Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion

Clinical practice recommendations for management of lateropulsion after stroke determined by a Delphi expert panel

Objective: People exhibiting post-stroke lateropulsion actively push their body across the midline to the more affected side and/or resist weight shift toward the less affected side. Despite its prevalence and associated negative rehabilitation outcomes, no clinical practice guidelines exist for the rehabilitation of poststroke lateropulsion. We aimed to develop consensus-based clinical practice recommendations for managing post-stroke lateropulsion using an international expert panel. Design: This Delphi panel process conformed with Guidance on Conducting and Reporting Delphi Studies recommendations. Participants: Panel members had demonstrated clinical and/or scientific background in the rehabilitation of people with post-stroke lateropulsion. Main Measures: The process consisted of four electronic survey rounds. Round One consisted of 13 open questions. Subsequent rounds ascertained levels of agreement with statements derived from Round One. Consensus was defined a priori as ≥75% agreement (agree or strongly agree), or ≥70% agreement after excluding ‘unsure’ responses. Results: Twenty participants completed all four rounds. Consensus was achieved regarding a total of 119 recommendations for rehabilitation approaches and considerations for rehabilitation delivery, positioning, managing fear of falling and fatigue, optimal therapy dose, and discharge planning. Statements for which ‘some agreement’ (50%–74% agreement) was achieved and those for which recommendations remain to be clarified were recorded. Conclusions: These recommendations build on existing evidence to guide the selection of interventions for post-stroke lateropulsion. Future research is required to elaborate specific rehabilitation strategies, consider the impact of additional cognitive and perceptual impairments, describe positioning options, and detail optimal therapy dose for people with lateropulsion

Post-stroke lateropulsion terminology: Pushing for agreement amongst experts

Post-stroke lateropulsion is prevalent. The global inconsistency in terminology used to describe the condition presents obstacles in accurately comparing research results, reaching consensus on use of measurement tools, agreeing upon a consistent approach to rehabilitation, and translating research to clinical practice. Commencing in 2021, 20 international experts undertook a Delphi Process that aimed to compile clinical practice recommendations for the rehabilitation of lateropulsion. As a part of the process, the panel agreed to aim to reach consensus regarding terminology used to describe the condition. Improved understanding of the condition could lead to improved management, which will enhance patient outcomes after stroke and increase efficiency of healthcare resource utilisation. While consensus was not reached, the panel achieved some agreement that ‘lateropulsion’ is the preferred term to describe the phenomenon of ‘active pushing of the body across the midline toward the more affected side, and / or actively resisting weight shift toward the less affected side’. This group recommends that ‘lateropulsion\u27 is used in future research and in clinical practice

Probabilistic Model Checking: Formalisms and Algorithms for Discrete and Real-Time Systems

. This paper considers the problem of verification of probabilistic systems, for both the discrete- and real-time cases. Probabilistic systems are modeled as Markov chains, concurrent Markov chains (i.e. MCs with non-determinism) or, in the real-time case, generalized semiMarkov processes (Markovian processes with time delays on events). Discrete-time specifications are described in the logics PTL (propositional temporal logic), ETL (extended temporal logic) or PCTL (probabilistic full computation tree logic), or as Buchi automata. Real-time specifications are described either in TCTL (timed computation tree logic) or as deterministic timed Muller automata. The problems considered are either the probabilistic satisfaction of a specification by a system (i.e., satisfaction with probability one) or the computation of the satisfaction probability. We give complexity results for the above problems and present some of the corresponding algorithms. Finally, we illustrate the approach with..