Publications by authors named "Bernd Hoffmann"

316 Publications

The Bank Vole ()-Small Animal Model for Hepacivirus Infection.

Viruses 2021 12 3;13(12). Epub 2021 Dec 3.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany.

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 () were susceptible to bank vole-associated and 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.
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http://dx.doi.org/10.3390/v13122421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8708279PMC
December 2021

Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Nature 2021 Dec 22. Epub 2021 Dec 22.

Institute of Virology and Immunology, Mittelhäusern, Switzerland.

Emerging variants of concern (VOC) drive the SARS-CoV-2 pandemic. Experimental assessment of replication and transmission of major VOC compared to progenitors are needed to understand successful emerging mechanisms of VOC. Here, we show that Alpha and Beta spike (S) proteins have a greater affinity to human angiotensin converting enzyme 2 (hACE2) receptor over the progenitor variant (wt-S) in vitro. Yet Alpha and wt-S had similar replication kinetics in human nasal airway epithelial cultures, whereas Beta was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over the progenitor variant (wt-S) in ferrets and two mouse models, where the substitutions in S were major drivers for fitness advantage. In hamsters, supporting high replication levels, Alpha and wt-S had comparable fitness. In contrast, Beta was outcompeted by Alpha and wt-S in hamsters and hACE2-expressing mice. Our study highlights the importance of using multiple models for complete fitness characterization of VOC and demonstrates adaptation of Alpha towards increased upper respiratory tract replication and enhanced transmission in vivo in restrictive models, whereas Beta fails to overcome contemporary strains in naïve animals.
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http://dx.doi.org/10.1038/s41586-021-04342-0DOI Listing
December 2021

Desmoplakin Maintains Transcellular Keratin Scaffolding and Protects From Intestinal Injury.

Cell Mol Gastroenterol Hepatol 2021 Dec 17. Epub 2021 Dec 17.

Department of Medicine III, Aachen, Germany. Electronic address:

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 (DSP) and the combined intestine-specific DSG2/DSP (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 DSP 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, DSP mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSP/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.
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http://dx.doi.org/10.1016/j.jcmgh.2021.12.009DOI Listing
December 2021

Optimizing Release of Nucleic Acids of and from FTA Cards.

Int J Mol Sci 2021 Nov 29;22(23). Epub 2021 Nov 29.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald, Germany.

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 (ASFV) positive EDTA blood and (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.
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http://dx.doi.org/10.3390/ijms222312915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8657678PMC
November 2021

Saving Resources: SARS-CoV-2 Diagnostics by Real-Time RT-PCR Using Reduced Reaction Volumes.

Diseases 2021 Nov 15;9(4). Epub 2021 Nov 15.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, 17493 Greifswald, Germany.

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.
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http://dx.doi.org/10.3390/diseases9040084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8628695PMC
November 2021

Outbreak of a Systemic Form of Camelpox in a Dromedary Herd () in the United Arab Emirates.

Viruses 2021 09 28;13(10). Epub 2021 Sep 28.

Central Veterinary Research Laboratory, Dubai 597, United Arab Emirates.

Camelpox virus (CMLV) is the causative agent of camelpox, which frequently occurs in the Old World camelids-rearing countries except for Australia. It has also been described in experimentally inoculated New World camelids. Camelpox outbreaks are often experienced shortly after the rainy season, which occurs twice a year on the Arabian Peninsula because of the increased density of the insect population, particularly mosquitos. A systemic form of camelpox outbreak in seven dromedary camels was diagnosed by histology, virus isolation, and PCR. A phylogenetic analysis using full length CMLV genomes of the isolated CMLV strains showed a single phylogenetic unit without any distinctive differences between them. The United Arab Emirates (UAE) isolate sequences showed phylogenetical relatedness with CMLV isolates from Israel with only minor sequence differences. Although the sequences of viruses from both countries were closely related, the disease manifestation was vastly different. Our study shows that the virulence is not only determined by genetic features of CMLV alone but may also depend on other factors such as unknown aspects of the host (e.g., age, overall fitness), management, and the environment.
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http://dx.doi.org/10.3390/v13101940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8541543PMC
September 2021

Review: Vaccines and Vaccination against Lumpy Skin Disease.

Vaccines (Basel) 2021 Oct 6;9(10). Epub 2021 Oct 6.

Institute of International Animal Health/One Health, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.

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.
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http://dx.doi.org/10.3390/vaccines9101136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8539040PMC
October 2021

NSCs Under Strain-Unraveling the Mechanoprotective Role of Differentiating Astrocytes in a Cyclically Stretched Coculture With Differentiating Neurons.

Front Cell Neurosci 2021 24;15:706585. Epub 2021 Sep 24.

Mechanobiology, Institute of Biological Information Processing (IBI-2), Research Centre Juelich, Juelich, Germany.

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.
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http://dx.doi.org/10.3389/fncel.2021.706585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8497758PMC
September 2021

Development and Validation of an ELISA for the Detection of Bluetongue Virus Serotype 4-Specific Antibodies.

Viruses 2021 08 31;13(9). Epub 2021 Aug 31.

UMR 1161 Virologie, Laboratory for Animal Health, INRAE, Department of Animal Health, Ecole Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, 94700 Maisons-Alfort, France.

In this article, we describe the development and evaluation of a double antigen sandwich enzyme-linked immunosorbent assay (ELISA) able to detect serotype 4-specific antibodies from BTV-4 infected or vaccinated animals using a recombinant BTV-4 VP2 protein. The coding sequence of VP2 was inserted into a pVote plasmid by recombination in the Gateway cloning system. Vaccinia virus (VacV) was used as a vector for the expression of the recombinant VP2. After production in BSR cells, recombinant VP2 was purified by immunoprecipitation using a FLAG tag and then used both as the coated ELISA antigen and as the HRP-tagged conjugate. The performance of the ELISA was evaluated with 1186 samples collected from BTV negative, infected or vaccinated animals. The specificity and sensitivity of the BTV-4 ELISA were above the expected standards for the detection of anti-BTV-4 VP2 antibodies in animals reared in Europe or in the Mediterranean basin. Cross-reactions were observed with reference sera for serotypes 10 and 20, and to a lesser extent with serotypes 12, 17 and 24, due to their genetic proximity to serotype 4. Nevertheless, these serotypes have never been detected in Europe and the Mediterranean area. This ELISA, which requires only the production of a recombinant protein, can be used to detect BTV serotype 4-specific antibodies and is therefore an attractive alternative diagnostic method to serum neutralization.
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http://dx.doi.org/10.3390/v13091741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8473233PMC
August 2021

A New Molecular Detection System for Canine Distemper Virus Based on a Double-Check Strategy.

Viruses 2021 08 18;13(8). Epub 2021 Aug 18.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health Südufer 10, 17493 Greifswald-Insel Riems, Germany.

Due to changing distemper issues worldwide and to inadequate results of an inter-laboratory study in Germany, it seems sensible to adapt and optimize the diagnostic methods for the detection of the canine distemper virus (CDV) to the new genetic diversity of virus strains. The goal of the project was the development, establishment and validation of two independent one-step reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) methods for the safe detection of CDV in domestic and wild animals. For this purpose, an existing CDV-RT-qPCR was decisively adapted and, in addition, a completely new system was developed. Both CDV-RT-qPCR systems are characterized by a very high, comparable analytical and diagnostic sensitivity and specificity and can be mutually combined with inhibition or extraction controls. The reduction in the master mix used allows for the parallel implementation of both CDV-RT-qPCR systems without significant cost increases. For validation of the new CDV-RT-qPCR duplex assays, a panel comprising 378 samples derived from Germany, several European countries and one African country were tested. A sensitivity of 98.9% and a specificity of 100% were computed for the new assays, thus being a reliable molecular diagnostic tool for the detection of CDV in domestic and wild animals.
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http://dx.doi.org/10.3390/v13081632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402888PMC
August 2021

Retrospective Enhanced Bat Lyssavirus Surveillance in Germany between 2018-2020.

Viruses 2021 08 3;13(8). Epub 2021 Aug 3.

Friedrich-Loeffler-Institut (FLI), 17493 Greifswald-Insel Riems, Germany.

Lyssaviruses are the causative agents for rabies, a zoonotic and fatal disease. Bats are the ancestral reservoir host for lyssaviruses, and at least three different lyssaviruses have been found in bats from Germany. Across Europe, novel lyssaviruses were identified in bats recently and occasional spillover infections in other mammals and human cases highlight their public health relevance. Here, we report the results from an enhanced passive bat rabies surveillance that encompasses samples without human contact that would not be tested under routine conditions. To this end, 1236 bat brain samples obtained between 2018 and 2020 were screened for lyssaviruses via several RT-qPCR assays. European bat lyssavirus type 1 (EBLV-1) was dominant, with 15 positives exclusively found in serotine bats () from northern Germany. Additionally, when an archived set of bat samples that had tested negative for rabies by the FAT were screened in the process of assay validation, four samples tested EBLV-1 positive, including two detected in . Subsequent phylogenetic analysis of 17 full genomes assigned all except one of these viruses to the A1 cluster of the EBLV-1a sub-lineage. Furthermore, we report here another Bokeloh bat lyssavirus (BBLV) infection in a Natterer's bat () found in Lower Saxony, the tenth reported case of this novel bat lyssavirus.
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http://dx.doi.org/10.3390/v13081538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402685PMC
August 2021

From Microspikes to Stress Fibers: Actin Remodeling in Breast Acini Drives Myosin II-Mediated Basement Membrane Invasion.

Cells 2021 08 4;10(8). Epub 2021 Aug 4.

Institute of Biological Information Processing 2 (IBI-2): Mechanobiology, Forschungszentrum Jülich, 52428 Jülich, Germany.

The cellular mechanisms of basement membrane (BM) invasion remain poorly understood. We investigated the invasion-promoting mechanisms of actin cytoskeleton reorganization in BM-covered MCF10A breast acini. High-resolution confocal microscopy has characterized actin cell protrusion formation and function in response to tumor-resembling ECM stiffness and soluble EGF stimulation. Traction force microscopy quantified the mechanical BM stresses that invasion-triggered acini exerted on the BM-ECM interface. We demonstrate that acini use non-proteolytic actin microspikes as functional precursors of elongated protrusions to initiate BM penetration and ECM probing. Further, these microspikes mechanically widened the collagen IV pores to anchor within the BM scaffold via force-transmitting focal adhesions. Pre-invasive basal cells located at the BM-ECM interface exhibited predominantly cortical actin networks and actin microspikes. In response to pro-invasive conditions, these microspikes accumulated and converted subsequently into highly contractile stress fibers. The phenotypical switch to stress fiber cells matched spatiotemporally with emerging high BM stresses that were driven by actomyosin II contractility. The activation of proteolytic invadopodia with MT1-MMP occurred at later BM invasion stages and only in cells already disseminating into the ECM. Our study demonstrates that BM pore-widening filopodia bridge mechanical ECM probing function and contractility-driven BM weakening. Finally, these EMT-related cytoskeletal adaptations are critical mechanisms inducing the invasive transition of benign breast acini.
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http://dx.doi.org/10.3390/cells10081979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394122PMC
August 2021

Strain-induced mechanoresponse depends on cell contractility and BAG3-mediated autophagy.

Mol Biol Cell 2021 10 11;32(20):ar9. Epub 2021 Aug 11.

Mechanobiology, Institute of Biological Information Processing, Forschungszentrum Jülich, 52428 Jülich, Germany.

Basically, all mammalian tissues are constantly exposed to a variety of environmental mechanical signals. Depending on the signal strength, mechanics intervenes in a multitude of cellular processes and is thus capable of inducing simple cellular adaptations but also complex differentiation processes and even apoptosis. The underlying recognition typically depends on mechanosensitive proteins, which most often sense the mechanical signal for the induction of a cellular signaling cascade by changing their protein conformation. However, the fate of mechanosensors after mechanical stress application is still poorly understood, and it remains unclear whether protein degradation pathways affect the mechanosensitivity of cells. Here, we show that cyclic stretch induces autophagosome formation in a time-dependent manner. Formation depends on the cochaperone BAG family molecular chaperone regulator 3 (BAG3) and thus likely involves BAG3-mediated chaperone-assisted selective autophagy. Furthermore, we demonstrate that strain-induced cell reorientation is clearly delayed upon inhibition of autophagy, suggesting a bidirectional cross-talk between mechanotransduction and autophagic degradation. The strength of the observed delay depends on stable adhesion structures and stress fiber formation in a Ras homologue family member A (RhoA)-dependent manner.
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http://dx.doi.org/10.1091/mbc.E21-05-0254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8684750PMC
October 2021

Maintenance of Ligament Homeostasis of Spheroid-Colonized Embroidered and Functionalized Scaffolds after 3D Stretch.

Int J Mol Sci 2021 Jul 30;22(15). Epub 2021 Jul 30.

Institute of Anatomy and Cell Biology, Paracelsus Medical University, Nuremberg and Salzburg, Prof. Ernst Nathan Str. 1, 90419 Nuremberg, Germany.

Anterior cruciate ligament (ACL) ruptures are usually treated with autograft implantation to prevent knee instability. Tissue engineered ACL reconstruction is becoming promising to circumvent autograft limitations. The aim was to evaluate the influence of cyclic stretch on lapine (L) ACL fibroblasts on embroidered scaffolds with respect to adhesion, DNA and sulphated glycosaminoglycan (sGAG) contents, gene expression of ligament-associated extracellular matrix genes, such as type I collagen, decorin, tenascin C, tenomodulin, gap junctional connexin 43 and the transcription factor Mohawk. Control scaffolds and those functionalized by gas phase fluorination and cross-linked collagen foam were either pre-cultured with a suspension or with spheroids of LACL cells before being subjected to cyclic stretch (4%, 0.11 Hz, 3 days). Stretch increased significantly the scaffold area colonized with cells but impaired sGAGs and decorin gene expression (functionalized scaffolds seeded with cell suspension). Stretching increased tenascin C, connexin 43 and Mohawk but decreased decorin gene expression (control scaffolds seeded with cell suspension). Pre-cultivation of functionalized scaffolds with spheroids might be the more suitable method for maintaining ligamentogenesis in 3D scaffolds compared to using a cell suspension due to a significantly higher sGAG content in response to stretching and type I collagen gene expression in functionalized scaffolds.
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http://dx.doi.org/10.3390/ijms22158204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8348491PMC
July 2021

Skin under Strain: From Epithelial Model Tissues to Adult Epithelia.

Cells 2021 07 20;10(7). Epub 2021 Jul 20.

Forschungszentrum Jülich, Institute of Biological Information Processing, IBI-2: Mechanobiology, 52428 Jülich, Germany.

Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues.
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http://dx.doi.org/10.3390/cells10071834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304960PMC
July 2021

Maintaining proteostasis under mechanical stress.

EMBO Rep 2021 08 26;22(8):e52507. Epub 2021 Jul 26.

Institute of Biology II, Faculty of Biology, and Signalling Research Centres BIOSS and CIBSS, Albert-Ludwigs-University Freiburg, Freiburg, Germany.

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.
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http://dx.doi.org/10.15252/embr.202152507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339670PMC
August 2021

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe.

Viruses 2021 06 28;13(7). Epub 2021 Jun 28.

Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald-Insel Riems, Germany.

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (, syn. ). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (, syn. ). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species ( and ) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.
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http://dx.doi.org/10.3390/v13071258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8310187PMC
June 2021

CVnCoV and CV2CoV protect human ACE2 transgenic mice from ancestral B BavPat1 and emerging B.1.351 SARS-CoV-2.

Nat Commun 2021 06 30;12(1):4048. Epub 2021 Jun 30.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.
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http://dx.doi.org/10.1038/s41467-021-24339-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245475PMC
June 2021

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini.

Int J Mol Sci 2021 Apr 12;22(8). Epub 2021 Apr 12.

Institute of Biological Information Processing 2 (IBI-2): Mechanobiology, Forschungszentrum Jülich, 52425 Jülich, Germany.

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.
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http://dx.doi.org/10.3390/ijms22083962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070162PMC
April 2021

Egyptian Fruit Bats () Were Resistant to Experimental Inoculation with Avian-Origin Influenza A Virus of Subtype H9N2, But Are Susceptible to Experimental Infection with Bat-Borne H9N2 Virus.

Viruses 2021 04 14;13(4). Epub 2021 Apr 14.

Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Insel Riems, 17493 Greifswald, Insel Riems, Germany.

Influenza A viruses (IAV) of subtype H9N2, endemic in world-wide poultry holdings, are reported to cause spill-over infections to pigs and humans and have also contributed substantially to recent reassortment-derived pre-pandemic zoonotic viruses of concern, such as the Asian H7N9 viruses. Recently, a H9N2 bat influenza A virus was found in Egyptian fruit bats (), raising the question of whether this bat species is a suitable host for IAV. Here, we studied the susceptibility, pathogenesis and transmission of avian and bat-related H9N2 viruses in this new host. In a first experiment, we oronasally inoculated six Egyptian fruit bats with an avian-related H9N2 virus (A/layer chicken/Bangladesh/VP02-plaque/2016 (H9N2)). In a second experiment, six Egyptian fruit bats were inoculated with the newly discovered bat-related H9N2 virus (A/bat/Egypt/381OP/2017 (H9N2)). While turned out to be refractory to an infection with H9N2 avian-type, inoculation with the bat H9N2 subtype established a productive infection in all inoculated animals with a detectable seroconversion at day 21 post-infection. In conclusion, Egyptian fruit bats are most likely not susceptible to the avian H9N2 subtype, but can be infected with fruit bat-derived H9N2. H9-specific sero-reactivities in fruit bats in the field are therefore more likely the result of contact with a bat-adapted H9N2 strain.
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http://dx.doi.org/10.3390/v13040672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070959PMC
April 2021

Putative Novel Atypical BTV Serotype '36' Identified in Small Ruminants in Switzerland.

Viruses 2021 04 21;13(5). Epub 2021 Apr 21.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.

We identified a putative novel atypical BTV serotype '36' in Swiss goat flocks. In the initial flock clinical signs consisting of multifocal purulent dermatitis, facial oedema and fever were observed. Following BTV detection by RT-qPCR, serotyping identified BTV-25 and also a putative novel BTV serotype in several of the affected goats. We successfully propagated the so-called "BTV-36-CH2019" strain in cell culture, developed a specific RT-qPCR targeting Segment 2, and generated the full genome by high-throughput sequencing. Furthermore, we experimentally infected goats with BTV-36-CH2019. Regularly, EDTA blood, serum and diverse swab samples were collected. Throughout the experiment, neither fever nor clinical disease was observed in any of the inoculated goats. Four goats developed BTV viremia, whereas one inoculated goat and the two contact animals remained negative. No viral RNA was detected in the swab samples collected from nose, mouth, eye, and rectum, and thus the experimental infection of goats using this novel BTV serotype delivered no indications for any clinical symptoms or vector-free virus transmission pathways. The subclinical infection of the four goats is in accordance with the reports for other atypical BTVs. However, the clinical signs of the initial goat flock did most likely not result from infection with the novel BTV-36-CH0219.
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http://dx.doi.org/10.3390/v13050721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143309PMC
April 2021

Probe-Based Real-Time qPCR Assays for a Reliable Differentiation of Capripox Virus Species.

Microorganisms 2021 Apr 6;9(4). Epub 2021 Apr 6.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.

Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.
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http://dx.doi.org/10.3390/microorganisms9040765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067474PMC
April 2021

Differentiation of Capripox Viruses by Nanopore Sequencing.

Vaccines (Basel) 2021 Apr 6;9(4). Epub 2021 Apr 6.

Division of Microbiology and Animal Hygiene, University of Goettingen, D-37077 Goettingen, Germany.

The genus capripoxvirus (CaPV), family , includes three virus species: goatpox virus (GPV), sheeppox virus (SPV) and lumpy skin disease virus (LSDV). CaPV causes disease outbreaks with consequent economic losses in Africa and the Middle East. LSDV has recently spread to Southeast Europe. As CaPVs share 96-97% genetic similarity along the length of the entire genome and are difficult to distinguish using serological assays, simple, reliable and fast methods for diagnosis and species differentiation are crucial in cases of disease outbreak. The present study aimed to develop a field-applicable CaPV differentiation method. Nanopore technology was used for whole genome sequencing. A local database of complete CaPV genomes and partial sequences of three genes (RPO30, P32 and GPCR) was established for offline Basic Local Alignment Search Tool (BLAST). Specificities of 98.04% in whole genome and 97.86% in RPO30 gene runs were obtained among the three virus species, while other databases were less specific. The total run time was shortened to approximately 2 h. Functionality of the developed procedure was proved by samples with high host background sequences. Reliable differentiation options for the quality and capacity of hardware, and sample quality of suspected cases, were derived from these findings. The whole workflow can be performed rapidly with a mobile suitcase laboratory and mini-computer, allowing application at the point-of-need with limited resource settings.
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http://dx.doi.org/10.3390/vaccines9040351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8067513PMC
April 2021

Cyclically stretched ACL fibroblasts emigrating from spheroids adapt their cytoskeleton and ligament-related expression profile.

Cell Tissue Res 2021 Jun 9;384(3):675-690. Epub 2021 Apr 9.

Institute of Anatomy and Cell Biology, Paracelsus Medical University, Prof.-Ernst-Nathan Str. 1, 90419, Nuremberg and Salzburg, Nuremberg, Germany.

Mechanical stress of ligaments varies; hence, ligament fibroblasts must adapt their expression profile to novel mechanomilieus to ensure tissue resilience. Activation of the mechanoreceptors leads to a specific signal transduction, the so-called mechanotransduction. However, with regard to their natural three-dimensional (3D) microenvironment cell reaction to mechanical stimuli during emigrating from a 3D spheroid culture is still unclear. This study aims to provide a deeper understanding of the reaction profile of anterior cruciate ligament (ACL)-derived fibroblasts exposed to cyclic uniaxial strain in two-dimensional (2D) monolayer culture and during emigration from 3D spheroids with respect to cell survival, cell and cytoskeletal orientation, distribution, and expression profile. Monolayers and spheroids were cultured in crosslinked polydimethyl siloxane (PDMS) elastomeric chambers and uniaxially stretched (14% at 0.3 Hz) for 48 h. Cell vitality, their distribution, nuclear shape, stress fiber orientation, focal adhesions, proliferation, expression of ECM components such as sulfated glycosaminoglycans, collagen type I, decorin, tenascin C and cell-cell communication-related gap junctional connexin (CXN) 43, tendon-related markers Mohawk and tenomodulin (myodulin) were analyzed. In contrast to unstretched cells, stretched fibroblasts showed elongation of stress fibers, cell and cytoskeletal alignment perpendicular to strain direction, less rounded cell nuclei, increased numbers of focal adhesions, proliferation, amplified CXN43, and main ECM component expression in both cultures. The applied cyclic stretch protocol evoked an anabolic response and enhanced tendon-related marker expression in ACL-derived fibroblasts emigrating from 3D spheroids and seems also promising to support in future tissue formation in ACL scaffolds seeded in vitro with spheroids.
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http://dx.doi.org/10.1007/s00441-021-03416-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211585PMC
June 2021

Introduction and spread of variegated squirrel bornavirus 1 (VSBV-1) between exotic squirrels and spill-over infections to humans in Germany.

Emerg Microbes Infect 2021 Dec;10(1):602-611

Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.

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 (). 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.
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http://dx.doi.org/10.1080/22221751.2021.1902752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018504PMC
December 2021

Swift and Reliable "Easy Lab" Methods for the Sensitive Molecular Detection of African Swine Fever Virus.

Int J Mol Sci 2021 Feb 25;22(5). Epub 2021 Feb 25.

Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, 17493 Greifswald-Insel Riems, Germany.

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.
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http://dx.doi.org/10.3390/ijms22052307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7956467PMC
February 2021

SARS-CoV-2 spike D614G change enhances replication and transmission.

Nature 2021 04 26;592(7852):122-127. Epub 2021 Feb 26.

Institute of Virology and Immunology (IVI), Mittelhäusern, Switzerland.

During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic. However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating.
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http://dx.doi.org/10.1038/s41586-021-03361-1DOI Listing
April 2021

A recombinase polymerase amplification assay for rapid detection of rabies virus.

Sci Rep 2021 02 4;11(1):3131. Epub 2021 Feb 4.

Virology Department, Institut Pasteur de Dakar, 36, Avenue Pasteur, 220, Dakar, Senegal.

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.
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http://dx.doi.org/10.1038/s41598-021-82479-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862592PMC
February 2021

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

PLoS Pathog 2021 01 26;17(1):e1009247. Epub 2021 Jan 26.

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872300PMC
January 2021
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