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RNA-seq data alignment of reads from the immune relevant tissues and cell lines in treated conditions: infection with IBDV, ALV-J, ILVV, LPS, H5N5/H5N1 or heat stress-induced conditions. The graph shows that also in these conditions, levels of chIFITM are lower compared to chIFITM2 and chIFITM3. Top panel, overall coverage. Bottom panel stack view of each chIFITM transcript
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Background
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...
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Citations
... This SNP has been correlated with worsened clinical outcomes for both seasonal influenza and H7N9 infections (11). When we examined the sequence of the IFITM3 gene as described by Smith et al. (2013) (39) and confirmed more recently by Bassano et al. (2017) (40) and despite an annotated presence of potential SNPs in the gene we detected no differences between susceptible and resistant chickens. We did however detect two SNPs in chicken IFITM1 which had a significant correlation with disease susceptibility (p=0.0263 and p=0.0012 for the first and second SNPs respectively). ...
... This SNP has been correlated with worsened clinical outcomes for both seasonal influenza and H7N9 infections (11). When we examined the sequence of the IFITM3 gene as described by Smith et al. (2013) (39) and confirmed more recently by Bassano et al. (2017) (40) and despite an annotated presence of potential SNPs in the gene we detected no differences between susceptible and resistant chickens. We did however detect two SNPs in chicken IFITM1 which had a significant correlation with disease susceptibility (p=0.0263 and p=0.0012 for the first and second SNPs respectively). ...
The zoonotic H7N9 avian influenza (AI) virus first emerged in 2013 as a low pathogenic (LPAI) strain, and has repeatedly caused human infection resulting in severe respiratory illness and a mortality of ~39% (>600 deaths) across five epidemic waves. This virus has circulated in poultry with little to no discernible clinical signs, making detection and control difficult. Contrary to published data, our group has observed a subset of specific pathogen free chickens infected with the H7N9 virus succumb to disease, showing clinical signs consistent with highly pathogenic AI (HPAI). Viral genome sequencing revealed two key mutations had occurred following infection in the haemagglutinin (HA 226 L>Q) and nucleoprotein (NP 373 A>T) proteins. We further investigated the impact of the NP mutation and demonstrated that only chickens bearing a single nucleotide polymorphism (SNP) in their IFITM1 gene were susceptible to the H7N9 virus. Susceptible chickens demonstrated a distinct loss of CD8⁺ T cells from the periphery as well as a dysregulation of IFNγ that was not observed for resistant chickens, suggesting a role for the NP mutation in altered T cell activation. Alternatively, it is possible that this mutation led to altered polymerase activity, as the mutation occurs in the NP 360-373 loop which has been previously show to be important in RNA binding. These data have broad ramifications for our understanding of the pathobiology of AI in chickens and humans and provide an excellent model for investigating the role of antiviral genes in a natural host species.
... Research has been carried out to better understand the molecular pathogenesis of the changes that mutations in the IFITM5 gene carry. Although BRIL expression has been demonstrated in bone tissues from rodents [30,38,39], humans [40,41], tammar wallaby [42], and chicken [43], a clear in vivo role for the protein is still largely elusive. One hypothesis put forward is that the acidic C-terminus of BRIL represents a calcium binding moiety, although no formal experimental validation has been performed [44,45]. ...
Osteogenesis imperfecta (OI) is a large group of genetically heterogeneous diseases resulting from decreased bone density and an abnormal microarchitecture, which are clinically manifested by abnormal bone fractures. A distinctive clinical feature of this group of diseases is the presence of spontaneous fractures and skeletal deformities. However, the clinical manifestations of different types of OI are characterized by marked polymorphism with variable severity of skeletal and extra-skeletal features. Previous studies have shown that a mutation (c.-14C>T) in the IFITM5 gene is responsible for autosomal dominant OI type V. However, the mutation has a variable expression pattern and marked clinical heterogeneity. In this study, a clinical and genetic analysis of 12 cases with molecularly confirmed OI type V from 12 unrelated families was performed. Significant clinical heterogeneity of the disease with the same molecular defect was detected. In six subjects (50%), there were no classic signs of OI type V (formation of a hyperplastic bone callus, calcification of the interosseous membrane and dislocation of the radial head). In all cases, the mutation occurred de novo.
... The clinical symptoms presented by infected birds are dependent on several factors including the strain of IBV, of which there are many genetic variants and serotypes. The majority of IBV strains, including the Massachusetts (Mass) serotype M41 produce prominent yet annotated in the Gallus gallus reference sequence, studies show the existence of chIFITM2 and suggest a hypothetical genetic structure of the locus based on the human syntenic genome region [51]. The mammalian IFITM proteins are relatively small (about 130 amino acids) [33] and share a topology defined by a conserved CD225 domain, consisting of two intramembrane (IM) regions and a conserved intracellular loop (CIL) [52]. ...
The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.
... Interferon-inducible transmembrane proteins (IFITMs) are small membrane-spanning proteins with high sequence conservation in different vertebrates. Human IFITMs mainly refers to IFITM1, IFITM2, IFITM3, IFITM5 and IFITM10 and these IFITM genes forms a locus located on chromosome 11 (Bassano et al., 2017). Among them, IFITM1, IFITM2 and IFITM3 were identified as innate antiviral factors against a broad spectrum of pathogenic viruses, especially enveloped RNA viral pathogens of 9 viral families e.g., Orthomyxoviruses (Influenza A virus, IAV), Filoviruses (Ebola virus, EBOV), Coronaviruses (severe acute respiratory syndrome coronavirus, SARS-CoV), Lentiviruses (human immunodeficiency virus, HIV), Marburg virus (MARV), Bunyaviruses (Rift Valley fever virus, RVFV) and Hantaan virus (HTNV), and relative more Flaviviridae members including hepatitis C virus (HCV), dengue virus (DENV), Zika Virus (ZIKV), West Nile virus (WNV), Yellow Fever Virus(YFV), Omsk Hemorrhagic Fever Virus (OHFV) (Liao et al., 2019;Zhao et al., 2019). ...
Japanese encephalitis virus (JEV) is an infectious pathogen spreading in a wide range of vertebrate species. Pigs are amplifying hosts of JEV and thought to be maintained in nature predominantly by avian-mosquito cycles. In the innate immune system, interferon-inducible transmembrane protein (IFITM) is a small transmembrane protein family and has been identified as the first line of defense against a broad range of RNA virus invasion. In this paper, we found that swine IFITM (sIFITM) could restrict the replication of both JEV vaccine strain and wild strain NJ-2008. The cysteine S-palmitoylation modification of sIFITM plays important roles in their anti-JEV effects and intracellular distributions. Our findings show the anti-JEV activities of swine interferon-inducible transmembrane proteins and broaden the antiviral spectrum of IFITM protein family. The preliminary exploration of S-palmitoylation modification of sIFITM may contribute to understanding of the antiviral molecular mechanism of sIFITM.
... Interestingly, the prediction revealed three differences in the IFITM3 protein between mammals and birds. However, this prediction was carried out on limited species of mammals and birds registered in GenBank, further confirmation in recently reported IFITM3 sequences is needed in the future (Bassano et al. 2017). In a previous topology study in the human IFITM3 protein, substantial evidence indicated that the human IFITM3 protein favors the cytosolic Nterminus. ...
The interferon-induced transmembrane protein 3 (IFITM3) gene is classified as a small interferon-stimulated gene and is associated with a broad spectrum of antiviral functions against several fatal enveloped viruses, including influenza A viruses (IAVs). The rs12252 single nucleotide polymorphism (SNP) of the IFITM3 gene in humans was associated with susceptibility to H1N1 influenza in a 2009 pandemic. In addition, overexpression of the IFITM3 protein potently inhibits the highly pathogenic avian influenza H5N1 virus in ducks and chickens. Although chickens are a major host of influenza viruses and the IFITM3 gene participates in the host antiviral system, studies on chicken IFITM3 gene are very rare. To investigate the genetic characteristics of the chicken IFITM3 gene, we performed direct sequencing and alignment in 108 Dekalb White and 72 Ross breeds. We also investigated the genotype and haplotype frequencies and linkage disequilibrium of the IFITM3 gene polymorphisms and evaluated whether the non-synonymous SNPs are deleterious. We found significantly different genotype, allele and haplotypes frequencies between two chicken breeds, Dekalb White and Ross. Furthermore, we compared and analyzed the promoter structure of the chicken IFITM3 gene with that of several species. We found that birds have a long C-terminal domain and inverted topology of the IFITM3 protein compared to mammals. We also identified fourteen genetic polymorphisms in the chicken IFITM3 gene. L100 M and N125H were predicted as ‘probably damaging’ and L100 M can alter the length of its conserved intracellular loop (CIL). Furthermore, chickens, but not mammals, contain CpG islands (CGIs) in this promoter region.
... We have recently showed that targeted pull down of the chIFITM locus can successfully and accurately characterize this locus without the need of performing whole genome sequencing [6]. Here we have used SureSelect probes to pull down the chIFITM locus in a total of 206 chickens, we have assembled the locus using as a reference a Red Jungle Fowl (Gallus gallus) we previously sequenced [6] and we performed genetic variation analysis using a modified version of the GATK Best Practice pipeline [7]. ...
... We have recently showed that targeted pull down of the chIFITM locus can successfully and accurately characterize this locus without the need of performing whole genome sequencing [6]. Here we have used SureSelect probes to pull down the chIFITM locus in a total of 206 chickens, we have assembled the locus using as a reference a Red Jungle Fowl (Gallus gallus) we previously sequenced [6] and we performed genetic variation analysis using a modified version of the GATK Best Practice pipeline [7]. Our results catalogue the first comprehensive list of SNVs and INDELs for the chIFITM locus as well as within and between groups of birds variation. ...
... Up to 5 μg of the extracted DNA were sent to the Wellcome Sanger Institute sequencing facility where the samples were sequenced using Illumina MiSeq sequencers, following targeted SureSelect pull-down of the locus of interest according to the manufacturer's protocol [6,8]. ...
Background
The interferon-induced transmembrane (IFITM) protein family comprises a class of restriction factors widely characterised in humans for their potent antiviral activity. Their biological activity is well documented in several animal species, but their genetic variation and biological mechanism is less well understood, particularly in avian species.
Results
Here we report the complete sequence of the domestic chicken Gallus gallus IFITM locus from a wide variety of chicken breeds to examine the detailed pattern of genetic variation of the locus on chromosome 5, including the flanking genes ATHL1 and B4GALNT4. We have generated chIFITM sequences from commercial breeds (supermarket-derived chicken breasts), indigenous chickens from Nigeria (Nsukka) and Ethiopia, European breeds and inbred chicken lines from the Pirbright Institute, totalling of 206 chickens. Through mapping of genetic variants to the latest chIFITM consensus sequence our data reveal that the chIFITM locus does not show structural variation in the locus across the populations analysed, despite spanning diverse breeds from different geographic locations. However, single nucleotide variants (SNVs) in functionally important regions of the proteins within certain groups of chickens were detected, in particular the European breeds and indigenous birds from Ethiopia and Nigeria. In addition, we also found that two out of four SNVs located in the chIFITM1 (Ser36 and Arg77) and chIFITM3 (Val103) proteins were simultaneously under positive selection.
Conclusions
Together these data suggest that IFITM genetic variation may contribute to the capacities of different chicken populations to resist virus infection.
Electronic supplementary material
The online version of this article (10.1186/s12864-019-5621-5) contains supplementary material, which is available to authorized users.
... One family of ISGs that functions as broad-spectrum inhibitors of viral replication is the interferon-inducible transmembrane protein (IFITM) family. IFITMs are functionally conserved across many species, including birds (9)(10)(11)(12), pigs (13,14), and bats (13). In most cases, this family of restriction factors blocks infection during virus entry into cells (15), although additional mechanisms have been proposed (16,17). ...
Host susceptibility to viral infection is multifactorial, but early control of viruses not previously encountered is predominantly mediated by the interferon-stimulated gene (ISG) family. There are upwards of 300 of these genes, the majority of which do not have a clearly defined function or mechanism of action. The cellular location of these proteins may have an important effect on their function. One ISG located at the plasma membrane is interferon-inducible transmembrane protein 1 (IFITM1). Here we demonstrate that IFITM1 can inhibit infection with a range of viruses that enter via the plasma membrane. Mutant IFITM1 proteins that were unable to localize to the plasma membrane did not restrict viral infection. We also observed for the first time that IFITM1 plays a role in vivo , and Ifitm1 −/− mice were more susceptible to viral lung infection. These data contribute to our understanding of how ISGs prevent viral infections.
... One of the problems facing researchers in this field is the incomplete genome build of many avian species, however, the IFITM locus in chickens has been resolved and there is now a contiguous sequence which will aid further research (Bassano et al., 2017). ...
... This level of genetic analysis has not been conducted in chickens because the chicken IFITM locus was only recently correctly annotated in the chicken genome (Bassano et al., 2017). ...
... Aligning the avian IFITMs sequence against the Galgal 5 reference genome was performed in order to assess the level of sequence conservation and to identify conserved regions which may be important for viral restriction. Furthermore, the novel sequencing strategy developed by Bassano et al. (2017) was used to sequence three avian cell lines in order to identify genetic variation that may influence the cell line specific chIFITMrestriction. In addition, the cellular localisation of these proteins was characterised in vitro alongside any cellular interacting partners that may influence restriction or may control how the genes are expressed under native conditions. ...
Interferon-inducible transmembrane (IFITM) proteins are host cell derived restriction factors. Mammalian IFITM proteins have been shown to confer antiviral resistance when challenged with a diverse range of both enveloped and non-enveloped viruses. Little characterisation has been undertaken to date with the specific aim of elucidating their function and the antiviral properties of the chicken IFITM (chIFITM) gene family. The chIFITM gene family contains four genes located within a 17kb region on Gallus gallus chromosome 5. Currently there is little information available about the sequence diversity of these genes, their expression profiles or the role that they may play in restricting avian viral pathogens. Data presented in this thesis outlines a novel DNA pull-down sequencing technique which has allowed for the generation of a high quality contiguous reference sequence, alongside targeted sequencing of chicken cell lines and ex vivo cell cultures. Studies in this thesis have established that the chIFITMs are interferon stimulated and have characterized their upregulation in response to viral challenge with influenza A virus (IAV) in ovo, in vivo and in vitro, alongside other avian viruses. Stably-overexpressing DF-1 (immortalized chick embryo fibroblast) cells that express chIFITM1, 2, 3 and 3MUT (C71/71A) have been generated. These cell lines have been challenged with avian viruses including diverse strains of IAV and infectious bronchitis virus (IBV) and this data demonstrates that the chIFITMs are able to restrict avian viruses in vitro. Moreover, novel interactions have been identified which may help to uncover a possible mechanism of action. Global food security and protection of livestock from infectious agents remains a key priority, both in the United Kingdom and Internationally. This study examines the role of chIFITM proteins during viral infections and highlights one potential method of safeguarding the poultry industry, ensuring continuity of global food security.
... Although BRIL expression has been demonstrated in bone tissues from rodents [1,2,6], humans [3,13], tammar wallaby [5], and chicken [17], a clear in vivo role for the protein is still largely elusive. One hypothesis put forward is that the acidic C-terminus of BRIL represents a calcium binding moiety, although no formal experimental validation has been performed [18,19]. ...
BRIL (bone-restricted IFITM-like), is a short transmembrane protein expressed almost exclusively in osteoblasts. Although much is known about its bone-restricted gene expression pattern and protein biochemical and topological features, little information is available for BRIL physiological function. Two autosomal dominant forms of osteogenesis imperfecta (OI) are caused by distinct, but recurrent mutations in the BRIL gene. Yet, the underlying mechanisms by which those mutations lead to OI are still poorly understood. A previous report indicated that BRIL knockout (KO) mice had bone deformities, shortened long bones, and reproductive problems. Here we generated and systematically analyzed the skeletal phenotype of a new global Bril KO/LacZ knockin mouse model. KO mice reproduced and thrived normally up to 12 month of age. The skeletal phenotype of KO and WT littermates was assessed at embryonic (E13.5 to E18.5) and postnatal (2 days, 3 weeks, 3 months and 8 months) time-points. Embryos from E13.5 through to E18.5 showed significant X-Gal staining in all skeletal elements without any apparent patterning anomalies. Although bone deformities were never observed at any postnatal ages, minor and transient differences were noted in terms of bone length and static uCT parameters, but not systematically across all ages and genders. These changes, however, were not accompanied by significant alteration in bone material properties as assessed by a 3-point bending test. In addition, no changes were detected in circulating serum markers of bone turnover (P1NP, CTX-I, and osteocalcin). Gene expression monitoring also revealed no major impact of the loss of BRIL. Further, when mice were challenged with a surgically-induced fracture in tibia, bones repaired equally well in the KO mice as compared to WT. Finally, we showed that BRIL C-terminus is not a bona fide binding site for calcium. In conclusion, our in depth analysis suggest that skeletal patterning, bone mass accrual and remodeling in mice proceeded independent of BRIL.
