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

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.

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.

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.