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Background During the ongoing Covid-19 pandemic caused by the emerging virus SARS-CoV-2, research in the field of coronaviruses has expanded tremendously. The genome of SARS-CoV-2 has rapidly acquired numerous mutations, giving rise to several Variants of Concern (VOCs) with altered epidemiological, immunological, and pathogenic properties. Methods As cell culture models are important tools to study viruses, we investigated replication kinetics and infectivity of SARS-CoV-2 in the African Green Monkey-derived Vero E6 kidney cell line and the two human cell lines Caco-2, a colon epithelial carcinoma cell line, and the airway epithelial carcinoma cell line Calu-3. We assessed viral RNA copy numbers and infectivity of viral particles in cell culture supernatants at different time points ranging from 2 to 96 h post-infection. Results We here describe a systematic comparison of growth kinetics of the five SARS-CoV-2 VOCs Alpha/B.1.1.7, Beta/B.1.351, Gamma/P.1, Delta/B.1.617.2, and Omicron/B.1.1.529 and a non-VOC/B.1.1 strain on three different cell lines to provide profound information on the differential behaviour of VOCs in different cell lines for researchers worldwide. We show distinct differences in viral replication kinetics of the SARS-CoV-2 non-VOC and five VOCs on the three cell culture models Vero E6, Caco-2, and Calu-3. Conclusion This is the first systematic comparison of all SARS-CoV-2 VOCs on three different cell culture models. This data provides support for researchers worldwide in their experimental design for work on SARS-CoV-2. It is recommended to perform virus isolation and propagation on Vero E6 while infection studies or drug screening and antibody-based assays should rather be conducted on the human cell lines Caco-2 and Calu-3.

A major ingredient in Higgs searches at the Tevatron and LHC is the elimination of backgrounds with jets. In current H → WW → ℓνℓν searches, jet algorithms are used to veto central jets to obtain a 0-jet sample, which is then analyzed to discover the Higgs signal. Imposing this tight jet veto induces large double logarithms which significantly modify the Higgs production cross section. These jet-veto logarithms are presently only accounted for at fixed order or with the leading-logarithmic summation from parton-shower Monte Carlos. Here we consider Higgs production with an inclusive event-shape variable for the jet veto, namely beam thrust , which has a close correspondence with a traditional p T jet veto. allows us to systematically sum the large jet-veto logarithms to higher orders and to provide better estimates for theoretical uncertainties. We present results for the 0-jet Higgs production cross section from gluon fusion at next-to-next-to-leading-logarithmic order (NNLL), fully incorporating fixed-order results at next-to-next-to-leading order (NNLO). At this order the scale uncertainty is 15−20%, depending on the cut, implying that a larger scale uncertainty should be used in current Tevatron bounds on the Higgs.

Summary Pseudomonas aeruginosa produces phenazine‐1‐carboxylic acid (PCA) and pyocyanin (PYO), which aid its anaerobic survival by mediating electron transfer to distant oxygen. These natural secondary metabolites are being explored in biotechnology to mediate electron transfer to the anode of bioelectrochemical systems. A major challenge is that only a small fraction of electrons from microbial substrate conversion is recovered. It remained unclear whether phenazines can re‐enter the cell and thus, if the electrons accessed by the phenazines arise mainly from cytoplasmic or periplasmic pathways. Here, we prove that the periplasmic glucose dehydrogenase (Gcd) of P. aeruginosa and P. putida is involved in the reduction of natural phenazines. PYO displayed a 60‐fold faster enzymatic reduction than PCA; PCA was, however, more stable for long‐term electron shuttling to the anode. Evaluation of a Gcd knockout and overexpression strain showed that up to 9% of the anodic current can be designated to this enzymatic reaction. We further assessed phenazine uptake with the aid of two molecular biosensors, which experimentally confirm the phenazines’ ability to re‐enter the cytoplasm. These findings significantly advance the understanding of the (electro) physiology of phenazines for future tailoring of phenazine electron discharge in biotechnological applications. Pseudomonas aeruginosa phenazines aid its anaerobic survival by mediating electron transfer to distant oxygen. It remained unclear whether phenazines can re‐enter the cell and thus, if the electrons accessed by the phenazines arise mainly from cytoplasmic or periplasmic pathways. Here, we prove that the periplasmic glucose dehydrogenase (Gcd) of P. aeruginosa and P. putida is involved in the reduction of natural phenazines, but we also experimentally confirm the phenazines’ ability to re‐enter the cytoplasm. ​

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis - and trans -acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses. IMPORTANCE Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses. Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.

The isolation and sequencing of new strains of Pseudomonas aeruginosa created an extensive dataset of closed genomes. Many of the publicly available genomes are only used in their original publication while additional in silico information, based on comparison to previously published genomes, is not being explored. In this study, we defined and investigated the genome of the environmental isolate P. aeruginosa KRP1 and compared it to more than 100 publicly available closed P. aeruginosa genomes. By using different genomic island prediction programs, we could identify a total of 17 genomic islands and 8 genomic islets, marking the majority of the accessory genome that covers ~ 12% of the total genome. Based on intra-strain comparisons, we are able to predict the pathogenic potential of this environmental isolate. It shares a substantial amount of genomic information with the highly virulent PSE9 and LESB58 strains. For both of these, the increased virulence has been directly linked to their accessory genome before. Hence, the integrated use of previously published data can help to minimize expensive and time consuming wetlab work to determine the pathogenetic potential.

Sufficient supply of oxygen is a major bottleneck in industrial biotechnological synthesis. One example is the heterologous production of rhamnolipids using Pseudomonas putida KT2440. Typically, the synthesis is accompanied by strong foam formation in the reactor vessel hampering the process. It is caused by the extensive bubbling needed to sustain the high respirative oxygen demand in the presence of the produced surfactants. One way to reduce the oxygen requirement is to enable the cells to use the anode of a bioelectrochemical system (BES) as an alternative sink for their metabolically derived electrons. We here used a P. putida KT2440 strain that interacts with the anode using mediated extracellular electron transfer via intrinsically produced phenazines, to perform heterologous rhamnolipid production under oxygen limitation. The strain P. putida RL-PCA successfully produced 30.4 ± 4.7 mg/L mono-rhamnolipids together with 11.2 ± 0.8 mg/L of phenazine-1-carboxylic acid (PCA) in 500-mL benchtop BES reactors and 30.5 ± 0.5 mg/L rhamnolipids accompanied by 25.7 ± 8.0 mg/L PCA in electrode containing standard 1-L bioreactors. Hence, this study marks a first proof of concept to produce glycolipid surfactants in oxygen-limited BES with an industrially relevant strain.

Rapid antigen tests (RATs) are an integral part of SARS-CoV-2 containment strategies. As emerging variants of concern (VOCs) displace the initially circulating strains, it is crucial that RATs do not fail to detect these new variants. In this study, four RATs for nasal swab testing were investigated using cultured strains of B.1.1 (non-VOC), B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta). Based on dilution series in cell culture medium and pooled saliva, the limit of detection of these RATs was determined in a laboratory setting. Further investigations on cross-reactivity were conducted using recombinant N-protein from seasonal human coronaviruses (hCoVs). RATs evaluated showed an overall comparable performance with cultured strains of the non-VOC B.1.1 and the VOCs Alpha, Beta, Gamma, and Delta. No cross-reactivity was detected with recombinant N-protein of the hCoV strains HKU1, OC43, NL63, and 229E. A continuous evaluation of SARS-CoV-2 RAT performance is required, especially with regard to evolving mutations. Moreover, cross-reactivity and interference with pathogens and other substances on the test performance of RATs should be consistently investigated to ensure suitability in the context of SARS-CoV-2 containment.