Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation.

During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and de novo lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

INTRODUCTION: Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. METHODS: We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. RESULTS: We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. CONCLUSION: In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

African swine fever virus - variants on the rise.

African swine fever virus (ASFV), a large and complex DNA-virus circulating between soft ticks and indigenous suids in sub-Saharan Africa, has made its way into swine populations from Europe to Asia. This virus, causing a severe haemorrhagic disease (African swine fever) with very high lethality rates in wild boar and domestic pigs, has demonstrated a remarkably high genetic stability for over 10 years. Consequently, analyses into virus evolution and molecular epidemiology often struggled to provide the genetic basis to trace outbreaks while few resources have been dedicated to genomic surveillance on whole-genome level. During its recent incursion into Germany in 2020, ASFV has unexpectedly diverged into five clearly distinguishable linages with at least ten different variants characterized by high-impact mutations never identified before. Noticeably, all new variants share a frameshift mutation in the 3' end of the DNA polymerase PolX gene O174L, suggesting a causative role as possible mutator gene. Although epidemiological modelling supported the influence of increased mutation rates, it remains unknown how fast virus evolution might progress under these circumstances. Moreover, a tailored Sanger sequencing approach allowed us, for the first time, to trace variants with genomic epidemiology to regional clusters. In conclusion, our findings suggest that this new factor has the potential to dramatically influence the course of the ASFV pandemic with unknown outcome. Therefore, our work highlights the importance of genomic surveillance of ASFV on whole-genome level, the need for high-quality sequences and calls for a closer monitoring of future phenotypic changes of ASFV.

Identification of Risk Factors for African Swine Fever: A Systematic Review.

African swine fever (ASF) is an internationally-spreading viral pig disease that severely damages agricultural pork production and trade economy as well as social welfare in disease-affected regions. A comprehensive understanding of ASF risk factors is imperative for efficient disease control. As the absence of effective ASF vaccines limits disease management options, the identification and minimisation of ASF-associated risk factors is critical to preventing ASF outbreaks. Here, we compile currently known potential ASF risk factors identified through a systematic literature review. We found 154 observation-based and 1239 potential ASF risk factors, which we were able to group into the following defined risk categories: 'ASF-virus', 'Biosecurity', 'Disease control', 'Environment', 'Husbandry', 'Movement', 'Network', 'Pig', 'Society' and 'Surveillance'. Throughout the epidemiological history of ASF there have been similar risk categories, such as 'Environment'-related risk factors, predominantly reported in the literature irrespective of the ASF situation at the time. While ASF risk factor reporting has markedly increased since 2010, the majority of identified risk factors overall have referred to domestic pigs. The reporting of risk factors for ASF in wild boar mostly commenced from 2016 onwards. The compendium of ASF risk factors presented herein defines our current knowledge of ASF risk factors, and critically informs ASF-related problem solving.

Efficacy of Liming Forest Soil in the Context of African Swine Fever Virus.

Since September 2020, Germany has experienced the first ever outbreak of African swine fever (ASF). The first known cases occurred exclusively in wild boar in forest areas in Brandenburg and Saxony; in July 2021, infected domestic pigs were also confirmed for the first time. As wild boar are considered the main reservoir for the virus in the European region, an effective interruption of this infection chain is essential. In particular, the removal and safe disposal of infected carcasses and the direct disinfection of contaminated, unpaved ground are priorities in this regard. For the disinfection, highly potent as well as environmentally compatible disinfectants must be used, which are neither influenced in their effectiveness by the soil condition nor by increased organic contamination. Thus, in this study, slaked lime, milk of lime and quicklime (1% to 10% solutions) were selected for efficacy testing against the test virus recommended by the German Veterinary Society (DVG), Modified Vaccinia Ankara virus (MVAV), and ASF virus (ASFV) in conjunction with six different forest soils from Saxony in two different soil layers (top soil and mineral soil) each. In summary, 10% of any tested lime type is able to inactivate both MVAV and ASFV under conditions of high organic load and independent of the water content of the soil. At least a 4 log reduction of the virus titer in all tested forest soil types and layers and by all applied lime types was observed. In conclusion, the high efficacy and suitability of all tested lime products against both viruses and in the presence of high organic load in forest soil can be confirmed and will help to control ASF spread.

Performance Characteristics of Real-Time PCRs for African Swine Fever Virus Genome Detection-Comparison of Twelve Kits to an OIE-Recommended Method.

African swine fever (ASF) is a major threat to pig production, and real-time PCR (qPCR) protocols are an integral part of ASF laboratory diagnosis. With the pandemic spread of ASF, commercial kits have risen on the market. In Germany, the kits have to go through an approval process and thus, general validation can be assumed. However, they have never been compared to each other. In this study, 12 commercial PCR kits were compared to an OIE-recommended method. Samples representing different matrices, genome loads, and genotypes were included in a panel that was tested under diagnostic conditions. The comparison included user-friendliness, internal controls, and the time required. All qPCRs were able to detect ASFV genome in different matrices across all genotypes and disease courses. With one exception, there were no significant differences when comparing the overall mean. The overall specificity was 100% (95% CI 87.66-100), and the sensitivity was between 95% and 100% (95% CI 91.11-100). As can be expected, variability concerned samples with low genome load. To conclude, all tests were fit for purpose. The test system can therefore be chosen based on compatibility and prioritization of the internal control system.

The Efficacy of Disinfection on Modified Vaccinia Ankara and African Swine Fever Virus in Various Forest Soil Types.

African swine fever (ASF) has become a global threat to the pig industry and wild suids. Within Europe, including Germany, affected wild boar populations play a major role. Fencing and carcass removal in combination with the reduction in environmental contamination are key to control further spread. The handling of the ASF virus (ASFV) is restricted to high-containment conditions in Germany. According to the regulation of the German Veterinarian Society (DVG), modified vaccinia Ankara virus (MVAV) is the virus of choice to determine the efficacy of disinfection for enveloped viruses. The aim of this study was to use the MVAV as a guide to select the best possible disinfectant solution and concentration for the inactivation of ASFV in soil. Both viruses were tested simultaneously. In this study, two layers (top and mineral soil) of soil types from six different locations in Saxony, Germany, were collected. The tenacity of ASFV and MVAV were tested at various time points (0.5 to 72 h). The capabilities of different concentrations of peracetic acid and citric acid (approx. 0.1 to 2%) to inactivate the viruses in the selected soil types with spiked high protein load were examined under appropriate containment conditions. Around 2-3 Log10 (TCID50) levels of reduction in the infectivity of both ASFV and MVAV were observed in all soil types starting after two hours. For MVAV, a 4 Log10 loss was recorded after 72 h. A total of 0.1% of peracetic acid (5 L/m2) was sufficient to inactivate the viruses. A 4 log10 reduction in the infectivity of MVAV was noticed by applying 1% citric acid, while a 2 log10 decline was recorded with ASFV. In conclusion, comparing MVAV to ASFV for efficacy screening of disinfectant solutions has revealed many similarities. Peracetic acid reduced the infectivity of both viruses independently of the soil type and the existence of a high organic soiling.

African Swine Fever in Wild Boar in Europe-A Review.

The introduction of genotype II African swine fever (ASF) virus, presumably from Africa into Georgia in 2007, and its continuous spread through Europe and Asia as a panzootic disease of suids, continues to have a huge socio-economic impact. ASF is characterized by hemorrhagic fever leading to a high case/fatality ratio in pigs. In Europe, wild boar are especially affected. This review summarizes the currently available knowledge on ASF in wild boar in Europe. The current ASF panzootic is characterized by self-sustaining cycles of infection in the wild boar population. Spill-over and spill-back events occur from wild boar to domestic pigs and vice versa. The social structure of wild boar populations and the spatial behavior of the animals, a variety of ASF virus (ASFV) transmission mechanisms and persistence in the environment complicate the modeling of the disease. Control measures focus on the detection and removal of wild boar carcasses, in which ASFV can remain infectious for months. Further measures include the reduction in wild boar density and the limitation of wild boar movements through fences. Using these measures, the Czech Republic and Belgium succeeded in eliminating ASF in their territories, while the disease spread in others. So far, no vaccine is available to protect wild boar or domestic pigs reliably against ASF.

Stability of African swine fever virus on spiked spray-dried porcine plasma.

African swine fever (ASF) is a viral disease that affects members of the Suidae family. The notifiable disease is considered a major threat to the pig industry, animal health, and food security worldwide. According to the European Food Safety Authority, ASF virus (ASFV) survival and transmission in feed and feed materials is a major research gap. Against this background, the objective of this study was to determine the survival of ASFV on spiked spray-dried porcine plasma (SDPP) when stored at two different temperatures. To this means, commercial SDPP granules were contaminated with high titers of ASFV in a worst-case external contamination scenario. Three samples per time point and temperature condition were subjected to blind passaging on macrophage cultures and subsequent haemadsorption test to determine residual infectivity. In addition, viral genome was detected by real-time PCR. The results indicate that heavily contaminated SDPP stored at 4°C remains infectious for at least 5 weeks. In contrast, spiked SDPP stored at room temperature displayed a distinct ASFV titer reduction after 1 week (>2.8 log levels) and complete inactivation after 2 weeks (>5.7 log levels). In conclusion, the residual risk of ASFV transmission through externally contaminated SDPP is low if SDPP is stored at room temperature (21 ± 2°C) for a period of at least 2 weeks before feeding.

African Swine Fever Laboratory Diagnosis-Lessons Learned from Recent Animal Trials.

African swine fever virus (ASFV) causes a hemorrhagic disease in pigs with high socio-economic consequences. To lower the impact of disease incursions, early detection is crucial. In the context of experimental animal trials, we evaluated diagnostic workflows for a high sample throughput in active surveillance, alternative sample matrices for passive surveillance, and lateral flow devices (LFD) for rapid testing. We could demonstrate that EDTA blood is significantly better suited for early ASFV detection than serum. Tissues recommended by the respective diagnostic manuals were in general comparable in their performance, with spleen samples giving best results. Superficial lymph nodes, ear punches, and different blood swabs were also evaluated as potential alternatives. In summary, all matrices yielded positive results at the peak of clinical signs and could be fit for purpose in passive surveillance. However, weaknesses were discovered for some matrices when it comes to the early phase of infection or recovery. The antigen LFD showed variable results with best performance in the clinical phase. The antibody LFD was quite comparable with ELISA systems. Concluding, alternative approaches are feasible but have to be embedded in control strategies selecting test methods and sample materials following a "fit-for-purpose" approach.

Joining the club: First detection of African swine fever in wild boar in Germany.

African swine fever (ASF) has spread across many countries in Europe since the introduction into Georgia in 2007. We report here on the first cases of ASF in wild boar detected in Germany close to the border with Poland. In addition to the constant risk of ASF virus (ASFV) spread through human activities, movements of infected wild boar also represent a route of introduction. Since ASF emerged in Western Poland in November 2019, surveillance efforts, in particular examination of wild boar found dead, were intensified in the regions of Germany bordering with Poland. The first case of ASF in wild boar in Germany was therefore detected by passive surveillance and confirmed on 10 September 2020. By 24 September 2020, 32 cases were recorded. Testing of samples from tissues of carcasses in different stages of decomposition yielded cycle threshold values from 18 to 36 in the OIE-recommended PCR, which were comparable between the regional and national reference laboratory. Blood swabs yielded reliable results, indicating that the method is suitable also under outbreak conditions. Phylogenetic analysis of the ASFV whole-genome sequence generated from material of the first carcass detected in Germany, revealed that it groups with ASFV genotype II including all sequences from Eastern Europe, Asia and Belgium. However, some genetic markers including a 14 bp tandem repeat duplication in the O174L gene were confirmed that have so far been detected only in sequences from Poland (including Western Poland). Epidemiological investigations that include estimated postmortem intervals of wild boar carcasses of infected animals suggest that ASFV had been introduced into Germany in the first half of July 2020 or even earlier.

Stability of African Swine Fever Virus in Soil and Options to Mitigate the Potential Transmission Risk.

Understanding African swine fever virus (ASFV) transmission is essential for strategies to minimize virus spread during an outbreak. ASFV can survive for extended time periods in animal products, carcasses, and the environment. While the ASFV genome was found in environments around infected farms, data on the virus survival in soil are scarce. We investigated different soil matrices spiked with ASFV-positive blood from infected wild boar to see if ASFV can remain infectious in the soil beneath infected carcasses. As expected, ASFV genome detection was possible over the entire sampling period. Soil pH, structure, and ambient temperature played a role in the stability of infectious ASFV. Infectious ASFV was demonstrated in specimens originating from sterile sand for at least three weeks, from beach sand for up to two weeks, from yard soil for one week, and from swamp soil for three days. The virus was not recovered from two acidic forest soils. All risk mitigation experiments with citric acid or calcium hydroxide resulted in complete inactivation. In conclusion, the stability of infectious ASFV is very low in acidic forest soils but rather high in sandy soils. However, given the high variability, treatment of carcass collection points with disinfectants should be considered.

Stability of African Swine Fever Virus in Carcasses of Domestic Pigs and Wild Boar Experimentally Infected with the ASFV "Estonia 2014" Isolate.

Europe is currently experiencing a long-lasting African swine fever (ASF) epidemic, both in domestic pigs and wild boar. There is great concern that carcasses of infected wild boar may act as long-term virus reservoirs in the environment. We evaluated the tenacity of ASF virus (ASFV) in tissues and body fluids from experimentally infected domestic pigs and wild boar, which were stored on different matrices and at different temperatures. Samples were analysed at regular intervals for viral genome and infectious virus. ASFV was most stable in spleen or muscles stored at -20 °C and in blood stored at 4 °C. In bones stored at -20 °C, infectious virus was detected for up to three months, and at 4 °C for up to one month, while at room temperature (RT), no infectious virus could be recovered after one week. Skin stored at -20 °C, 4 °C and RT remained infectious for up to three, six and three months, respectively. In urine and faeces, no infectious virus was recovered after one week, irrespective of the matrix. In conclusion, tissues and organs from decomposing carcasses that persist in the environment for a long time can be a source of infection for several months, especially at low temperatures.

Stability of African swine fever virus on heat-treated field crops.

African swine fever (ASF) is an infectious disease of pigs and represents a massive threat to animal health and the pig industry worldwide. The ASF virus (ASFV) is efficiently transmitted via blood and meat from infected animals and can be highly stable in the environment. There is therefore great concern about the potential role of contaminated raw materials used for feed or bedding in the spread of ASFV. Especially crops and derived products originating from areas with ASF in wild boar and thus with high environmental ASFV contamination may be a risk for virus introduction into domestic pig herds. However, little is known about the stability of ASFV on contaminated crops and possible inactivation methods. In this study, we tested the effect of drying and heat treatment on the inactivation of ASFV on six different types of field crops, namely wheat, barley, rye, triticale, corn, and peas, contaminated with infectious blood. Samples were analysed for the presence of viral DNA and infectious virus after 2 hr drying at room temperature or after drying and 1 hr exposure to moderate heat at a specific temperature between 40°C and 75°C. ASFV genome was detected in all samples by real-time polymerase chain reaction (PCR), including samples that had been dried for 2 hr and incubated for 1 hr at 75°C. On the other hand, no infectious virus could be detected after 2 hr drying using virus isolation in porcine macrophages in combination with the detection of ASFV by the haemadsorption test (HAT). We therefore conclude that the risk of ASFV transmission via contaminated crops is most likely low, if they are incubated for at least 2 hr minimum at room temperature. Nonetheless, to minimize the risk of transmission as much as possible crops from ASF-affected zones should not be used for pig feed.

The African swine fever virus isolate Belgium 2018/1 shows high virulence in European wild boar.

African swine fever (ASF) is one of the most important and complex viral diseases in domestic pigs and wild boar. Over the last decade, the disease has spread to several European and Asian countries and is now one of the major threats to profitable pig production worldwide. One of the more recently affected western countries is Belgium. To date, only wild boar are affected in a rather defined area in the Luxembourg region close to France, Luxembourg and Germany. While detailed sequence analyses were recently performed, biological characterization was still pending. Here, we report on the experimental inoculation of four sub-adult wild boar to further characterize the virus and its distribution in different tissues. After oronasal inoculation with the virus strain 'Belgium 2018/1', all animals developed an acute and severe disease course with typical pathomorphological and histopathological lesions. Organs and blood samples were positive in qPCR, haemadsorption test and antigen lateral flow devices (LFD). Virus and viral genome were also detected in genitals and accessory sex glands of two boars. There were no antibodies detectable in commercial antibody ELISAs, antibody LFDs and indirect immunoperoxidase tests. Thus, the genotype II ASF virus isolate 'Belgium 2018/1' showed a highly virulent phenotype in European wild boar similar to parental viruses like Armenia 2007 and other previously characterized ASFV strains. The study also provided a large set of well-characterized sample materials for test validation and assay harmonization.

Novel insights into the genetic background of genetically modified mice.

Unclear or misclassified genetic background of laboratory rodents or a lack of strain awareness causes a number of difficulties in performing or reproducing scientific experiments. Until now, genetic differentiation between strains and substrains of inbred mice has been a challenge. We have developed a screening method for analyzing inbred strains regarding their genetic background. It is based on 240 highly informative short tandem repeat (STR) markers covering the 19 autosomes as well as X and Y chromosomes. Combination of analysis results for presence of known C57BL/6 substrain-specific mutations together with autosomal STR markers and the Y-chromosomal STR-haplotype provides a comprehensive snapshot of the genetic background of mice. In this study, the genetic background of 72 mouse lines obtained from 18 scientific institutions in Germany and Austria was determined. By analyzing only 3 individuals per genetically modified line it was possible to detect mixed genetic backgrounds frequently. In several lines presence of a mispairing Y chromosome was detected. At least every second genetically modified line displayed a mixed genetic background which could lead to unexpected and non-reproducible results, irrespective of the investigated gene of interest.

Characterization of Simbu serogroup virus infections in type I interferon receptor knock-out mice.

In late 2011, Schmallenberg virus (SBV), a novel, arthropod-borne, teratogenic orthobunyavirus, emerged near the German/Dutch border and thereafter spread rapidly throughout the continent thereby causing great economic losses in European livestock. SBV mainly infects ruminants and closely related viruses such as Sabo virus (SABOV), Simbu virus (SIMBUV) and Sathuperi virus (SATV) have been isolated from their insect-vectors or putative ruminant hosts. However, information about their pathogenesis and in vivo studies with SABOV, SIMBUV, and SATV are scarce. As experimental infections of ruminants are comprehensive and time-consuming, an SBV small animal model was assessed regarding its suitability for studying Simbu viruses. Adult type I interferon deficient mice (IFNAR-/-) were subcutaneously infected with the Simbu serogroup members SABOV, SIMV and SATV, respectively, and compared to SBV-infected mice. All animals were clinically, virologically, serologically, and pathologically examined. The clinical signs were mainly characterised by the loss of body weight and by paralysis. In blood, and samples from the spleen and brain, high loads of viral genome were detected using newly developed real-time PCR assays. The most common histologic lesions included meningo-encephalomyelitis, perivascular cuffing of lymphocytes and macrophages, neuronal degeneration and gliosis. These lesions have also been described in foetuses after transplacental infection with SBV. In-situ hybridisation signals were widely distributed in multiple neurons of the brain and spinal cord in all examined, inoculated mice. In conclusion, IFNAR-/- mice are a suitable animal model for pathogenesis studies of a broad range of Simbu serogroup viruses since all the viruses examined displayed a common pattern of viral organ and tissue distribution in this mouse model.

Large deletion on the Y-chromosome long arm (Yq) of C57BL/6JBomTac inbred mice.

The Y-chromosome of mice has a crucial role in sex determination, gender ratio equilibrium as well as male fertility, and is moreover involved in behavioral, immunological, and cardiovascular traits. During routine short tandem repeat genotyping of C57BL/6 substrains, a unique deletion on the Y-chromosome long arm of males from the commercially available inbred substrain C57BL/6JBomTac was identified. In this study, the deletion was confirmed by fluorescence in situ hybridization on metaphase spreads and the extent of the deletion was assessed using position-specific genetic markers. It covers 40 Mbp of the Y-chromosome long arm, ranging from at least 6.57 to 46.73 Mbp. Therefore, C57BL/6JBomTac might be a valuable model system for Y-chromosome research. A deletion spanning almost half of the Y-chromosome long arm should not be neglected regarding the evaluation of scientific experiments. Our data are in line with others that it is of major importance that the usage of mice strains requires the exact nomenclature including the name of the substrain.

Efficacy Assessment of Nucleic Acid Decontamination Reagents Used in Molecular Diagnostic Laboratories.

The occurrence of nucleic acid cross contamination in the laboratory resulting in false positive results of diagnostic samples is seriously problematic. Despite precautions to minimize or even avoid nucleic acid cross contaminations, it may appear anyway. Until now, no standardized strategy is available to evaluate the efficacy of commercially offered decontamination reagents. Therefore, a protocol for the reliable determination of nucleic acid decontamination efficacy using highly standardized solution and surface tests was established and validated. All tested sodium hypochlorite-based reagents proved to be highly efficient in nucleic acid decontamination even after short reaction times. For DNA Away, a sodium hydroxide-based decontamination product, dose- and time-dependent effectiveness was ascertained. For two other commercial decontamination reagents, the phosphoric acid-based DNA Remover and the non-enzymatic reagent DNA-ExitusPlus™ IF, no reduction of amplifiable DNA/RNA was observed. In conclusion, a simple test procedure for evaluation of the elimination efficacy of decontamination reagents against amplifiable nucleic acid is presented.

Lagos bat virus transmission in an Eidolon helvum bat colony, Ghana.

A brain sample of a straw-coloured fruit bat (Eidolon helvum) from Ghana without evident signs of disease tested positive by generic Lyssavirus RT-PCR and direct antigen staining. Sequence analysis confirmed the presence of a Lagos bat virus belonging to phylogenetic lineage A. Virus neutralization tests using the isolate with sera from the same group of bats yielded neutralizing antibodies in 74% of 567 animals. No cross-neutralization was observed against a different Lagos bat virus (lineage B).