During hematoma formation following injury, an inflammatory reaction ensues as an initial step in the healing process. As granulation tissue matures, revascularization is a prerequisite for successful healing. The hypothesis of this study was that scarless tissue reconstitution in the regenerative bone healing process is dependent on a balanced immune reaction that initiates revasculatory steps. To test this hypothesis, cellular composition and expression profiles of a bone hematoma (regenerative, scarless) was compared with a muscle soft tissue hematoma (healing with a scar) in a sheep model. Upregulation of regulatory T helper cells and anti-inflammatory cytokine expression (IL-10) coincided with an upregulation of angiogenic factors (HIF1α and HIF1α regulated genes) in the regenerative bone hematoma but not in the soft tissue hematoma. These results indicate that the timely termination of inflammation and early onset of revascularization are interdependent and essential for a regenerative healing process. Prolonged pro-inflammatory signaling occurring in a delayed bone-healing model supports the finding that timely termination of inflammation furthers the regenerative process. Differing cellular compositions are due to different cell sources invading the hematoma, determining the ensuing cytokine expression profile and thus paving the path for regenerative healing in bone or the formation of scar tissue in muscle injury.

Rotaviruses, the single most important agents of acute severe gastroenteritis in children, are nonenveloped viruses formed by a three-layered capsid that encloses a genome formed by 11 segments of double-stranded RNA. The mechanism of entry of these viruses into the host cell is not well understood. The best-studied strain, RRV, which is sensitive to neuraminidase (NA) treatment of the cells, uses integrins ␣2␤1 and ␣v␤3 and the heat shock protein hsc70 as receptors and enters MA104 cells through a non-clathrin-, non-caveolin-mediated pathway that depends on a functional dynamin and on the presence of cholesterol on the cell surface. In this work, using a combination of pharmacological, biochemical, and genetic approaches, we compared the entry characteristics of four rotavirus strains known to have different receptor requirements. We chose four rotavirus strains that represent all phenotypic combinations of NA resistance or sensitivity and integrin dependence or independence. We found that even though all the strains share their requirements for hsc70, dynamin, and cholesterol, three of them differ from the simian strain RRV in the endocytic pathway used. The human strain Wa, porcine strain TFR-41, and bovine strain UK seem to enter the cell through clathrin-mediated endocytosis, since treatments that inhibit this pathway block their infectivity; consistent with this entry route, these strains were sensitive to changes in the endosomal pH. The inhibition of other endocytic mechanisms, such as macropinocytosis or caveola-mediated uptake, had no effect on the internalization of the rotavirus strains tested here.

While recently we have learned much about the viral and cellular proteins involved in the initial attachment of rotaviruses to MA104 cells, the mechanism by which these viruses reach the interior of the cell is poorly understood. For this study, we observed the effects of drugs and of dominant-negative mutants, known to impair clathrin-mediated endocytosis and endocytosis mediated by caveolae, on rotavirus cell infection. Rotaviruses were able to enter cells in the presence of compounds that inhibit clathrin-mediated endocytosis as well as cells overexpressing a dominant-negative form of Eps15, a protein crucial for the assembly of clathrin coats. We also found that rotaviruses infected cells in which caveolar uptake was blocked; treatment with the cholesterol binding agents nystatin and filipin, as well as transfection of cells with dominant-negative caveolin-1 and caveolin-3 mutants, had no effect on rotavirus infection. Interestingly, cells treated with methyl-␤-cyclodextrin, a drug that sequesters cholesterol from membranes, and cells expressing a dominant-negative mutant of the large GTPase dynamin, which is known to function in several membrane scission events, were not infected by rotaviruses, indicating that cholesterol and dynamin play a role in the entry of rotaviruses.The initial steps in a viral infection involve the specific attachment of the viral particle to a receptor(s) on the cell surface, followed by internalization of the virus into the cell and the subsequent uncoating of the virion to release the active transcription complex. These events are essential for the suc-

Summary Alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the class II virus fusion protein E1, an elongated molecule with three extramembrane domains (DI–III). E1 drives fusion by inserting its fusion peptide loop into the target membrane and refolding to a hairpin-like trimer in which DIII moves toward the target membrane and packs against the central trimer. Three-dimensional structures provide static pictures of prefusion and postfusion E1 but do not explain this transition. Using truncated forms of E1, we reconstituted a low-pH-dependent intermediate composed of trimers of DI/II. Unexpectedly, DI/II trimers were stable in the absence of DIII. Once formed at a low pH, DI/II trimers efficiently and specifically bound recombinant DIII through a pH-independent reaction. Even in the absence of DIII, DI/II trimers interacted to form hexagonal lattices and to cause membrane deformation and tubulation. These studies identify a prefusion intermediate in class II membrane fusion.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease-19 (COVID-19), has emerged as the cause of a global pandemic. We used RNA sequencing to analyze 286 nasopharyngeal (NP) swab and 53 whole-blood (WB) samples from 333 patients with COVID-19 and controls. Overall, a muted immune response was observed in COVID-19 relative to other infections (influenza, other seasonal coronaviruses, and bacterial sepsis), with paradoxical down-regulation of several key differentially expressed genes. Hospitalized patients and outpatients exhibited up-regulation of interferon-associated pathways, although heightened and more robust inflammatory responses were observed in hospitalized patients with more clinically severe illness. Two-layer machine learning–based host classifiers consisting of complete (>1000 genes), medium (<100), and small (<20) gene biomarker panels identified COVID-19 disease with 85.1–86.5% accuracy when benchmarked using an independent test set. SARS-CoV-2 infection has a distinct biosignature that differs between NP swabs and WB and can be leveraged for COVID-19 diagnosis.