Restriction of avian viruses by host interferon-inducible transmembrane proteins (IFITMs).

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine

Abstract

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Planned Impact

With current global production of 55 billion chickens annually, poultry products are the main source of animal protein for human consumption worldwide. The sustainability of this major food resource is now a global research priority. The UK poultry industry currently contributes around £3.4bn to the economy. Avian viruses, such as avian influenza, Infectious Bursal Disease Virus and Infectious Bronchitis Virus create major challenges to poultry health through loss of productivity and mortality, and have concomitant effects on the global poultry industry through a reduction in the output of poultry meat and eggs. Developing efficient control strategies against these viral diseases will not only of benefit Western societies, but also alleviate poverty in developing countries, where these diseases are widespread, causing devastating effects on poultry farming.

The primary focus of this study will be the academic work necessary to underpin and facilitate a range of benefits for different stakeholders. The discovery of the role of IFITM proteins as broad spectrum, anti-viral agents for avian viruses will provide new insights into innate immunity and potentially novel tools and breading programs with which to counter viral pathogens of poultry. The work will create opportunities for medium term industrial outcomes from this project, and we will formulate plans to engage industry in the project. One of the main beneficiaries of this work will be the livestock industry, and specifically poultry breeding companies. Two of the world leading companies, Aviagen and Cobb-Vantress are partners in this Animal Health Research club and thus results generated by this project has a great potential to benefit their global economic performance, and specifically the economic competitiveness of the UK poultry industry. Worldwide, it is likely that the EU and UK policy makers in Animal Health will have an interest in the results due to the impact that the poultry industry has on food sustainability. Outreach activities will be conducted to ensure that stakeholders benefit directly from the project, by gaining a deeper understanding how we intend to turn this research into practice.

We will have impact on the professional development of scientist involved in this work. In addition to laboratory- and animal-based training, the staff employed on this project will have access to training courses on scientific methods, technical writing, presentation skills, ethics, and transferable skills courses provided by the Pirbright Institute and WTSI. They will also train new staff members and visitors to perpetuate skills and benefit from working with leading poultry companies during the project. Throughout the project we will utilise e-outreach by providing a description and updates of the project for a lay public through the Pirbright Institute and Sanger websites. IAH will also produce a video describing our work for a general audience that will be released during year one (a basic introduction to the project) and year three (the results of the project and future utility). We will engage with the General Public through the provision of press releases to media outlets at the initiation of the project; to both promote the objectives of BBSRC's Animal Health Research Club, and to explore this area of science in an understandable fashion. Scientists employed during the project will also be encouraged to take part in public engagement. Dr Fife is an active STEM ambassador, which creates opportunities to inspire young people and develop their creativity, problem-solving and employability skills the UK's future competitiveness. Prof Kellam gives science presentations to junior and secondary school children and all researchers will be encouraged to participate in STEM activities including school visits.
 
Description Genomic characterization of the chicken interferon-inducible transmembrane gene locus

The interferon-inducible transmembrane (IFITM) protein family are cellular restriction factors that inhibit the entry of different viruses by preventing efficient virus-cell fusion. We recently identified these genes in chickens and showed that like the human orthologous genes they are able to restrict influenza A virus and lyssaviruses. This ability to restrict different viruses gives rise to the question if whether variants of the IFITM genes exist in different breeds of chickens resulting therefore in altered virus susceptibility.

Chicken IFITM genes (chIFITM) are clustered on chromosome 5 and they are flanked by the ATHL1 and B4GALNT4 genes, however, the current chicken genome is poorly assembled in this region. Using Illumina MiSeq and Pacific Bioscience (PacBio) sequencing platforms we sequenced a BAC encompassing the chIFITM locus and we show the complete sequence for the IFITM1, 2, 3, 5, ATHL1 and B4GALNT4 genes. Using the PacBio sequencing results we were able to close all of the gaps within the region analysed showing that several gaps were misassembled in the original reference genome.

This work was presented by Irene Bassano (funded by this award) at the Microbiology Society Annual 2016 conference

This work has been further extended in a publication, Bassano I, Ong SH, Lawless N, Whitehead T, Fife M, Kellam P. Accurate characterization of the IFITM locus using MiSeq and PacBio sequencing shows genetic variation in Galliformes.
BMC Genomics. 2017 May 30;18(1):419.


Interferon inducible transmembrane (IFITM) proteins are effectors of the immune system widely characterized for their role in restricting infection by diverse enveloped and non-enveloped viruses. The chicken IFITM (chIFITM) genes are clustered on chromosome 5 and to date four genes have been annotated, namely chIFITM1, chIFITM3, chIFITM5 and chIFITM10. However, due to poor assembly of this locus in the Gallus Gallus v4, accurate characterization has so far proven problematic. Recently, a new chicken reference genome assembly Gallus Gallus v5 was generated using Sanger, 454, Illumina and PacBio sequencing technologies identifying considerable differences in the chIFITM locus over the previous genome releases. Here we re-sequenced the locus using both Illumina MiSeq and PacBio RS II sequencing technologies and confirmed the Gallus Gallus v5 consensus except for two insertions of 5 and 1 base pair within the chIFITM3 and B4GALNT4 genes, respectively, and a single base pair deletion within the B4GALNT4 gene. We then mapped RNA-seq data from the European Nucleotide Archive (ENA) to this finalized chIFITM locus and show chIFITM2 and chIFITM3 expression in numerous tissue types of different chicken breeds and avian cell lines, while the expression of the putative chIFITM1 is limited to the testis, caecum and ileum tissues. Using SureSelect probes capture probes designed to the finalized chIFITM locus, we sequenced the locus of a different chicken breed, a White Leghorn, revealing a single amino acid substitution of A63V in the CIL domain of IFITM2 compared to Red Jungle fowl and 13, 13 and 11 differences between IFITM1, 2 and 3 of chickens and turkeys, respectively. Locus resequencing using these capture probes and RNA-seq based expression analysis will allow the further characterization of genetic diversity within Galliformes.


Using these methods we are now analysing the extend of genetic variation in inbred, commercial and outbred chickens. The results of such variation analysis are being passed onto Prof Mark Fifie and the award BB/L003996/1 for analysis of the functional consequences of observed genetic variation.

This work has been extended in a large scale sequencing study of the avian IFITM locus in the paper (submitted) 'Comparative analysis of targeted avian pull-down genome reveals an insight into the evolution of the chicken IFITM locus'. Using the pull down method we sequence 218 diverse and commercial chickens to examine the detailed pattern of genetic variation at the IFITM locus on chicken chromosome 5 , including the flanking genes ATHL1 and B4GALNT4 but with a focus on the chIFITM1, 2, 3 and 5 genes. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breast), ancient bone samples, indigenous chickens from Nigeria and Ethiopia, European breeds and inbred lines from the Pirbright Institute. We have mapped genetic variants to the latest chIFITM consensus sequence and discovered SNPs previously not annotated. Our data reveal that the chIFITM locus does not show significant variation in its general structure across the population analysed, despite spanning breeds belonging to different period and geographic locations. However, SNPs in functionally important regions of the proteins within individual groups were detected, suggesting a pattern of evolution peculiar for a specific population, in particular the European breeds and indigenous birds. Of these, of interest is a SNP in the IFITM3 protein which appears to be also under positive selection. Our results indicate that IFITM genetic variation may contribute to the differing capacities of distinct chicken populations to resist a wide range of viruses and other pathogens.

This work was published as: Bassano I, Ong SH, Sanz-Hernandez M, Vinkler M, Kebede A, Hanotte O, Onuigbo E, Fife M, Kellam P. Comparative analysis of the chicken IFITM locus by targeted genome sequencing reveals evolution of the locus and positive selection in IFITM1 and IFITM3. BMC Genomics. 2019;20(1): 272.

These results complete specific aim 2: What is the level of expression and genetic variation at the IFITM locus within inbred, commercial and outbred broiler and layer breeds?
Exploitation Route Patent filed by the Pirbright. Institute: Avian cells for improved virus production (US20160108359A1)

Abstract
The present Invention provides as avian cell in which the expression or activity of one or more of the following genes, or a homologue thereof: Chicken IFITM 1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3) is reduced. The invention also provides methods for passaging viruses in avian cells, embryos and/or avian cell lines which have reduced expression of one or more IFITM genes and methods which involve investigating the sequence of one or more of the following genes, or a homologue thereof: Chicken IFITM1 (SEQ ID No. 1); Chicken IFITM2 (SEQ ID No. 2) and Chicken IFITM3 (SEQ ID No. 3).
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Awareness in the agriculture industry through the Annual Animal Research Club grant holders meeting of the work being undertaken in this project.
First Year Of Impact 2016
Sector Agriculture, Food and Drink