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Recent studies have revealed that the long-recognized link between the historically defined Fusobacterium nucleatum group and colorectal cancer is largely driven by Fusobacterium animalis. This species, along with two others (Fusobacterium polymorphum and Fusobacterium vincentii), was recently reclassified as distinct from F. nucleatum, highlighting functional divergence within this group. Due to their close genetic relatedness, traditional partial 16S rRNA gene sequencing lacks the resolution to reliably distinguish these species. Nevertheless, accurate species-level identification remains essential in cancer-associated microbiome research. Here, we demonstrate that full-length 16S rRNA sequencing using Oxford Nanopore Technology, combined with a novel custom demultiplexing software, enables robust species-level discrimination within the Fusobacterium genus. Our approach accurately classified clinically relevant Fusobacterium species and recovered their expected proportions from whole cells, DNA mixtures, and clinical CRC specimens. This method provides high-resolution profiling to elucidate species-specific roles of Fusobacterium in colorectal cancer.

Tick-borne encephalitis virus (TBEV) is a neurotropic flavivirus that causes thousands of human infections annually. Viral tropism in the brain is determined by the presence of necessary receptors, entry factors, and the ability of the virus to overcome host defenses. The viral structural proteins, pre-membrane (prM), and envelope (E) play an important role in receptor binding, membrane fusion, particle maturation, and antibody neutralization. To understand how these proteins influence virus distribution and tropism in the brain, we generated a chimeric virus harboring the prM and ectodomain of E from TBEV in the background of the low-pathogenic Langat virus (LGTV). We solved the atomic structures of both the chimeric virus and LGTV to compare them to the known TBEV structure. We show that this chimeric virus remains low-pathogenic, while being structurally and antigenically similar to TBEV. Using 3D optical whole brain imaging combined with immunohistochemistry, we found that both LGTV and the chimeric virus primarily infect the cerebral cortex, with no significant differences in their localization or tropism. In contrast, TBEV shows high infection of the cerebellum and a strong preference toward Purkinje cells, indicating that factors other than the prM and E proteins are important for determining TBEV tropism in the brain. Together, this provides new insights into the roles of the structural and non-structural proteins of tick-borne flaviviruses. IMPORTANCE: Although an effective vaccine exists, there is no treatment for those infected by the tick-borne encephalitis virus (TBEV). This study aimed to better understand how the virus's surface proteins influence viral tropism and pathogenicity. We created a chimeric virus with prM and E proteins of TBEV in the genetic background of the low-pathogenic Langat virus (LGTV). The chimeric virus remained low pathogenic, similar to LGTV. Both viruses infected similar brain regions, while TBEV showed a strong preference for the cerebellum and Purkinje cells. This means that other parts of the virus, such as non-structural proteins or NCR, likely decide how the virus behaves in the brain. This study also presents the first cryogenic electron microscopy structure of LGTV, the first whole-brain imaging of TBEV infection in mouse brain, and a new model system to study surface proteins in tick-borne flaviviruses-laying groundwork for future studies on viral tropism, antibody cross-reactivity, and virus-receptor interaction.

Single-cell RNA-seq methods can be used to delineate cell types and states at unprecedented resolution but do little to explain why certain genes are expressed. Single-cell ATAC-seq and multiome (ATAC + RNA) have emerged to give a complementary view of the cell state. It is however unclear what additional information can be extracted from ATAC-seq data besides transcription factor binding sites. Here, we show that ATAC-seq telomere-like reads counter-inituively cannot be used to infer telomere length, as they mostly originate from the subtelomere, but can be used as a biomarker for chromatin condensation. Using long-read sequencing, we further show that modern hyperactive Tn5 does not duplicate 9 bp of its target sequence, contrary to common belief. We provide a new tool, Telomemore, which can quantify nonaligning subtelomeric reads. By analyzing several public datasets and generating new multiome fibroblast and B-cell atlases, we show how this new readout can aid single-cell data interpretation. We show how drivers of condensation processes can be inferred, and how it complements common RNA-seq-based cell cycle inference, which fails for monocytes. Telomemore-based analysis of the condensation state is thus a valuable complement to the single-cell analysis toolbox.

African Swine Fever (ASF) is caused by a DNA virus (AFSV) maintained and transmitted by the Argasid ticks. The re-emergence of the disease in Africa coupled with its rapid spread globally is a threat to the pig industry, food security and livelihoods. The ecology and epidemiology of the ASFV sylvatic cycle, especially in the face of changing land use and land cover, further compounds the menace and impacts of this disease in Kenya. The study aimed to determine the occurrence and distribution of ASFV seroprevalence in warthog populations, the tick vectors and extent of tick infestation of warthog burrows, and the genotypes of ASFV in soft ticks in Kenya. Warthogs from different parts of Kenya were captured and venous blood was centrifuged to harvest sera. Warthog burrows were examined for their conditions and to extract ticks. Sera were analyzed for antibodies against ASFV using a commercial ELISA kit coated with p32 ASFV recombinant protein. Ticks were pooled, DNA extracted and the p72 gene of the ASFV was amplified by qPCR and conventional PCR. The overall seroprevalence of ASFV in warthogs was 87.5 %. A total of 228 warthog burrows were examined and 2154 argasid ticks were extracted from the burrows. Tick pools from Kigio Farm and Lewa Wildlife Conservancies were ASFV-positive by qPCR and conventional PCR. ASFV was further confirmed by the Twist Comprehensive Viral Research Panel (TCVRP), which also identified the argasid ticks as Ornithodoros porcinus. The ticks were infected with virus genotype IX, and their occurrence overlaps with regions of previous ASF outbreaks in domestic pigs. Further, Viruses that could be tick endosymbionts/commensals or due to bloodmeal were detected in ticks by TCVRP; Porcine type-C oncovirus; Pandoravirus neocaledonia; Choristoneura fumiferana granulovirus; Enterobacteria phage p7; Leporid herpesvirus 4 isolate; 5; Human Lymphotropic virus; Human herpesvirus 5. In conclusion, our results suggest that infected Ornithodoros spp. seems to have a rich virome, which has not been explored but could be exploited to inform ASF control in Kenya. Further, the ecology of Ornithodoros spp. and burrow-use dynamics are complex and more studies are needed to understand these dynamics, specifically in the spread of ASFV at the interface of wild and domestic pigs. Further, our results provide evidence of genotype IX ASFV sylvatic cycle which through O. porcinus tick transmission has resulted in high exposure of adult common warthogs. Finally, the co-circulation of ASFV genotype IX in the same location with past ASF outbreaks in domestic pigs and presently in ticks brings to focus the role of the interface and ticks on virus transmission to pigs and warthogs.

Orthohantaviruses, transmitted primarily by rodents, cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus pulmonary syndrome in the Americas. These viruses, with documented human-to-human transmission, exhibit a wide case-fatality rate, 0.5–40 %, depending on the virus species, and no vaccine or effective treatment for severe Orthohantavirus infections exists. In Europe, the Puumala virus (PUUV), carried by the bank vole Myodes glareolus, causes a milder form of HFRS. Despite the reliance on serology and PCR for diagnosis, the three genomic segments of Swedish wild-type PUUV have yet to be completely sequenced.

We have developed a targeted hybrid-capture method aimed at comprehensive genomic sequencing of wild-type PUUV isolates and the identification of other Orthohantaviruses. Our custom-designed panel includes >11,200 probes covering the entire Orthohantavirus genus. Using this panel, we sequenced complete viral genomes from bank vole lung tissue, human plasma samples, and cell-cultured reference strains. Analysis revealed that Swedish PUUV isolates belong to the Northern Scandinavian lineage, with nucleotide diversity ranging from 2.8 % to 3.7 % among them. Notably, no significant genotypic differences were observed between the viral sequences from reservoirs and human cases except in the nonstructural protein.

Despite the high endemicity of PUUV in Northern Sweden, these are the first complete Swedish wild-type PUUV genomes and substantially increase our understanding of PUUV evolution and epidemiology. The panel's sensitivity enables genomic sequencing of human samples with viral RNA levels reflecting the natural progression of infection and underscores our panel's diagnostic value, and could help to uncover novel Orthohantavirus transmission routes.

Background: Mounting evidence suggests a significant role of the gut microbiota in the development and progression of colorectal cancer (CRC). In particular, an over-representation of oral pathogens has been linked to CRC. The aim of this study was to further investigate the faecal microbial landscape of CRC patients, with a focus on the oral pathogens Parvimonas micra and Fusobacterium nucleatum.

Methods: In this study, 16S rRNA sequencing was conducted using faecal samples from CRC patients (n = 275) and controls without pathological findings (n = 95).

Results: We discovered a significant difference in microbial composition depending on tumour location and microsatellite instability (MSI) status, with P. micra, F. nucleatum, and Peptostreptococcus stomatis found to be more abundant in patients with MSI tumours. Moreover, P. micra and F. nucleatum were associated with a cluster of CRC-related bacteria including Bacteroides fragilis as well as with other oral pathogens such as P. stomatis and various Porphyromonas species. This cluster was distinctly different in the control group, suggesting its potential linkage with CRC.

Conclusions: Our results suggest a similar distribution of several CRC-associated bacteria within CRC patients, underscoring the importance of considering the concomitant presence of bacterial species in studies investigating the mechanisms of CRC development and progression.