The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Regulation of conceptus interferon-tau gene subtypes expressed in the uterus during the peri-implantation period of cattle.

Conceptus interferon tau (IFNT), produced by the embryonic trophectoderm, is known as a major signaling protein essential for the process of maternal recognition of pregnancy in ruminants. Similar to other IFN gene families such as IFNA and IFNB, multiple IFNT genes exist. The number of IFNT genes actively transcribed and regulated in conceptuses of cattle has, however, not been well characterized. In this study, IFNT transcripts in utero were studied through the use of next generation sequencer. Among 38 IFN genes registered and eight annotated as IFNT, only two transcripts, IFNT1 and IFNTc1, were found in conceptuses in utero. Relative abundance of transcription factor(s) involved in the regulation of IFNT genes were investigated by real-time PCR. Transcriptional activity of IFNT1 and IFNTc1 were investigated using bovine non-trophoblast ear fibroblast (EF) cells, which were co-transfected with luciferase reporter constructs with upstream (-631 to -51) promoter regions of IFNT1 or IFNTc1 and various transcription factor expression plasmids, CDX2, AP1 (JUN), ETS2 and/or CREBBP. CDX2 with AP1 and ETS2 was found to increase luciferase activity of IFNT1 and IFNTc1 approximately 14- and 11-fold, respectively, in EF cells, which do not express the CDX2 gene. These results indicated that two isoforms of conceptus IFNT genes of cattle could be regulated differently in utero. Furthermore, IFNT1 and IFNTc1 were found to have similar antiviral activity, suggesting that both IFNT genes could function to increase conceptus signaling to the uterine endometrium for the process of maternal recognition of pregnancy during the pre-implantation period.

Interferons and viruses: an evolutionary arms race of molecular interactions.

Over half a century has passed since interferons (IFNs) were discovered and shown to inhibit virus infection in cultured cells. Since then, researchers have steadily brought to light the molecular details of IFN signaling, catalogued their pleiotropic effects on cells, and harnessed their therapeutic potential for a variety of maladies. While advances have been plentiful, several fundamental questions have yet to be answered and much complexity remains to be unraveled. We explore the current knowledge surrounding four main questions: are type I IFN subtypes differentially produced in response to distinct pathogens? How are IFN subtypes distinguished by cells? What are the mechanisms and consequences of viral antagonism? Lastly, how can the IFN response be harnessed to improve vaccine efficacy?

The molecular basis for functional plasticity in type I interferon signaling.

Type I interferons (IFNs) are best known for their role in innate immunity, but they are also involved in other functions including immunomodulation, restricting proliferation, cancer surveillance, and the regulation of the adaptive immune response. All these responses are mediated through the interaction with a single cell surface receptor, albeit at different ligand and receptor concentrations, ligand subtypes, and time of activation. Here we review the functional plasticity of IFN signaling from a quantitative perspective, showing how variations in different ingredients of the system lead to differential IFN responses and how cells tune the system to maximize efficiency while minimizing detrimental effects. We present a basic model wherein the integrated action of different feedback mechanisms can provide sufficient temporal control to differentially drive cellular decisions.

Transcriptional regulation of two conceptus interferon tau genes expressed in Japanese black cattle during peri-implantation period.

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0  =  day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions -637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.

Distinct evolution process among type I interferon in mammals.

Interferon (IFN) is thought to play an important role in the vertebrate immune system, but systemic knowledge of IFN evolution has yet to be elucidated. To evaluate the phylogenic distribution and evolutionary history of type I IFNs, 13genomes were searched using BLASTn program, and a phylogenetic tree of vertebrate type I IFNs was constructed. In the present study, an IFNδ-like gene in the human genome was identified, refuting the concept that humans have no IFNδ genes, and other mammalian IFN genes were also identified. In the phylogenetic tree, the mammalian IFNß, IFNɛ, and IFNκ formed a clade separate from the other mammalian type I IFNs, while piscine and avian IFNs formed distinct clades. Based on this phylogenetic analysis and the various characteristics of type I IFNs, the evolutionary history of type I IFNs was further evaluated. Our data indicate that an ancestral IFNα-like gene forms a core from which new IFNs divided during vertebrate evolution. In addition, the data suggest how the other type I IFNs evolved from IFNα and shaped the complex type I IFN system. The promoters of type I IFNs were conserved among different mammals, as well as their genic regions. However, the intergenic regions of type I IFN clusters were not conserved among different mammals, demonstrating a high selection pressure upon type I IFNs during their evolution.

Mechanisms and implications of age-associated impaired innate interferon secretion by dendritic cells: a mini-review.

Initial secretion of interferons by innate immune cells such as dendritic cells is crucial for protection against infections as well as for alerting and activating the downstream immune responses. The secretion of innate interferons, both type I and type III, by dendritic cells is severely impaired in aged subjects. This review focuses on the mechanisms responsible for the reduced interferon secretion by dendritic cells and the role this plays in the increased susceptibility of the elderly to infections, particularly of the respiratory mucosa.

Teleost fish interferons and their role in immunity.

Interferons (IFNs) are the hallmark of the vertebrate antiviral system. Two of the three IFN families identified in higher vertebrates are now known to be important for antiviral defence in teleost fish. Based on the cysteine patterns, the fish type I IFN family can be divided into two subfamilies, which possibly interact with distinct receptors for signalling. The fish type II IFN family consists of two members, IFN-γ with similar functions to mammalian IFN-γ and a teleost specific IFN-γ related (IFN-γrel) molecule whose functions are not fully elucidated. These two type II IFNs also appear to bind to distinct receptors to exert their functions. It has become clear that fish IFN responses are mediated by the host pattern recognition receptors and an array of transcription factors including the IFN regulatory factors, the Jak/Stat proteins and the suppressor of cytokine signalling (SOCS) molecules.

Chiropteran types I and II interferon genes inferred from genome sequencing traces by a statistical gene-family assembler.

BACKGROUND: The rate of emergence of human pathogens is steadily increasing; most of these novel agents originate in wildlife. Bats, remarkably, are the natural reservoirs of many of the most pathogenic viruses in humans. There are two bat genome projects currently underway, a circumstance that promises to speed the discovery host factors important in the coevolution of bats with their viruses. These genomes, however, are not yet assembled and one of them will provide only low coverage, making the inference of most genes of immunological interest error-prone. Many more wildlife genome projects are underway and intend to provide only shallow coverage. RESULTS: We have developed a statistical method for the assembly of gene families from partial genomes. The method takes full advantage of the quality scores generated by base-calling software, incorporating them into a complete probabilistic error model, to overcome the limitation inherent in the inference of gene family members from partial sequence information. We validated the method by inferring the human IFNA genes from the genome trace archives, and used it to infer 61 type-I interferon genes, and single type-II interferon genes in the bats Pteropus vampyrus and Myotis lucifugus. We confirmed our inferences by direct cloning and sequencing of IFNA, IFNB, IFND, and IFNK in P. vampyrus, and by demonstrating transcription of some of the inferred genes by known interferon-inducing stimuli. CONCLUSION: The statistical trace assembler described here provides a reliable method for extracting information from the many available and forthcoming partial or shallow genome sequencing projects, thereby facilitating the study of a wider variety of organisms with ecological and biomedical significance to humans than would otherwise be possible.

Overview of the biology of type I interferons.

Type I interferons are pleiotropic cytokines with antiviral, antitumor and immunoregulatory functions. An aspect of their complex biology is the paradox that, depending on context, type I interferons can be anti-inflammatory and tissue protective or can be proinflammatory and promote autoimmunity. Along these lines, the activation of type I interferon pathways is effective in suppressing disease activity in patients with multiple sclerosis and in animal models of arthritis and colitis, while there is an expectation that blockade of the same pathways will be beneficial in the treatment of patients with systemic lupus erythematosus.

Characterization and virus-induced expression profiles of the porcine interferon-omega multigene family.

Interferon-omega is a member of the type I interferon family. In this work, 8 functional porcine interferon-omega genes and 4 pseudogenes present on porcine chromosome 1 were identified in the porcine genome database by BLAST scanning. Their genetic and genomic characteristics were investigated using bioinformatics tools. Then the PoIFN-omega functional subtype genes were isolated and expressed in BHK-21 cells. The PoIFN-omega subtypes possessed about 10(4) to 10(5) units of antiviral activity per milliliter. PoIFN-omega 7 had the highest antiviral activity, about 20 times that of PoIFN-omega 4, which had the lowest antiviral activity. Differential expression of the subtypes was detected in PK15 cells and porcine peripheral blood mononuclear cells (PBMCs) in response to pseudorabies virus and poly(I).poly(C). Expression of PoIFN-omega 2/-omega 6 was up-regulated to the greatest extent by virus infection.

Characterization and expression analysis of type I interferon in common carp Cyprinus carpio L.

The type I interferon (I-IFN) gene has recently been cloned and sequenced in the common carp species Cyprinus carpio L. Carp I-IFN cDNA is composed of 675 base pairs and is translated into a protein of 186 amino acid residues. The carp I-IFN encodes a predicted signal peptide of 23 amino acid residues and contains the I-IFN family signature His(140)-Trp(158). Analysis of the homology between carp I-IFN and other known I-IFN and type II interferon (II-IFN) family members has revealed significant similarities to grass carp I-IFN. Phylogenetic analysis demonstrated that carp I-IFN clusters with I-IFN in teleosts, away from the other II-IFN family members. In addition, the gene structure for carp I-IFN is composed of 5 exons and 4 introns, a composition that is similar to that of the teleost I-IFN gene. RT-PCR analysis did not reveal gene expression in un-stimulated tissues including intestine, liver, gill, head kidney, muscle, spleen, mid-kidney and skin. However, I-IFN expression levels increased following stimulation with imiquimod in the head kidney cells. Furthermore, recombinant carp I-IFN protein (mature form) produced via the cell-free protein synthesis system stimulated the expression of the interferon-inducible Mx gene in the head kidney cells.

Characterization of the bovine type I IFN locus: rearrangements, expansions, and novel subfamilies.

BACKGROUND: The Type I interferons (IFN) have major roles in the innate immune response to viruses, a function that is believed to have led to expansion in the number and complexity of their genes, although these genes have remained confined to single chromosomal region in all mammals so far examined. IFNB and IFNE define the limits of the locus, with all other Type I IFN genes except IFNK distributed between these boundaries, strongly suggesting that the locus has broadened as IFN genes duplicated and then evolved into a series of distinct families. RESULTS: The Type I IFN locus in Bos taurus has undergone significant rearrangement and expansion compared to mouse and human, however, with the constituent genes separated into two sub-loci separated by >700 kb. The IFNW family is greatly expanded, comprising 24 potentially functional genes and at least 8 pseudogenes. The IFNB (n = 6), represented in human and mouse by one copy, are also present as multiple copies in Bos taurus. The IFNT, which encode a non-virally inducible, ruminant-specific IFN secreted by the pre-implantation conceptus, are represented by three genes and two pseudogenes. The latter have sequences intermediate between IFNT and IFNW. A new Type I IFN family (IFNX) of four members, one of which is a pseudogene, appears to have diverged from the IFNA lineage at least 83 million years ago, but is absent in all other sequenced genomes with the possible exception of the horse, a non-ruminant herbivore. CONCLUSION: In summary, we have provided the first comprehensive annotation of the Type I IFN locus in Bos taurus, thereby providing an insight into the functional evolution of the Type I IFN in ruminants. The diversity and global spread of the ruminant species may have required an expansion of the Type I IFN locus and its constituent genes to provide broad anti-viral protection required for foraging and foregut fermentation.

A novel type I IFN-producing cell subset in murine lupus.

Excess type I IFNs (IFN-I) have been linked to the pathogenesis of systemic lupus erythematosus (SLE). Therapeutic use of IFN-I can trigger the onset of SLE and most lupus patients display up-regulation of a group of IFN-stimulated genes (ISGs). Although this "IFN signature" has been linked with disease activity, kidney involvement, and autoantibody production, the source of IFN-I production in SLE remains unclear. 2,6,10,14-Tetramethylpentadecane-induced lupus is at present the only model of SLE associated with excess IFN-I production and ISG expression. In this study, we demonstrate that tetramethylpentadecane treatment induces an accumulation of immature Ly6C(high) monocytes, which are a major source of IFN-I in this lupus model. Importantly, they were distinct from IFN-producing dendritic cells (DCs). The expression of IFN-I and ISGs was rapidly abolished by monocyte depletion whereas systemic ablation of DCs had little effect. In addition, there was a striking correlation between the numbers of Ly6C(high) monocytes and the production of lupus autoantibodies. Therefore, immature monocytes rather than DCs appear to be the primary source of IFN-I in this model of IFN-I-dependent lupus.

Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates.

In this report, three type I IFN genes were identified in rainbow trout (rt) Oncorhynchus mykiss and are classified into two groups based on their primary protein sequences: group I containing two cysteine residues; and group II containing four cysteines residues. The group I rtIFNs were induced in fibroblasts (RTG-2 cells), macrophages (RTS-11 cells), and head kidney leukocytes when stimulated with polyinosinic:polycytidylic acid, whereas group II IFN was up-regulated in head kidney leukocytes but not in RTG-2 and RTS-11 cells. Recombinant group I rtIFNs were potent at inducing Mx expression and eliciting antiviral responses, whereas recombinant group II rtIFN was poor in these activities. That two subgroups of type I IFN exist in trout prompted a survey of the genomes of several fish species, including zebrafish, medaka, threespine stickleback and fugu, the amphibian Xenopus tropicalis, the monotreme platypus and the marsupial opossum, to gain further insight into possible IFN evolution. Analysis of the sequences confirmed that the new IFN subgroup found in trout (group II IFN) exists in other fish species but was not universally present in fish. The IFN genes in amphibians were shown for the first time to contain introns and to conserve the four cysteine structure found in all type I IFNs except IFN-betaepsilon and fish group I IFN. The data overall support the concept that different vertebrate groups have independently expanded their IFN types, with deletion of different pairs of cysteines apparent in fish group I IFN and IFN-betaepsilon of mammals.

Modification of TLR-induced activation of human dendritic cells by type I IFN: synergistic interaction with TLR4 but not TLR3 agonists.

Upon detection of direct and indirect signs of infection, dendritic cells (DC) undergo functional changes that modify their ability to elicit immune responses. Type I interferon (IFN-alpha/beta), which includes a large family of closely related infection-inducible cytokines, represents one indirect signal that can act as a DC stimulus. We have investigated the ability of IFN-alpha/beta subtypes to affect DC function and to influence DC responses to Toll-like receptor (TLR) agonists (i.e., direct infection-associated signals). Subtle differences were observed among 15 subtypes of IFN-alpha/beta in the ability to stimulate expression of maturation markers and chemokines by human monocyte-derived DC, with IFN-omega being the most unique in its effects. Pre-treatment with IFN-alpha/beta did not alter the ability of DC to mature in response to subsequent contact with TLR agonists, but did modulate their secretion of chemokines. Conversely, IFN-alpha/beta was shown to act synergistically with TLR4 but not TLR3 agonists for the induction of maturation and chemokine production when DC were exposed to IFN-alpha/beta and TLR ligands simultaneously. Taken together, these results indicate a complex role for IFN-alpha/beta in regulating DC function during the course an infection, which varies according to IFN-alpha/beta subtype and the timing of exposure to other stimuli.

Identification of interferon-tau isoforms expressed by the peri-implantation goat (Capra hircus) conceptus.

Interferon-tau (IFN-tau) is the maternal recognition of pregnancy factor in pecoran ruminants. The aims of this study were to identify the various IFN-tau transcripts in the peri-implantation caprine (ca) conceptus and to compare these nucleotide sequences phylogenetically with established mRNA sequences from the goat. Conceptuses (n = 5) were collected from Boer crossbred and Angora female goats by laparotomy at days 17 and 18 of pregnancy. Total cellular RNA was extracted and RT-PCR was performed by standard procedures using a DNA polymerase with proofreading activity and gene-specific primers complimentary to non-coding regions of the published caIFN-tau sequence. Nine distinct nucleotide sequences were isolated that encode five distinct caIFN-tau proteins. These caIFN-tau have greater sequence homology with ovine IFN-tau (94-96% nucleotide identity; 90-93% amino acid identity) than with bovine IFN-tau (<92% nucleotide identity; <85% amino acid identity). The novel caIFN-tau isoforms contained pronounced nucleotide and amino acid sequence identity with one another (97-99% nucleotide identity; 94-99% amino acid identity) but only moderate sequence identity with the previously identified caIFN-tau (94-96% nucleotide identity; 87-90% amino acid identity). In conclusion, multiple caIFN-tau mRNA species are expressed during peri-implantation conceptus development and distinct clusters of caIFN-tau genes appear to have evolved in this species.

Diversity and relatedness among the type I interferons.

Type I interferons (IFNs) include the IFN-alpha family of subtypes, IFN-beta, IFN-omega, IFN-tau, IFN-kappa, IFN-lambda, and IFN-zeta. IFN genes lack introns and encode secretory signal peptide sequences that are proteolytically cleaved prior to secretion from the cell. In contrast to the approximately 50% amino acid sequence identity among the human IFN-alpha subtypes, human IFN-alphas share approximately 22% identity with human IFN-beta and 37% identity with human IFN-omega. Many of the conserved residues among the type I IFNs are implicated in receptor recognition and structural integrity. This report provides an update on the gene annotations for the mouse and human IFN gene clusters on chromosome 4 and 9, respectively, with accompanying amino acid sequence alignments. Based on sequence identities, a phylogenic tree analysis for the different mammalian Type I IFNs is also presented, showing the high degree of relatedness among these IFNs. Notably, sequence alignment of the different human and mouse IFN promoter regions reveals different signature patterns for transcription factor binding sites, implying different inducers might differentially activate the transcription of the different IFNs.

Transient transfection of mouse fibroblasts with type I interferon transgenes provides various degrees of protection against herpes simplex virus infection.

Type I interferons (IFN) constitute one of the initial and most potent components of the innate immune response against viral infections. While there is only one IFN-beta gene, there are several IFN-alpha genes whose products act through the same receptor calling into question the role of these gene products against viral infection. The focus of the present study was to compare the anti-viral state of cells transiently transfected with different murine type I IFN transgenes including IFN-alpha1, -alpha4, -alpha5, -alpha6, -alpha9, and IFN-beta. Transfected cells produced biologically active IFN ranging from 6 to 46 units/ml. L929 and 3T12.3 cells transfected with the IFN-beta transgene consistently showed a 2-4 fold reduction in herpes simplex virus type 1 (HSV-1) and HSV-2 viral titers compared with cells transfected with the IFN-alpha transgenes which were much less consistent based on HSV species and cell type. Parallel with the reduction in viral titers, cells transfected with the IFN-beta transgene showed the complete absence or significant reduction in viral immediate early, early, and late gene expression. Collectively, the results suggest that the IFN-beta transgene is superior to IFN-alpha transgenes against HSV infection in vitro in part due to a reduction in viral gene expression. These results indicate events downstream of the type I IFN receptor distinguish between the subtypes of IFN-alpha species relative to the activation of genes ultimately responsible for the establishment of the anti-HSV state.