Comparison of CpG- and UpA-mediated restriction of RNA virus replication in mammalian and avian cells and investigation of potential ZAP-mediated shaping of host transcriptome compositions.

The ability of zinc finger antiviral protein (ZAP) to recognize and respond to RNA virus sequences with elevated frequencies of CpG dinucleotides has been proposed as a functional part of the vertebrate innate immune antiviral response. It has been further proposed that ZAP activity shapes compositions of cytoplasmic mRNA sequences to avoid self-recognition, particularly mRNAs for interferons (IFNs) and IFN-stimulated genes (ISGs) expressed during the antiviral state. We investigated whether restriction of the replication of mutants of influenza A virus (IAV) and the echovirus 7 (E7) replicon with high CpG and UpA frequencies varied in different species of mammals and birds. Cell lines from different bird orders showed substantial variability in restriction of CpG-high mutants of IAV and E7 replicons, whereas none restricted UpA-high mutants, in marked contrast to universal restriction of both mutants in mammalian cells. Dinucleotide representation in ISGs and IFN genes was compared with those of cellular transcriptomes to determine whether potential differences in inferred ZAP activity between species shaped dinucleotide compositions of highly expressed genes during the antiviral state. While mammalian type 1 IFN genes typically showed often profound suppression of CpG and UpA frequencies, there was no oversuppression of either in ISGs in any species, irrespective of their ability to restrict CpG- or UpA-high mutants. Similarly, genome sequences of mammalian and avian RNA viruses were compositionally equivalent, as were IAV strains recovered from ducks, chickens and humans. Overall, we found no evidence for host variability in inferred ZAP function shaping host or viral transcriptome compositions.

Adaptation and Cryptic Pseudogenization in Penguin Toll-Like Receptors.

Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.

Understanding the evolution of immune genes in jawed vertebrates.

Driven by co-evolution with pathogens, host immunity continuously adapts to optimize defence against pathogens within a given environment. Recent advances in genetics, genomics and transcriptomics have enabled a more detailed investigation into how immunogenetic variation shapes the diversity of immune responses seen across domestic and wild animal species. However, a deeper understanding of the diverse molecular mechanisms that shape immunity within and among species is still needed to gain insight into-and generate evolutionary hypotheses on-the ultimate drivers of immunological differences. Here, we discuss current advances in our understanding of molecular evolution underpinning jawed vertebrate immunity. First, we introduce the immunome concept, a framework for characterizing genes involved in immune defence from a comparative perspective, then we outline how immune genes of interest can be identified. Second, we focus on how different selection modes are observed acting across groups of immune genes and propose hypotheses to explain these differences. We then provide an overview of the approaches used so far to study the evolutionary heterogeneity of immune genes on macro and microevolutionary scales. Finally, we discuss some of the current evidence as to how specific pathogens affect the evolution of different groups of immune genes. This review results from the collective discussion on the current key challenges in evolutionary immunology conducted at the ESEB 2021 Online Satellite Symposium: Molecular evolution of the vertebrate immune system, from the lab to natural populations.

Repertoire analysis of γδ T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires.

BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

Evidence of Pathogen-Induced Immunogenetic Selection across the Large Geographic Range of a Wild Seabird.

Over evolutionary time, pathogen challenge shapes the immune phenotype of the host to better respond to an incipient threat. The extent and direction of this selection pressure depend on the local pathogen composition, which is in turn determined by biotic and abiotic features of the environment. However, little is known about adaptation to local pathogen threats in wild animals. The Gentoo penguin (Pygoscelis papua) is a species complex that lends itself to the study of immune adaptation because of its circumpolar distribution over a large latitudinal range, with little or no admixture between different clades. In this study, we examine the diversity in a key family of innate immune genes-the Toll-like receptors (TLRs)-across the range of the Gentoo penguin. The three TLRs that we investigated present varying levels of diversity, with TLR4 and TLR5 greatly exceeding the diversity of TLR7. We present evidence of positive selection in TLR4 and TLR5, which points to pathogen-driven adaptation to the local pathogen milieu. Finally, we demonstrate that two positively selected cosegregating sites in TLR5 are sufficient to alter the responsiveness of the receptor to its bacterial ligand, flagellin. Taken together, these results suggest that Gentoo penguins have experienced distinct pathogen-driven selection pressures in different environments, which may be important given the role of the Gentoo penguin as a sentinel species in some of the world's most rapidly changing environments.

Risk awareness during operation of analytical flow cytometers and implications throughout the COVID-19 pandemic.

The COVID-19 pandemic has brought biosafety to the forefront of many life sciences. The outbreak has compelled research institutions to re-evaluate biosafety practices and potential at-risk areas within research laboratories and more specifically within Shared Resource Laboratories (SRLs). In flow cytometry facilities, biological safety assessment encompasses known hazards based on the biological sample and associated risk group, as well as potential or unknown hazards, such as aerosol generation and instrument "failure modes." Cell sorting procedures undergo clearly defined biological safety assessments and adhere to well-established biosafety guidelines that help to protect SRL staff and users against aerosol exposure. Conversely, benchtop analyzers are considered low risk due to their low sample pressure and enclosed fluidic systems, although there is little empirical evidence to support this assumption of low risk. To investigate this, we evaluated several regions on analyzers using the Cyclex-d microsphere assay, a recently established method for cell sorter aerosol containment testing. We found that aerosol and/or droplet hazards were detected on all benchtop analyzers predominantly during operation in "failure modes." These results indicate that benchtop analytical cytometers present a more complicated set of risks than are commonly appreciated.

Biosafety during a pandemic: shared resource laboratories rise to the challenge.

Biosafety has always been an important aspect of daily work in any research institution, particularly for cytometry Shared Resources Laboratories (SRLs). SRLs are common-use spaces that facilitate the sharing of knowledge, expertise, and ideas. This sharing inescapably involves contact and interaction of all those within this working environment on a daily basis. The current pandemic caused by SARS-CoV-2 has prompted the re-evaluation of many policies governing the operations of SRLs. Here we identify and review the unique challenges SRLs face in maintaining biosafety standards, highlighting the potential risks associated with not only cytometry instrumentation and samples, but also the people working with them. We propose possible solutions to safety issues raised by the COVID-19 pandemic and provide tools for facilities to adapt to evolving guidelines and future challenges.

Molecular archaeoparasitology identifies cultural changes in the Medieval Hanseatic trading centre of Lübeck.

Throughout history, humans have been afflicted by parasitic worms, and eggs are readily detected in archaeological deposits. This study integrated parasitological and ancient DNA methods with a large sample set dating between Neolithic and Early Modern periods to explore the utility of molecular archaeoparasitology as a new approach to study the past. Molecular analyses provided unequivocal species-level parasite identification and revealed location-specific epidemiological signatures. Faecal-oral transmitted nematodes (Ascaris lumbricoides and Trichuris trichiura) were ubiquitous across time and space. By contrast, high numbers of food-associated cestodes (Diphyllobothrium latum and Taenia saginata) were restricted to medieval Lübeck. The presence of these cestodes and changes in their prevalence at approximately 1300 CE indicate substantial alterations in diet or parasite availability. Trichuris trichiura ITS-1 sequences grouped into two clades; one ubiquitous and one restricted to medieval Lübeck and Bristol. The high sequence diversity of T.tITS-1 detected in Lübeck is consistent with its importance as a Hanseatic trading centre. Collectively, these results introduce molecular archaeoparasitology as an artefact-independent source of historical evidence.

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

Monitoring chicken flock behaviour provides early warning of infection by human pathogen Campylobacter.

Campylobacter is the commonest bacterial cause of gastrointestinal infection in humans, and chicken meat is the major source of infection throughout the world. Strict and expensive on-farm biosecurity measures have been largely unsuccessful in controlling infection and are hampered by the time needed to analyse faecal samples, with the result that Campylobacter status is often known only after a flock has been processed. Our data demonstrate an alternative approach that monitors the behaviour of live chickens with cameras and analyses the 'optical flow' patterns made by flock movements. Campylobacter-free chicken flocks have higher mean and lower kurtosis of optical flow than those testing positive for Campylobacter by microbiological methods. We show that by monitoring behaviour in this way, flocks likely to become positive can be identified within the first 7-10 days of life, much earlier than conventional on-farm microbiological methods. This early warning has the potential to lead to a more targeted approach to Campylobacter control and also provides new insights into possible sources of infection that could transform the control of this globally important food-borne pathogen.

Bovine γδ T cells are a major regulatory T cell subset.

In humans and mice, γδ T cells represent <5% of the total circulating lymphocytes. In contrast, the γδ T cell compartment in ruminants accounts for 15-60% of the total circulating mononuclear lymphocytes. Despite the existence of CD4(+)CD25(high) Foxp3(+) T cells in the bovine system, these are neither anergic nor suppressive. We present evidence showing that bovine γδ T cells are the major regulatory T cell subset in peripheral blood. These γδ T cells spontaneously secrete IL-10 and proliferate in response to IL-10, TGF-ß, and contact with APCs. IL-10-expressing γδ T cells inhibit Ag-specific and nonspecific proliferation of CD4(+) and CD8(+) T cells in vitro. APC subsets expressing IL-10 and TFG-ß regulate proliferation of γδ T cells producing IL-10. We propose that γδ T cells are a major regulatory T cell population in the bovine system.

TLR15 is unique to avian and reptilian lineages and recognizes a yeast-derived agonist.

The TLRs represent a family of pattern recognition receptors critical in the induction of vertebrate immune responses. Between 10 and 13 different TLR genes can be identified in each vertebrate species, with many represented as orthologous genes in different species. The agonist specificity of orthologous TLR is also highly conserved. In contrast, TLR15 can only be identified in avian and reptilian genomes, suggesting that this receptor arose ~320 million years ago after divergence of the bird/reptile and mammalian lineages. Transfection of a constitutively active form of chicken TLR15 led to NF-κB activation in HEK293 cells and induced cytokine mRNA upregulation in chicken cell lines. Full-length TLR15 mediated NF-κB induction in response to lysates from yeast, but not those derived from viral or bacterial pathogens, or a panel of well-characterized TLR agonists. TLR15 responses were induced by whole-cell lysates derived from Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, but not zymosan preparations from S. cerevisiae. The ability of yeast lysate to activate TLR15-dependent NF-κB pathways (in transfection assays) or stimulate IL-1ß mRNA upregulation in chicken macrophages was abrogated by heat inactivation or pre-exposure of the lysate to PMSF. Identification of yeast as an agonist source for TLR15 provides a functional framework for consideration of this TLR within the context of pattern recognition receptor evolution and may impact on the development of novel adjuvants.

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαß+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRß repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite.

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

A genetic and functional relationship between T cells and cellular proliferation in the adult hippocampus.

Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.

Impact of host age on viral and bacterial communities in a waterbird population.

Wildlife harbour pathogens that can harm human or livestock health and are the source of most emerging infectious diseases. It is rarely considered how changes in wildlife population age-structures or how age-stratified behaviours might alter the level of pathogen detection within a species, or risk of spillover to other species. Micro-organisms that occur in healthy animals can be an important model for understanding and predicting the dynamics of pathogens of greater health concern, which are hard to study in wild populations due to their relative rarity. We therefore used a metagenomic approach to jointly characterise viral and prokaryotic carriage in faeces collected from a healthy wild bird population (Cygnus olor; mute swan) that has been subject to long-term study. Using 223 samples from known individuals allowed us to compare differences in prokaryotic and eukaryotic viral carriage between adults and juveniles at an unprecedented level of detail. We discovered and characterised 77 novel virus species, of which 21% belong putatively to bird-infecting families, and described the core prokaryotic microbiome of C. olor. Whilst no difference in microbiota diversity was observed between juveniles and adult individuals, 50% (4/8) of bird-infecting virus families (picornaviruses, astroviruses, adenoviruses and bornaviruses) and 3.4% (9/267) of prokaryotic families (including Helicobacteraceae, Spirochaetaceae and Flavobacteriaceae families) were differentially abundant and/or prevalent between juveniles and adults. This indicates that perturbations that affect population age-structures of wildlife could alter circulation dynamics and spillover risk of microbes, potentially including pathogens.

Ancient chicken remains reveal the origins of virulence in Marek's disease virus.

The pronounced growth in livestock populations since the 1950s has altered the epidemiological and evolutionary trajectory of their associated pathogens. For example, Marek's disease virus (MDV), which causes lymphoid tumors in chickens, has experienced a marked increase in virulence over the past century. Today, MDV infections kill >90% of unvaccinated birds, and controlling it costs more than US$1 billion annually. By sequencing MDV genomes derived from archeological chickens, we demonstrate that it has been circulating for at least 1000 years. We functionally tested the Meq oncogene, one of 49 viral genes positively selected in modern strains, demonstrating that ancient MDV was likely incapable of driving tumor formation. Our results demonstrate the power of ancient DNA approaches to trace the molecular basis of virulence in economically relevant pathogens.

Reverse cope elimination of hydroxylamines and alkenes or alkynes: theoretical investigation of tether length and substituent effects.

Quantum mechanical calculations have been used to study the intramolecular additions of hydroxylamines to alkenes and alkynes ("reverse Cope eliminations"). In intermolecular reverse Cope eliminations, alkynes are more reactive than alkenes. However, competition experiments have shown that tethering the hydroxylamine to the alkene or alkyne can reverse the reactivity order from that normally observed. The exact outcome depends on the length of the tether. In agreement with experiment, a range of density functional theory methods and CBS-QB3 calculations predict that the activation energies for intramolecular reverse Cope eliminations follow the order 6-exo-dig < 5-exo-trig < 5-exo-dig ≈ 7-exo-dig. The order of the barriers for the 5-, 6-, and 7-exo-dig reactions of alkynes arises mainly from differences in tether strain in the transition states (TSs), but is also influenced by the TS interaction between the hydroxylamine and alkyne. Cyclization onto an alkene in the 5-exo-trig fashion incurs slightly less tether strain than a 6-exo-dig alkyne cyclization, but its activation energy is higher because the hydroxylamine fragment must distort more before the TS is reached. If the alkene terminus is substituted with two methyl groups, the barrier becomes so much higher that it is also disfavored compared to the 5- and 7-exo-dig cyclizations.

Induction of lymphomas by inoculation of Marek's disease virus-derived lymphoblastoid cell lines: prevention by CVI988 vaccination.

Lymphoblastoid cell lines 265(L) and 990(O) are monoclonal lymphomas, derived respectively from liver and ovarian tumours, generated in inbred P-line (MHC B(19)/B(19)) chickens infected with RB-1B strain of Marek's disease virus (MDV) and pRB-1B5 BAC clone respectively. These were inoculated into inbred, MDV-susceptible, P-line chickens by intra-venous or intra-abdominal routes. Additional groups of birds were vaccinated using 1000 plaque-forming units of CVI988 vaccine 8 days prior to inoculation of the cell lines. Non-vaccinated birds developed visceral Marek's disease tumours with an increased rate 30 to 60 days post inoculation. Vaccination prevented tumour and disease development in challenged birds. TCRß repertoire analysis by spectratyping and sequencing of the inoculum was used to track tumour identity in primary tumours and tumour cell lines derived from inoculated birds. These data revealed that the tumours were a consequence of de novo virus infection and not metastasis and expansion of the inoculated tumour cells. Moreover, the data showed that the two MDV-derived cell lines were not transplantable even in syngeneic P-line birds. The data also demonstrated the application of spectratyping as a tool to track tumour identity in lymphoma transplantation studies.