Characterising and risk assessing the evolving zoonotic and pandemic potential of swine influenza A(H1N1) viruses endemic in Europe and China
Lead Research Organisation:
Imperial College London
Department Name: Infectious Disease
Abstract
A swine influenza virus caused the 2009 pandemic. Among all influenza viruses, swine influenza viruses are those considered to have greatest chance of causing the next pandemic, particularly one strain called 'Eurasian avian-like swine influenza virus H1N1' , EAH1N1. EAH1N1 originally jumped from birds into pigs in the late 1970s where it has circulated ever since. EAH1N1 is among the most prevalent swine influenza viruses in Europe and China, where it circulates alongside descendants of the 2009 pandemic influenza virus (pH1N1) - which jumped back into pigs shortly after 2009. In many places EAH1N1 and pH1N1 have exchanged genes generating viruses that may have even higher pandemic potential.
In this proposal we aim to better understand how influenza viruses jump into, and adapt to new hosts, using EAH1N1 as a specific example. Additionally, we will risk assess different EAH1N1 viruses from around the world to determine if certain strains may be more likely to become future pandemics. Finally, we want to identify host proteins that these viruses rely on to efficiently infect their host, with the long-term strategy of using these identified proteins to generate gene-edited animals which are resistant to influenza virus.
This work will be undertaken using a mix of phylogenetics and bioinformatics to choose relevant current strains to test, and to identify mutations in circulating viruses associated with mammalian adaptation. We will generate recombinant viruses for a comprehensively risk assessment and to validate if the mutations that we identified enhance replication in swine or human cells. This work will involve studies in primary cells and transmission studies in pigs, or in ferrets which are the gold standard model for airborne transmissibility of influenza viruses in humans. We will also perform large scale screens using technology such as CRISPR to identify proteins in pigs which are responsible for efficient virus replication or adaptation. Identification of these proteins will pave the way to future strategies for controlling these viruses in pigs, for example by using targeted gene-editing technology to generate pigs which are resistant to influenza infection.
This UK/China collaboration brings together scientists from two of the biggest pig producers in the world (China and Europe), which both have high levels of circulating swine influenza viruses to work on a global problem with a timely and comprehensive approach. This will allow for a globally relevant risk assessment to be undertaken, and proper comparisons of viruses from the different continents to be made. This work will be valuable to both the agricultural industry, but also public health bodies and those preparing for future pandemics.
In this proposal we aim to better understand how influenza viruses jump into, and adapt to new hosts, using EAH1N1 as a specific example. Additionally, we will risk assess different EAH1N1 viruses from around the world to determine if certain strains may be more likely to become future pandemics. Finally, we want to identify host proteins that these viruses rely on to efficiently infect their host, with the long-term strategy of using these identified proteins to generate gene-edited animals which are resistant to influenza virus.
This work will be undertaken using a mix of phylogenetics and bioinformatics to choose relevant current strains to test, and to identify mutations in circulating viruses associated with mammalian adaptation. We will generate recombinant viruses for a comprehensively risk assessment and to validate if the mutations that we identified enhance replication in swine or human cells. This work will involve studies in primary cells and transmission studies in pigs, or in ferrets which are the gold standard model for airborne transmissibility of influenza viruses in humans. We will also perform large scale screens using technology such as CRISPR to identify proteins in pigs which are responsible for efficient virus replication or adaptation. Identification of these proteins will pave the way to future strategies for controlling these viruses in pigs, for example by using targeted gene-editing technology to generate pigs which are resistant to influenza infection.
This UK/China collaboration brings together scientists from two of the biggest pig producers in the world (China and Europe), which both have high levels of circulating swine influenza viruses to work on a global problem with a timely and comprehensive approach. This will allow for a globally relevant risk assessment to be undertaken, and proper comparisons of viruses from the different continents to be made. This work will be valuable to both the agricultural industry, but also public health bodies and those preparing for future pandemics.
Technical Summary
To achieve the objectives of this proposal we will
a) Perform detailed bioinformatics and phylogenetic analysis on publicly available swine influenza virus genomes to i) identify representative circulating strains for downstream risk assessment and ii) identify potential markers of ongoing adaptation in swine, for example by identifying convergent mutations that have appeared multiple times across the phylogenetic tree.
b) Generated recombinant viruses using reverse genetics. These rescued viruses and mutants therefor will then be tested in many assays such as:
- Minireplicon assays that measure virus polymerase activity in avian, swine and human cells.
- Virus growth kinetics in avian, human or pig cell lines
- Virus growth kinetics in avian, human or swine explants or primary cell cultures.
c) Tested a small panel of the viruses for virus kinetics, pathogenicity, transmissibility and adaptation in mice, pigs or ferrets. Outputs from these experiments will include virus shedding, weight loss, exhaled virus, virus lung titres and tropism, fever, clinical signs and adaptation of viruses measured by deep sequencing.
d) Perform genome wide screens to identify host and restriction factors that impact influenza virus replication in swine. These screens will combine pull downs and mass spec of viral proteins with known adaptations added to them, as well as several different CRISPR strategies including traditional knock out as well as CRISPR gene activation.
Proteins from these screens will be validated on their impact on influenza replication using siRNA, clonal CRISPR or overexpression. The strongest hits will be taken further to study protein orthologues or paralogues ability to impact flu replication, as well as deep mutational scanning to identify regions of the protein vital for modulating influenza replication.
a) Perform detailed bioinformatics and phylogenetic analysis on publicly available swine influenza virus genomes to i) identify representative circulating strains for downstream risk assessment and ii) identify potential markers of ongoing adaptation in swine, for example by identifying convergent mutations that have appeared multiple times across the phylogenetic tree.
b) Generated recombinant viruses using reverse genetics. These rescued viruses and mutants therefor will then be tested in many assays such as:
- Minireplicon assays that measure virus polymerase activity in avian, swine and human cells.
- Virus growth kinetics in avian, human or pig cell lines
- Virus growth kinetics in avian, human or swine explants or primary cell cultures.
c) Tested a small panel of the viruses for virus kinetics, pathogenicity, transmissibility and adaptation in mice, pigs or ferrets. Outputs from these experiments will include virus shedding, weight loss, exhaled virus, virus lung titres and tropism, fever, clinical signs and adaptation of viruses measured by deep sequencing.
d) Perform genome wide screens to identify host and restriction factors that impact influenza virus replication in swine. These screens will combine pull downs and mass spec of viral proteins with known adaptations added to them, as well as several different CRISPR strategies including traditional knock out as well as CRISPR gene activation.
Proteins from these screens will be validated on their impact on influenza replication using siRNA, clonal CRISPR or overexpression. The strongest hits will be taken further to study protein orthologues or paralogues ability to impact flu replication, as well as deep mutational scanning to identify regions of the protein vital for modulating influenza replication.
