Molecular Evolution of the Porcine Type I Interferon Family: Subtype-Specific Expression and Antiviral Activity

Type I interferons (IFNs), key antiviral cytokines, evolve to adapt with ever-changing viral threats during vertebrate speciation. Due to novel pathogenic pressure associated with Suidae speciation and domestication, porcine IFNs evolutionarily engender both molecular and functional diversification, which have not been well addressed in pigs, an important livestock species and animal model for biomedical sciences. Annotation of current swine genome assembly Sscrofa10.2 reveals 57 functional genes and 16 pseudogenes of type I IFNs. Subfamilies of multiple IFNA, IFNW and porcine-specific IFND genes are separated into four clusters with ∼60 kb intervals within the IFNB/IFNE bordered region in SSC1, and each cluster contains mingled subtypes of IFNA, IFNW and IFND. Further curation of the 57 functional IFN genes indicates that they include 18 potential artifactual duplicates. We performed phylogenetic construction as well as analyses of gene duplication/conversion and natural selection and showed that porcine type I IFN genes have been undergoing active diversification through both gene duplication and conversion. Extensive analyses of the non-coding sequences proximal to all IFN coding regions identified several genomic repetitive elements significantly associated with different IFN subtypes. Family-wide studies further revealed their molecular diversity with respect to differential expression and restrictive activity on the resurgence of a porcine endogenous retrovirus. Based on predicted 3-D structures of representative animal IFNs and inferred activity, we categorized the general functional propensity underlying the structure-activity relationship. Evidence indicates gene expansion of porcine type I IFNs. Genomic repetitive elements that associated with IFN subtypes may serve as molecular signatures of respective IFN subtypes and genomic mechanisms to mediate IFN gene evolution and expression. In summary, the porcine type I IFN profile has been phylogenetically defined family-wide and linked to diverse expression and antiviral activity, which is important information for further biological studies across the porcine type I IFN family.     

Type III IFNs in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity

Bats are known to harbor a number of emerging and re-emerging zoonotic viruses, many of which are highly pathogenic in other mammals but result in no clinical symptoms in bats. The ability of bats to coexist with viruses may be the result of rapid control of viral replication early in the immune response. IFNs provide the first line of defense against viral infection in vertebrates. Type III IFNs (IFN-λs) are a recently identified IFN family that share similar antiviral activities with type I IFNs. To our knowledge, we demonstrate the first functional analysis of type III IFNs from any species of bat, with the investigation of two IFN-λ genes from the pteropid bat, Pteropus alecto. Our results demonstrate that bat type III IFN has similar antiviral activity to type I and III IFNs from other mammals. In addition, the two bat type III IFNs are differentially induced relative to each other and to type I IFNs after treatment or transfection with synthetic dsRNA. Infection with the bat paramyxovirus, Tioman virus, resulted in no upregulation of type I IFN production in bat splenocytes but was capable of inducing a type III IFN response in three of the four bats tested. To our knowledge, this is the first report to describe the simultaneous suppression of type I IFN and induction of type III IFN after virus infection. These results may have important implications for the role of type III IFNs in the ability of bats to coexist with viruses.     

Cloning and Molecular Evolution of the Aldehyde Dehydrogenase 2 Gene (Aldh2) in Bats (Chiroptera)

Old World fruit bats (Pteropodidae) and New World fruit bats (Phyllostomidae) ingest significant quantities of ethanol while foraging. Mitochondrial aldehyde dehydrogenase (ALDH2, encoded by the Aldh2 gene) plays an important role in ethanol metabolism. To test whether the Aldh2 gene has undergone adaptive evolution in frugivorous and nectarivorous bats in relation to ethanol elimination, we sequenced part of the coding region of the gene (1,143 bp, ~73 % coverage) in 14 bat species, including three Old World fruit bats and two New World fruit bats. Our results showed that the Aldh2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to Old World fruit bats and New World fruit bats. Further research is needed to determine whether other genes involved in ethanol metabolism have been the targets of positive selection in frugivorous and nectarivorous bats.     

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.     

Natural selection in the evolution of SARS-CoV-2 in bats created a generalist virus and highly capable human pathogen

Virus host shifts are generally associated with novel adaptations to exploit the cells of the new host species optimally. Surprisingly, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has apparently required little to no significant adaptation to humans since the start of the Coronavirus Disease 2019 (COVID-19) pandemic and to October 2020. Here we assess the types of natural selection taking place in Sarbecoviruses in horseshoe bats versus the early SARS-CoV-2 evolution in humans. While there is moderate evidence of diversifying positive selection in SARS-CoV-2 in humans, it is limited to the early phase of the pandemic, and purifying selection is much weaker in SARS-CoV-2 than in related bat Sarbecoviruses. In contrast, our analysis detects evidence for significant positive episodic diversifying selection acting at the base of the bat virus lineage SARS-CoV-2 emerged from, accompanied by an adaptive depletion in CpG composition presumed to be linked to the action of antiviral mechanisms in these ancestral bat hosts. The closest bat virus to SARS-CoV-2, RmYN02 (sharing an ancestor about 1976), is a recombinant with a structure that includes differential CpG content in Spike; clear evidence of coinfection and evolution in bats without involvement of other species. While an undiscovered “facilitating” intermediate species cannot be discounted, collectively, our results support the progenitor of SARS-CoV-2 being capable of efficient human–human transmission as a consequence of its adaptive evolutionary history in bats, not humans, which created a relatively generalist virus.

A study of the natural origins of SARS-CoV-2 reveals very little adaptive evolution occurring since it emerged in humans, but strong evolutionary signals in the bat virus lineage from which SARS-CoV-2 arose. Evolution in bats involved lineage-specific depletion of CpG nucleotides (linked to host anti-viral molecules), and clear evidence of recombination across these lineages, supporting bat host species’ influence on the ancestral viruses.     

Rapid Evolution of PARP Genes Suggests a Broad Role for ADP-Ribosylation in Host-Virus Conflicts

Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic ‘arms races’ with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in ‘housekeeping’ functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our evolutionary analyses suggest that addition, recognition and removal of ADP-ribosylation is a critical, underappreciated currency in host-virus conflicts.     

Evolution of the interferon alpha gene family in eutherian mammals

Interferon alpha (IFNA) genes code for proteins with important signaling roles during the innate immune response. Phylogenetically, IFNA family members in eutherians (placental mammals) cluster together in a species-specific manner except for closely related species (i.e. Homo sapiens and Pan troglodytes) where gene-specific clustering is evident. Previous research has been unable to clarify whether gene conversion or recent gene duplication accounts for gene-specific clustering, partly because the similarity of members of the IFNA family within species has made it historically difficult to identify the exact composition of IFNA gene families. IFNA gene families were fully characterized in recently available genomes from Canis familiaris, Macaca mulatta, P. troglodytes and Rattus norvegicus, and combined with previously characterized IFNA gene families from H. sapiens and Mus musculus, for the analysis of both whole and partial gene conversion events using a variety of statistical methods. Gene conversion was inferred in every eutherian species analyzed and comparison of the IFNA gene family locus between primate species revealed independent gene duplication in M. mulatta. Thus, both gene conversion and gene duplication have shaped the evolution of the IFNA gene family in eutherian species. Scenarios may be envisaged whereby the increased production of a specific IFN-alpha protein would be beneficial against a particular pathogenic infection. Gene conversion, similar to duplication, provides a mechanism by which the protein product of a specific IFNA gene can be increased.     

A survey of type I interferons from a marsupial and monotreme: implications for the evolution of the type I interferon gene family in mammals

Sequence data for type I interferons (IFNs) have previously only been available for birds and eutherian ('placental') mammals, but not for the other two groups of extant mammals, the marsupials and monotremes. This has left a large gap in our knowledge of the evolutionary and functional relationships of what is a complex gene family in eutherians. In this study, a PCR-based survey of type I IFN genes from a marsupial, the tammar wallaby (Macropus eugenii), and a monotreme, the short-beaked echidna (Tachyglossus aculeatus), was conducted. Along with Southern blot and phylogenetic analysis, this revealed a large number of type I IFN genes for the wallaby, rivalling that of eutherians, but relatively few type I IFN genes in the echidna. The wallaby genes include both IFNA and IFNB orthologues, indicating that the gene duplication leading to these subtypes occurred prior to the divergence of marsupials and eutherians some 130 million years ago. Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules. For example, this expansion in complexity may have, at least partially, facilitated the evolution of viviparity in marsupials and eutherians. Other evolutionary aspects of these sequences are also discussed.     

Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.     

Diversification of takeout, a male-biased gene family in Drosophila

The display of courtship behavior has evolved in response to sexual selection driven by competition to obtain mates. Sexually dimorphic mate selection rituals are likely controlled at least in part by genes with sex-biased patterns of expression. In Drosophila melanogaster, male courtship behavior has been well described and consists of a series of stereotyped behaviors. The takeout gene is predominantly expressed in males and affects male courtship behavior. In this study, we examine the patterns of expression and evolution in takeout and the family of related proteins. We show that a number of genes in the takeout gene family show male-biased expression in D. melanogaster, largely in non-reproductive tissues. Phylogenetic analysis reveals that this gene family is conserved across insects. As expected for genes with male-biased expression, we also find evidence of positive selection in some lineages. Our results suggest that the genes in this family may have evolutionarily conserved sex specific roles in male mating behavior across insects.     

Parallel Evolution of the Glycogen Synthase 1 (Muscle) Gene Gys1 Between Old World and New World Fruit Bats (Order: Chiroptera)

Glycogen synthase, which catalyzes the synthesis of glycogen, is especially important for Old World (Pteropodidae) and New World (Phyllostomidae) fruit bats that ingest high-carbohydrate diets. Glycogen synthase 1, encoded by the Gys1 gene, is the glycogen synthase isozyme that functions in muscles. To determine whether Gys1 has undergone adaptive evolution in bats with carbohydrate-rich diets, in comparison to insect-eating sister bat taxa, we sequenced the coding region of the Gys1 gene from 10 species of bats, including two Old World fruit bats (Pteropodidae) and a New World fruit bat (Phyllostomidae). Our results show no evidence for positive selection in the Gys1 coding sequence on the ancestral Old World and the New World Artibeus lituratus branches. Tests for convergent evolution indicated convergence of the sequences and one parallel amino acid substitution (T395A) was detected on these branches, which was likely driven by natural selection.     

Differential Expression of Meis2, Mab21l2 and Tbx3 during Limb Development Associated with Diversification of Limb Morphology in Mammals

Bats are the only mammals capable of self-powered flight using wings. Differing from mouse or human limbs, four elongated digits within a broad wing membrane support the bat wing, and the foot of the bat has evolved a long calcar that spread the interfemoral membrane. Our recent mRNA sequencing (mRNA-Seq) study found unique expression patterns for genes at the 5′ end of the Hoxd gene cluster and for Tbx3 that are associated with digit elongation and wing membrane growth in bats. In this study, we focused on two additional genes, Meis2 and Mab21l2, identified from the mRNA-Seq data. Using whole-mount in situ hybridization (WISH) we validated the mRNA-Seq results for differences in the expression patterns of Meis2 and Mab21l2 between bat and mouse limbs, and further characterize the timing and location of the expression of these two genes. These analyses suggest that Meis2 may function in wing membrane growth and Mab21l2 may have a role in AP and DV axial patterning. In addition, we found that Tbx3 is uniquely expressed in the unique calcar structure found in the bat hindlimb, suggesting a role for this gene in calcar growth and elongation. Moreover, analysis of the coding sequences for Meis2, Mab21l2 and Tbx3 showed that Meis2 and Mab21l2 have high sequence identity, consistent with the functions of genes being conserved, but that Tbx3 showed accelerated evolution in bats. However, evidence for positive selection in Tbx3 was not found, which would suggest that the function of this gene has not been changed. Together, our findings support the hypothesis that the modulation of the spatiotemporal expression patterns of multiple functional conserved genes control limb morphology and drive morphological change in the diversification of mammalian limbs.     

The evolution of bat nucleic acid‐sensing Toll‐like receptors

We characterized the nucleic acid‐sensing Toll‐like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid‐sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand‐binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand‐binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long‐term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order‐specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.     

The Glycogen Synthase 2 Gene (Gys2) Displays Parallel Evolution Between Old World and New World Fruit Bats

Frugivorous and nectarivorous bats rely largely on hepatic glycogenesis and glycogenolysis for postprandial blood glucose disposal and maintenance of glucose homeostasis during short time starvation, respectively. The glycogen synthase 2 encoded by the Gys2 gene plays a critical role in liver glycogen synthesis. To test whether the Gys2 gene has undergone adaptive evolution in bats with carbohydrate-rich diets in relation to their insect-eating sister taxa, we sequenced the coding region of the Gys2 gene in a number of bat species, including three Old World fruit bats (OWFBs) (Pteropodidae) and two New World fruit bats (NWFBs) (Phyllostomidae). Our results showed that the Gys2 coding sequences are highly conserved across all bat species we examined, and no evidence of positive selection was detected in the ancestral branches leading to OWFBs and NWFBs. Our explicit convergence test showed that posterior probabilities of convergence between several branches of OWFBs, and the NWFBs were markedly higher than that of divergence. Three parallel amino acid substitutions (Q72H, K371Q, and E666D) were detected among branches of OWFBs and NWFBs. Tests for parallel evolution showed that two parallel substitutions (Q72H and E666D) were driven by natural selection, while the K371Q was more likely to be fixed randomly. Thus, our results suggested that the Gys2 gene has undergone parallel evolution on amino acid level between OWFBs and NWFBs in relation to their carbohydrate metabolism.     

Polymorphism in the interferon-alpha gene family.

A pronounced genetic polymorphism of the interferon type I gene family has been assumed on the basis of RFLP analysis of the genomic region as well as the large number of sequences published compared to the number of loci. However, IFNA2 is the only locus that has been carefully analyzed concerning gene frequency, and only naturally occurring rare alleles have been found. We have extended the studies on a variation of expressed sequences by studying the IFNA1, IFNA2, IFNA10, IFNA13, IFNA14, and IFNA17 genes. Genomic white-blood-cell DNA from a population sample of blood donors and from a family material were screened by single-nucleotide primer extension (allele-specific primer extension) of PCR fragments. Because of sequence similarities, in some cases "nested" PCR was used, and, when applicable, restriction analysis or control sequencing was performed. All individuals carried the interferon-alpha 1 and interferon-alpha 13 variants but not the LeIF D variant. At the IFNA2 and IFNA14 loci only one sequence variant was found, while in the IFNA10 and IFNA17 groups two alleles were detected in each group. The IFNA10 and IFNA17 alleles segregated in families and showed a close fit to the Hardy-Weinberg equilibrium. There was a significant linkage disequilibrium between IFNA10 and IFNA17 alleles. The fact that the extent of genetic polymorphism was lower than expected suggests that a majority of the previously described gene sequences represent nonpolymorphic rare mutants that may have arisen in tumor cell lines.     

Interferon Induced IFIT Family Genes in Host Antiviral Defense

Secretion of interferons (IFNs) from virus-infected cells is a hallmark of host antiviral immunity and in fact, IFNs exert their antiviral activities through the induction of antiviral proteins. The IFN-induced protein with tetratricopeptide repeats (IFITs) family is among hundreds of IFN-stimulated genes. This family contains a cluster of duplicated loci. Most mammals have IFIT1, IFIT2, IFIT3 and IFIT5; however, bird, marsupial, frog and fish have only IFIT5. Regardless of species, IFIT5 is always adjacent to SLC16A12. IFIT family genes are predominantly induced by type I and type III interferons and are regulated by the pattern recognition and the JAK-STAT signaling pathway. IFIT family proteins are involved in many processes in response to viral infection. However, some viruses can escape the antiviral functions of the IFIT family by suppressing IFIT family genes expression or methylation of 5'' cap of viral molecules. In addition, the variants of IFIT family genes could significantly influence the outcome of hepatitis C virus (HCV) therapy. We believe that our current review provides a comprehensive picture for the community to understand the structure and function of IFIT family genes in response to pathogens in human, as well as in animals.     

The first evidence of positive selection in peptidoglycan recognition protein (PGRP) genes of Crassostrea gigas

The oyster Crassostrea gigas is thought to have developed effective immunity to potentially harmful pathogens while under continuous exposure to marine microorganisms; however, the evolutionary mechanisms by which such immunity developed has not been understood. To understand the evolution of immunity, we characterized the family of peptidoglycan recognition proteins in the oyster (CgPGRPs). These proteins are crucial pattern recognition receptors for peptidoglycans (PGNs) and thereby, for activating the innate immune response of host. Herein, we identify seven new CgPGRP genes. Phylogenetic analysis of the seven new and five previously reported CgPRGP genes reveals that the CgPRGP gene family can be clustered into two groups, CgPRGPS and CgPRGPL. Moreover, the CgPRGPS group can be further divided into five subgroups. A codon-substitution model and three likelihood ratio tests (LRTs) suggest that seven sites in the CgPGRP family of genes have been subjected to strong positive selection (ω = 3.035–4.143), Three dimensional modeling revealed that these sites are found primarily at the periphery of coils and α-helices rather than in β-strands, perhaps allowing PGRP to adapt to, and recognize, variability of PGN structure. In conclusion, our studies provide the first evidence of positive Darwinian selection in the CgPGRP family, contributing to a better understanding of the adaptive mechanism of host-pathogens interaction in marine mollusks.     

Episodes of adaptive evolution of mitochondrial genome in asiatic salamanders (Amphibia,Caudata, Hynobiidae)

To elucidate the effect of natural selection on the evolution of mitochondrial DNA (mtDNA) in Asiatic salamanders of the family Hynobiidae, nucleotide sequences of 12 protein-coding genes were analyzed. Using a mixed effects model of evolution, it was found that, in spite of the pronounced effect of negative selection on the mtDNA evolution in Hynobiidae (which is typical for the animals in general), two phylogenetic clusters, the West Asian one, represented by the genera Ranodon and Paradactylodon, and North Eurasian one, represented by the genus Salamandrella, were formed under the influence of episodic positive selection. Analysis of protein sequences encoded by the mitochondrial genome also supported the influence of positive selection on the evolution of Hynobiidae at some stages of their cladogenesis. It is suggested that the signatures of adaptive evolution detected in the mtDNA of Hynobiidae were determined by the complex and long-lasting history of their formation, accompanied by adaptation to the changing environment.