Influenza A viruses are amongst the most challenging viruses for both human and animal health. The diversity of hosts that they can infect and the frequency of inter-species transmission events maintain the threat of the emergence of strains with high efficiency of spread among humans that could result in pandemics. Since 2013, H5Nx reassortants viruses (derived from HPAI H5N1clade 220.127.116.11 viruses) spread panzootically all over the world. In France, the H5N8 strain resulted in the culling of millions of ducks and in massive economic losses for the poultry industry. Our project aims at a better understanding of the emergence of reassortants viruses from the avian reservoir and notably of the role of the segment 2, encoding PB1, the viral polymerase and the factor of virulence PB1-F2. Taking advantage of this natural reassortment event, we propose to combine different approaches to study this phenomenom occurring in the field: a phylogenetic analysis of segment 2 of avian viruses and a characterization of its ability to emerge by reassortment events, a study of the role of PB1-F2 on pathogenicity and transmission ability of the reassortants viruses (using H5N8 Tarn virus and its variants produced by reverse genetic). The propensity of fibrillization of avian PB1-F2 will be assessed in vitro.. Using mouse and ducks model of infection, the pathogenic potential of PB1-F2 and the inflammatory response of the host will be explored in order to decipher the mechanism of action of PB1-F2. Finally, the detection of PB1-F2 fibers will be performed at the single-cell level and in animal models (infected tissues) using innovative methods (synchrotron experiments).
The REACTIV project addresses a critical question considering the constant threat of emergence of IAV with pandemic potential: what is the importance of segment 2 and of PB1-F2 in reassortment event and adaptation to mammalian host? And what is the role of PB1-F2 in such event and in the pathogenicity of the emerging viruses?
The REACTIV project is ambitious and innovative in many aspects but above all it will be the first study combining at the same time a phylogenetic approach with an investigation of the consequences of specific sequence signatures in PB1-F2 on the transmissibility and pathogenesis of IAVs. Several studies aimed at studying the different aspects of the biology of segment 2 and of PB1-F2 but very few combined different approaches and it remains very difficult to correlate the data altogether to obtain a clear picture, notably about the “real” function of PB1-F2. Indeed, PB1-F2 is characterized by a multifunctional feature depending on the viral strain, the cell type infected and the host. Thus, it is necessary to develop a multidisciplinary project not to extrapolate putative exceptions into rules as it was often done with PB1-F2. The REACTIV project also provides a unique opportunity to work with H5N8 field isolates causing the on-going outbreaks in Europe and to study the evolution of viruses obtained through reassortment events and expressing or not PB1-F2.
Few studies have compared the biological properties associated with avian virus segments 2 and determined whether and how this segment poses a higher risk in mammals for transmission to human and virulence. Our project has 2 main objectives: understanding the involvement of the segment 2 of avian viruses in the adaptation of reassortants viruses in human, and decipher the function and contribution of PB1-F2 in the viral fitness and pathogenicity within avian and human hosts.
Our project operational objectives are as follows:
1 - Phylogenetic analysis of segment 2 of avian viruses circulating worldwide and linking a host determinant to a sequence feature
2 - Evaluation of the impact of PB1-F2 on pathogenicity and transmission ability of the reassortants viruses
3 - Study of the implication of avian PB1-F2 (recombinant and virus-expressed) in the pathogenicity of the virus and comparison of the inflammatory response in relevant animal models (duck and mouse)
4 - Structural characterization of PB1-F2 in vitro and in vivo using innovative methods such as FT-IR, DUV microscopy and cryo soft X-ray tomography (synchrotron SOLEIL)
The questions raised in the project REACTIV will be addressed by using diverse and complementary methodologies: bioinformatics tools to identify segment 2 sequence diversity associated with host species, experimental in vivo infections and measurement of their transmissibility, generation of reassortant viruses with avian virus segment 2 expressing or not PB1-F2, and measurement of virulence parameters in animal models. A collection of recombinant PB1-F2 will be produced and purified  for structural resolution of oligomerized complex, quantification of the inflammatory response triggered in animal models.
In REACTIV, we will work with the french field H5N8 virus (referred as "Tarn", non-fucntional truncated PB1-F2), the variant H5N8 Tarn with a restored PB1-F2 (H5N8+F2) and the HRN8 Czech reassortant virus (harbouring a full length PB1-F2). The introduction of mutations identified in WP1 in variants H5N8 viruses raises the question of gain of function. However, no change in the transmissibility of the virus (avian HA) is foreseen and thus we are in agreement with the last revision of the US moratorium on "gain of function" experiments.
All experiments will be carried out under strict biosafety conditions by fully trained personnel in biosafety level 3 laboratories and animal facilities available in Jouy-en-Josas, Toulouse and Memphis (USA).
The collaborative REACTIV project can be divided into 4 interconnected work packages and contains 5 work packages that are summarized below:
Work Package 0 (WP0): Project coordination, management and research exploitation
The objectives of WP0 are to monitor the progress of work towards the milestones and objectives of the work plan and to ensure compliance to the contractual obligations of the Grant Agreement, to disseminate the results to all key external stakeholders, to organize annual Consortium Meetings, steer the dissemination activities, and be in charge of intellectual property (IP) and Ethical issues.
Work Package 1 (WP1): Phylogenetic analysis of segment 2 of circulating avian influenza virus – identification of host determinant signature
Task 1.1: Assess the host determinants on segment 2 (PB1) and the host specific signatures of PB1-F2
Task 1.2: Assess the host determinism of known PB1-F2 amino acids signatures
Work Package 2 (WP2): Evaluation of the impact of PB1-F2 on pathogenicity and transmission ability of the reassortants viruses
Task 2.1: Impact of specific PB1-F2 amino acids signatures on replication in vitro
Task 2.2: Impact of specific PB1-F2 amino acids signatures on pathogenicity and transmission
Work Package 3 (WP3):Study of the Inflammatory response associated to PB1-F2 protein variants
Task 3.1: In vivo monitering of the inflammation in IAV-infected mice
Task 3.2: Study of the contribution of PB1-F2 fibers in the inflammatory response
Task 3.3: Histological study of PB1-F2-mediated inflammation
Task 3.4: Study of the immune response in ducks
Work Package 4 (WP4): Structural Characterization of PB1-F2 in vivo and in vitro
Task 4.1: Revelation of PB1-F2 aggregated structures in avian IAV-infected and -transfected cells using FT-IR and DUV microscopies
Task 4.2: Detection of PB1-F2 aggregated structures in lungs of IAV-infected mice and ducks using FT-IR and DUV microscopies
Task 4.3: Cryo soft-X-ray tomography of IAV-infected cells: 3D-reconstruction of IAV-infected cells and structural study of fibrillated PB1-F2
REACTIV is a very challenging and ambitious program, realistic and feasible considering the existing interconnexions and complementary skills of the members of the consortium and of the gathered preliminary results and established models: animal models (NF-kB-luciferase mice, neutropenic mice, MAVS and ASC-/- mice), reverse genetic system, recombinant protein engineering, model of inflammation (IVIS imaging, qRT-PCR, histology), transmissibility assays (ducks and ferrets) and SOLEIL experiments.
The consortium established for this interdisciplinary project gathers all the skills that are necessary to understand the host-specific determinism of PB1-F2 and its impact on transmissibility and pathogenicity of IAV viruses. The combined expertise ranges from phylogenetics, virology (including reverse genetic technology), molecular biology, cellular biology, protein engineering, Infra-red and DUV microspectroscopies, structural biology (Cryo soft X-ray tomography), animal model infectiology (viral challenge, in vivo imaging, plethysmography, anatomo-pathology) up to the study of inflammatory response, and are shared by both young and leading scientists with broad and unique experience.
Scientific, technical and economical applications
Zoonotic influenza A viruses sporadically infect humans and may cause severe respiratory disease and fatalities. Fortunately, most of these viruses do not have the ability to efficiently spread among humans and to subsequently cause a pandemic. However, adaptation of these zoonotic viruses to humans by mutation or reassortment with human influenza A viruses may result in airborne transmissible viruses with pandemic potential. Segment 2 is of particular interest because of its implication in the emergence of all the pandemic viruses documented and it encodes 2 proteins, PB1 and PB1-F2 recognized as virulence factors of IAVs. Moreover, the three influenza pandemics of the 20th century were caused by viruses expressing a functional PB1-F2, in contrast with the 2009 pandemic virus. The hemagglutinin (HA) and the polymerase complex are classically considered as the main virulence determinants of IAV. However, the acquisition of the polybasic cleavage site for HA or of described mutations in PB1 and NA are not sufficient for immediate transformation into a highly pathogenic strain. For instance, HA of the most deadly 1918 virus did not contain a polybasic site but had a special PB1-F2 signature. The REACTIV project is a rare opportunity to shed light on PB1-F2 structure and function during infection in a host-dependent manner, and notably in the avian host. A better understanding of the determinants and mechanisms of reassortment of segment 2 and the implication of PB1-F2, as biomarker of virulence, in such an event should aid in assessing the risks posed by avian influenza viruses to human health: our proposal stands within a “One Health” perspective. REACTIV may help predict the pandemic potential of IAV emerging strain and develop new diagnostic tools as biosensors detecting PB1-F2 cytotoxic oligomers. The Consortium behind this proposal will go beyond the state-of-the-art by using multidisciplinary fields to achieve the project objectives. Our project indeed consists of a study of molecular machineries (i.e. viral genome to viral protein to virus) at a different scale (cellular environment but also in animal models) by using physics, computing, phylogenetics, molecular, cellular and structural biology, imaging and the development of original analytical techniques such as FT-IR microspectroscopy (Synchrotron radiation experiments).The impact of the REACTIV project on public health is clear. Our findings will contribute to pandemic preparedness. We will indeed be able to provide valuable pieces of information for experts to score likelihood and impact risks of our virus (es) of interest: genomic characteristics, infection in animals and geographic distribution in animal (elements of the likelihood of risk scoring) (WHO TIPRA). Our project will thus contribute to the calculation of the overall virus risk scores (Figure 2). In addition, and maybe more at the European scale, our project will provide valuable information on the impact of reassortment events on progeny HPAI H5N8 viruses in Europe and contribute assessing their putative pandemics risk while they continue causing outbreaks on the continent.
Taken together, our project perfectly fulfils the requirements of “Défi 4, Vie, Santé et Bien-être; Environnement Santé - Maladies Emergentes et réémergentes.” and it will allow carrying out an ambitious but realistic research project at the national scale with a complementary consortium.