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Abstract Fusobacteria have been associated to different diseases, including colorectal cancer (CRC), but knowledge of which taxonomic groups contribute to specific conditions is incomplete. We analyzed the genetic diversity and relationships within the Fusobacterium genus. We report recent and ancestral recombination in core genes, indicating that fusobacteria have mosaic genomes and emphasizing that taxonomic demarcation should not rely on single genes/gene regions. Across databases, we found ample evidence of species miss-classification and of undescribed species, which are both expected to complicate disease association. By focusing on a lineage that includes F. periodonticum/pseudoperiodonticum and F. nucleatum, we show that genomes belong to four modern populations, but most known species/subspecies emerged from individual ancestral populations. Of these, the F. periodonticum/pseudoperiodonticum population experienced the lowest drift and displays the highest genetic diversity, in line with the less specialized distribution of these bacteria in oral sites. A highly drifted ancestral population instead contributed genetic ancestry to a new species, which includes genomes classified within the F. nucleatum animalis diversity in a recent CRC study. Thus, evidence herein calls for a re-analysis of F. nucleatum animalis features associated to CRC. More generally, our data inform future molecular profiling approaches to investigate the epidemiology of Fusobacterium-associated diseases.

The zinc-finger antiviral protein (ZAP) is an innate immunity sensor of non-self nucleic acids. Its antiviral activity is exerted through the physical interaction with different cofactors, including TRIM25, Riplet and KHNYN. Cellular proteins that interact with infectious agents are expected to be engaged in genetic conflicts that often result in their rapid evolution. To test this possibility and to identify the regions most strongly targeted by natural selection, we applied in silico molecular evolution tools to analyze the evolutionary history of ZAP and cofactors in four mammalian groups. We report evidence of positive selection in all genes and in most mammalian groups. On average, the intrinsically disordered regions (IDRs) embedded in the four proteins evolve significantly faster than folded domains and most positively selected sites fall within IDRs. In ZAP, the PARP domain also shows abundant signals of selection, and independent evolution in different mammalian groups suggests modulation of its ADP-ribose binding ability. Detailed analyses of the biophysical properties of IDRs revealed that chain compaction and conformational entropy are conserved across mammals. The IDRs in ZAP and KHNYN are particularly compact, indicating that they may promote phase separation (PS). In line with this hypothesis, we predicted several PS-promoting regions in ZAP and KHNYN, as well as in TRIM25. Positively selected sites are abundant in these regions, suggesting that PS may be important for the antiviral functions of these proteins and the evolutionary arms race with viruses. Our data shed light into the evolution of ZAP and cofactors and indicate that IDRs represent central elements in host-pathogen interactions.

ABSTRACT CpG and UpA dinucleotides are under-represented in vertebrate genomes, whereas most invertebrates only show a bias against UpA. RNA viruses are thought to have evolved genomes that resemble the dinucleotide composition of their hosts, possibly to avoid restriction by the zinc-finger antiviral protein (ZAP). By performing a comprehensive analysis of RNA viruses, we show that, whereas UpA dinucleotides are similarly under-represented irrespective of viral genome composition or host, important differences are observed for CpG. The tendency for vertebrate-infecting viruses to have stronger CpG bias than invertebrate-infecting viruses is not universal. Rather, it is mainly driven by single-stranded (ss) RNA(+) viruses. Conversely, ssRNA(−) viruses have a dinucleotide composition that is unrelated to the host clade. Also, these viruses, especially those in the order Bunyavirales, are extremely CpG-depleted. By focusing on specific viral families, we also show that, even for vertebrate ssRNA(+) viruses, ZAP is unlikely to be a driver of CpG depletion. Consistently, CpG dinucleotides tend to be preferentially depleted in A/U-rich contexts in both vertebrate- and invertebrate-infecting viruses. Finally, within the same viral genomes, individual viral open reading frames (ORFs) can display different CpG content. Analysis of SARS-CoV-2 revealed a remarkable depletion of CpG dinucleotides in ORF1ab and S, but not in N and M. Thus, these results do not support the view that an adaptive shift for CpG depletion in the SARS-CoV-2 lineage occurred as an innate immunity evasion strategy. Our data provide a better understanding of viral evolution and inform approaches based on the modulation of CpG to generate attenuated viruses. IMPORTANCE Akin to a molecular signature, dinucleotide composition can be exploited by the zinc-finger antiviral protein (ZAP) to restrict CpG-rich (and UpA-rich) RNA viruses. ZAP evolved in tetrapods, and it is not encoded by invertebrates and fish. Because a systematic analysis is missing, we analyzed the genomes of RNA viruses that infect vertebrates or invertebrates. We show that vertebrate single-stranded (ss) RNA(+) viruses and, to a lesser extent, double-stranded RNA viruses tend to have stronger CpG bias than invertebrate viruses. Conversely, ssRNA(−) viruses have similar dinucleotide composition whether they infect vertebrates or invertebrates. Analysis of ssRNA(+) viruses that infect mammals, reptiles, and fish indicated that ZAP is unlikely to be a major driver of CpG depletion. We also show that, compared to other coronaviruses, the genome of SARS-CoV-2 is not homogeneously CpG-depleted. Our study provides new insights into virus evolution and strategies for recoding RNA virus genomes.

Optical genome mapping (OGM), which allows analysis of ultra-high molecular weight (UHMW) DNA molecules, represents a response to the restriction created by short-read next-generation-sequencing, even in cases where the causative variant is a neutral copy-number-variant insensitive to quantitative investigations. This study aimed to provide a molecular diagnosis to a boy with Marfan syndrome (MFS) and intellectual disability (ID) carrying a de novo translocation involving chromosomes 3, 4, and 13 and a 1.7 Mb deletion at the breakpoint of chromosome 3. No FBN1 alteration explaining his Marfan phenotype was highlighted. UHMW gDNA was isolated from both the patient and his parents and processed using OGM. Genome assembly was followed by variant calling and annotation. Multiple strategies confirmed the results. The 3p deletion, which disrupted ROBO2, (MIM*602431) included three copy-neutral insertions. Two came from chromosome 13; the third contained 15q21.1, including the FBN1 from intron-45 onwards, thus explaining the MFS phenotype. We could not attribute the ID to a specific gene variant nor to the reshuffling of topologically associating domains (TADs). Our patient did not have vesicular reflux-2, as reported by missense alterations of ROBO2 (VUR2, MIM#610878), implying that reduced expression of all or some isoforms has a different effect than some of the point mutations. Indeed, the ROBO2 expression pattern and its role as an axon-guide suggests that its partial deletion is responsible for the patient’s neurological phenotype. Conclusion: OGM testing 1) highlights copy-neutral variants that could remain invisible if no loss of heterozygosity is observed and 2) is mandatory before other molecular studies in the presence of any chromosomal rearrangement for an accurate genotype-phenotype relationship.

Abstract Vaccinia virus (VACV) was used for smallpox eradication, but its ultimate origin remains unknown. The genetic relationships among vaccine stocks are also poorly understood. We analyzed 63 vaccine strains with different origin, as well horsepox virus (HPXV). Results indicated the genetic diversity of VACV is intermediate between variola and cowpox viruses, and that mutation contributed more than recombination to VACV evolution. STRUCTURE identified 9 contributing subpopulations and showed that the lowest drift was experienced by the ancestry components of Tian Tan and HPXV/Mütter/Mulford genomes. Subpopulations that experienced very strong drift include those that contributed the ancestry of MVA and IHD-W, in good agreement with the very long passage history of these vaccines. Another highly drifted population contributed the full ancestry of viruses sampled from human/cattle infections in Brazil and, partially, to IOC clones, strongly suggesting that the recurrent infections in Brazil derive from the spillback of IOC to the feral state.

Summary: Coronaviruses encode a variable number of accessory proteins that are involved in host-virus interaction, suppression of immune responses, or immune evasion. SARS-CoV-2 encodes at least twelve accessory proteins, whose roles during infection have been studied. Nevertheless, the role of the ORF3c accessory protein, an alternative open reading frame of ORF3a, has remained elusive. Herein, we show that the ORF3c protein has a mitochondrial localization and alters mitochondrial metabolism, inducing a shift from glucose to fatty acids oxidation and enhanced oxidative phosphorylation. These effects result in increased ROS production and block of the autophagic flux. In particular, ORF3c affects lysosomal acidification, blocking the normal autophagic degradation process and leading to autolysosome accumulation. We also observed different effect on autophagy for SARS-CoV-2 and batCoV RaTG13 ORF3c proteins; the 36R and 40K sites are necessary and sufficient to determine these effects.

ABSTRACT The ongoing worldwide monkeypox outbreak is caused by viral lineages (globally referred to as hMPXV1) that are related to but distinct from clade IIb MPXV viruses transmitted within Nigeria. Analysis of the genetic differences has indicated that APOBEC-mediated editing might be responsible for the unexpectedly high number of mutations observed in hMPXV1 genomes. Here, using 1,624 publicly available hMPXV1 sequences, we analyzed the mutations that accrued between 2017 and the emergence of the current predominant variant (B.1), as well as those that that have been accumulating during the 2022 outbreak. We confirmed an overwhelming prevalence of C-to-T and G-to-A mutations, with a sequence context (5′-TC-3′) consistent with the preferences of several human APOBEC3 enzymes. We also found that mutations preferentially occur in highly expressed viral genes, although no transcriptional asymmetry was observed. A comparison of the mutation spectrum and context was also performed against the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV), as well as fowlpox virus (FWPV). The results indicated that in VARV genomes, C-to-T and G-to-A changes were more common than the opposite substitutions, although the effect was less marked than for hMPXV1. Conversely, no preference toward C-to-T and G-to-A changes was observed in CPXV and FWPV. Consistently, the sequence context of C-to-T changes confirmed a preference for a T in the −1 position for VARV, but not for CPXV or FWPV. Overall, our results strongly support the view that, irrespective of the transmission route, orthopoxviruses infecting humans are edited by the host APOBEC3 enzymes. IMPORTANCE Analysis of the viral lineages responsible for the 2022 monkeypox outbreak suggested that APOBEC enzymes are driving hMPXV1 evolution. Using 1,624 public sequences, we analyzed the mutations that accumulated between 2017 and the emergence of the predominant variant and those that characterize the last outbreak. We found that the mutation spectrum of hMPXV1 has been dominated by TC-to-TT and GA-to-AA changes, consistent with the editing activity of human APOBEC3 proteins. We also found that mutations preferentially affect highly expressed viral genes, possibly because transcription exposes single-stranded DNA (ssDNA), a target of APOBEC3 editing. Notably, analysis of the human-specific variola virus (VARV) and the zoonotic cowpox virus (CPXV) indicated that in VARV genomes, TC-to-TT and GA-to-AA changes are likewise extremely frequent. Conversely, no preference toward TC-to-TT and GA-to-AA changes is observed in CPXV. These results suggest that APOBEC3 proteins have an impact on the evolution of different human-infecting orthopoxviruses.

The global outbreak of human monkeypox virus (hMPXV1) in 2022 highlighted the usefulness of dermatological manifestations for its diagnosis. Infection by the human monkeypox virus thus necessitated inclusion in the diagnostic repertoire of dermatopathology. To assess the histopathological and microscopical findings of cutaneous lesions related to hMPXV infection, we analyzed skin biopsies from patients with positive MPXV DNA polymerase chain reaction presenting with a typical course of hMPXV1 infection. The most prominent histopathological findings were ascribable to a pustular stage in which epidermal necrosis with areas of non-viable keratinocytes and a “shadow cell” appearance were evident; in some cases, the deep portion of the hair follicle and the acrosyringial epithelium were affected. The main cytopathic modifications included ballooning keratinocytes, followed by Guarnieri bodies and a ground glass appearance of the keratinocytes’ nuclei, together with a dense mixed inflammatory cell infiltrate with prominent neutrophil exocytosis. Transmission electron microscopy analysis demonstrated viral particle aggregates in the cytoplasm of keratinocytes, without any involvement of the nucleus. Interestingly, we also found the presence of viral particles in infected mesenchymal cells, although to a lesser extent than in epithelial cells. Through this study, we contributed to expanding the histological and microscopic knowledge of the human mpox virus, a key step to understanding current and potential future trends of the disease, as well as of other Orthopoxvirus infections.

Background: AIM2 is a key cytoplasmatic pathogen-sensor that detects foreign DNA from viruses and bacteria; it can also recognize damaged or anomalous presence of DNA, promoting inflammasome assembly and activation with the secretion of IL-1β, thus sustaining a chronic inflammatory state, potentially leading to the onset of autoinflammatory skin diseases. Given the implication of the IL-1β pathway in the pathogenesis of syndromic hidradenitis suppurativa (HS), an autoinflammatory immune-mediated skin condition, the potential involvement of AIM2 was investigated. Methods: Sequencing of the whole coding region of the AIM2 gene, comprising 5′- and 3′ UTR and a region upstream of the first exon of ~800 bp was performed in twelve syndromic HS patients. Results: Six out of twelve syndromic HS patients carried a heterozygous variant c.−208 A ≥ C (rs41264459), located on the promoter region of the AIM2 gene, with a minor allele frequency of 0.25, which is much higher than that reported in 1000 G and GnomAD (0.075 and 0.094, respectively). The same variant was found at a lower allelic frequency in sporadic HS and isolated pyoderma gangrenosum (PG) (0.125 and 0.065, respectively). Conclusion: Our data suggest that this variant might play a role in susceptibility to develop syndromic forms of HS but not to progress to sporadic HS and PG. Furthermore, epigenetic and/or somatic variations could affect AIM2 expression leading to different, context-dependent responses.

Human viral hepatitis, a major cause of morbidity and mortality worldwide, is caused by highly diverse viruses with different genetic, ecological, and pathogenetic features. Technological advances that allow throughput sequencing of viral genomes, as well as the development of computational tools to analyze such genome data, have largely expanded our knowledge on the host range and evolutionary history of human hepatitis viruses. Thus, with the exclusion of hepatitis D virus, close or distant relatives of these human pathogens were identified in a number of domestic and wild mammals. Also, sequences of human viral strains isolated from different geographic locations and over different time-spans have allowed the application of phylogeographic and molecular dating approaches to large viral phylogenies. In this review, we summarize the most recent insights into our understanding of the evolutionary events and ecological contexts that determined the origin and spread of human hepatitis viruses. Keywords: Human hepatitis virus, Zoonosis, Molecular dating, Host switch

Four endemic coronaviruses infect humans and cause mild symptoms. Because previous analyses were based on a limited number of sequences and did not control for effects that affect molecular dating, we re-assessed the timing of endemic coronavirus emergence. After controlling for recombination, selective pressure, and molecular clock model, we obtained similar tMRCA (time to the most recent common ancestor) estimates for the four coronaviruses, ranging from 72 (HCoV-229E) to 54 (HCoV-NL63) years ago. The split times of HCoV-229E and HCoV-OC43 from camel alphacoronavirus and bovine coronavirus were dated ~268 and ~99 years ago. The split times of HCoV-HKU1 and HCoV-NL63 could not be calculated, as their zoonoticic sources are unknown. To compare the timing of coronavirus emergence to that of another respiratory virus, we recorded the occurrence of influenza pandemics since 1500. Although there is no clear relationship between pandemic occurrence and human population size, the frequency of influenza pandemics seems to intensify starting around 1700, which corresponds with the initial phase of exponential increase of human population and to the emergence of HCoV-229E. The frequency of flu pandemics in the 19th century also suggests that the concurrence of HCoV-OC43 emergence and the Russian flu pandemic may be due to chance.

Human cytomegalovirus (HCMV) infection often leads to systemic disease in immunodeficient patients and congenitally infected children. Despite its clinical significance, the exact mechanisms contributing to HCMV pathogenesis and clinical outcomes have yet to be determined. One of such mechanisms involves HCMV-mediated NK cell immune response, which favors viral immune evasion by hindering NK cell-mediated cytolysis. This process appears to be dependent on the extent of HCMV genetic variation as high levels of variability in viral genes involved in immune escape have an impact on viral pathogenesis. However, the link between viral genome variations and their functional effects has so far remained elusive. Thus, here we sought to determine whether inter-host genetic variability of HCMV influences its ability to modulate NK cell responses to infection. For this purpose, five HCMV clinical isolates from a previously characterized cohort of pediatric patients with confirmed HCMV congenital infection were evaluated by next-generation sequencing (NGS) for genetic polymorphisms, phylogenetic relationships, and multiple-strain infection. We report variable levels of genetic characteristics among the selected clinical strains, with moderate variations in genome regions associated with modulation of NK cell functions. Remarkably, we show that different HCMV clinical strains differentially modulate the expression of several ligands for the NK cell-activating receptors NKG2D, DNAM-1/CD226, and NKp30. Specifically, the DNAM-1/CD226 ligand PVR/CD155 appears to be predominantly upregulated by fast-replicating (“aggressive”) HCMV isolates. On the other hand, the NGK2D ligands ULBP2/5/6 are downregulated regardless of the strain used, while other NK cell ligands (i.e., MICA, MICB, ULBP3, Nectin-2/CD112, and B7-H6) are not significantly modulated. Furthermore, we show that IFN-γ; production by NK cells co-cultured with HCMV-infected fibroblasts is directly proportional to the aggressiveness of the HCMV clinical isolates employed. Interestingly, loss of NK cell-modulating genes directed against NK cell ligands appears to be a common feature among the “aggressive” HCMV strains, which also share several gene variants across their genomes. Overall, even though further studies based on a higher number of patients would offer a more definitive scenario, our findings provide novel mechanistic insights into the impact of HCMV genetic variability on NK cell-mediated immune responses.

Cytomegaloviruses (order Herpesvirales) display remarkable species-specificity as a result of long-term co-evolution with their mammalian hosts. Human cytomegalovirus (HCMV) is exquisitely adapted to our species and displays high genetic diversity. We leveraged information on inter-species divergence of primate-infecting cytomegaloviruses and intra-species diversity of clinical isolates to provide a genome-wide picture of HCMV adaptation across different time-frames. During adaptation to the human host, core viral genes were commonly targeted by positive selection. Functional characterization of adaptive mutations in the primase gene (UL70) indicated that selection favored amino acid replacements that decrease viral replication in human fibroblasts, suggesting evolution towards viral temperance. HCMV intra-species diversity was largely governed by immune system-driven selective pressure, with several adaptive variants located in antigenic domains. A significant excess of positively selected sites was also detected in the signal peptides (SPs) of viral proteins, indicating that, although they are removed from mature proteins, SPs can contribute to viral adaptation. Functional characterization of one of these SPs indicated that adaptive variants modulate the timing of cleavage by the signal peptidase and the dynamics of glycoprotein intracellular trafficking. We thus used evolutionary information to generate experimentally-testable hypotheses on the functional effect of HCMV genetic diversity and we define modulators of viral phenotypes.

Known multiple sclerosis (MS) susceptibility variants can only explain half of the disease’s estimated heritability, whereas low-frequency and rare variants may partly account for the missing heritability. Thus, here we sought to determine the occurrence of rare functional variants in a large Italian MS multiplex family with five affected members. For this purpose, we combined linkage analysis and next-generation sequencing (NGS)-based whole exome and whole genome sequencing (WES and WGS, respectively). The genetic burden attributable to known common MS variants was also assessed by weighted genetic risk score (wGRS). We found a significantly higher burden of common variants in the affected family members compared to that observed among sporadic MS patients and healthy controls (HCs). We also identified 34 genes containing at least one low-frequency functional variant shared among all affected family members, showing a significant enrichment in genes involved in specific biological processes—particularly mRNA transport—or neurodegenerative diseases. Altogether, our findings point to a possible pathogenic role of different low-frequency functional MS variants belonging to shared pathways. We propose that these rare variants, together with other known common MS variants, may account for the high number of affected family members within this MS multiplex family.

Abstract In addition to high intelligence, humans evolved specialized social-cognitive skills, which are specifically affected in children with autism spectrum disorder (ASD). Genes affected in ASD represent suitable candidates to study the evolution of human social cognition. We performed an evolutionary analysis on 68 genes associated to neurodevelopmental disorders; our data indicate that genetic diversity was shaped by distinct selective forces, including natural selection and introgression from archaic hominins. We discuss the possibility that segregation distortion during spermatogenesis accounts for a subset of ASD mutations. Finally, we detected modern-human-specific alleles in DYRK1A and TCF4. These variants are located within regions that display chromatin features typical of transcriptional enhancers in several brain areas, strongly suggesting a regulatory role. These SNPs thus represent candidates for association with neurodevelopmental disorders, and await experimental validation in future studies.

The Betacoronavirus genus of mammal-infecting viruses includes three subgenera (Sarbecovirus, Embecovirus, and Merbecovirus), in which most known human coronaviruses, including SARS-CoV-2, cluster. Coronaviruses are prone to host shifts, with recombination and positive selection possibly contributing to their high zoonotic potential. We analyzed the role of these two forces in the evolution of viruses belonging to the Betacoronavirus genus. The results showed that recombination has been pervasive during sarbecovirus evolution, and it is more widespread in this subgenus compared to the other two. In both sarbecoviruses and merbecoviruses, recombination hotspots are clearly observed. Conversely, positive selection was a less prominent force in sarbecoviruses compared to embecoviruses and merbecoviruses and targeted distinct genomic regions in the three subgenera, with S being the major target in sarbecoviruses alone. Overall, the results herein indicate that Betacoronavirus subgenera evolved along different trajectories, which might recapitulate their host preferences or reflect the origins of the presently available coronavirus sequences.

Cytomegaloviruses (CMVs) infect many mammals, including humans and non–human primates (NHPs). Human cytomegalovirus (HCMV) is an important opportunistic pathogen among immunocompromised patients and represents the most common infectious cause of birth defects. HCMV possesses a large genome and very high genetic diversity. NHP–infecting CMVs share with HCMV a similar genomic organization and coding content, as well as the course of viral infection. Recent technological advances have allowed the sequencing of several HCMV strains from clinical samples and provided insight into the diversity of NHP–infecting CMVs. The emerging picture indicates that, with the exclusion of core genes (genes that have orthologs in all herpesviruses), CMV genomes are relatively plastic and diverse in terms of gene content, both at the inter– and at the intra–species level. Such variability most likely underlies the strict species–specificity of these viruses, as well as their ability to persist lifelong and with relatively little damage to their hosts. However, core genes, despite their strong conservation, also represented a target of adaptive evolution and subtle changes in their coding sequence contributed to CMV adaptation to different hosts. Indubitably, important knowledge gaps remain, the most relevant of which concerns the role of viral genetics in HCMV–associated human disease.

Lassa mammarenavirus (LASV) is the etiologic agent of Lassa fever. In endemic regions in West Africa, LASV genetic diversity tends to cluster by geographic area. Seven LASV lineages are recognized, but the role of viral genetic determinants on disease presentation in humans is uncertain. We investigated the geographic structure and distribution of LASV in West Africa. We found strong spatial clustering of LASV populations, with two major east–west and north–south diversity gradients. Analysis of ancestry components indicated that known LASV lineages diverged from an ancestral population that most likely circulated in Nigeria, although alternative locations, such as Togo, cannot be excluded. Extant sequences carrying the largest contribution of this ancestral population include the prototype Pinneo strain, the Togo isolates, and a few viruses isolated in Nigeria. The LASV populations that experienced the strongest drift circulate in Mali and the Ivory Coast. By focusing on sequences form a single LASV sublineage (IIg), we identified an ancestry component possibly associated with protection from a fatal disease outcome. Although the same ancestry component tends to associate with lower viral loads in plasma, the small sample size requires that these results are treated with extreme caution.

Iron is an essential micronutrient for most living species. In mammals, hemoglobin (Hb) stores more than two thirds of the body's iron content. In the bloodstream, haptoglobin (Hp) and hemopexin (Hpx) sequester free Hb or heme. Pathogenic microorganisms usually acquire iron from their hosts and have evolved complex systems of iron piracy to circumvent nutritional immunity. Herein, we performed an evolutionary analysis of genes coding for mammalian heme-binding proteins and heme-scavengers in pathogen species. The underlying hypothesis is that these molecules are engaged in a molecular arms race. We show that positive selection drove the evolution of mammalian Hb and Hpx. Positively selected sites in Hb are located at the interaction surface with Neisseria meningitidis heme scavenger HpuA and with Staphylococcus aureus iron-regulated surface determinant B (IsdB). In turn, positively selected sites in HpuA and IsdB are located in the flexible protein regions that contact Hb. A residue in Hb (S45H) was also selected on the Caprinae branch. This site stabilizes the interaction with Trypanosoma brucei hemoglobin-haptoglobin (HbHp) receptor (TbHpHbR), a molecule that also mediates trypanosome lytic factor (TLF) entry. In TbHpHbR, positive selection drove the evolution of a variant (L210S) which allows evasion from TLF but reduces affinity for HbHp. Finally, selected sites in Hpx are located at the interaction surface with the Haemophilus influenzae hemophore HxuA, which in turn displays fast evolving sites at the Hpx-binding interface. These results shed light into host-pathogens conflicts and establish the importance of nutritional immunity as an evolutionary force.

Hepatitis C virus (HCV) belongs to the Hepacivirus genus and is genetically heterogeneous, with seven major genotypes further divided into several recognized subtypes. HCV origin was previously dated in a range between ∼200 and 1000 years ago. Hepaciviruses have been identified in several domestic and wild mammals, the largest viral diversity being observed in bats and rodents. The closest relatives of HCV were found in horses/donkeys (equine hepaciviruses, EHV). However, the origin of HCV as a human pathogen is still an unsolved puzzle. Using a selection-informed evolutionary model, we show that the common ancestor of extant HCV genotypes existed at least 3000 years ago (CI: 3192–5221 years ago), with the oldest genotypes being endemic to Asia. EHV originated around 1100 CE (CI: 291–1640 CE). These time estimates exclude that EHV transmission was mainly sustained by widespread veterinary practices and suggest that HCV originated from a single zoonotic event with subsequent diversification in human populations. We also describe a number of biologically important sites in the major HCV genotypes that have been positively selected and indicate that drug resistance-associated variants are significantly enriched at positively selected sites. HCV exploits several cell-surface molecules for cell entry, but only two of these (CD81 and OCLN) determine the species-specificity of infection. Herein evolutionary analyses do not support a long-standing association between primates and hepaciviruses, and signals of positive selection at CD81 were only observed in Chiroptera. No evidence of selection was detected for OCLN in any mammalian order. These results shed light on the origin of HCV and provide a catalog of candidate genetic modulators of HCV phenotypic diversity.

The Flavivirus genus comprises several human pathogens such as dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Although ZIKV usually causes mild symptoms, growing evidence is linking it to congenital birth defects and to increased risk of Guillain-Barré syndrome. ZIKV encodes a polyprotein that is processed to produce three structural and seven nonstructural (NS) proteins. We investigated the evolution of the viral polyprotein in ZIKV and in related flaviviruses (DENV, Spondweni virus, and Kedougou virus). After accounting for saturation issues, alignment uncertainties, and recombination, we found evidence of episodic positive selection on the branch that separates DENV from the other flaviviruses. NS1 emerged as the major selection target, and selected sites were located in immune epitopes or in functionally important protein regions. Three of these sites are located in an NS1 region that interacts with structural proteins and is essential for virion biogenesis. Analysis of the more recent evolutionary history of ZIKV lineages indicated that positive selection acted on NS5 and NS4B, this latter representing the preferential target. All selected sites were located in the N-terminal portion of NS4B, which inhibits interferon response. One of the positively selected sites (26M/I/T/V) in ZIKV also represents a selection target in sylvatic DENV2 isolates, and a nearby residue evolves adaptively in JEV. Two additional positively selected sites are within a protein region that interacts with host (e.g. STING) and viral (i.e. NS1, NS4A) proteins. Notably, mutations in the NS4B region of other flaviviruses modulate neurovirulence and/or neuroinvasiveness. These results suggest that the positively selected sites we identified modulate viral replication and contribute to immune evasion. These sites should be prioritized in future experimental studies. However, analyses herein detected no selective events associated to the spread of the Asian/American ZIKV lineage.

The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage. We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.

The HAVCR2 gene encodes TIM-3, an immunoglobulin superfamily member expressed by exhausted CD8+ T cells during chronic viral infection. We investigated whether genetic variation at HAVCR2 modulates the susceptibility to HIV-1 acquisition; specifically we focused on a 3' UTR variant (rs4704846, A/G) that represents a natural selection target. We genotyped rs4704846 in three independent cohorts of HIV-1 exposed seronegative (HESN) individuals with different geographic origin (Italy and Spain) and distinct route of exposure to HIV-1 (sexual and injection drug use). Matched HIV-1 positive subjects and healthy controls were also analyzed. In all case-control cohorts the minor G allele at rs4704846 was more common in HIV-1 infected individuals than in HESN, with healthy controls showing intermediate frequency. Results from the three association analyses were combined through a random effect meta-analysis, which revealed no heterogeneity among samples (Cochrane's Q, p value = 0.89, I2 = 0) and yielded a p value of 6.8 ×10(-4). The minor G allele at rs4704846 was found to increase HAVCR2 expression after in vitro HIV-1 infection. Thus, a positively selected polymorphism in the 3' UTR, which modulates HAVCR2 expression, is associated with the susceptibility to HIV-1 infection. These data warrant further investigation into the role of TIM-3 in the prevention and treatment of HIV-1/AIDS.

The ERAP1 gene encodes an aminopeptidase involved in antigen processing. A functional polymorphism in the gene (rs30187, Arg528Lys) associates with susceptibility to ankylosying spondylitis (AS), whereas a SNP in the interacting ERAP2 gene increases susceptibility to another inflammatory autoimmune disorder, Crohn's disease (CD). We analysed rs30187 in 572 Italian patients with CD and in 517 subjects suffering from multiple sclerosis (MS); for each cohort, an independent sex- and age-matched control group was genotyped. The frequency of the 528Arg allele was significantly higher in both disease cohorts compared to the respective control population (for CD, OR = 1.20 95%CI: 1.01-1.43, p = 0.036; for RRMS, OR = 1.26; 95%CI: 1.04-1.51, p = 0.01). Meta-analysis with the Wellcome Trust Cases Control Consortium GWAS data confirmed the association with MS (p(meta) = 0.005), but not with CD. In AS, the rs30187 variant has a predisposing effect only in an HLA-B27 allelic background. It remains to be evaluated whether interaction between ERAP1 and distinct HLA class I alleles also affects the predisposition to MS, and explains the failure to provide definitive evidence for a role of rs30187 in CD. Results herein support the emerging concept that a subset of master-regulatory genes underlay the pathogenesis of autoimmunity.

Previous genome-wide scans of positive natural selection in humans have identified a number of non-neutrally evolving genes that play important roles in skin pigmentation, metabolism, or immune function. Recent studies have also shown that a genome-wide pattern of local adaptation can be detected by identifying correlations between patterns of allele frequencies and environmental variables. Despite these observations, the degree to which natural selection is primarily driven by adaptation to local environments, and the role of pathogens or other ecological factors as selective agents, is still under debate. To address this issue, we correlated the spatial allele frequency distribution of a large sample of SNPs from 55 distinct human populations to a set of environmental factors that describe local geographical features such as climate, diet regimes, and pathogen loads. In concordance with previous studies, we detected a significant enrichment of genic SNPs, and particularly non-synonymous SNPs associated with local adaptation. Furthermore, we show that the diversity of the local pathogenic environment is the predominant driver of local adaptation, and that climate, at least as measured here, only plays a relatively minor role. While background demography by far makes the strongest contribution in explaining the genetic variance among populations, we detected about 100 genes which show an unexpectedly strong correlation between allele frequencies and pathogenic environment, after correcting for demography. Conversely, for diet regimes and climatic conditions, no genes show a similar correlation between the environmental factor and allele frequencies. This result is validated using low-coverage sequencing data for multiple populations. Among the loci targeted by pathogen-driven selection, we found an enrichment of genes associated to autoimmune diseases, such as celiac disease, type 1 diabetes, and multiples sclerosis, which lends credence to the hypothesis that some susceptibility alleles for autoimmune diseases may be maintained in human population due to past selective processes.

Viruses have exerted a constant and potent selective pressure on human genes throughout evolution. We utilized the marks left by selection on allele frequency to identify viral infection-associated allelic variants. Virus diversity (the number of different viruses in a geographic region) was used to measure virus-driven selective pressure. Results showed an excess of variants correlated with virus diversity in genes involved in immune response and in the biosynthesis of glycan structures functioning as viral receptors; a significantly higher than expected number of variants was also seen in genes encoding proteins that directly interact with viral components. Genome-wide analyses identified 441 variants significantly associated with virus-diversity; these are more frequently located within gene regions than expected, and they map to 139 human genes. Analysis of functional relationships among genes subjected to virus-driven selective pressure identified a complex network enriched in viral products-interacting proteins. The novel approach to the study of infectious disease epidemiology presented herein may represent an alternative to classic genome-wide association studies and provides a large set of candidate susceptibility variants for viral infections.

Stem cell populations have been shown to be extremely versatile: they can generate differentiated cells specific to the tissue in which they reside and descendents that are of different germ layer origin. This raises the possibility of obtaining neuronal cells from new biological source of the same adult human subjects. In this study, we found that epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) cooperated to induce the proliferation, self-renewal, and expansion of neural stem cell-like population isolated from several newborn and adult mouse tissues: muscle and hematopoietic tissues. This population, in both primary culture and secondary expanded clones, formed spheres of undifferentiated cells that were induced to differentiate into neurons, astrocytes, and oligodendrocytes. Brain engraftment of the somatic-derived neural stem cells generated neuronal phenotypes, demonstrating the great plasticity of these cells with potential clinical application.