Friedrich-Loeffler-Institut

No widely available ovine monocyte/macrophage cell system exists. We present an affinity magnetic separation method combining gradient separation and magnetic bead selection to isolate pure primary ovine monocytes from blood, yielding large quantities of viable primary macrophages suitable for microbiological infection studies using reservoir-specific host cells targeted by intracellular pathogens.

The zoonotic Orthoflavivirus Usutu virus (USUV) is distributed throughout Germany, putting hosts at a considerable risk of infection nationwide. Besides birds as reservoir hosts, a broad range of accidental hosts is suspected. However, there are few reports documenting the progression of USUV-associated disease. This case report describes the course of fatal USUV infections in three harbor seals (Phoca vitulina) from a rescue center on the North Sea coast in Germany. Corresponding samples were analyzed using (histo-)pathological, immunohistochemical, molecular and phylogenetic methods. The most prevalent findings in clinically affected animals were neurological signs and non-suppurative encephalitis. All animals were found dead or had been euthanized due to animal welfare reasons within 30 h after the onset of clinical signs. Blood samples taken from another 37 young harbor seals from the same rescue center in the same year revealed two further asymptomatic USUV RNA and antibody-positive animals. The sequences were found to belong to USUV lineages Europe 2 and Africa 3, which are known to circulate in birds in Germany. This case report highlights the importance of USUV as a potential diagnosis for neurological impairments in marine mammals and documents the first cases of USUV infection in harbor seals.

The 2024 oral rabies vaccination (ORV) campaign in Namibia’s Zambezi region aimed to address persistent rabies transmission through a large-scale effort combining direct dog vaccination and owner-assisted distribution. A total of 9,393 baits were distributed, and the campaign demonstrated feasibility and operational efficiency, achieving an estimated vaccination coverage of 47.9% of dogs in just four days, increasing to 56.8% when accounting for prior parenteral vaccination efforts. Spatial analysis revealed an average vaccination coverage of 60% across 10 × 10 km grid cells, and encompassed 81% of the human population of the Zambezi region within the vaccination coverage area. Post-vaccination surveys indicated a rise in vaccination rates (54.5% of dogs vaccinated, up from 18.9%) and lower presence of rabies. Improved education and awareness campaigns, targeted interventions in low-coverage areas, and enhanced surveillance are recommended to further reduce rabies incidence. The findings highlight the promise and proficiency of ORV campaigns in resource-constrained settings and demonstrate that its application can be key for sustained rabies control efforts in Namibia.

There is increasing evidence that over‐production of reactive species (RS) constitutes a significant physiological cost for migrating birds. The current view entails that RS during migration originate primarily from increased metabolic activity associated with prolonged flights and refueling. In this review, we argue that there is another substantial, yet mostly overlooked, source for RS in migrating birds: infections. Specifically, we highlight that acute and chronic infections during migration are the norm rather than the exception, and that those infections can also result in harmful RS production, either via immune system activation or via the pathogen itself. Thus, infections may impose further oxidative costs on migrating individuals in addition to those directly caused by migratory flights and refueling. Depending on the type of infection and the associated immune responses, these costs may be substantial. Therefore, both migration‐ and infection‐related oxidative challenges should be considered when trying to explain variation in (individual) migration strategies and in coping mechanisms used to mitigate oxidative damage. We explore the ecological and evolutionary consequences of infection during migration in relation to oxidative stress. Specific research on the processes and mechanisms involved in immune‐oxidative stress may lead to fundamental advances in our understanding of the ecology and evolution of migration and residency.

Background Cutaneous melanoma (CM) is the most aggressive form of skin cancer, with an increasing incidence in recent years.Immunotherapy is considered the most promising therapeutic option for late-stage CM.However, resistance occurs in 40-65% of the cases and 20-30% of responding patients develop secondary resistance, mainly due to the immunosuppression imposed by the tumor microenvironment.For this reason, it is essential to investigate the complex interactions between immune cells and tumors.Methods So far, most of the studies in this context have relied on in vivo models.However, these models present both scientific and ethical limitations.In our lab, we have established an in vitro CM skin model that fully recapitulates skin pathophysiology.Therefore, our objective is now to optimize an immune competent in vitro CM skin model to study the crosstalk between tumor cells and the immune system, and to investigate the mechanisms underlying immunotherapy response and resistance.For this, we developed an in vitro CM skin model composed by a dermis equivalent made with fibroblasts and the extracellular matrix they secrete, and an epidermis equivalent composed by CM cells and keratinocytes that stratify into all the layers of the epidermis.Once the model was fully established, we added to the medium PBMCs previously isolated from healthy donors.To determine the best conditions that allow the immune cells to infiltrate the skin equivalent and migrate toward the CM cells, we tested different immune/CM cell ratios (3:1; 10:1; 30:1) and different incubation times (2, 3 and 4 days).In parallel, as a control condition, we performed the same model but without immune cells.After fixation, the models have been processed for paraffin embedding (for histological analysis with haematoxylin and eosin staining (H&E)) and cryopreservation (for immunofluorescence microscopy analysis).Results It is possible to recognize by H&E staining the presence of small round cells in the dermis of the model to which PBMCs were added for 4 days at a 30:1 ratio of PBMCs/CM cells, consistent with the morphology of this type of cells.This observation suggests that there was infiltration of PBMCs into the CM skin model.However, since H&E staining is not sufficient to discriminate between PBMCs and the other cell types, we intend to perform immunofluorescence using an antibody that recognizes CD45, a marker that is specifically expressed by PBMCs. ConclusionThe results obtained are promising due to the presence in the dermis of small, rounded cells that are morphologically compatible with PBMCs, suggesting their infiltration in the model.However, immunofluorescence staining is essential to confirm the results.Once established, this model will provide a clinically relevant platform to identify predictive biomarkers of immunotherapy response and novel therapeutic strategies to overcome immunotherapy resistance in CM.P08.

Diagnostics and prevention of COVID-19 are essential for controlling the spread of the virus and reducing mortality rates. As SARS-CoV-2 surface proteins are susceptible to mutations, the nucleocapsid protein (NP) with its highly conserved gene sequence is an attractive target for studying virus-host interactions. NP plays a key role in the coronavirus life cycle, modulating viral RNA packaging, transcription, and assembly. In addition, its abundant expression during infection makes it a valuable diagnostic marker. NP is involved in modulating the host's innate immunity; however, the cellular mechanisms of its pathogenicity are not yet fully understood. This study developed and characterized murine monoclonal antibodies (MAbs) specific to the SARS-CoV-2 NP to investigate its antigenic regions and utilize the MAbs in virus-detecting systems or cellular NP blocking assays. The MAbs showed cross-reactivity with Omicron NP, recognizing epitopes within functionally active domains. They also identified NP in SARS-CoV-2-infected cells, supporting their feasibility in future immunoassays. Additionally, the ability to inhibit NP-cell interaction was assessed, with MAbs 4B3, 7F10, 16D9, and 18A8 found to reduce NP internalization. Overall, this study provides well-characterized tools for investigating SARS-CoV-2 antigenicity and pathogenicity and demonstrates the functional potential of the generated MAbs in studying NP-mediated host cell interactions.

The diet of wild non-human primates is still largely unknown, but advances in molecular techniques using DNA metabarcoding allow obtaining such information much faster and in more detail than until recently. The genus Trachypithecus is the most speciose and most broadly distributed genus among Asian colobines, a group of highly folivorous primates. The genus contains four species groups of which three occur in rainforest habitats while the fourth, the T. francoisi group, is restricted to limestone habitat. To characterize the food plant composition of langurs living in different habitats, we employed a metabarcoding approach and analyzed monthly samples (n = 419) collected over a 1-year period from T. crepusculus and T. germaini, members of the T. obscurus and T. cristatus groups, respectively, and T. delacouri and T. hatinhensis, both members of the T. francoisi group. By identifying plant taxa primarily to the genus level, we found that langur species consume a large number of different plants, with diets comprising of 122 - 129 plant genera across 59 - 73 families. While both overall dietary diversity and the most common plants consumed differed among the four species, we found no clear distinction between rainforest and limestone langurs. The four langur species also differed in their reaction to seasonal changes, as some species consumed different plants during dry and wet season, whereas others showed little change in plant consumption over the year. This study improves understanding of Trachypithecus dietary composition and represents the first dietary inventory of T. crepusculus in Vietnam.

Lassa virus (LASV) is the causative agent of Lassa fever, which causes thousands of deaths every year within the endemic regions of West Africa. Despite its discovery over half a century ago, our knowledge regarding basic steps in the LASV life cycle remains limited. In order to simplify studying basic principles of the LASV life cycle, we developed a novel transcription- and replication-competent virus-like particle (trVLP) system that can model the whole virus life cycle using only authentic LASV components but without the need for a maximum containment laboratory. As a proof-of-concept we used this system, together with classical minigenome systems, to determine the functional contributions of highly conserved amino acids within the mammarenavirus matrix protein Z. We could demonstrate that residues L71 and P72 are essential for the role of Z in inhibiting viral RNA synthesis, with residues R16, D22, K68 and T73 also contributing to this function. Additionally, we showed that L71-T73 mediate interaction of Z with the viral nucleoprotein (NP), which is also supported by residue G2. Given the critical role of these interactions in the virus life cycle, understanding these amino acids’ functions will help to define potential targets for the development of urgently needed countermeasures.

ABSTRACTRecently, cases of human infection with rat hepatitis E virus (ratHEV, Rocahepevirus ratti) have been reported worldwide. Due to the significant genetic differences between ratHEV and human HEV genotypes 1–4 (Paslahepevirus balayani), current HEV diagnostic assays are unable to detect ratHEV. The aim was to establish and validate a laboratory‐developed ratHEV RT‐qPCR assay for use with human plasma and stool samples on a fully automated, high‐throughput platform. Published primers and probes were optimized for use on cobas 5800/6800/8800 systems using European Union In Vitro Diagnostics Regulation (IVDR)‐grade reagents, including an RNA full‐process inhibition control. Analytical sensitivity (21 repeats), linear range (five repeats) and precision (three repeats over 3 days) were evaluated using viral particles from cell culture (MN450851.1). The inclusivity was verified using DNA oligonucleotides and known positive samples (rat liver and human serum). The limits of detection were 98.9 copies/ml in plasma and 60.3 copies/ml in stool, and the assay showed excellent linearity over at least 5 log (r2: 0.991 in plasma and 0.9989 in stool) and high precision (< 0.62 ct). The assay reliably detected different ratHEV C1 subgenotypes, returning positive results for all 11 rat liver samples and one known ratHEV RNA‐positive human plasma sample, while no false positives were detected in the broad cross‐reactivity set (n = 41). In the pilot ratHEV surveillance cohort, 1.1% of plasma samples (n = 1999) were positive for HEV RNA, but none were positive for ratHEV RNA. Our new, fully automated, lab‐developed ratHEV assay can be used in compliance with the IVDR for routine human diagnostics. Further studies are needed to determine the clinical relevance in different human cohorts.