Hanno Schmidt

We report the complete genome sequence of the attenuated human cytomegalovirus strain TR-VAC, developed for subviral particle vaccine production. Oxford Nanopore duplex sequencing confirmed all engineered modifications, including UL130 repair, UL25 stop codons, ddFKBP insertion, GFP deletion, and retention of the bacterial artificial chromosome backbone, without large-scale structural rearrangements.

Genetic characterization of pathogen isolates is increasingly important in healthcare settings, yet the bioinformatic workflows required can be complex and resource-intensive. Here we analyzed 19 environmental Legionella pneumophila isolates using whole genome sequencing (WGS) and compared results from an established multi-step bioinformatic pipeline with several accessible, single-step analysis tools. All approaches produced consistent clustering patterns, resolving the isolates into two major genetic clusters. No clear association between spatial sampling distance and genetic relatedness was observed within this dataset: one cluster was detected in two settlements 50 km apart, while both clusters also co-occurred on a single hospital floor. Across this dataset, all WGS-based approaches provided clustering resolution broadly comparable to MLST/cgMLST. These findings indicate that accessible WGS analysis tools can reproduce the main genomic relationships inferred by more complex workflows while reducing analytical complexity and required bioinformatic expertise. Based on these observations, we discuss how rapid low-complexity WGS workflows may support future same-day bacterial isolate characterization in applied settings.

Within the last 800 million years, animals evolved a vast range of diversity of species exhibiting an enormous disparity of forms and lifestyles. The process involved an increase in complexity from life forms with few cell types to organisms with many hundreds of cell types. However, neither genome size nor number of protein-coding genes can explain these differences, and their biological basis remains elusive. Yet, recent studies suggest that the evolution of complexity is closely linked to the acquisition of a class of noncoding gene regulators called microRNAs. To test this hypothesis, we investigated the association between loss of organismal complexity and microRNAs in Syndermata, an invertebrate group including free-living wheel animals (Monogononta, Bdelloidea), epibiotic Seisonidea, and endoparasitic thorny-headed worms (Acanthocephala). Analyses of genomic, transcriptomic, and morphological data of altogether 25 syndermatan species revealed strong correlations of microRNA losses with reductions of protein-coding genes and morphological traits. The hierarchical pattern sums up to ∼85% loss of microRNAs and a ∼50% loss of conserved metazoan core genes (Benchmarking Universal Single-Copy Orthologs) on the lineage to thorny-headed worms. Extraordinarily reduced microRNA complements were confirmed by small RNA sequencing data. Endoparasitic Acanthocephala was additionally distinguished by the most morphological reductions of ancestral features, such as the digestive tract. Together, we observed that reductions of ∼400 protein-coding genes and 10 metazoan core genes tended to accompany the loss of single microRNA families. Furthermore, 4 microRNA families and 34 metazoan core genes appeared to be associated, on average, with the reduction of a single morphological trait.

Infection of cells with the human cytomegalovirus (HCMV) triggers the expression of interferon-stimulated genes (ISGs). ISGs encode proteins with antiviral functions, such as inhibiting viral replication, promoting cell death of infected cells and enhancing immune responses. HCMV has evolved mechanisms to evade the antiviral effects of ISGs. The viral proteins encoded by the viral genes US7, US8, and US9 have been shown to interfere with interferon induction. US7 to US9 are embedded in a cluster of HCMV genes, termed US2 to US11. The individual members of this gene family interfere on multiple levels with innate and adaptive immune responses to HCMV infection. Using viral mutants with different deletions in US2 to US11, we addressed the question if genes other than US7 to US9 would also influence the IFN responses. Surprisingly, deletion of the complete US2 to US11 gene region led to reduced levels of selected ISGs. Cells infected with viruses in which individual US2 to US11 genes were deleted showed a less pronounced reduction of the selected ISGs. The experiments including RNA-seq analyses indicate that genes of the US2 to US11 gene family have a complex interaction with the IFN-ISG response which is likely regulated on the level of ISG protein stability. As US2–US11 are dispensable for replication in cell culture, the genomic region was frequently used for the insertion of bacterial artificial chromosome vectors in the process of cloning the complete HCMV genome. The results shown here must be considered when viruses derived from BACs with US2–US11 deletions are used and whether appropriate controls must be applied.

Immunocompromised patients struggle to adequately clear viral infections, offering the virus the opportunity to adapt to the immune system in the host. Here we present a case study of a patient undergoing allogeneic hematopoietic stem cell transplantation with a 521-day follow-up of a SARS-CoV-2 infection with the BF.7.21 variant. Virus samples from five time points were submitted to whole genome sequencing. Between the first detection of SARS-CoV-2 infection and its clearance, the patient’s virus population acquired 34 amino acid substitutions and 8 deletions in coding regions. With 11 amino acid substitutions in the receptor binding domain of the virus’ spike protein, substitutions were 15 times more abundant than expected for a random distribution in this highly functional region. Amongst them were the substitutions S:K417T, S:N440S, S:K444R, S:V445A, S:G446N, S:L452Q, S:N460K, and S:E484V at positions that are notorious for their resistance-mediating effects. The substitution patterns found indicate ongoing adaptive evolution.

Figure: Graphical abstract During the last 800 million years of evolution animals radiated into a vast range of diversity of species and disparity of forms and lifestyles. The process involved a near hierarchical increase in complexity from life forms with few cell types to organisms with many hundreds of cell-types. However, neither genome size nor number of protein-coding genes can explain these differences and their biological basis remains elusive. Yet, recent studies have suggested that the evolution of complexity is closely linked to the acquisition of a class of protein coding gene-regulators called microRNAs. In a regressive approach, to investigate the association between loss of organismal complexity and microRNAs, we here studied Syndermata, an invertebrate group including free-living rotifers (Monogononta, Bdelloidea), the epibiotic Seisonidea and the endoparasitic Acanthocephala. Genomic, transcriptomic and morphological characterization and comparisons across 25 syndermatan species revealed a strong correlation between loss of microRNAs, loss of protein-coding genes and decreasing morphological complexity. The near hierarchical loss extends to ∼85% loss of microRNAs and a ∼50% loss of BUSCO genes in the endoparasitic Acanthocephala, the most reduced group we studied. Together, the loss of ∼400 protein-coding genes and ∼10 metazoan core gene losses went along with one microRNA family loss. Furthermore, the loss of ∼4 microRNA families or ∼34 metazoan core genes associated with one lost morphological feature. These are the first quantitative insights into the regulatory impact of microRNAs on organismic complexity as a predictable consequence in regressive evolution of parasites.

The basal splits within Syndermata probably date back hundreds of millions of years, and extant syndermatans greatly differ in reproduction modes (parthenogenesis, metagenesis, heterosexuality) and lifestyles (free-living, epizoic, endoparasitic). Against this background, the present investigation aims to reconstruct genome and transcriptome metrics in the last common ancestor (LCA) of Syndermata. We especially annotated nuclear genome assemblies of one representative, each, of monogononts (Brachionus plicatilis), bdelloids (Adineta vaga), seisonids (Seison nebaliae), and acanthocephalans (Pomphorhynchus laevis), S1 and additionally considered previously published data. Present results suggest that the syndermatan LCA possessed a compact nuclear genome of 50–100 Mb with a coding portion of ≥ 40%. Its ~ 17,000 protein-coding genes probably had about 4–6 exons and 3–5 introns on average. Long interspersed nuclear elements (LINEs) should have occupied 4–5% of the ancestral genome. Based on this, independent evolutionary trajectories involving gene loss, genome duplication, and repeat proliferation resulted in greatly varying genomes within the clade. Nevertheless, we identified 2114 clusters containing proteins of all four syndermatan taxa. About 200 of these clusters lacked sufficiently similar counterparts in the platyhelminths Macrostomum lignano and Schmidtea mediterranea. Gene ontologies assigned to these clusters might reference the muscular pharynx, light perception, and transposition, amongst others.

In 2022, a series of human monkeypox cases in multiple countries led to the largest and most widespread outbreak outside the known endemic areas. Genomic surveillance is of utmost importance to control such outbreaks. To this end, we performed Nanopore Whole Genome Sequencing of a local monkeypox sample on a PromethION 24. Adaptive sampling was applied for in silico depletion of the human host genome, allowing for the enrichment of low abundance viral DNA without a priori knowledge of sample composition. Nanopore sequencing allowed for high viral genome coverage, tracking of sample composition during sequencing, strain determination, and preliminary assessment of mutational pattern. Nanopore sequencing is a highly versatile method to characterize a virus in real-time without pre-sequencing target enrichment.

Background: With the expansion of animal production, parasitic helminths are gaining increasing economic importance. However, application of several established deworming agents can harm treated hosts and environment due to their low specificity. Furthermore, the number of parasite strains showing resistance is growing, while hardly any new anthelminthics are being developed. Here, we present a bioinformatics workflow designed to reduce the time and cost in the development of new strategies against parasites. The workflow includes quantitative transcriptomics and proteomics, 3D structure modeling, binding site prediction, and virtual ligand screening. Its use is demonstrated for Acanthocephala (thorny-headed worms) which are an emerging pest in fish aquaculture. We included three acanthocephalans (Pomphorhynchus laevis, Neoechinorhynchus agilis,Neoechinorhynchus buttnerae) from four fish species (common barbel, European eel, thinlip mullet, tambaqui).Results: The workflow led to eleven highly specific candidate targets in acanthocephalans. The candidate targets showed constant and elevated transcript abundances across definitive and accidental hosts, suggestive of constitutive expression and functional importance. Hence, the impairment of the corresponding proteins should enable specific and effective killing of acanthocephalans. Candidate targets were also highly abundant in the acanthocephalan body wall, through which these gutless parasites take up nutrients. Thus, the candidate targets are likely to be accessible to compounds that are orally administered to fish. Virtual ligand screening led to ten compounds, of which five appeared to be especially promising according to ADMET, GHS, and RO5 criteria: tadalafil, pranazepide, piketoprofen, heliomycin, and the nematicide derquantel.Conclusions: The combination of genomics, transcriptomics, and proteomics led to a broadly applicable procedure for the cost- and time-saving identification of candidate target proteins in parasites. The ligands predicted to bind can now be further evaluated for their suitability in the control of acanthocephalans. The workflow has been deposited at the Galaxy workflow server under the URLtinyurl.com/yx72rda7.