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Biology subjects

Beer, M.

Publications and source records attributed to Beer, M..

At least 55 records · Page 3Linked to original sources

Emergence of bluetongue virus serotype 3 in western Germany, October 2023, and ad-hoc monitoring in Culicoides biting midges

Bluetongue virus serotype 3 emerged in October 2023 in Germany, where Schmallenberg virus is enzootic. BTV-3 was detected in a pool of Culicoides biting midges at the same time as infections were reported from ruminants. Schmallenberg virus was frequently found in vector pools, reflecting the epidemiological situation. One sentence summary lineCoinciding with the emergence and low-level circulation of bluetongue virus serotype 3 in ruminants in Germany in October 2023, the virus was detected in a pool of Culicoides biting midges of the Obsoletus Group, while enzootic Schmallenberg virus was found in numerous midge pools.

microbiology↗

SARS-CoV-2 and other coronaviruses in rats, Berlin, Germany, 2023

We tested 130 rats trapped in Berlin for coronaviruses. Antibodies against SARS-CoV-2 were detected in a single animal only, but not in further 66 rats from the same location, speaking against virus circulation in the rat population. All animals tested negative for SARS-CoV-2 by RT-PCR. However, rodent-associated alphacoronaviruses were found.

microbiology↗

Pathogen-prey-predator relations of avian raptors during epizootics of highly pathogenic avian influenza virus HPAIV H5N1 (clade 2.3.4.4b) in Germany

Transition of highly pathogenic clade 2.3.4.4b H5 avian influenza virus (HPAIV) from epizootic to enzootic status in Northern European countries was associated with severe losses and even mass mortalities among various wild bird species. Both avian and mammalian raptors hunting infected debilitated birds or scavenging on virus-contaminated avian carcasses contracted HPAIV infection. This precarious pathogen-prey-predator relation further worsened when in 2021 and 2022 outbreaks in Germany overlapped with the hatching season of avian raptor species. Retro- and prospective surveillance revealed avian raptors as important indicators of HPAIV and its genetic diversity on the one hand. On the other hand, their role as victims of HPAIV is stipulated. The first case of an HPAIV H5N1-related death of a white-tailed sea eagle (Haliaeetus albicilla; WTSE) hatch in Germany, 2021, followed by several such cases in 2022, and a low overall seropositivity rate of 5.0-7.9% among WTSE nestlings, raised fears of a serious negative impact on reproduction rates of WTSEs and other birds of prey when HPAIV becomes enzootic in an ecosystem. However, comparably stable breeding success of WTSE in the study area in 2022 and a potentially evolving natural immunity raises hope for a less severe long-term impact. Article impact statementAdapted surveillance measures were developed to assess risks for the conservation of avian raptors due to the panzootic spread of HPAIV.

microbiology↗

Small in size but huge as reservoir -- insights into the virome of European white-toothed shrews

While the virome and immune system of bats and rodents have been extensively studied, comprehensive data are lacking for insectivores. Anthropogenic land use and outdoor recreational activities may lead to an expansion of the human-shrew interface with the risk of zoonotic infections, as reported for Borna disease virus 1. We investigated the virosphere of four white-toothed shrew species from Central Europe, addressing the One Health concept of spillover prevention. A high diversity of viruses was identified, including several co-infections. Whole genomes were generated for novel species of paramyxoviruses (n=3), nairoviruses (n=2) and hepevirus. Phylogenetically, they are closely related to WHO priority diseases, such as henipaviruses. High viral loads of orthoparamyxoviruses were detected in kidneys, in well-perfused organs for orthonairoviruses, and an association with liver and intestine was identified for orthohepevirus. Our study highlights the virus diversity present in shrews, not only in biodiversity hotspots but also in industrialised countries.

evolutionary biology↗

Electric fields determine carbapenemase activity in class A β-lactamases

Antimicrobial resistance is a public health crisis. Limited understanding of the catalytic drivers in resistance-mediating enzymes such as {beta}-lactamases hinders our ability to combat this crisis. Here, we dissect the catalytic contributions of active-site electric fields in class A {beta}-lactamases. We studied the enzymatic hydrolysis of a carbapenem antibiotic by QM/MM molecular dynamics simulations and quantified active-site fields with a custom-made script. We discovered that the fields correlate well with activity and identified seven positions, some distal, that distinguish efficient carbapenemases. Electric-field analysis may help predict the activity of {beta}-lactamases and guide antibiotic and enzyme design. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/565607v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@15ee41aorg.highwire.dtl.DTLVardef@d1a11corg.highwire.dtl.DTLVardef@a83bd7org.highwire.dtl.DTLVardef@f0abdf_HPS_FORMAT_FIGEXP M_FIG C_FIG Electric field script: www.github.com/bunzela/FieldTools

biochemistry↗

No evidence for highly pathogenic avian influenza virus H5N1 (clade 2.3.4.4b) in the Antarctic region during the austral summer 2022/23

The current highly pathogenic avian influenza H5N1 panzootic has substantial impacts on wild birds and marine mammals. Although major outbreaks occurred in South America, incursion to Antarctica emerged late in the breeding season of 2023/2024 and was confined the wider region of the Antarctic Peninsula. To infer potential underlying processes, we compiled H5N1 surveillance from Antarctica and Sub-Antarctic Islands prior to the first confirmed cases.

microbiology↗

ZeptoCTC - Sensitive Protein Analysis of True Single Cell Lysates using Reverse Phase Protein Arrays (RPPA)

Circulating Tumor Cells (CTCs) are commonly analyzed through genomic profiling, which does not capture posttranslational and functional alterations of encoded proteins. To address this limitation, we developed ZeptoCTC, a single-cell protein analysis workflow that combines established technologies for single-cell isolation and sensitive Reverse Phase Protein Array (RPPA) analysis to assess multiple protein expression and activation in individual CTCs. The workflow involves single cell labeling, isolation, lysis, and printing of the true single cell lysates onto a ZeptoChip using a modified micromanipulator CellCelectorTM. Subsequently, the printed lysates undergo fluorescence immunoassay RPPA protein detection using a ZeptoReader followed by signal quantification with Image J software. ZeptoCTC was successfully optimized, beginning with the measurement of EpCAM protein expression--a standard marker for CTC detection. As expected, mean fluorescence signals for EpCAM levels were significantly higher in single MCF-7 cells compared to MDA-MB-231 cells. Next, Capivasertib-treated MCF-7 cells exhibited an approximately 2-fold increase in the pAkt/Akt ratio compared to non-treated control cells. This finding was consistent with a co-performed western blot analysis of pooled MCF-7 cells. Application of ZeptoCTC to the analysis of single CTCs derived from a metastasized breast cancer (MBC) patient indicated a significantly higher level of pAkt, accompanied by a corresponding increase in pErk level when compared to patient-matched WBC. Finally, the current workflow successfully indicated the detectable pAkt and Akt signal difference in CTCs from two MBC patients: one with an Akt1 wild-type genotype, and the other harboring approximately 80% Akt1(E17K) mutated CTCs. The mutated CTCs revealed clearly elevated pAkt levels (1.8-fold), along with an even more strongly elevated total Akt (3.4-fold) when compared to the respective signals measured in wild-type CTCs. In conclusion, ZeptoCTC is a highly sensitive method for measuring the expression and phosphorylation of treatment-relevant proteins in key cancer-driving signaling pathways from true single cell samples.

molecular biology↗

Diving Deep into Fish Bornaviruses: Uncovering Hidden Diversity and Transcriptional Strategies through Comprehensive Data Mining

Recently, we discovered two novel orthobornaviruses in colubrid and viperid snakes using an in silico data mining approach. Here, we present the results of a screening of more than 100,000 nucleic acid sequence datasets of fish samples from the Sequence Read Archive (SRA) for potential bornaviral sequences. We discovered the potentially complete genomes of seven bornaviruses in datasets from osteichthyans and chondrichthyans. Four of these are likely to represent novel species within the genus Cultervirus, and we propose that one genome represents a novel genus within the family of Bornaviridae. Specifically, we identified sequences of W[u]han sharpbelly bornavirus (WhSBV) in sequence data from the widely used grass carp liver and kidney cell lines L8824 and CIK, respectively. A complete genome of Murray-Darling carp bornavirus (MDCBV) was identified in sequence data from a goldfish (Carassius auratus). The newly discovered little skate bornavirus (LSBV), identified in the little skate (Leucoraja erinacea) dataset, contained a novel and unusual genomic architecture (N-Vp1-Vp2-X-P-G-M-L), as compared to other bornaviruses. Its genome is thought to encode two additional open reading frames (tentatively named Vp1 and Vp2), which appear to represent ancient duplications of the gene encoding for the viral glycoprotein (G). The datasets also provided insights into the possible transcriptional gradients of these bornaviruses and revealed previously unknown splicing mechanisms.

evolutionary biology↗

Growth media affects susceptibility of air-lifted human nasal epithelial cell cultures to SARS-CoV2, but not Influenza A, virus infection.

Primary differentiated human epithelial cell cultures have been widely used by researchers to study viral fitness and virus-host interactions, especially during the COVID19 pandemic. These cultures recapitulate important characteristics of the respiratory epithelium such as diverse cell type composition, polarization, and innate immune responses. However, standardization and validation of these cultures remains an open issue. In this study, two different expansion medias were evaluated and the impact on the resulting differentiated culture was determined. Use of both Airway and Ex Plus media types resulted in high quality, consistent cultures that were able to be used for these studies. Upon histological evaluation, Airway-grown cultures were more organized and had a higher proportion of basal progenitor cells while Ex Plus-grown cultures had a higher proportion terminally differentiated cell types. In addition to having different cell type proportions and organization, the two different growth medias led to cultures with altered susceptibility to infection with SARS-CoV-2 but not Influenza A virus. RNAseq comparing cultures grown in different growth medias prior to differentiation uncovered a high degree of differentially expressed genes in cultures from the same donor. RNAseq on differentiated cultures showed less variation between growth medias but alterations in pathways that control the expression of human transmembrane proteases including TMPRSS11 and TMPRSS2 were documented. Enhanced susceptibility to SARS-CoV-2 cannot be explained by altered cell type proportions alone, rather serine protease cofactor expression also contributes to the enhanced replication of SARS-CoV-2 as inhibition with camostat affected replication of an early SARS-CoV-2 variant and a Delta, but not Omicron, variant showed difference in replication efficiency between culture types. Therefore, it is important for the research community to standardize cell culture protocols particularly when characterizing novel viruses.

microbiology↗

Highly pathogenic avian influenza A virus (HPAIV) H5N1 infection in two European grey seals (Halichoerus grypus) with encephalitis.

Recent reports documenting sporadic infections in carnivorous mammals worldwide with highly pathogenic avian influenza virus (HPAIV) H5N1 clade 2.3.4.4b have raised concerns about the potential risk of adaptation to sustained transmission in mammals, including humans. We report H5N1 clade 2.3.4.4b infection of two grey seals (Halichoerus grypus) from coastal waters of The Netherlands and Germany in December 2022 and February 2023, respectively. Histological and immunohistochemical investigations showed in both animals a non-suppurative and necrotizing encephalitis with viral antigen restricted to the neuroparenchyma. Whole genome sequencing showed the presence of HPAIV H5N1 clade 2.3.4.4b strains in brain tissue, which were closely related to sympatric avian influenza viruses. Viral RNA was also detected in the lung of the seal from Germany by real-time quantitative PCR. No other organs tested positive. The mammalian adaptation PB2-E627K mutation was identified in approximately 40% of the virus population present in the brain tissue of the German seal. Retrospective screening for nucleoprotein specific antibodies, of sera collected from 251 seals sampled in this region from 2020 to 2023, did not show evidence of influenza A virus specific antibodies. Similarly, screening by reverse transcription PCR of lung and brain tissue of 101 seals that had died along the Dutch coast in the period 2020-2021, did not show evidence of influenza virus infection. Collectively, these results indicate that individual seals are sporadically infected with HPAIV-H5N1 clade 2.3.4.4b, resulting in an encephalitis in the absence of a systemic infection, and with no evidence thus far of onward spread between seals.

ecology↗

Single-cycle SARS-CoV-2 vaccine elicits high protection and sterilizing immunity in hamsters

Vaccines have been central in ending the COVID-19 pandemic, but newly emerging SARS-CoV-2 variants increasingly escape first-generation vaccine protection. To fill this gap, live particle-based vaccines mimicking natural infection aim at protecting against a broader spectrum of virus variants. We designed "single-cycle SARS-CoV-2 viruses" (SCVs) that lack essential viral genes, possess superior immune-modulatory features and provide an excellent safety profile in the Syrian hamster model. Full protection of all intranasally vaccinated animals was achieved against an autologous challenge with SARS-CoV-2 virus using an Envelope-gene-deleted vaccine candidate. By deleting key immune-downregulating genes, sterilizing immunity was achieved with an advanced candidate without virus spread to contact animals. Hence, SCVs have the potential to induce a broad and durable protection against COVID-19 superior to a natural infection.

molecular biology↗

Dogs and cats are less susceptible to the omicron variant of concern of SARS-CoV-2 - a field study

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic of unprecedented extent. Beside humans, a number of animal species can be infected, however, in some species differing susceptibilities were observed depending on the virus variant. Here, we serologically investigated cats and dogs living in households with human COVID-19 patients. The study was conducted during the transition period from delta as the dominating variant of concern (VOC) to omicron (BA.1/BA.2) to investigate the frequency of virus transmission of both VOCs from infected owners to their pets. The animal sera were tested by surrogate virus neutralization tests (sVNT) using either the original receptor-binding domain (RBD), enabling the detection of antibodies against the delta variant, or an omicron-specific RBD. Of the 290 canine samples, 20 tested positive by sVNT, but there were marked differences between the sampling time, and, related thereto, the virus variants, the dogs had contact to. While in November 2021 infected owners led to 50% seropositive dogs (18/36), only 0.8% (2/254) of animals with household contacts to SARS-CoV-2 between December 2021 and April 2022 tested positive. In all cases, the positive reaction was recorded against the original RBD. For cats, a similar pattern was seen, as in November 2021 38.1% (16/42) tested positive and between December 2021 and March 2022 only 5.0% (10/199). The markedly reduced ratio of seropositive animals during the period of omicron circulation suggests a considerably lower susceptibility of dogs and cats to this VOC. To examine the effect of BA.2, BA.4 and BA.5 omicron subvariants, sera taken in the second and third quarter of 2022 from randomly selected cats were investigated. 2.3% (11/372) tested seropositive and all of them showed a stronger reaction against the original RBD, further supporting the assumption of a lower susceptibility of companion animals to the omicron VOC.

microbiology↗

Assessment of Immunogenicity and Efficacy of CV0501 mRNA-based Omicron COVID-19 Vaccination in Small Animal Models

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron and its subvariants (BA.2, BA.4, BA.5) represent the most commonly circulating variants of concern (VOC) in the coronavirus disease 2019 (COVID-19) pandemic in 2022. Despite high vaccination rates with approved SARS-CoV-2 vaccines encoding the ancestral spike (S) protein, these Omicron subvariants have collectively resulted in increased viral transmission and disease incidence. This necessitates the development and characterization of vaccines incorporating later emerging S proteins to enhance protection against VOC. In this context, bivalent vaccine formulations may induce broad protection against VOC and potential future SARS-CoV-2 variants. Here, we report preclinical data for a lipid nanoparticle (LNP)-formulated RNActive(R) N1-methylpseudouridine (N1m{Psi}) modified mRNA vaccine (CV0501) based on our second-generation SARS-CoV-2 vaccine CV2CoV, encoding the S protein of Omicron BA.1. The immunogenicity of CV0501, alone or in combination with a corresponding vaccine encoding the ancestral S protein (ancestral N1m{Psi}), was first measured in dose-response and booster immunization studies performed in Wistar rats. Both monovalent CV0501 and bivalent CV0501/ancestral N1m{Psi} immunization induced robust neutralizing antibody titers against the BA.1, BA.2 and BA.5 Omicron subvariants, in addition to other SARS-CoV-2 variants in a booster immunization study. The protective efficacy of monovalent CV0501 against live SARS-CoV-2 BA.2 infection was then assessed in hamsters. Monovalent CV0501 significantly reduced SARS-CoV-2 BA.2 viral loads in the airways, demonstrating protection induced by CV0501 vaccination. CV0501 has now advanced into human Phase 1 clinical trials (ClinicalTrials.gov Identifier: NCT05477186).

immunology↗

West Nile Virus spread in Europe - phylogeographic pattern analysis and key drivers

Spread and emergence of West Nile virus (WNV) in Europe have been very different from those observed in North America. Here, we describe key drivers by combining viral genome sequences with epidemiological data and possible factors of spread into phylodynamic models. WNV in Europe has greater lineage diversity than other regions of the world, suggesting repeated introductions and local amplification. Among the six lineages found in Europe, WNV-2a is predominant, has spread to at least 14 countries and evolved into two major co-circulating clusters (A and B). Both of these seem to originate from regions of Central Europe. Viruses of Cluster A emerged earlier and have spread towards the west of Europe with higher genetic diversity. Amongst multiple drivers, high agriculture activities were associated with both spread direction and velocity. Our study suggests future surveillance activities should be strengthened in Central Europe and Southeast European countries, and enhanced monitoring should be targeted to areas with high agriculture activities.

evolutionary biology↗

Tautomer-specific deacylation and {Omega}-loop flexibility explain carbapenem-hydrolyzing, broad-spectrum activity of the KPC-2 β-lactamase

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-{beta}-lactamase (SBL) responsible for extensive {beta}-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate {beta}-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent {beta}-lactams, evade activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently catalyze deacylation of carbapenem-derived acyl-enzymes. We present high-resolution (1.25-1.4 [A]) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin) and carbapenems (imipenem, meropenem and ertapenem), obtained utilizing an isosteric deacylation-deficient mutant (E166Q). Mobility of the {Omega}-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different {beta}-lactams. Carbapenem-derived acyl-enzyme structures reveal predominance of the {Delta}1-(2R) imine tautomer, except for the imipenem acyl-enzyme, which is present in dual occupancy in both {Delta}1-(2R) and (2S) configurations. Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of deacylation of the KPC-2:meropenem acyl-enzyme, using an adaptive string method (ASM), show that the {Delta}1-(2R) isomer has a 7 kcal/mol higher barrier for the (rate-determining) formation of the tetrahedral deacylation intermediate than the {Delta}2 tautomer. The simulations identify tautomer-specific differences in hydrogen bonding networks involving the carbapenem C-3 carboxylate and the deacylating water, that, together with stabilization by protonated N-4 of accumulating negative charge during oxyanion formation, accelerate deacylation of the {Delta}2-enamine acyl-enzyme compared to the {Delta}1-imine. Taken together, our data show how the flexible {Omega}-loop helps confer broad spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the {Delta}2-enamine acyl-enzyme tautomer. Differentiation of the barriers associated with deacylation of these subtly different {beta}-lactam isomers further identifies ASM as a sensitive method for calculation of reaction energetics that can accurately model turnover and, potentially, predict the impact of substrate modifications or point mutations upon activity.

biochemistry↗

An advanced sequence clustering and designation workflow reveals the enzootic maintenance of a dominant West Nile virus subclade in Germany

West Nile virus (WNV) is the most widespread arthropod-borne (arbo) virus and the primary cause of arboviral encephalitis globally. Members of WNV species genetically diverged and are classified into different hierarchical groups below species rank. However, the demarcation criteria for allocating WNV sequences into these groups remain individual, inconsistent, and the use of names for different levels of the hierarchical levels is unstructured. In order to have an objective and comprehensible grouping of WNV sequences, we developed an advanced grouping workflow using the "affinity propagation clustering"-algorithm and newly included the "agglomerative hierarchical clustering"-algorithm for the allocation of WNV sequences into different groups below species rank. In addition, we propose to use a fixed set of terms for the hierarchical naming of WNV below species level and a clear decimal numbering system to label the determined groups. For validation, we applied the refined workflow to WNV sequences that have been previously grouped into various lineages, clades, and clusters in other studies. Although our workflow regrouped some WNV sequences, overall, it generally corresponds with previous groupings. We employed our novel approach to the sequences from the WNV circulation in Germany 2020, primarily from WNV-infected birds and horses. Besides two newly defined minor (sub)clusters comprising only of three sequences each, subcluster 2.5.3.4.3c was the predominant WNV sequence group detected in Germany from 2018-20. This predominant subcluster was also associated with at least five human WNV-infections in 2019-20. In summary, our analyses imply that the genetic diversity of the WNV population in Germany is shaped by enzootic maintenance of the dominant WNV subcluster accompanied by sporadic incursions of other rare clusters and subclusters. Moreover, we show that our refined approach for sequence grouping yields meaningful results. Although we primarily aimed at a more detailed WNV classification, the presented workflow can also be applied to the objective genotyping of other virus species.

genetics↗

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.

genomics↗

Cold-passaged isolates and bat-swine influenza A chimeric viruses as modified live-attenuated vaccines against influenza A viruses in pigs

Swine influenza A virus (swIAV) infections in pig populations cause considerable morbidity and economic losses. Frequent reverse zoonotic incursions of human IAV boost reassortment opportunities with authentic porcine and avian-like IAV in swine herds potentially enhancing zoonotic and even pre-pandemic potential. Vaccination using adjuvanted inactivated full virus vaccines is frequently used in attempting control of swIAV infections. Accelerated antigenic drift of swIAV in large swine holdings and interference of maternal antibodies with vaccine in piglets can compromise these efforts. Potentially more efficacious modified live-attenuated vaccines (MLVs) bear the risk of reversion of MLV to virulence. Here we evaluated new MLV candidates based on cold-passaged swIAV or on reassortment-incompetent bat-IAV-swIAV chimeric viruses. Serial cold-passaging of various swIAV subtypes did not yield unambiguously temperature-sensitive mutants although safety studies in mice and pigs suggested some degree of attenuation. Chimeric bat-swIAV expressing the hemagglutinin and neuraminidase of an avian-like H1N1, in contrast, proved to be safe in mice and pigs, and a single nasal inoculation induced protective immunity against homologous challenge in pigs. Reassortant-incompetent chimeric bat-swIAV vaccines could aid in reducing the amount of swIAV circulating in pig populations, thereby increasing animal welfare, limiting economic losses and lowering the risk of zoonotic swIAV transmission.

microbiology↗