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Background Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity. Methods We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. Results Capture with the Twist CVRP increased sensitivity by at least 10–100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600–6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. Conclusions Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.

Paramutation involves the transfer of a repressive epigenetic mark from a silent allele to an active homolog and, consequently, non-Mendelian inheritance. In tomato, the sulfurea (sulf) paramutation is associated with a high level of CHG hypermethylation in a region overlapping with the transcription start site (TSS) of the SlTAB2 gene that affects chlorophyll synthesis. The CCG subcontext hypermethylation is under-represented at this region relative to CTG or CAG, implicating the CHROMOME-THYLTRANSFERASE3a (CMT3) in paramutation at this locus. Consistent with this interpretation, loss of CMT3 function leads to loss of the sulf chlorosis, the associated CHG hypermethylation, and paramutation. Loss of KRYPTONITE (KYP) histone methyltransferase function has a similar effect linked to reduced H3K9me2 at the promoter region of SlTAB2 and a shift in higher order chromatin structure at this locus. Mutation of the largest subunit of RNA polymerase V (PolV) in contrast does not affect sulf paramutation. These findings indicate the involvement of a CMT3/KYP–dependent feedback loop rather than the PolV-dependent pathway leading to RNA-directed DNA methylation (RdDM) in the maintenance of paramutation.

Paramutation involves the transfer of a repressive epigenetic mark from a silent allele to an active homologue and, consequently, non-Mendelian inheritance. In tomato the sulfurea (sulf) paramutation is associated with a high level of CHG hypermethylation in a region overlapping the transcription start site of the SlTAB2 gene that affects chlorophyll synthesis. The CCG sub-context hypermethylation is under-represented at this region relative to CTG or CAG implicating the CHROMOMETHYLTRANSFERASE3 (CMT3) in paramutation at this locus. Consistent with this interpretation, loss of CMT3 function leads to loss of the sulf chlorosis, the associated CHG hypermethylation and paramutation. Loss of KRYPTONITE (KYP) histone methyl transferase function has a similar effect linked to reduced H3K9me2 at the promoter region of SlTAB2 and a shift in higher order chromatin structure at this locus. Mutation of the largest subunit of RNA polymerase V (PolV) in contrast does not affect sulf paramutation. These findings indicate the involvement of a CMT3/KYP dependent feedback rather than the PolV-dependent pathway leading to RNA directed DNA methylation (RdDM) in the maintenance of paramutation.

Insects are particularly vulnerable to rapid environmental changes, which are disproportionally affecting high latitudes. Increased temperature could influence insect species differentially and reshape assemblages over time. We quantified temporal assemblage turnover of Arctic Diptera (flies) in the Muscidae, one of the most diverse and abundant families of Arctic insects, using time series data from Zackenberg, north-east Greenland. We measured temporal patterns of abundance, diversity, and composition of muscid assemblages in wet fen, mesic and arid heath habitats from yearly collections spanning 1996–2014 and tested their relationship to climate. A total of 18 385 individuals representing 16 species of muscid flies were identified. A significant decrease of 80% of total muscid abundance was observed during the study period. Species richness declined in each habitat type but this trend was not significant across habitats. The number of common and abundant species also decreased significantly over time across habitats revealing a temporal modification of species evenness. Significant temporal changes in composition observed in the wet fen and across habitats were mainly driven by a change in relative abundance of certain species rather than by species replacement. Shift in composition in each habitat and decline in muscid abundance across habitats were associated with summer temperature, which has significantly increased over the study period. However, relationships between temperature and muscid abundance at the species level were noticeable for a few species only. Significant directional change in composition was documented in the wet fen but no biotic homogenization across habitats was observed. As one of the few studies of species-level changes in abundance, diversity and composition of an insect taxon in the Arctic over the past two decades, our study shows that habitat types may modulate insect species responses to recent climate change and that contrasting species responses can alter species assemblages within a few decades.

Methane is produced in the rumen of ruminant livestock by methanogens and is a major contributor to agricultural greenhouse gases. Vaccination against ruminal methanogens could reduce methane emissions by inducing antibodies in saliva which enter the rumen and impair ability of methanogens to produce methane. Presently, it is not known if vaccination can induce sufficient amounts of antibody in the saliva to target methanogen populations in the rumen and little is known about how long antibody in the rumen remains active. In the current study, sheep were vaccinated twice at a 3-week interval with a model methanogen antigen, recombinant glycosyl transferase protein (rGT2) formulated with one of four adjuvants: saponin, Montanide ISA61, a chitosan thermogel, or a lipid nanoparticle/cationic liposome adjuvant (n = 6/formulation). A control group of sheep (n = 6) was not vaccinated. The highest antigen-specific IgA and IgG responses in both saliva and serum were observed with Montanide ISA61, which promoted levels of salivary antibodies that were five-fold higher than the second most potent adjuvant, saponin. A rGT2-specific IgG standard was used to determine the level of rGT2-specific IgG in serum and saliva. Vaccination with GT2/Montanide ISA61 produced a peak antibody concentration of 7 × 1016 molecules of antigen-specific IgG per litre of saliva, and it was estimated that in the rumen there would be more than 104 molecules of antigen-specific IgG for each methanogen cell. Both IgG and IgA in saliva were shown to be relatively stable in the rumen. Salivary antibody exposed for 1-2 hours to an in vitro simulated rumen environment retained approximately 50% of antigen-binding activity. Collectively, the results from measuring antibody levels and stablility suggest a vaccination-based mitigation strategy for livestock generated methane is in theory feasible.

DNA‐based vaccines, alone or in combination with other sub‐unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacteriam bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85–A) and arget(ESAT‐6) produced measurable interferon‐gamma (IFN‐γ) responses to CD4+ T‐cell epitope‐peptide recall antigens in vitro. The magnitude of these responses was enhanced by co‐delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)‐1α or interleukin(IL)‐7), although they did not the match responses observed in mice that received Bacille Calmette−Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85–A) invoked higher IFN‐γ levels, with the most immunogenic regime of Ag85 or ESAT or IL‐7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA‐prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

Animal models indicate that exposure to environmental strains of mycobacteria can modulate immune responses and influence the effectiveness of live mycobacterial vaccines. Here, we describe that between the two recently reported Mycobacterium avium isolates, strain WAg 206 (but not strain WAg 207) interferes with human monocyte‐derived dendritic cell (MDDC) activation. WAg 206, unlike WAg 207, did not elicit inflammatory cytokine production (TNFα, IL‐1β, IL‐12) or costimulatory molecule expression (HLA‐DR, CD83, CD80, CD86) by human MDDCs in vitro. These data highlight the potential for environmental mycobacteria to modulate immune responses in humans, and suggest a mechanism by which earlier exposure to such microbes may compromise the efficacy of live mycobacterial vaccines, as has been observed in some human BCG vaccine trials.

Bacillus Calmette Guerin (BCG) has been reported to show variable efficacy as a vaccine against tuberculosis. We demonstrated that the secretion of biologically active IL‐2 (rBCG/IL‐2), but not IL‐18 (rBCG/IL‐18), by BCG improves its ability to induce and maintain a strong type 1 immune response in BALB/c mice. rBCG/IL‐2 induced significantly higher Ag‐specific proliferative responses, high IFN‐γ production and serum titres of IgG2a 16 weeks after vaccination. This immune profile was correlated to an increased rate of clearance of non‐pathogenic mycobacteria (live BCG delivered intranasally). Surprisingly, however, this strong type 1 immune profile induced no greater protective immunity against aerosol challenge with virulent Mycobacterium bovis than that induced by normal BCG (nBCG). By comparison, vaccination with rBCG/IL‐18 was found to induce significantly less IFN‐γ production in splenic lymphocytes than nBCG. This impaired induction of IFN‐γ was correlated to a significantly lower protective efficacy against M. bovis challenge, as compared to nBCG. The data suggest that manipulation of the immune response to tuberculosis and tuberculosis vaccines will require a more complete understanding of the factors that are important in generating a protective immune response.