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The effect of the PB2 627K mutation on highly pathogenic H5N1 avian influenza virus in the avian host - SD0504

Description
Many modern H5N1 avian influenza (AI) viruses (e.g. A/turkey/Turkey/1/05; ty/05) carry a mammalian adaptation motif PB2 627K, compared to the classic avian signature PB2 627E. This is the first time that avian viruses have circulated with mammalian adapting mutations. We hypothesized that the genetic makeup of avian viruses of other lineages meant that this type of mutation had not emerged before because it carried a fitness costs in the avian host. Through in vivo and in vitro models and using reverse genetics we have already demonstrated modern H5N1 viruses tolerate PB2 627K with no loss or gain of fitness. More recently, we found that a classic H5N1 virus from the 1990s (called 50-92) could be engineered to have the PB2 627K mutation but that the virus reverted back to the avian signature E627 during chicken egg passage. This work makes up the bulk of the PhD thesis to date, however there remains some outstanding pieces of work that are necessary in order to complete thesis chapters and to enable publication in two peer reviewed journals. These are as follows:

• Completion of PB2 627K investigations in order to identify viral genes (other than PB2) that may support the PB2 627 phenotypes, including those observed in our previous in vivo experiments :
Understanding the epidemiology of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses is critically important due to the high impact of disease outbreaks in both domestic and wild bird populations. In addition, the pandemic potential of these viruses must be assessed due to their associated high mortality. We have identified the effect of a mammalian adaptation, PB2 627K, (currently present in some circulating H5N1 viruses) on the avian host and compared it to a historical H5N1. Our evidence suggests some modern H5N1 viruses have evolved to tolerate the 627K mutation, providing crucial knowledge to surveillance activities and efficient disease control. Intriguingly, we identified a cost associated with this mutation in our historical H5N1 virus. The mechanism behind this is unknown, yet we have preliminary evidence that suggests a novel finding. We hypothesise a role in virus packaging. This work will examine the constellation of virus genes behind our observation using reverse genetics recombinant influenza viruses. In vitro experiments plus virus sequencing will determine the phenotype of our viruses. This information will contribute further knowledge for influenza surveillance activities and additional data for pandemic prediction models.

Experiments will include the completion of a reverse genetics virus panel comprised of mis-matched 50-92 and ty/05 gene segments in combination with the PB2 627K mutation. Recombinant viruses will be passaged in embryonated chicken and duck eggs and resultant viruses sequenced, especially with regards to the 627 amino acid site.
• Assessment of avian influenza virus host range restriction by apoptosis of infected cells:
Avian influenza viruses often exhibit strong host restriction between avian species. The mechanisms behind this observation are unclear. Further understanding of this frequently reported phenomenon is essential in order to inform risk pathways of avian influenza viruses and susceptible wild and domestic host bird species. Preliminary results, by our collaborators at Nottingham University, suggest certain avian influenza viruses may be restricted in duck species by inducing apoptosis. Conversely, some avian influenza viruses successfully replicate by inhibiting apoptosis in duck cells. In order to confirm and understand this observation, we plan to infect chicken and duck cells with recombinant 50-92 and ty/05 viruses and measure apoptosis by measuring caspase activity levels. By examining the propensity to cause apoptosis we will map the gene segment responsible of influenza for this observed host restriction. This fundamental information will aid in influenza surveillance.

• Understanding the mechanism of a novel HA virulence motif in the 50-92 H5N1 virus:
We have identified a novel HA virulence motif from a H5N1 viruses isolated in England, 1991. We plan to further investigate the mechanism behind our observations. Results suggest the pathogenesis of this virus is controlled by the fusion of the HA gene, therefore we will carry out in vitro fusion assays using our panel of reverse genetics viruses. This knowledge will contribute to our understanding of H5N1 virulence determinants and modes of action. These results will be published in a scientific journal.
Objective
This work hopes to identify markers that can be used to assess the virulence potential of avian influenza, so assisting predictive modelling which may be used to judge the potential impact of newly emerging variants and inform downstream policy decisions on vaccination. The identified regions on the genome could also be used as candidate areas for future targeting as part of novel vaccine development.
Time-Scale and Cost
From: 2013

To: 2013

Cost: £9,179
Contractor / Funded Organisations
A H V L A (Animal Health and Veterinary Laboratories Agency - AHVLA)
Keywords
Avian Infectious Influenza