TY - JOUR AB - UNLABELLED: The rapid increase in antibiotic resistance has led to a renewed interest in phage therapy, which has created a need for the establishment of phage collections in order to preserve diverse phages and to reduce the delivery time to patients. However, there are currently no standard methods for the long-term preservation of phages. We assessed the stability of four different phages under distinct storage conditions, including different temperatures, storage solutions, concentrations, and with or without cryoprotectant. We found that the type of storage buffer has a significant impact on phage stability, followed by the storage temperature. Phages demonstrated higher viability in lysogeny broth (LB) than saline-magnesium (SM) buffer without gelatin. We also observed a higher sensitivity to freezing in tailed phages with longer tails, such as T4. Ultimately, we found that all four phages maintained high stability after snap freezing, followed by storage at -80°C using LB as a storage buffer without cryoprotectant. IMPORTANCE: Phage therapy, which involves treating bacterial infections using bacteriophages (phage), has shown promise as an alternative to antibiotics and can offer a solution for treating infections caused by antibiotic-resistant bacteria. However, phages are not conventional drugs and can lose their viability when stored under unsuitable conditions. Their high diversity makes finding a standard storage method for long-term preservation challenging. Here, we studied the stability of phages under different storage conditions and identified key factors affecting their viability. We have also identified a specific storage condition that can effectively preserve a wide range of phage morphotypes for over 2 years. AU - Huang, W. AU - Khan Mirzaei, M. AU - Deng, L. C1 - 74126 C2 - 57314 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Comparative evaluation of long-term preservation methods for morphologically distinct bacteriophages. JO - Microbiol. Spectr. VL - 13 IS - 7 PB - Amer Soc Microbiology PY - 2025 SN - 2165-0497 ER - TY - JOUR AB - The genus Rhodococcus is recognized for its potential to degrade a large range of aromatic substances, including plant-derived phenolic compounds. We used comparative genomics in the context of the broader Rhodococcus pan-genome to study genomic traits of two newly described Rhodococcus strains (type-strain Rhodococcus pseudokoreensis R79T and Rhodococcus koreensis R85) isolated from apple rhizosphere. Of particular interest was their ability to degrade phenolic compounds as part of an integrated approach to treat apple replant disease (ARD) syndrome. The pan-genome of the genus Rhodococcus based on 109 high-quality genomes was open with a small core (1.3%) consisting of genes assigned to basic cell functioning. The range of genome sizes in Rhodococcus was high, from 3.7 to 10.9 Mbp. Genomes from host-associated strains were generally smaller compared to environmental isolates which were characterized by exceptionally large genome sizes. Due to large genomic differences, we propose the reclassification of distinct groups of rhodococci like the Rhodococcus equi cluster to new genera. Taxonomic species affiliation was the most important factor in predicting genetic content and clustering of the genomes. Additionally, we found genes that discriminated between the strains based on habitat. All members of the genus Rhodococcus had at least one gene involved in the pathway for the degradation of benzoate, while biphenyl degradation was mainly restricted to strains in close phylogenetic relationships with our isolates. The ~40% of genes still unclassified in larger Rhodococcus genomes, particularly those of environmental isolates, need more research to explore the metabolic potential of this genus.IMPORTANCERhodococcus is a diverse, metabolically powerful genus, with high potential to adapt to different habitats due to the linear plasmids and large genome sizes. The analysis of its pan-genome allowed us to separate host-associated from environmental strains, supporting taxonomic reclassification. It was shown which genes contribute to the differentiation of the genomes based on habitat, which can possibly be used for targeted isolation and screening for desired traits. With respect to apple replant disease (ARD), our isolates showed genome traits that suggest potential for application in reducing plant-derived phenolic substances in soil, which makes them good candidates for further testing against ARD. AU - Benning, S. AU - Pritsch, K. AU - Radl, V. AU - Siani, R. AU - Wang, Z. AU - Schloter, M. C1 - 70057 C2 - 55387 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - (Pan)genomic analysis of two Rhodococcus isolates and their role in phenolic compound degradation. JO - Microbiol. Spectr. VL - 12 IS - 4 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - SARS-CoV-2 spreads pandemically since 2020; in 2021, effective vaccinations became available and vaccination campaigns commenced. Still, it is hard to track the spread of the infection or to assess vaccination success in the broader population. Measuring specific anti-SARS-CoV-2 antibodies is the most effective tool to track the spread of the infection or successful vaccinations. The need for venous-blood sampling however poses a significant barrier for large studies. Dried-blood-spots on filter-cards (DBS) have been used for SARS-CoV-2 serology in our laboratory, but so far not to follow quantitative SARS-CoV-2 anti-spike reactivity in a longitudinal cohort. We developed a semi-automated protocol or quantitative SARS-CoV-2 anti-spike serology from self-sampled DBS, validating it in a cohort of matched DBS and venous-blood samples (n = 825). We investigated chromatographic effects, reproducibility, and carry-over effects and calculated a positivity threshold as well as a conversion formula to determine the quantitative binding units in the DBS with confidence intervals. Sensitivity and specificity reached 96.63% and 97.81%, respectively, compared to the same test performed in paired venous samples. Between a signal of 0.018 and 250 U/mL, we calculated a correction formula. Measuring longitudinal samples during vaccinations, we demonstrated relative changes in titers over time in several individuals and in a longitudinal cohort over four follow-ups. DBS sampling has proven itself for anti-nucleocapsid serosurveys in our laboratory. Similarly, anti-spike high-throughput DBS serology is feasible as a complementary assay. Quantitative measurements are accurate enough to follow titer dynamics in populations also after vaccination campaigns. This work was supported by the Bavarian State Ministry of Science and the Arts; LMU University Hospital, LMU Munich; Helmholtz Center Munich; University of Bonn; University of Bielefeld; German Ministry for Education and Research (proj. nr.: 01KI20271 and others) and the Medical Biodefense Research Program of the Bundeswehr Medical Service. Roche Diagnostics provided kits and machines for analyses at discounted rates. The project is funded also by the European-wide Consortium ORCHESTRA. The ORCHESTRA project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 101016167. The views expressed in this publication are the sole responsibility of the author, and the Commission is not responsible for any use that may be made of the information it contains.IMPORTANCESARS-CoV-2 has been spreading globally as a pandemic since 2020. To determine the prevalence of SARS-CoV-2 antibodies among populations, the most effective public health tool is measuring specific anti-SARS-CoV-2 antibodies induced by infection or vaccination. However, conducting large-scale studies that involve venous-blood sampling is challenging due to the associated feasibility and cost issues. A more cost-efficient and less invasive method for SARS-CoV-2 serological testing is using Dried-Blood-Spots on filter cards (DBS). In this paper, we have developed a semi-automated protocol for quantifying SARS-CoV-2 anti-spike antibodies from self-collected DBS. Our laboratory has previously successfully used DBS sampling for anti-nucleocapsid antibody surveys. Likewise, conducting high-throughput DBS serology for anti-spike antibodies is feasible as an additional test that can be performed using the same sample preparation as the anti-nucleocapsid analysis. The quantitative measurements obtained are accurate enough to track the dynamics of antibody levels in populations, even after vaccination campaigns. SARS-CoV-2 has been spreading globally as a pandemic since 2020. To determine the prevalence of SARS-CoV-2 antibodies among populations, the most effective public health tool is measuring specific anti-SARS-CoV-2 antibodies induced by infection or vaccination. However, conducting large-scale studies that involve venous-blood sampling is challenging due to the associated feasibility and cost issues. A more cost-efficient and less invasive method for SARS-CoV-2 serological testing is using Dried-Blood-Spots on filter cards (DBS). In this paper, we have developed a semi-automated protocol for quantifying SARS-CoV-2 anti-spike antibodies from self-collected DBS. Our laboratory has previously successfully used DBS sampling for anti-nucleocapsid antibody surveys. Likewise, conducting high-throughput DBS serology for anti-spike antibodies is feasible as an additional test that can be performed using the same sample preparation as the anti-nucleocapsid analysis. The quantitative measurements obtained are accurate enough to track the dynamics of antibody levels in populations, even after vaccination campaigns. AU - Castelletti, N. AU - Paunovic, I.* AU - Rubio-Acero, R.* AU - Beyerl, J.* AU - Plank, M.* AU - Reinkemeyer, C.* AU - Kroidl, I.* AU - Noreña, I.* AU - Winter, S.* AU - Olbrich, L.* AU - Janke, C.* AU - Hoelscher, M.* AU - Wieser, A.* C1 - 70411 C2 - 55526 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - A Dried Blood Spot protocol for high-throughput quantitative analysis of SARS-CoV-2 RBD serology based on the Roche Elecsys system. JO - Microbiol. Spectr. VL - 12 IS - 4 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - UNLABELLED: The key to a curative treatment of hepatitis B virus (HBV) infection is the eradication of the intranuclear episomal covalently closed circular DNA (cccDNA), the stable persistence reservoir of HBV. Currently, established therapies can only limit HBV replication but fail to tackle the cccDNA. Thus, novel therapeutic approaches toward curative treatment are urgently needed. Recent publications indicated a strong association between the HBV core protein SUMOylation and the association with promyelocytic leukemia nuclear bodies (PML-NBs) on relaxed circular DNA to cccDNA conversion. We propose that interference with the cellular SUMOylation system and PML-NB integrity using arsenic trioxide provides a useful tool in the treatment of HBV infection. Our study showed a significant reduction in HBV-infected cells, core protein levels, HBV mRNA, and total DNA. Additionally, a reduction, albeit to a limited extent, of HBV cccDNA could be observed. Furthermore, this interference was also applied for the treatment of an established HBV infection, characterized by a stably present nuclear pool of cccDNA. Arsenic trioxide (ATO) treatment not only changed the amount of expressed HBV core protein but also induced a distinct relocalization to an extranuclear phenotype during infection. Moreover, ATO treatment resulted in the redistribution of transfected HBV core protein away from PML-NBs, a phenotype similar to that previously observed with SUMOylation-deficient HBV core. Taken together, these findings revealed the inhibition of HBV replication by ATO treatment during several steps of the viral replication cycle, including viral entry into the nucleus as well as cccDNA formation and maintenance. We propose ATO as a novel prospective treatment option for further pre-clinical and clinical studies against HBV infection. IMPORTANCE: The main challenge for the achievement of a functional cure for hepatitis B virus (HBV) is the covalently closed circular DNA (cccDNA), the highly stable persistence reservoir of HBV, which is maintained by further rounds of infection with newly generated progeny viruses or by intracellular recycling of mature nucleocapsids. Eradication of the cccDNA is considered to be the holy grail for HBV curative treatment; however, current therapeutic approaches fail to directly tackle this HBV persistence reservoir. The molecular effect of arsenic trioxide (ATO) on HBV infection, protein expression, and cccDNA formation and maintenance, however, has not been characterized and understood until now. In this study, we reveal ATO treatment as a novel and innovative therapeutic approach against HBV infections, repressing viral gene expression and replication as well as the stable cccDNA pool at low micromolar concentrations by affecting the cellular function of promyelocytic leukemia nuclear bodies. AU - Hofmann, S.* AU - Luther, J.* AU - Plank, V.* AU - Oswald, A.* AU - Mai, J.* AU - Simons, I.M.* AU - Miller, J.* AU - Falcone, V.* AU - Hansen-Palmus, L.* AU - Hengel, H.* AU - Nassal, M.* AU - Protzer, U. AU - Schreiner, S.* C1 - 70441 C2 - 55629 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Arsenic trioxide impacts hepatitis B virus core nuclear localization and efficiently interferes with HBV infection. JO - Microbiol. Spectr. VL - 12 IS - 5 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - In our study, we aimed to explore the genomic and phenotypic traits of Priestia megaterium strain B1, which was isolated from root material of healthy apple plants, to adapt to the endophytic lifestyle and promote plant growth. We identified putative genes encoding proteins involved in chemotaxis, flagella biosynthesis, biofilm formation, secretory systems, detoxification, transporters, and transcription regulation. Furthermore, B1 exhibited both swarming and swimming motilities, along with biofilm formation. Both genomic and physiological analyses revealed the potential of B1 to promote plant growth through the production of indole-3-acetic acid and siderophores, as well as the solubilization of phosphate and zinc. To deduce potential genomic features associated with endophytism across members of P. megaterium strains, we conducted a comparative genomic analysis involving 27 and 31 genomes of strains recovered from plant and soil habitats, respectively, in addition to our strain B1. Our results indicated a closed pan genome and comparable genome size of strains from both habitats, suggesting a facultative host association and adaptive lifestyle to both habitats. Additionally, we performed a sparse Partial Least Squares Discriminant Analysis to infer the most discriminative functional features of the two habitats based on Pfam annotation. Despite the distinctive clustering of both groups, functional enrichment analysis revealed no significant enrichment of any Pfam domain in both habitats. Furthermore, when assessing genetic elements related to adaptation to endophytism in each individual strain, we observed their widespread presence among strains from both habitats. Moreover, all members displayed potential genetic elements for promoting plant growth.IMPORTANCEBoth genomic and phenotypic analyses yielded valuable insights into the capacity of P. megaterium B1 to adapt to the plant niche and enhance its growth. The comparative genomic analysis revealed that P. megaterium members, whether derived from soil or plant sources, possess the essential genetic machinery for interacting with plants and enhancing their growth. The conservation of these traits across various strains of this species extends its potential application as a bio-stimulant in diverse environments. This significance also applies to strain B1, particularly regarding its application to enhance the growth of plants facing apple replant disease conditions. AU - Mahmoud, F.M. AU - Pritsch, K. AU - Siani, R. AU - Benning, S. AU - Radl, V. AU - Kublik, S. AU - Bunk, B.* AU - Spröer, C.* AU - Schloter, M. C1 - 70899 C2 - 55972 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Comparative genomic analysis of strain Priestia megaterium B1 reveals conserved potential for adaptation to endophytism and plant growth promotion. JO - Microbiol. Spectr. VL - 12 IS - 8 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - UNLABELLED: Staphylococcus aureus strains exhibit varying associations with atopic dermatitis (AD), but the genetic determinants underpinning the pathogenicity are yet to be fully characterized. To reveal the genetic differences between S. aureus strains from AD patients and healthy individuals (HE), we developed and employed a random forest classifier to identify potential marker genes responsible for their phenotypic variations. The classifier was able to effectively distinguish strains from AD and HE. We also uncovered strong links between certain marker genes and phage functionalities, with phage holin emerging as the most pivotal differentiating factor. Further examination of S. aureus gene content highlighted the genetic diversity and functional implications of prophages in driving differentiation between strains from AD and HE. The HE group exhibited greater gene content diversity, largely influenced by their prophages. While strains from both AD and HE universally housed prophages, those in the HE group were distinctively higher at the strain level. Moreover, although prophages in the HE group exhibited variously higher enrichment of differential functions, the AD group displayed a notable enrichment of virulence factors within their prophages, underscoring the important contribution of prophages to the pathogenesis of AD-associated strains. Overall, prophages significantly shape the genetic and functional profiles of S. aureus strains, shedding light on their pathogenic potential and elucidating the mechanisms behind the phenotypic variations in AD and HE environments. IMPORTANCE: Through a nuanced exploration of Staphylococcus aureus strains obtained from atopic dermatitis (AD) patients and healthy controls (HE), our research unveils pivotal genetic determinants influencing their pathogenic associations. Utilizing a random forest classifier, we illuminate distinct marker genes, with phage holin emerging as a critical differential factor, revealing the profound impact of prophages on genetic and pathogenic profiles. HE strains exhibited a diverse gene content, notably shaped by unique, heightened prophages. Conversely, AD strains emphasized a pronounced enrichment of virulence factors within prophages, signifying their key role in AD pathogenesis. This work crucially highlights prophages as central architects of the genetic and functional attributes of S. aureus strains, providing vital insights into pathogenic mechanisms and phenotypic variations, thereby paving the way for targeted AD therapeutic approaches and management strategies by demystifying specific genetic and pathogenic mechanisms. AU - Wang, Z. AU - Peng, X. AU - Hülpüsch, C. AU - Khan Mirzaei, M. AU - Reiger, M. AU - Traidl-Hoffmann, C. AU - Deng, L. AU - Schloter, M. C1 - 71158 C2 - 56007 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Distinct prophage gene profiles of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals. JO - Microbiol. Spectr. VL - 12 IS - 8 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - Atopic dermatitis (AD) is the most common chronic inflammatory skin disease worldwide and is characterized by a complex interplay with skin microbiota, with Staphylococcus aureus often abnormally more abundant in AD patients than in healthy individuals (HE). S. aureus harbors diverse strains with varied genetic compositions and functionalities, which exhibit differential connections with the severity of AD. However, the differences in S. aureus strains between AD and HE remain unclear, with most variations seen at a specific geographic level, implying spontaneous adaptations rather than systematic distinctions. This study presents genomic and functional differences between these S. aureus strains from AD and HE on both global and local levels. We observed reduced gene content diversity but increased functional variation in the global AD-associated strains. Two additional AD-dominant clusters emerged, with Cluster 1 enriched in transposases and Cluster 2 showcasing genes linked to adaptability and antibiotic resistance. Particularly, robust evidence illustrates that the lantibiotic operon of S. aureus, involved in the biosynthesis of lantibiotics, was acquired via horizontal gene transfer from environmental bacteria. Comparisons of the gene abundance profiles in functional categories also indicate limited zoonotic potential between human and animal isolates. Local analysis mirrored global gene diversity but showed distinct functional variations between AD and HE strains. Overall, this research provides foundational insights into the genomic evolution, adaptability, and antibiotic resistance of S. aureus, with significant implications for clinical microbiology.IMPORTANCEOur study uncovers significant genomic variations in Staphylococcus aureus strains associated with atopic dermatitis. We observed adaptive evolution tailored to the disease microenvironment, characterized by a smaller pan-genome than strains from healthy skin both on the global and local levels. Key functional categories driving strain diversification include "replication and repair" and "transporters," with transposases being pivotal. Interestingly, the local strains predominantly featured metal-related genes, whereas global ones emphasized antimicrobial resistances, signifying scale-dependent diversification nuances. We also pinpointed horizontal gene transfer events, indicating interactions between human-associated and environmental bacteria. These insights expand our comprehension of S. aureus's genetic adaptation in atopic dermatitis, yielding valuable implications for clinical approaches. AU - Wang, Z. AU - Hülpüsch, C. AU - Fösel, B. AU - Traidl-Hoffmann, C. AU - Reiger, M. AU - Schloter, M. C1 - 71502 C2 - 56218 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Genomic and functional divergence of Staphylococcus aureus strains from atopic dermatitis patients and healthy individuals: Insights from global and local scales. JO - Microbiol. Spectr. VL - 12 IS - 10 PB - Amer Soc Microbiology PY - 2024 SN - 2165-0497 ER - TY - JOUR AB - Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA. AU - Hofmann, S.* AU - Plank, V.* AU - Groitl, P.* AU - Skvorc, N.* AU - Hofmann, K.* AU - Luther, J.* AU - Ko, C. AU - Zimmerman, P.* AU - Bruss, V. AU - Stadler, D. AU - Carpentier, A.* AU - Rezk, S.* AU - Nassal, M.* AU - Protzer, U. AU - Schreiner, S. C1 - 67922 C2 - 54400 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - SUMO modification of hepatitis B virus core mediates nuclear entry, promyelocytic leukemia nuclear body association, and efficient formation of covalently closed circular DNA. JO - Microbiol. Spectr. VL - 11 IS - 3 PB - Amer Soc Microbiology PY - 2023 SN - 2165-0497 ER - TY - JOUR AB - Due to rising antibiotic resistance, there is an urgent need for different treatment options for multidrug-resistant infections. One alternative under investigation is phage therapy, which uses phages to treat bacterial infections. Although phages are highly abundant in the environment, not all phages are suitable for phage therapy, and finding efficient phages that lack undesirable traits such as bacterial virulence factors is challenging. Here, we developed a targeted single-phage isolation method to detect and isolate phages of interest and to characterize their kinetics in a high-throughput manner. This assay has also revealed cell-to-cell variations at a single-cell level among cells infected with the same phage species, as well as among cells infected with different phage species. IMPORTANCE The spread of multidrug-resistant bacteria is a global human health threat, and without immediate action we are fast approaching a postantibiotic era. One possible alternative to antibiotics is the use of phages, that is, bacterial viruses. However, the isolation of phages that effectively kill their target bacteria has proven challenging. In addition, isolated phages must go through significant characterization before their efficacy is measured. The method developed in this work can isolate single phage particles on the basis of their similarity to previously characterized phages while excluding those with known undesirable traits, such as bacterial toxins, as well as characterizing their kinetics. Using this method, we revealed significant cell-to-cell variations in phage kinetics at a single-cell level among highly virulent phages. These results shed some light on unknown phage-bacterium interactions at the single-cell level. AU - Unterer, M. AU - Mirzaei, M.K. AU - Deng, L. C1 - 68439 C2 - 55156 CY - 1752 N St Nw, Washington, Dc 20036-2904 Usa TI - Targeted single-phage isolation reveals phage-dependent heterogeneous infection dynamics. JO - Microbiol. Spectr. VL - 11 IS - 3 PB - Amer Soc Microbiology PY - 2023 SN - 2165-0497 ER - TY - JOUR AB - The continuous genetic evolution of SARS-CoV-2 resulting in the immune escape variant of concern (VoC) Omicron poses a challenge to rapid and accurate diagnosis of infection, especially in high-risk groups such as healthcare workers. We performed a clinical study to determine the diagnostic accuracy and robustness of a second-generation rapid antigen test compared to a first-generation rapid antigen test with an RT-qPCR-based assay as gold standard, for early detection of infections with SARS-CoV-2 Omicron VoC. A total of 428 healthcare workers with COVID-19-associated symptoms or during routine testing participated in the study and completed a questionnaire on infection-associated symptoms, previous SARS-CoV-2 infections, and vaccination status. All participants performed a second- and first-generation rapid antigen test on the day of presentation and repeated the test 2 days later, and a diagnostic SARS-CoV-2 RT-qPCR assay was performed. qPCR-confirmed SARS-CoV-2 infections (n = 104) with Omicron VoC (BA.2, BA.4, BA.5) were detected by the first-generation rapid antigen test with a sensitivity of 83.7% (95% CI 75.12%-90.18%), whereas the second-generation rapid antigen test performed with a sensitivity of 89.4% (95% CI 81.9%-94.6%). Increased sensitivity of the second-generation rapid antigen test led to earlier detection of SARS-CoV-2 infection, while lower test sensitivity of the first-generation rapid antigen test was compensated by repeated testing 2 days later. Moreover, direct in vitro comparison revealed a lower limit of detection for the second-generation rapid antigen test for isolates of currently circulating Omicron sub-lineages BA.5.2, BQ.1, and XBB.1. IMPORTANCE The results from this study demonstrate the usefulness of a second-generation rapid antigen test for early detection of infection with the SARS-CoV-2 Omicron variant of concern (VoC) and reveal a higher sensitivity to detect immune escape Omicron VoCs compared to a first-generation rapid antigen test (89.4% vs 83.7%) in the high-risk group of healthcare workers. AU - Wettengel, J.* AU - Strehle, K.* AU - von Lucke, C.* AU - Roggendorf, H.* AU - Jeske, S.D.* AU - Christa, C.* AU - Zelger, O.* AU - Haller, B.* AU - Protzer, U. AU - Knolle, P.A.* C1 - 68658 C2 - 54863 TI - Improved detection of infection with SARS-CoV-2 Omicron variants of concern in healthcare workers by a second-generation rapid antigen test. JO - Microbiol. Spectr. VL - 11 IS - 6 PY - 2023 SN - 2165-0497 ER - TY - JOUR AB - Promyelocytic leukemia nuclear bodies (PML-NBs) were considered to maintain antiviral capacity, as these spherical complexes are antagonized by viruses. Actual work provides evidence, that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Several early HAdV proteins target PML-NBs, such as E4orf3 that promotes redistribution into track-like structures. PML-associated dependency factors that enhance viral gene expression, such as Sp100A remain in the nuclear tracks while restrictive factors, such as Daxx, are inhibited by either proteasomal degradation or relocalization to repress antiviral functions. Here, we did a comprehensive analysis of nuclear PML isoforms during HAdV infection. Our results show cell line specific differences as PML isoforms differentially regulate productive HAdV replication and progeny production. Here, we identified PML-II as a dependency factor that supports viral progeny production, while PML-III and PML-IV suppress viral replication. In contrast, we identified PML-I as a positive regulator and PML-V as a restrictive factor during HAdV infection. Solely PML-VI was shown to repress adenoviral progeny production in both model systems. We showed for the first time, that HAdV can reorganize PML-NBs that contain PML isoforms other then PML-II. Intriguingly, HAdV was not able to fully disrupt PML-NBs composed out of the PML isoforms that inhibit viral replication, while PML-NBs composed out of PML isoforms with beneficial influence on the virus formed tracks in all examined cells. In sum, our findings clearly illustrate the crucial role of PML-track formation in efficient viral replication. IMPORTANCE Actual work provides evidence that PML-NB-associated factors might also be beneficial for distinct viral processes indicating why genomes and replication centers of nuclear replicating viruses are often found juxtaposed to PML-NBs. Alternatively spliced PML isoforms I-VII are expressed from one single pml gene containing nine exons and their transcription is tightly controlled and stimulated by interferons and p53. Several early HAdV proteins target PML-NBs, such as E4orf3, promoting redistribution into track-like structures. Our comprehensive studies indicate a diverging role of PML isoforms throughout the course of productive HAdV infection in either stably transformed human lung (H1299) or liver (HepG2) cells, in which we observed a multivalent regulation of HAdV by all six PML isoforms. PML-I and PML-II support HAdV-mediated track formation and efficient formation of viral replication centers, thus promoting HAdV productive infection. Simultaneously, PML-III, -IV,-V, and -VI antagonize viral gene expression and particle production. AU - Mai, J.* AU - Stubbe, M.* AU - Hofmann, S.* AU - Masser, S.* AU - Dobner, T.* AU - Boutell, C.* AU - Groitl, P.* AU - Schreiner, S. C1 - 65432 C2 - 52678 TI - PML alternative splice products differentially regulate HAdV productive infection. JO - Microbiol. Spectr. VL - 10 IS - 4 PY - 2022 SN - 2165-0497 ER - TY - JOUR AB - Quick and accurate detection of neutralizing antibodies (nAbs) against yellow fever is essential in serodiagnosis during outbreaks for surveillance and to evaluate vaccine efficacy in population-wide studies. All of this requires serological assays that can process a large number of samples in a highly standardized format. Albeit being laborious, time-consuming, and limited in throughput, the classical plaque reduction neutralization test (PRNT) is still considered the gold standard for the detection and quantification of nAbs due to its sensitivity and specificity. Here, we report the development of an alternative fluorescence-based serological assay (SNTFLUO) with an equally high sensitivity and specificity that is fit for high-throughput testing with the potential for automation. Finally, our novel SNTFLUO was cross-validated in several reference laboratories and against international WHO standards, showing its potential to be implemented in clinical use. SNTFLUO assays with similar performance are available for the Japanese encephalitis, Zika, and dengue viruses amenable to differential diagnostics. IMPORTANCE Fast and accurate detection of neutralizing antibodies (nAbs) against yellow fever virus (YFV) is key in yellow fever serodiagnosis, outbreak surveillance, and monitoring of vaccine efficacy. Although classical PRNT remains the gold standard for measuring YFV nAbs, this methodology suffers from inherent limitations such as low throughput and overall high labor intensity. We present a novel fluorescence-based serum neutralization test (SNTFLUO) with equally high sensitivity and specificity that is fit for processing a large number of samples in a highly standardized manner and has the potential to be implemented for clinical use. In addition, we present SNTFLUO assays with similar performance for Japanese encephalitis, Zika, and dengue viruses, opening new avenues for differential diagnostics. AU - Rasulova, M.* AU - Vercruysse, T.* AU - Paulissen, J.* AU - Coun, C.* AU - Suin, V.* AU - Heyndrickx, L.* AU - Ma, J.* AU - Geerts, K.* AU - Timmermans, J.* AU - Mishra, N.* AU - Li, L.H.* AU - Kum, D.B.* AU - Coelmont, L.* AU - Van Gucht, S.* AU - Karimzadeh, H. AU - Thorn-Seshold, J. AU - Rothenfußer, S. AU - Ariën, K.K.* AU - Neyts, J.* AU - Dallmeier, K.* AU - Thibaut, H.J.* C1 - 65460 C2 - 52694 TI - A high-throughput yellow fever neutralization assay. JO - Microbiol. Spectr. VL - 10 IS - 3 PY - 2022 SN - 2165-0497 ER -