Rapid acquisition of mammalian characteristics by avian influenza virus in single host infections.

The natural host for influenza A viruses are wild aquatic birds where large numbers of different subtypes continuously circulating causing little disease. Wild birds are known to spread avian influenza virus (AIV) to farmed poultry species causing outbreaks that have significant production and economic consequence. In recent years there have been increased reports of AIV outbreaks in farmed poultry, including the currently circulating strains H5N8 and H7N9. Outbreaks of AIV in poultry increases the opportunity of cross-species infections into humans because of close proximity in a farming situation or the live bird markets of South East Asia of infected birds with humans. For AIV to cause sustained human infections and onward transmission critical virus characteristics' must undergo adaptation. One characteristic is the ability of the viral polymerase to replicate the viral genome effectively in mammalian cells at the low temperatures found in the human upper respiratory tract. There are several well defined mutations that can occur in the AIV polymerase proteins that enhances activity in mammalian cells. Analysis of the polymerase protein sequence from databases and our preliminary data show that for some AIV strains there are low frequencies of these mutations in viral isolates from avian hosts but that in the same viral strains isolated from mammalian hosts these adapting mutations appear at high frequency. This suggests that upon infection of a mammalian host by certain AIV strains rapid adaptation to enhance virus replication occurs in the individual infected. In this project we will make use of recombinant AIVs to measure if rapid adaptation to a mammalian host, the mouse, can occur in the infected individual before the virus is cleared by the immune system. We will infect mice with pertinent AIV strains and sequence the viral genetic material recovered from the infected mice at multiple time-points after initial infection to characterise any changes to the virus and determine when they appear. H9N2 viruses are endemic in poultry in vast swathes of south and central Asia. H9N2 viruses donated the internal genetic segments to the H7N9 avian influenza strain that is currently causing numerous poultry and human infections in China. Both H7N9 and H9N2 have been reported to rapidly adapt polymerase genes in the initial mammalian host therefore there may be characteristics of the internal H9N2 genes that predispose them to this rapid adaption. We will assess the viral characteristics of polymerase fidelity and baseline replication activity in mammalian cells of the H9N2 viral polymerase genes and comparison these to other important AIV subtypes. We will manipulate the fidelity of the viral polymerase to ask if this alters the ability to rapidly adapt. The host immune response to influenza virus infection ultimately resolves infection, so the ability of a virus to suppress a host immune response may affect the accumulation of a viral genome containing adaptation mutations. We will therefore also investigate different AIV strain ability to suppress the host immune responses use genetic manipulation to generate viruses that have this characteristic altered. In this way we will understand if specific AIV characteristics that are encoded in the virus genome can change the propensity to undergo rapid adaptation in mammalian hosts. Our aim is to provide fundamental knowledge about the ease of mammalian adaptation in an individual host for serious AIV strains that cause frequent poultry outbreaks and shed some light on the mechanism that facilitate this phenomenon. It is hoped this research will have an impact on the decisions that policymakers in the area of avian influenza control in poultry will make which often have far-reaching economic and societal costs.

Recently increased outbreaks globally in poultry of avian influenza virus (AIV) has resulted in the sporadically infection of humans. In order for human pandemic emergence, AIVs infecting humans must already be sufficiently able, or must rapidly adapt to overcome species barrier constraints and transmit to a subsequent human host before the infection is cleared. This proposal will aim to understand AIV genetics that facilitate the rapid acquisition of mammalian adaptation characteristics in a single host infection. Our first objective will accurately determine how quickly viruses containing H9N2 internal genetic constellations adapt to mammalian hosts. We will generate recombinant AIVs via reverse genetics, whereby the HA and NA surface glycoproteins will be of a vaccine strain and the internal genetic segments cassettes of various H9N2 and H7N9 genes. Viruses will be inoculated into mice and lungs sampled daily. RNA recovered from lung homogenates will be deep sequenced and mutations arising will be characterised for adaptation. Relative viral fitness and the rapidity of the accumulation of mutations will be measured and compared amongst the viral strains. Our second objective will extend our studies to other important AIVs such as the currently circulating, H5N8 and H5N6. Finally we will investigate predisposing features of viral genetic constellations that may trigger adaptation at a more rapid rate. Polymerase fidelity measurements will be made by deep sequencing primer ID tagged replicon products, generated by different AIV polymerases. An increased fidelity mutant of H7N9 through a V43I mutation in the PB1 gene will be generated to compare the rate of mammalian adaption when viral population diversity is reduced. In addition the ability to control the innate response and allow accumulation of mutation during infection in the individual host will be measured. H7N9 mutation of NS1 to increase control of IFNb responses through binding of CPSF30 will be utilised.

The global poultry industry is worth greater than £50 billion and it is estimated that by 2020 poultry will be the dominant meat industry globally. Frequent outbreaks of avian influenza virus into poultry and the subsequent mitigation controls such as de-flocking of farms, restrictions on movement and trade including the closures of live bird markets in South East Asia causes significant economic concern for the industry. Such controls must balance against the risk to human health by the avian influenza strains that are infecting poultry species. In this proposal we aim to understand the risk of different avian influenza strains to undergo rapid mammalian adaptation in the first mammalian host encountered. We will also investigate specific virus characteristics may have on the differences in the propensity of strains to acquire these adaptations rapidly. The outcomes from this programme of work will be of great interest to those involved in determining national and international health policy by providing scientific data about the risks posed by currently circulating avian influenza strains to mutate upon zoonotic infection of humans. Such information can be used to guide policy on mitigation scenarios and provide the relevant incentive to comply with measures prescribed. As such our data will help to inform poultry farmers and the general public with accessible fundamental scientific knowledge about the requirement of control measures facilitating increased willingness to respond to them. This project will identify which avian currently circulating virus strains are most at risk of rapid mammalian adaptation and thus the data produced will feed into the 'One medicine agenda' linking animal disease and human zoonotic disease. Information about potentially predisposing genetic viral gene constellations for rapid adaptation can be taken up by multiple influenza surveillance networks such as those run by the OIE/FAO (OFFLU) and WHO (Global Influenza Surveillance and Response System (GISRS) and European Influenza Surveillance Network (EISN)) as well as national animal and human health programmes based at the Animal Health and Veterinary Laboratories Agency (AHVLA) and Public Health England (PHE).