humans.

Academia and Training
The multidisciplinary nature of this project will provide opportunities for broad training to all staff including other members and students of the institution ('strengthen the research community in the areas of disease and pest resistance of farmed animals through interdisciplinary research and the provision of training'). Results with respect to the evolution of the HPAI phenotype, the pathogenesis, transmissibility and immunogenicity of the variant strains will be of interest to a wide scientific community and will be published in peer-reviewed journals and presented at national and international scientific meetings.


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Lycett SJ (2019) A brief history of bird flu. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Stevens M (2016) Professor Pete Kaiser 1964-2016. in Avian pathology : journal of the W.V.P.A

Description In headline form, the major findings of the project were:

We found a way to engineer avian influenza virus into a form that is highly immunogenic, infectious, non-lethal but does not transmit between chickens; in other words, an effective live-attenuated vaccine virus.

The mutations we introduced into segment 7 (which encodes the viral matrix protein and two forms of an ion channel) which prevented the virus from transmitting between chickens also change the morphology of the virus particle, suggesting a link between the shape of the viruses and how well they transmit.

In more detail:

1) The project revolved around the study of a highly pathogenic strain of avian influenza from a 1983 outbreak in Pennsylvania, USA which we predicted would have the unusual property of expressing two forms of the viral ion channel, M2 and M42. We confirmed this prediction and furthermore, found that engineering the virus to only express one form or the other altered virus properties such that it remains infectious in chickens, but no longer transmits between birds in close contact. This property would be beneficial for live attenuated vaccine viruses. We therefore tested whether our mutant viruses would work as vaccines in a series of vaccine/challenge experiments, which showed that they gave complete protection from lethal challenge with antigenically homologous and heterologous strains of highly pathogenic avian influenza, as well as significantly reducing virus shedding from the challenged animals. We conclude that this represents a useful strategy for designing a live attenuated influenza vaccine for veterinary purposes, and idea which is currently the subject of a patent application.

2)The intracellular localisation of the influenza virus M2 protein (a key protein involved in virus entry and exit from cells that is also a drug target) is controlled by a leucine residue at position 4 in the ectodomain.

3) The type of virus particle (either spherical or long filaments) produced by the particular strains of avian influenza being studied in this proposal are controlled by the identity of a single amino acid in the viral matrix protein, which differs between the highly pathogenic strain and its precursor low pathogenicity strain.

4) The morphology of the virus particle is further affected by alterations to M2 or M42 expression, by more subtle variation in the ellipticity of the virus particles.

5) Overall, the work supports the hypothesis that the shape of influenza virus particles is important for how well the virus transmits between hosts and gives us hypotheses for further research.
Exploitation Route The key translational finding from SA4 is a means of engineering an avian influenza A virus with desirable properties for a live attenuated vaccine for poultry and potentially other species. We are attempting to patent this work and are investigating routes to further developing the idea.

The key overall scientific finding of the project comes from identifying mutations that hinder virus transmission between animals. The factors that affect influenza virus transmissibility is a hugely important topic which is poorly understood. Our project gives a route to further understanding this
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

Description H2020
Amount € 5,500,000 (EUR)
Organisation European Commission H2020 
Sector Public
Country Belgium
Start 05/2017 
End 04/2022
Description USDA-SEPRL 
Organisation U.S. Department of Agriculture USDA
Department Agricultural Research Service
Country United States 
Sector Public 
PI Contribution The grant is a joint one with the USDA - the BBSRC contributes the UK half of the funding
Collaborator Contribution As above, the USDA also contributes direct funding to the partner laboratory (PIs Dr Darrell Kapcyzinski and Mary Pantin-Jackwood). Their laboratory also benefits from core funding that provides further in kind benefits to the project.
Impact Only tangible outcome so far are meeting abstracts.
Start Year 2015
Description Science in the Saddle 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact A guided bike ride around Midlothian county, interspersed with stops and short talks by scientists, usually connected with sites of local scientific interest.

Sadly the event had to be cancelled on the day because of awful weather, but we will run it again in 2019
Year(s) Of Engagement Activity 2018