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Abstract Borna disease virus 1 (BoDV-1) is the causative agent of Borna disease, a fatal neurologic disorder of domestic mammals and humans, resulting from spill-over infection from its natural reservoir host, the bicolored white-toothed shrew (Crocidura leucodon). The known BoDV-1-endemic area is remarkably restricted to parts of Germany, Austria, Switzerland and Liechtenstein. To gain comprehensive data on its occurrence, we analysed diagnostic material from suspected BoDV-1-induced encephalitis cases based on clinical and/or histopathological diagnosis. BoDV-1 infection was confirmed by RT-qPCR in 207 domestic mammals, 28 humans and seven wild shrews. Thereby, this study markedly raises the number of published laboratory-confirmed human BoDV-1 infections and provides a first comprehensive summary. Generation of 136 new BoDV-1 genome sequences from animals and humans facilitated an in-depth phylogeographic analysis, allowing for the definition of risk areas for zoonotic BoDV-1 transmission and facilitating the assessment of geographical infection sources. Consistent with the low mobility of its reservoir host, BoDV-1 sequences showed a remarkable geographic association, with individual phylogenetic clades occupying distinct areas. The closest genetic relatives of most human-derived BoDV-1 sequences were located at distances of less than 40 km, indicating that spill-over transmission from the natural reservoir usually occurs in the patient´s home region.

In October 2023, bluetongue virus serotype 3 (BTV-3) emerged in Germany, where Schmallenberg virus is enzootic. We detected BTV-3 in 1 pool of Culicoides biting midges collected at the time ruminant infections were reported. Schmallenberg virus was found in many vector pools. Vector trapping and analysis could elucidate viral spread.

Abstract Background Astrocytes are the most abundant cell type of the central nervous system and are fundamentally involved in homeostasis, neuroprotection, and synaptic plasticity. This regulatory function of astrocytes on their neighboring cells in the healthy brain is subject of current research. In the ischemic brain we assume disease specific differences in astrocytic acting. The renin–angiotensin–aldosterone system regulates arterial blood pressure through endothelial cells and perivascular musculature. Moreover, astrocytes express angiotensin II type 1 and 2 receptors. However, their role in astrocytic function has not yet been fully elucidated. We hypothesized that the angiotensin II receptors impact astrocyte function as revealed in an in vitro system mimicking cerebral ischemia. Astrocytes derived from neonatal wistar rats were exposed to telmisartan (angiotensin II type 1 receptor-blocker) or PD123319 (angiotensin II type 2 receptor-blocker) under normal conditions (control) or deprivation from oxygen and glucose. Conditioned medium (CM) of astrocytes was harvested to elucidate astrocyte-mediated indirect effects on microglia and cortical neurons. Result The blockade of angiotensin II type 1 receptor by telmisartan increased the survival of astrocytes during ischemic conditions in vitro without affecting their proliferation rate or disturbing their expression of S100A10, a marker of activation. The inhibition of the angiotensin II type 2 receptor pathway by PD123319 resulted in both increased expression of S100A10 and proliferation rate. The CM of telmisartan-treated astrocytes reduced the expression of pro-inflammatory mediators with simultaneous increase of anti-inflammatory markers in microglia. Increased neuronal activity was observed after treatment of neurons with CM of telmisartan- as well as PD123319-stimulated astrocytes. Conclusion Data show that angiotensin II receptors have functional relevance for astrocytes that differs in healthy and ischemic conditions and effects surrounding microglia and neuronal activity via secretory signals. Above that, this work emphasizes the strong interference of the different cells in the CNS and that targeting astrocytes might serve as a therapeutic strategy to influence the acting of glia-neuronal network in de- and regenerative context.

Bluetongue (BT) and Epizootic Hemorrhagic Disease (EHD) are two notifiable animal diseases transmitted to ruminants by small hematophagous midges belonging to the Culicoides genus. The etiological agents, Bluetongue virus (BTV) and Epizootic hemorrhagic disease virus (EHDV), are both members of the Sedoreoviridae family and Orbivirus genus, which include double-stranded (ds) RNA segmented genomes (10 segments). By the end of the summer 2023, first's outbreaks of EHD were reported from the south west of France, concurrently with unexpectedly severe BT cases in Central France and Corsica. Within a few weeks, numerous BT and EHD outbreaks were recorded with significant sanitary and economic impact on cattle and sheep farms (no sanitary impact of EHD for sheep). Using a customized SISPA approach and the nanopore sequencing technology we successfully recovered genomic sequences from viral isolates and blood samples from infected animals from EHD and BT outbreaks. Three different viruses were responsible for these outbreaks: EHDV-8, BTV-8 and BTV-4. The EHDV-8 strain detected in France corresponded to the strain circulating in Tunisia, Sardinia and Spain since 2021 and 2022. A new BTV-8 strain of unknown origin, clearly different from the enzootic strain circulating in France since 2015, was responsible of the BT outbreaks in domestic ruminants in 2023 on both mainland France and Corsica. A second BTV, BTV-4, also involved in BT outbreaks in Corsica, corresponded to a BTV-4 strain occasionally detected on Corsica island since 2016, suggesting either a new introduction of this strain or a silent circulation on the field. The exceptional nature of orbivirus epizootics in France in 2023, including new introduction, emergence or incursions, raises numerous questions regarding BTV and EHDV dynamics and epidemiology and stresses out the need to identify factors involved in these emergences.

IntroductionTick-borne pathogens, such as Borreliella spp., Rickettsia spp., and Anaplasma spp., are frequently detected in Germany. They circulate between animals and tick vectors and can cause mild to severe diseases in humans. Knowledge about distribution and prevalence of these pathogens over time is important for risk assessment of human and animal health.MethodsIxodes ricinus nymphs were collected at different locations in 2009/2010 and 2019 in Germany and analyzed for tick-borne pathogens by real-time PCR and sequencing.ResultsBorreliella spp. were detected with a prevalence of 11.96% in 2009/2010 and 13.10% in 2019 with B. afzelii and B. garinii as dominant species. Borrelia miyamotoi was detected in seven ticks and in coinfection with B. afzelii or B. garinii. Rickettsia spp. showed a prevalence of 8.82% in 2009/2010 and 1.68% in 2019 with the exclusive detection of R. helvetica. The prevalence of Anaplasma spp. was 1.00% in 2009/2010 and 7.01% in 2019. A. phagocytophilum was detected in seven tick samples. None of the nymphs were positive for C. burnetii.DiscussionHere, observed changes in prevalence were not significant after a decade but require longitudinal observations including parameters like host species and density, climatic factors to improve our understanding of tick-borne diseases.

Epizootic hemorrhagic disease (EHD) is an infectious, non-contagious viral disease seriously affecting cattle and some wild ruminants and has a worldwide distribution. All viruses can be subdivided into “Eastern” and “Western” topotypes according to geographic distribution via the phylogenetic analysis of internal genes. In Israel, during the last decade, three outbreaks were registered: caused by EHDV-6 in 2015, by EHDV-1 in 2016, and by EHDV-7 in 2020. Additionally, RNA of EHDV-8 was found in imported calves from Portugal in 2023. During the same period in other countries of the region, non-Israeli-like EHDV-6 and EHDV-8 were identified. Full genome sequencing, BLAST, and phylogenetic analyses of the locally and globally known EHDV genomes allowed us to presume the probable route and origin of these viruses detected in Israel. Thus, EHDV-6 has probably been circulating in the region for a long period when EHDV-1 and -8 appeared here for the last years, while their route of introduction into the new areas was probably natural; all of them belonged to the “Western” topotype. In contrast, EHDV-7 probably had the “Eastern”, anthropogenic origin. Data from the study can facilitate the evaluation of the appearance or reappearance of EHDVs in the Mediterranean area and enhance the planning of prevention measures.

Bovine ephemeral fever (BEF) is an arthropod-borne viral disease, which frequently causes significant epizootics in susceptible water buffalo and cattle in Africa, Australia, Asia and the Middle East. In the current study, a two-stage protocol for BEFV viral isolation was developed. Data on the clinical signs, geographic distribution and phylogenetic analysis of BEFV strains isolated in Israel in 2015, 2018, 2021 and 2023 were summarized. It was found that during 2015–2021, all BEF outbreaks were caused by local BEFV strains, whereas the epizootic of BEFV in 2023 was caused by a new “Mayotte-like” BEFV strain. A comparison of bluetongue (BT) and BEF outbreaks during 2023 in Israel demonstrated that the incidence of BEFV was 2.21 times higher and its pathogenicity was more serious for the cattle population compared to that caused by BTVs. A phylogenetic analysis of Israeli and global BEFV revealed the emergence of non-local strains in new areas. This finding suggests that BEFV can no longer be classified based only upon geographic distribution. Considering a phylogenetic, genetic and proteomic analysis of all available BEFV strains, we suggest classifying them as a single serotype, which includes four lineages.

Here, we report the results of a monitoring study of bat viruses in Austria to strengthen the knowledge of circulating viruses in Austrian bat populations. In this study, we analyzed 618 oropharyngeal and rectal swab samples from 309 bats and 155 pooled tissue samples from dead bats. Samples were collected from 18 different bat species from multiple locations in Austria, from November 2015 to April 2018, and examined for astroviruses, bornaviruses, coronaviruses, hantaviruses, morbilliviruses, orthomyxoviruses (influenza A/C/D viruses), pestiviruses and rhabdoviruses (lyssaviruses) using molecular techniques and sequencing. Using RT-qPCR, 36 samples revealed positive or suspicious results for astroviruses, Brno-hantaviruses, and coronaviruses in nine different bat species. Further sequencing revealed correspondent sequences in five samples. In contrast, none of the tested samples was positive for influenza viruses A/C/D, bornaviruses, morbilliviruses, lyssaviruses, or pestiviruses.

In situ cryo-electron tomography (cryo-ET) is the most current, state-of-the-art technique to study cell machinery in its hydrated near-native state. The method provides ultrastructural details at sub-nanometer resolution for many components within the cellular context. Making use of recent advances in sample preparation techniques and combining this method with correlative light and electron microscopy (CLEM) approaches have enabled targeted molecular visualization. Nevertheless, the implementation has also added to the complexity of the workflow and introduced new obstacles in the way of streamlining and achieving high throughput, sample yield, and sample quality. Here, we report a detailed protocol by combining multiple newly available technologies to establish an integrated, high-throughput, optimized, and streamlined cryo-CLEM workflow for improved sample yield.Key features• PRIMO micropatterning allows precise cell positioning and maximum number of cell targets amenable to thinning with cryo focused-ion-beam–scanning electron microscopy.• CERES ice shield ensures that the lamellae remain free of ice contamination during the batch milling process.• METEOR in-chamber fluorescence microscope facilitates the targeted cryo focused-ion-beam (cryo FIB) milling of these targets.• Combining the three technologies into one cryo-CLEM workflow maximizes sample yield, throughput, and efficiency.Graphical overview

Abstract Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known. Here we show that in stratifying epithelia the polarity protein aPKCλ controls the reorganization from stress fibers to cortical actomyosin during differentiation and upward movement of cells. Without aPKC, stress fibers are retained resulting in increased contractile prestress. This aberrant stress is counterbalanced by reorganization and bundling of keratins, thereby increasing mechanical resilience. Inhibiting contractility in aPKCλ−/− cells restores normal cortical keratin networks but also normalizes resilience. Consistently, increasing contractile stress is sufficient to induce keratin bundling and enhance resilience, mimicking aPKC loss. In conclusion, our data indicate that keratins sense the contractile stress state of stratified epithelia and balance increased contractility by mounting a protective response to maintain tissue integrity.

Concussion, caused by a rotational acceleration/deceleration injury mild enough to avoid structural brain damage, is insufficiently captured in recent preclinical models, hampering the relation of pathophysiological findings on the cellular level to functional and behavioral deficits. We here describe a novel model of unrestrained, single vs. repetitive concussive brain injury (CBI) in male C56Bl/6j mice. Longitudinal behavioral assessments were conducted for up to seven days afterward, alongside the evaluation of structural cerebral integrity by in vivo magnetic resonance imaging (MRI, 9.4 T), and validated ex vivo by histology. Blood–brain barrier (BBB) integrity was analyzed by means of fluorescent dextran- as well as immunoglobulin G (IgG) extravasation, and neuroinflammatory processes were characterized both in vivo by positron emission tomography (PET) using [18F]DPA-714 and ex vivo using immunohistochemistry. While a single CBI resulted in a defined, subacute neuropsychiatric phenotype, longitudinal cognitive testing revealed a marked decrease in spatial cognition, most pronounced in mice subjected to CBI at high frequency (every 48 h). Functional deficits were correlated to a parallel disruption of the BBB, (R2 = 0.29, p < 0.01), even detectable by a significant increase in hippocampal uptake of [18F]DPA-714, which was not due to activation of microglia, as confirmed immunohistochemically. Featuring a mild but widespread disruption of the BBB without evidence of macroscopic damage, this model induces a characteristic neuro-psychiatric phenotype that correlates to the degree of BBB disruption. Based on these findings, the BBB may function as both a biomarker of CBI severity and as a potential treatment target to improve recovery from concussion.

A comprehensive characterisation of Omicron-BA.1 and VOC Delta in numerous in vitro and in vivo models uncovers the factors that led to its global dominance.

Background & Aims: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell–cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome–keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. Methods: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8–yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. Results: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8–yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. Conclusions: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.

Efficient and reliable transfer of nucleic acids for therapy applications is a major challenge. Stabilization of lipo- and polyplexes has already been successfully achieved by PEGylation. This modification reduces the interaction with serum proteins and thus prevents the lipoplexes from being cleared by the reticuloendothelial system. Problematically, this stabilization of lipoplexes simultaneously leads to reduced transfer efficiencies compared to non-PEGylated complexes. However, this reduction in transfer efficiency can be used to advantage since additional modification of PEGylated lipoplexes with functional groups enables improved selective transfer into target cells. Cancer cells overexpress folate receptors because of a significantly increased need of folate due to high cell proliferation rates. Thus, additional folate functionalization of PEGylated lipoplexes improves uptake into cancer cells. We demonstrate herein that NHS coupling chemistries can be used to modify two commercially available transfection reagents (Fuse-It-DNA and Lipofectamine® 3000) with NHS-PEG-folate for increased uptake of nucleic acids into cancer cells. Lipoplex characterization and functional analysis in cultures of cancer- and healthy cells clearly demonstrate that functionalization of PEGylated lipoplexes offers a promising method to generate efficient, stable and selective nucleic acid transfer systems.

The peste des petits ruminant (PPR) virus is a transboundary virus found in small domestic ruminants that causes high morbidity and mortality in naive herds. PPR can be effectively controlled and eradicated by vaccinating small domestic ruminants with a live-attenuated peste des petits ruminant virus (PPRV) vaccine, which provides long-lasting immunity. We studied the potency and safety of a live-attenuated vaccine in goats by detecting their cellular and humoral immune responses. Six goats were subcutaneously vaccinated with a live-attenuated PPRV vaccine according to the manufacturer’s instructions, and two goats were kept in contact. Following vaccination, the goats were monitored daily, and we recorded their body temperature and clinical score. Heparinized blood and serum were collected for a serological analysis, and swab samples and EDTA blood were collected to detect the PPRV genome. The safety of the used PPRV vaccine was confirmed by the absence of PPR-related clinical signs, a negative pen-side test, a low virus genome load as detected with RT-qPCR on the vaccinated goats, and the lack horizontal transmission between the in-contact goats. The strong humoral and cellular immune responses detected in the vaccinated goats showed that the live-attenuated PPRV vaccine has a strong potency in goats. Therefore, live-attenuated vaccines against PPR can be used to control and eradicate PRR.

Emerging SARS-CoV-2 variants with mutations in the spike protein raise concerns regarding vaccine efficacy. Here, the authors show that two spike encoding mRNA vaccines in preclinical and clinical development protect human ACE2 mice from the B.1.351 variant of concern and ancestral B BavPat1.

Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 µm; diameter: 10 µm, distance: 5 µm). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15–1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150–200 nN). Within the same continuous microtissues, the pillar-ring topography induced the growth of bilayered cell tubes. The low effective pillar stiffness overwrote cellular sensing of the high substrate stiffness and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11–15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states during microtissue development and homeostasis. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility, hence microtissue geometry and function.

The efficient and biocompatible transfer of nucleic acids into mammalian cells for research applications or medical purposes is a long-standing, challenging task. Viral transduction is the most efficient transfer system, but often entails high safety levels for research and potential health impairments for patients in medical applications. Lipo- or polyplexes are commonly used transfer systems but result in comparably low transfer efficiencies. Moreover, inflammatory responses caused by cytotoxic side effects were reported for these transfer methods. Often accountable for these effects are various recognition mechanisms for transferred nucleic acids. Using commercially available fusogenic liposomes (Fuse-It-mRNA), we established highly efficient and fully biocompatible transfer of RNA molecules for in vitro as well as in vivo applications. We demonstrated bypassing of endosomal uptake routes and, therefore, of pattern recognition receptors that recognize nucleic acids with high efficiency. This may underlie the observed almost complete abolishment of inflammatory cytokine responses. RNA transfer experiments into zebrafish embryos and adult animals fully confirmed the functional mechanism and the wide range of applications from single cells to organisms.

Sheeppox virus (SPPV) (genus Capripoxvirus, family Poxviridae) infections are a highly virulent and contagious disease of sheep with a high morbidity and mortality, especially in naïve populations and young animals. For the control of SPPV, homologous and heterologous live-attenuated vaccines are commercially available. In our study, we compared a commercially available live-attenuated lumpy skin disease virus (LSDV) vaccine strain (Lumpyvax) with our recently developed inactivated LSDV vaccine candidate regarding their protective efficacy against SPPV in sheep. Both vaccines were proven to be safe in sheep, and neither clinical signs nor viremia could be detected after vaccination and challenge infection. However, the local replication of the challenge virus in the nasal mucosa of previously vaccinated animals was observed. Because of the advantages of an inactivated vaccine and its heterologous protection efficacy against SPPV in sheep, our inactivated LSDV vaccine candidate is a promising additional tool for the prevention and control of SPPV outbreaks in the future.

Abstract Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

The variegated squirrel bornavirus 1 (VSBV-1) is a recently discovered emerging viral pathogen which causes severe and eventually fatal encephalitis in humans after contact to exotic squirrels in private holdings and zoological gardens. Understanding the VSBV-1 epidemiology is crucial to develop, implement, and maintain surveillance strategies for the detection and control of animal and human infections. Based on a newly detected human encephalitis case in a zoological garden, epidemiological squirrel trade investigations and molecular phylogeny analyses of VSBV-1 with temporal and spatial resolution were conducted. Phylogenetic analyses indicated a recent emergence of VSBV-1 in European squirrel holdings and several animal–animal and animal–human spill-over infections. Virus phylogeny linked to squirrel trade analysis showed the introduction of a common ancestor of the known current VSBV-1 isolates into captive exotic squirrels in Germany, most likely by Prevost’s squirrels (Callosciurus prevostii). The links of the animal trade between private breeders and zoos, the likely introduction pathway of VSBV-1 into Germany, and the role of a primary animal distributor were elucidated. In addition, a seroprevalence study was performed among zoo animal caretakers from VSBV-1 affected zoos. No seropositive healthy zoo animal caretakers were found, underlining a probable high-case fatality rate of human VSBV-1 infections. This study illustrates the network and health consequences of uncontrolled wild pet trading as well as the benefits of molecular epidemiology for elucidation and future prevention of infection chains by zoonotic viruses. To respond to emerging zoonotic diseases rapidly, improved regulation and control strategies are urgently needed.

Abstract Background The electromechanical function of myocardial tissue depends on the intercellular communication between cardiomyocytes (CMs) as well as their crosstalk with other cell types. Cell injury, and subsequent death trigger inflammation as in myocardial infarction (MI) resulting in myocardial remodeling. Although mechanisms underlying myocardial cell death have been studied so far, the signaling events following single cell death and spontaneous response of connected cells in the myocardial tissue is still barely understood. Methods Here, we investigated the effect of laser-induced single cell death on Calcium (Ca2+) concentrations and transport in myocardial cell clusters in vitro. Spatial and temporal changes in intracellular Ca2+ concentrations [Ca2+]i were studied using a fluorescent calcium indicator, Fluo-4AM. Spontaneous signaling events following cell death were studied in rat embryonic cardiomyocytes and non-myocytes using separate cell culture systems. Results Cell death triggered spontaneous increase in intracellular Ca2+ levels ([Ca2+]i) of surrounding cells. The spread of the observed propagating Ca2+ signal was slow and sustained in myocytes while it was rapid and transient in fibroblasts (Fbs). Further, sustained high Ca2+ levels temporarily impaired the contractility in CMs. The cell-type specific effect of ablation was confirmed using separate cultures of CMs and Fbs. Comparing Ca2+ propagation speed in myocytes and fibroblasts, we argue for a diffusion-driven Ca2+ propagation in myocytes, but not in fibroblasts. Radial and sequential Ca2+ diffusion across the CMs through cell–cell contacts and presence of Cx43-based intercellular junctions indicated a gap junction flow of Ca2+. Conclusions These findings illustrate the spontaneous Ca2+-mediated functional interplay in myocardial cell clusters upon mechanical injury and, further, the difference in Ca2+ signaling in cardiomyocytes and fibroblasts. Video Abstract

Abstract Background Bavaria, a large federal state in Germany, has been declared free from infections with Bovine Alphaherpesvirus 1 (BoHV-1) in 2011. To maintain this status the cattle population is monitored for antibodies against BoHV-1 regularly. Several years ago, infrequent but recurrent problems in this sero-surveillance were statistically put into correlation with the presence of antibodies against Bovine Alphaherpesvirus 2 (BoHV-2). In Europe, BoHV-2 is primarily known as the agent causing bovine herpes mammillitis. However, very little information about BoHV-2 infections in Bavaria is available so far. Therefore, the aims of this study were to determine BoHV-2 seroprevalences and to detect virus genomes in potential clinical samples. Results 6801 blood sera of healthy cattle from all over Bavaria were tested for antibodies against BoHV-2, revealing an overall seroprevalence of 5.51%. Interestingly, seroprevalences markedly varied between the North and the South of Bavaria, namely from 0.42 to 11.17%. Concurrently, the previously reported relation between the epidemiologically inexplicable sero-reactivities in BoHV-1 ELISAs and the presence of BoHV-2 infections were statistically corroborated in this study. To detect BoHV-2 genomes a fast and sensitive real time PCR was established. Using a multiple PCR strategy, tissue samples from skin lesions at relevant localizations, corresponding lymph nodes, and trigeminal ganglia from 111 animals, as well as nasal swabs from 918 bovines with respiratory symptoms were tested. However, BoHV-2 genomes were not detected in any of these samples. Conclusions BoHV-2 antibodies were found in samples from bovines all over Bavaria, albeit with an explicit South-North-divide. BoHV-2 genomes, however, could not be detected in any of the analyzed samples, indicating that acute clinical cases as well as obvious virus reactivation are relatively rare. Consequently, the future spread of BoHV-2 infections throughout Bavaria, particularly, after eradicating BoHV-1, has to be further monitored.

Abstract Background The detection of antibodies against capripoxvirus has become easier with a commercially available ELISA validated for serum and plasma. In order to explore its suitability for immunological investigations on alternative samples, this study targeted milk as sample matrix available through non-invasive sampling. Methods Samples for this study were collected from dairy cows vaccinated against LSD in an area without reported LSD virus circulation. Paired serum and milk (individual and bulk) samples were tested by ELISA without and with modifications of the sample incubation time for the milk samples. For the evaluation of the test specificity, 352 milk samples from a milk repository in Germany were used as negative control. Receiver operating characteristic analysis was performed for determination of the Youden index and determination of the most suitable cut-off value for maximum specificity. Results From 154 analyzed serum samples from Serbia, 75 were detected as positive in the ELISA. Sensitivity and specificity of the ELISA test for milk samples reached values of 88 to 91% using Youden criteria. A cut-off of 10 was determined aiming for maximum specificity. This cut-off value was used for further analysis. Using the protocol for serum, out of 154 milk samples, 38 were detected as positive, number of positive detected milk samples increase up to 48 with modified protocol. Milk samples from Germany reacted negative, except two samples that had borderline results using modified protocol. Significant statistical difference (p

Abstract Background Pneumonia and stomatitis represent severe and often fatal diseases in different captive snakes. Apart from bacterial infections, paramyxo-, adeno-, reo- and arenaviruses cause these diseases. In 2014, new viruses emerged as the cause of pneumonia in pythons. In a few publications, nidoviruses have been reported in association with pneumonia in ball pythons and a tiger python. The viruses were found using new sequencing methods from the organ tissue of dead animals. Methods Severe pneumonia and stomatitis resulted in a high mortality rate in a captive breeding collection of green tree pythons. Unbiased deep sequencing lead to the detection of nidoviral sequences. A developed RT-qPCR was used to confirm the metagenome results and to determine the importance of this virus. A total of 1554 different boid snakes, including animals suffering from respiratory diseases as well as healthy controls, were screened for nidoviruses. Furthermore, in addition to two full-length sequences, partial sequences were generated from different snake species. Results The assembled full-length snake nidovirus genomes share only an overall genome sequence identity of less than 66.9% to other published snake nidoviruses and new partial sequences vary between 99.89 and 79.4%. Highest viral loads were detected in lung samples. The snake nidovirus was not only present in diseased animals, but also in snakes showing no typical clinical signs. Conclusions Our findings further highlight the possible importance of snake nidoviruses in respiratory diseases and proof multiple circulating strains with varying disease potential. Nidovirus detection in clinical healthy individuals might represent testing during the incubation period or reconvalescence. Our investigations show new aspects of nidovirus infections in pythons. Nidoviruses should be included in routine diagnostic workup of diseased reptiles.

Israel is endemic to bluetongue virus (BTV). The introduction of novel-for-the-region arboviruses have been recorded annually in recent years. In 2019, previously non-reported in-the-country BTV-1 and BTV-9 were identified. BTV-1 caused a single-season outbreak, probably linked to mild infection in ruminants. BTV-9 was retrospectively detected in the field samples collected from August 2018 until 2020. It was the dominant serotype in 2019, out of the six serotypes recorded during that calendar year. Clinical manifestation of the disease in cases diagnosed with BTV-9 were compared to those in cases determined to have BTV-1. BLAST and phylogenetic analyses of BTV-1 showed that the nucleotide (nt) sequence coding the viral outer protein 1 (VP2) determining the serotype is closely related to BTV-1 isolated in Sudan in 1987, and the coding sequence of the outer protein 2 (VP5) is related to South African BTV-1 from 2017. A probable common ancestor with Libyan BTV-9 strains isolated in 2008 was seen in an analysis of Israeli BTV-9 nt sequences. Notably, the outbreak-caused BTV-9 strains collected in 2019 exhibited a distinct level of genetic reassortment with local Israeli strains compared to BTV-9 strains registered in 2018 and 2020.

African swine fever (ASF) is a contagious viral hemorrhagic disease that affects domestic pigs and wild boar. The disease is notifiable to the World Organization of Animal Health (WOAH), and causes significant deaths and economic losses. There is currently no fully licensed vaccine available. As a result, early identification of the causative agent, ASF virus (ASFV), is crucial for the implementation of control measures. PCR and real-time PCR are the WOAH-recommended standard methods for the direct detection of ASFV. However, under special field conditions or in simple or remote field laboratories, there may be no sophisticated equipment or even stable electricity available. Under these circumstances, point-of-care systems can be put in place. Along these lines, a previously published, rapid, reliable, and electricity-free extraction method (TripleE) was used to isolate viral nucleic acid from diagnostic specimens. With this tool, nucleic acid extraction from up to eight diagnostic samples can be realized in one run in less than 10 min. In addition, the possibility of completely omitting viral DNA extraction was analyzed with so-called direct real-time PCR protocols using ASFV original samples diluted to 1:40 in RNase-free water. Furthermore, three real-time PCR cyclers, developed for use under field conditions (IndiField, Liberty16 and UF-300 GenecheckerTM), were comparatively applied for the sensitive high-speed detection of ASFV genomes, with overall PCR run times between 20 and 54 min. Depending on the viral DNA extraction/releasing method used and the point-of-care cycler applied, a total time for detection of 30 to 60 min for up to eight samples was feasible. As expected, the limitations in analytical sensitivity were positively correlated to the analysis time. These limitations are acceptable for ASFV diagnostics due to the expected high ASFV genome loads in diseased animals or carcasses.

The neural stem cell (NSC) niche is a highly vascularized microenvironment that supplies stem cells with relevant biological and chemical cues. However, the NSCs’ proximity to the vasculature also means that the NSCs are subjected to permanent tissue deformation effected by the vessels’ heartbeat-induced pulsatile movements. Cultivating NSCs under common culture conditions neglects the—yet unknown—influence of this cyclic mechanical strain on neural stem cells. Under the hypothesis that pulsatile strain should affect essential NSC functions, a cyclic uniaxial strain was applied under biomimetic conditions using an in-house developed stretching system based on cross-linked polydimethylsiloxane (PDMS) elastomer. While lineage commitment remained unaffected by cyclic deformation, strain affected NSC quiescence and cytoskeletal organization. Unexpectedly, cyclically stretched stem cells aligned in stretch direction, a phenomenon unknown for other types of cells in the mammalian organism. The same effect was observed for young astrocytes differentiating from NSCs. In contrast, young neurons differentiating from NSCs did not show mechanoresponsiveness. The exceptional orientation of NSCs and young astrocytes in the stretch direction was blocked upon RhoA activation and went along with a lack of stress fibers. Compared to postnatal astrocytes and mature neurons, NSCs and their young progeny displayed characteristic and distinct mechanoresponsiveness. Data suggest a protective role of young astrocytes in mixed cultures of differentiating neurons and astrocytes by mitigating the mechanical stress of pulsatile strain on developing neurons.

Lumpy skin disease (LSD) emerged in Bangladesh in mid-2019, leading to great economic losses for cattle farmers. This study describes the recent occurrence of the LSDV in Bangladesh and examines the clinical manifestation of the disease in local cattle breeds, characteristic epidemiological features, and pathological findings in affected animals. In addition, a full-genome sequencing of two local LSDV isolates was carried out. A total of 565 animals from 88 households were investigated, and 165 samples (skin lesions, saliva, nasal discharge, feces, and milk) were collected for virus detection. Pathology and immunohistochemistry were performed on nodule biopsies. Fever, nodular skin lesions, and swelling of the joints were the most common clinical manifestations. Skin lesions had a higher concentration of viral DNA compared to other sample types and were therefore selected for virus isolation and characterization. Pathology of the LSD skin nodules comprised a granulomatous reaction in the dermis and hypodermis that extended to the surrounding tissues. Development of the skin lesions started with swelling of keratinocytes with cytoplasmic vacuolation, vasculitis, panniculitis, thrombosis, and infarction. Altogether, the LSDV produced transmural, hemorrhagic, necrotizing, proliferative and ulcerative dermatitis. The LSD viral antigen was detected occasionally in the macrophages, epithelial cells, and vascular smooth muscle cells. The complete genome sequence analysis revealed that the two Bangladeshi field strains (BD-V392.1 and BD-V395.1) were distinct from the contemporary field strains and were closely related to the ancestral African Neethling strain. The findings of this study will improve the diagnosis, monitoring, and control of LSD in Bangladesh.

Six different biosample collection cards, often collectively referred to as FTA (Flinders Technology Associates) cards, were compared for their ability to inactivate viruses and stabilize viral nucleic acid for molecular testing. The cards were tested with bluetongue virus, foot-and-mouth disease virus (FMDV), small ruminant morbillivirus (peste des petits ruminants virus), and lumpy skin disease virus (LSDV), encompassing non-enveloped and enveloped representatives of viruses with double-stranded and single-stranded RNA genomes, as well as an enveloped DNA virus. The cards were loaded with virus-containing cell culture supernatant and tested after one day, one week, and one month. The inactivation of the RNA viruses was successful for the majority of the cards and filters. Most of them completely inactivated the viruses within one day or one week at the latest, but the inactivation of LSDV presented a greater challenge. Three of the six cards inactivated LSDV within one day, but the others did not achieve this even after an incubation period of 30 days. Differences between the cards were also evident in the stabilization of nucleic acid. The amount of detectable viral genome on the cards remained approximately constant for all viruses and cards over an incubation period of one month. With some cards, however, a bigger loss of detectable nucleic acid compared with a directly extracted sample was observed. Using FMDV, it was confirmed that the material applied to the cards was sufficiently conserved to allow detailed molecular characterization by sequencing. Furthermore, it was possible to successfully recover infectious FMDV by chemical transfection from some cards, confirming the preservation of full-length RNAs.

Abstract Background In the presented study we investigated the development of the humoral immune response against LSDV during the process of re-vaccination of cattle over a time span of 5 months. In addition, the performance of different serological techniques for antibody detection against LSDV was compared. For sample collection, an area without previous LSD outbreak reports in Serbia was selected. Seventy-nine cattle from twenty farms vaccinated in 2016 and re-vaccinated in 2017 were included in the study. Two farms from the same area with good calving management were selected for investigation of passive LSDV antibody transfer from vaccinated mothers to new-borne calves. Results All investigated cattle were healthy on the day of vaccination and during the whole study. Swelling at the injection site or other side effects of vaccination did not occur after re-vaccination in the study. Detection of LSD-specific antibodies was performed with the standard serological methods VNT and IFAT as well as a commercially available Capripox double antigen multi-species-ELISA. Capripoxvirus-specific antibodies were detected 46 to 47 weeks after vaccination in 2016, with VNT in 35.06% and with IFAT and ELISA in 33.77%. A secondary response was observed in all three tests 1 month after re-vaccination with a significant increase in seropositive animals compared to the results before re-vaccination. With all applied serological methods, the number of animals testing positive was significantly higher at 1 and 5 months post re-vaccination than before re-vaccination. No significant statistical difference (p > 0.05) was observed between the results of all three tests used. The sensitivity and specificity of ELISA was estimated to be SeELISA 91% and SpELISA 87% calculated by the results of VNT and SeELISA 88% and SpELISA 76% calculated by the results of IFAT. Passive antibody transfer from vaccinated mothers to new-born calves was investigated at 14 days after birth. Discrepancies for the detection of LSDV specific antibodies between cows and newborn calves at the age of 14 days were observed in VNT and IFAT, but not in ELISA. Conclusion Of all tests used the commercially available ELISA shows to be the most useful for high throughput analysis compared to VNT or IFAT.

Peste des Petits Ruminants (PPR) is a transboundary contagious disease in domestic small ruminants. Infections with the small ruminant morbillivirus (SRMV) were regularly found in wildlife, with unknown roles in PPR epidemiology. In order to access infection dynamics and virulence, we infected German Edelziege goats intranasally with a SRMV isolate that originated from Barbary sheep from an outbreak in the United Arab Emirates. Six goats were infected with cell culture-isolated SRMV, and two goats were kept in contact. Goats were daily monitored, and clinical score was recorded. EDTA blood, nasal, conjunctival and rectal swab samples were collected for the detection of SRMV genome load and serum for serological analysis. Short incubation period in infected (4 to 5 dpi) as well as in contact goats (9 dpi) was followed by typical clinical signs related to PPR. The highest viral load was detectable in conjunctival and nasal swab samples with RT-qPCR and rapid pen-side test. Specific antibodies were detected at 7 dpi in infected and 14 dpi in contact goats. In general, high virulence and easy transmission of the virus originated from wildlife in domestic goats was observed. The virus isolate belongs to Asian lineage IV, genetically related to Chinese and Mongolian strains.

Safe sample transport is of great importance for infectious diseases diagnostics. Various treatments and buffers are used to inactivate pathogens in diagnostic samples. At the same time, adequate sample preservation, particularly of nucleic acids, is essential to allow an accurate laboratory diagnosis. For viruses with single-stranded RNA genomes of positive polarity, such as foot-and-mouth disease virus (FMDV), however, naked full-length viral RNA can itself be infectious. In order to assess the risk of infection from inactivated FMDV samples, two animal experiments were performed. In the first trial, six cattle were injected with FMDV RNA (isolate A22/IRQ/24/64) into the tongue epithelium. All animals developed clinical disease within two days and FMDV was reisolated from serum and saliva samples. In the second trial, another group of six cattle was exposed to FMDV RNA by instilling it on the tongue and spraying it into the nose. The animals were observed for 10 days after exposure. All animals remained clinically unremarkable and virus isolation as well as FMDV genome detection in serum and saliva were negative. No transfection reagent was used for any of the animal inoculations. In conclusion, cattle can be infected by injection with naked FMDV RNA, but not by non-invasive exposure to the RNA. Inactivated FMDV samples that contain full-length viral RNA carry only a negligible risk of infecting animals.

Capripox virus-induced diseases are commonly described as the most serious poxvirus diseases of production animals, as they have a significant impact on national and global economies. Therefore, they are classified as notifiable diseases under the guidelines of the World Organization for Animal Health (OIE). Controlling lumpy skin disease viral infections is based on early detection, slaughter of affected herds, and ring vaccinations. Until now, only live attenuated vaccines have been commercially available, which often induce adverse effects in vaccinated animals. Furthermore, their application leads to the loss of the “disease-free” status of the respective country. For these reasons, inactivated vaccines have increasingly generated interest. Since 2016, experimental studies have been published showing the high efficacy of inactivated capripox virus vaccines. In the present study, we examined the minimum protective dose of a BEI-inactivated LSDV-Serbia field strain adjuvanted with a low-molecular-weight copolymer adjuvant. Unexpectedly, even the lowest dose tested, with a virus titer of 104 CCID50 before inactivation, was able to provide complete clinical protection in all vaccinated cattle. Moreover, none of the vaccinated cattle showed viremia or viral shedding, indicating the high efficacy of the prototype vaccine even with a relatively low antigen amount.

Eradication of small ruminant morbillivirus (PPRV) is targeted for 2030. PPRV lineage IV is found in much of Asia and Africa. We used PPRV lineage IV strain Kurdistan/2011 in transmission trials to investigate the role of pigs, wild boar, and small ruminants as PPRV reservoirs. Suids were a possible source of infection.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Lumpy skin disease (LSD) is a viral disorder of cattle caused by the lumpy skin disease virus (LSDV) which can induce severe infections leading to high economic losses. Being of African origin, the first LSD outbreaks in Europe occurred in Greece and later in the Balkan region. Little is known about the mode of transmission, especially in relation to the potential role of arthropods vectors. The purpose of our study was to investigate the role of Stomoxys calcitrans in the transmission of LSDV and their presence at different farms in Switzerland. Laboratory-reared flies were exposed to LSDV spiked-blood and incubated under a realistic fluctuating temperature regime. Body parts, regurgitated blood, and faecal samples were analysed by qPCR for the presence of viral DNA and infectious virus at different time points post-feeding (p.f.). LSDV DNA was detected in heads, bodies, and regurgitated blood up to three days p.f. and up to two days p.f. in the faeces. Infectious virus was isolated from bodies and faeces up to two days and in the regurgitated blood up to 12 h p.f. There was no increase in viral load, consolidating the role of S. calcitrans as mechanical vectors for LSDV. Stomoxys flies were present at all eight farms investigated, including a farm located at 2128 m asl. The persistence of LSDV in S. calcitrans in combination with the long flight ranges of this abundant and widespread fly might have implications on LSD epidemiology and on implementing control measures during disease outbreaks.

A proficiency test was performed to verify that the regional veterinary laboratories in Germany can provide reliable foot-and-mouth disease virus (FMDV) diagnostics. Overall, 24 samples were to be analyzed for FMDV-specific nucleic acids by real-time RT-PCR, and 16 samples had to be tested by ELISA for antibodies against non-structural proteins of FMDV. For both methods, a range of dilutions of the original materials (inactivated FMDV vaccine or convalescent serum from infected animals, respectively) was prepared, and negative samples were included as well. All 23 participating laboratories were able to detect FMDV genome down to a dilution of 1:100,000 of the vaccine preparation. Even at a dilution of 1:1,000,000, FMDV genome was detected by more than half of the participants. With the antibody ELISA, all sera were correctly identified by all participating laboratories. No false-positive results were returned with either method. All participating laboratories were found to be fully proficient in FMDV diagnostics.

The complexity of the current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method (easy express extraction, called TripleE) as a centrifugation-free and electricity-free nucleic acid isolation method. The procedure is based on the well-established magnetic-bead extraction technology using an in-house self-made magnetic 8-channel and a rod cover. With this extraction system, nucleic acids can be isolated with two simple and universal protocols. One method was designed for the extraction of the nucleic acid in resource-limited “easy labs”, and the other method can be used for RNA/DNA extraction in the field for so-called molecular “pen-side tests”. In both scenarios, users can extract up to 8 samples in 6 to 10 min, without the need for any electricity, centrifuges or robotic systems. In order to evaluate and compare both methods, clinical samples from various viruses (African swine fever virus; lumpy skin disease virus; peste des petits ruminants virus; bluetongue virus), matrices and animals were tested and compared with standard magnetic-bead nucleic acid extraction technology based on the KingFisher platform. Hence, validation data were generated by evaluating two DNA viruses as well as one single-stranded and one double-stranded RNA virus. The results showed that the fast, easy, portable and electricity-free extraction protocols allowed rapid and reliable nucleic acid extraction for a variety of viruses and most likely also for other pathogens, without a substantial loss of sensitivity compared to standard procedures. The speed and simplicity of the methods make them ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings.

In recent years, numerous atypical Bluetongue virus (BTV) strains have been discovered all around the world. Atypical BTV strains are phylogenetically distinct from the classical BTV serotypes 1–24 and differ in terms of several biological features. For the first time, the atypical strains BTV-25-GER2018 and BTV-33-MNG3/2016 as well as the re-emerged classical strain BTV-8-GER2018 were evaluated comparatively in a pathogenesis study in goats—the natural host of atypical BTV. A substantial number of in-contact animals were included in this study to detect potential contact transmissions of the virus. After infection, EDTA blood, ocular, nasal and oral swab samples as well as serum were collected regularly and were used for virological and serological analyses, respectively. Our study showed differences in the immunological reaction between the two atypical BTV strains (no group-specific antibody detection) and the classical BTV strain BTV-8-GER2018 (group-specific antibody detection). Furthermore, we observed an increase in the total WBC count (neutrophils and lymphocytes) in goats infected with the atypical BTV strains. No horizontal transmission was seen for all three strains. Our study suggests that the atypical BTVs used in the trial differ from classical BTVs in their immunopathogenesis. However, no evidence of direct contact transmission was found.

Feto-maternal communication in the dog involves the differentiation of stromal cells into decidual cells. As the only placental cells expressing the nuclear progesterone (P4) receptor (PGR), decidual cells play crucial roles in the maintenance and termination of pregnancy. Accordingly, to investigate possible PGR-mediated mechanisms in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with functional PGR-blockers, mifepristone and aglepristone. Effects on decidualization markers, epithelial and mesenchymal factors, and markers of cellular viability were assessed. Decidualization increased the expression of PTGES, PGR, IGF1, and PRLR, along with ECM1, COL4 and CX43, but downregulated IGF2. DUS cells retained their mesenchymal character, and the expression of COL4 indicated the mesenchymal-epithelial transformation. Antigestagen treatment decreased the availability of PTGES, PRLR, IGF1 and PGR. Furthermore, antigestagens decreased the mRNA and protein expression of CX43, and transcriptional levels of ECM1 and COL4. Additionally, antigestagens increased levels of activated-CASP3 (a proapoptotic factor), associated with lowered levels of PCNA (a proliferation marker). These data reveal important aspects of the functional involvement of PGR in canine decidual cells, regarding the expression of decidualization markers and acquisition of epithelial-like characteristics. Some of these mechanisms may be crucial for the maintenance and/or termination of canine pregnancy.

Microglia—the brain’s primary immune cells—exert a tightly regulated cascade of pro- and anti-inflammatory effects upon brain pathology, either promoting regeneration or neurodegeneration. Therefore, harnessing microglia emerges as a potential therapeutic concept in neurological research. Recent studies suggest that—besides being affected by chemokines and cytokines—various cell entities in the brain relevantly respond to the mechanical properties of their microenvironment. For example, we lately reported considerable effects of elasticity on neural stem cells, regarding quiescence and differentiation potential. However, the effects of elasticity on microglia remain to be explored.Under the hypothesis that the elasticity of the microenvironment affects key characteristics and functions of microglia, we established an in vitro model of primary rat microglia grown in a polydimethylsiloxane (PDMS) elastomer-based cell culture system. This way, we simulated the brain’s physiological elasticity range and compared it to supraphysiological stiffer PDMS controls. We assessed functional parameters of microglia under “resting” conditions, as well as when polarized towards a pro-inflammatory phenotype (M1) by lipopolysaccharide (LPS), or an anti-inflammatory phenotype (M2) by interleukin-4 (IL-4). Microglia viability was unimpaired on soft substrates, but we found various significant effects with a more than two-fold increase in microglia proliferation on soft substrate elasticities mimicking the brain (relative to PDMS controls). Furthermore, soft substrates promoted the expression of the activation marker vimentin in microglia. Moreover, the M2-marker CD206 was upregulated in parallel to an increase in the secretion of Insulin-Like Growth Factor-1 (IGF-1). The upregulation of CD206 was abolished by blockage of stretch-dependent chloride channels. Our data suggest that the cultivation of microglia on substrates of brain-like elasticity promotes a basic anti-inflammatory activation state via stretch-dependent chloride channels. The results highlight the significance of the omnipresent but mostly overlooked mechanobiological effects exerted on microglia and contribute to a better understanding of the complex spatial and temporal interactions between microglia, neural stem cells, and glia, in health and disease.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost’s squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost’s squirrel. Antibodies against bornaviruses were detected in the patient’s cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.

Abstract Background Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. Methods Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. Results Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A24-2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. Conclusion The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization profile were unaffected, but the 146S particle yield differed for one of the tested serotypes.

Many people worldwide suffer from hepatitis C virus (HCV) infection, which is frequently persistent. The lack of efficient vaccines against HCV and the unavailability of or limited compliance with existing antiviral therapies is problematic for health care systems worldwide. Improved small animal models would support further hepacivirus research, including development of vaccines and novel antivirals. The recent discovery of several mammalian hepaciviruses may facilitate such research. In this study, we demonstrated that bank voles (Clethrionomys glareolus) were susceptible to bank vole-associated Hepacivirus F and Hepacivirus J strains, based on the detection of hepaciviral RNA in 52 of 55 experimentally inoculated voles. In contrast, interferon α/β receptor deficient C57/Bl6 mice were resistant to infection with both bank vole hepaciviruses (BvHVs). The highest viral genome loads in infected voles were detected in the liver, and viral RNA was visualized by in situ hybridization in hepatocytes, confirming a marked hepatotropism. Furthermore, liver lesions in infected voles resembled those of HCV infection in humans. In conclusion, infection with both BvHVs in their natural hosts shares striking similarities to HCV infection in humans and may represent promising small animal models for this important human disease.

Lumpy skin disease virus (LSDV), together with sheeppox virus and goatpox virus, belong to the genus Capripoxvirus within the family Poxviridae. Collectively, they are considered the most serious poxvirus diseases of agricultural livestock. Due to their severe clinical course and consequent loss of production, as well as high mortality of naïve small and large ruminant populations, they are known to have a significant impact on the economy and global trade restrictions of affected countries. Therefore, all capripox diseases are classified as notifiable under the guidelines of the World Organization of Animal Health (OIE). Since the 1970s, several outbreaks of LSD have been recorded in Nigeria. Until now, only a little information on the virus strains leading to the reported outbreaks have been published, dealing mainly with the phylogenetic relationship of those strains and the description of field outbreaks. During the present study, we experimentally infected cattle with a low-passage Nigerian LSDV strain isolated from a skin sample of LSD positive cattle in Nigeria in 2018. Clinical, molecular and serological data indicate that this LSDV isolate is highly pathogenic in cattle since it induced a severe clinical course and approximately 33% mortality in naïve Holstein Friesian cattle after experimental infection.

FTA cards and related products simplify the collection, transport, and transient storage of biological sample fluids. Here, we have compared the yield and quality of DNA and RNA released from seven different FTA cards using seven releasing/extraction methods with eleven experimental eluates. For the validation, dilution series of African swine fever virus (ASFV) positive EDTA blood and Influenza A virus (IAV) positive allantoic fluid were used. Based on our data, we conclude that direct PCR amplification without the need for additional nucleic acid extraction and purification could be suitable and more convenient for ASFV DNA release from FTA cards. In contrast, IAV RNA loads can be amplified from FTA card punches if a standard extraction procedure including a lysis step is applied. These differences between the amplifiable viral DNA and RNA after releasing and extraction are not influenced by the type of commercial FTA card or the eleven different nucleic acid releasing procedures used for the comparative analyses. In general, different commercial FTA cards were successfully used for the storage and recovery of the ASFV and IAV genetic material suitable for PCR. Nevertheless, the usage of optimized nucleic acid releasing protocols could improve the recovery of the viral genome of both viruses. Here, the application of Chelex® Resin 100 buffer mixed with 1 × Tris EDTA buffer (TE, pH 8.0) or with TED 10 (TE buffer and Dimethylsulfoxid) delivered the best results and can be used as a universal method for releasing viral DNA and RNA from FTA cards.

Since the beginning of 2020, the betacoronavirus SARS-CoV-2 is causing a global pandemic of an acute respiratory disease termed COVID-19. The diagnostics of the novel disease is primarily based on direct virus detection by RT-PCR; however, the availability of test kits may become a major bottleneck, when millions of tests are performed per week. To increase the flexibility of SARS-CoV-2 diagnostics, three real-time RT-PCR assays listed on the homepage of the World Health Organization were selected and investigated regarding their compatibility with three different RT-PCR kits. Furthermore, the reaction volume of the PCR chemistry was reduced up to half of the original protocol to make the individual reactions more cost- and resource-effective. When testing dilution series of culture-grown virus, nearly identical quantification cycle values (Cq) were obtained for all RT-PCR assay/chemistry combinations. Regarding the SARS-CoV-2 detection in clinical samples, agreeing results were obtained for all combinations for virus negative specimens and swabs containing high to medium viral genome loads. In cases of very low SARS-CoV-2 genome loads (Cq > 36), inconsistent results were observed, with some test runs scoring negative and some positive. However, no preference of a specific target within the viral genome (E, RdRp, or N) or of a certain chemistry was seen. In summary, a reduction of the reaction volume and the type of PCR chemistry did not influence the PCR sensitivity.

The geographical distribution of lumpy skin disease (LSD), an economically important cattle disease caused by a capripoxvirus, has reached an unprecedented extent. Vaccination is the only way to prevent the spread of the infection in endemic and newly affected regions. Yet, in the event of an outbreak, selection of the best vaccine is a major challenge for veterinary authorities and farmers. Decision makers need sound scientific information to support their decisions and subsequent actions. The available vaccine products vary in terms of quality, efficacy, safety, side effects, and price. The pros and cons of different types of live attenuated and inactivated vaccines, vaccination strategies, and associated risks are discussed. Seroconversion, which typically follows vaccination, places specific demands on the tools and methods used to evaluate the effectiveness of the LSD vaccination campaigns in the field. We aimed to give a comprehensive update on available vaccines and vaccination against LSD, to better prepare affected and at-risk countries to control LSD and ensure the safe trade of cattle.