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SARS-CoV-2 cell entry starts with membrane attachment and ends with spike-protein (S) catalyzed membrane fusion depending on two cleavage steps, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time 3D single virion tracking, we show fusion and genome penetration requires virion exposure to an acidic milieu of pH 6.2-6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2 overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2 expressing cells in the acidic milieu of the nasal cavity.Significance StatementInfection by SARS-CoV-2 depends upon the S large spike protein decorating the virions and is responsible for receptor engagement and subsequent fusion of viral and cellular membranes allowing release of virion contents into the cell. Using new single particle imaging tools, to visualize and track the successive steps from virion attachment to fusion, combined with chemical and genetic perturbations of the cells, we provide the first direct evidence for the cellular uptake routes of productive infection in multiple cell types and their dependence on proteolysis of S by cell surface or endosomal proteases. We show that fusion and content release always require the acidic environment from endosomes, preceded by liberation of the S1 fragment which depends on ACE2 receptor engagement.One sentence summaryDetailed molecular snapshots of the productive infectious entry pathway of SARS-CoV-2 into cells

Center rot of onion, caused by Pantoea ananatis, is an economically important disease in onion production in Georgia and elsewhere in the United States. Growers rely on frequent foliar applications of bactericides and, in some cases, plant defense inducers to manage this disease. However, regular prophylactic application of these chemicals is not cost-effective and may not be environmentally friendly. Thrips (Thrips tabaci and Frankliniella fusca) are vectors of P. ananatis, and their feeding may compromise the effectiveness of foliar applications against P. ananatis. In this study, foliar treatments with acibenzolar-S-methyl (Actigard 50WG), cupric hydroxide (Kocide 3000), and Actigard plus Kocide were evaluated for their effectiveness in the presence and absence of thrips infestation at two critical onion growth stages: bulb initiation and bulb swelling. Onion growth stage had no impact on the effectiveness of either Kocide or Actigard. In the absence of thrips, Kocide application resulted in reduced center rot incidence compared with Actigard, regardless of the growth stage. However, when thrips were present, the efficacy of both Kocide and Actigard was reduced, with bulb incidence not significantly different from the nontreated control. In independent greenhouse studies in the presence or absence of thrips, it was observed that use of protective chemicals (Kocide, Actigard, and their combinations) at different rates also affected pathogen progression into internal neck tissue and incidence of bulb rot. These results suggest that thrips infestation can reduce the efficacy of protective chemical treatments against P. ananatis. Thrips feeding on onion foliage and resulting feeding scars could facilitate P. ananatis entry and subsequently compromise the efficacy of protective chemical treatments. Therefore, an effective center rot management strategy should likely include thrips management in addition to bactericides at susceptible growth stages of onion.

The global emergence of many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants jeopardizes the protective antiviral immunity induced following infection or vaccination. To address the public health threat caused by the increasing SARS-CoV-2 genomic diversity, the National Institute of Allergy and Infectious Diseases (NIAID) within the National Institutes of Health (NIH) established the SARS-CoV-2 Assessment of Viral Evolution (SAVE) program. This effort was designed to provide a real-time risk assessment of SARS-CoV-2 variants potentially impacting transmission, virulence, and resistance to convalescent and vaccine-induced immunity. The SAVE program serves as a critical data-generating component of the United States Government SARS-CoV-2 Interagency Group to assess implications of SARS-CoV-2 variants on diagnostics, vaccines, and therapeutics and for communicating public health risk. Here we describe the coordinated approach used to identify and curate data about emerging variants, their impact on immunity, and effects on vaccine protection using animal models. We report the development of reagents, methodologies, models, and pivotal findings facilitated by this collaborative approach and identify future challenges. This program serves as a template for the response against rapidly evolving pandemic pathogens by monitoring viral evolution in the human population to identify variants that could erode the effectiveness of countermeasures.

Streptomyces and other closely-related actinobacteria are important sources of bioactive molecules. Streptomyces synthetic biology and genetics empower therapeutic and agrichemical development through strain improvement and biosynthetic understanding. Such efforts rely on the availability of developed molecular toolsets. Among these tools, vectors that enable combinatorial chromosomal manipulations are particularly desirable. Towards developing tools for facile multiplex engineering, we herein describe the development of new integrating vectors derived from BD1 subgroup actinophage OzzyJ (ϕOZJ). By demonstrating the transformation of several Streptomyces spp. using ϕOZJ-derived vectors, we reveal their potential for strain engineering. We further report the development of new ϕC31 and ϕBT1-based vectors having orthogonal resistance, replication and integration features for concomitant transformation with our ϕOZJ-derived vectors. Importantly, the resulting compatible vector panel enabled us to demonstrate the transfer of up to three plasmids each into Streptomyces venezuelae, Streptomyces roseosporus and Streptomyces pristinaespiralis during a single conjugation experiment. To our knowledge this is the first documentation of conjugation-mediated multiplex plasmid transformation, a useful approach for rapid combinatorial strain development.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape1–3, have activity against diverse sarbecoviruses4–7, and be highly protective through viral neutralization8–11 and effector functions12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E128) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics. A survey of SARS-CoV-2 RBD antibodies identifies those with activity against diverse SARS-CoV-2 variants and SARS-related coronaviruses, highlighting epitopes and features to prioritize in antibody and vaccine development.

Rapidly-emerging variants jeopardize antibody-based countermeasures against SARS-CoV-2. While recent cell culture experiments have demonstrated loss of potency of several anti-spike neutralizing antibodies against SARS-CoV-2 variant strains1-3, the in vivo significance of these results remains uncertain. Here, using a panel of monoclonal antibodies (mAbs) corresponding to many in advanced clinical development by Vir Biotechnology, AbbVie, AstraZeneca, Regeneron, and Lilly we report the impact on protection in animals against authentic SARS-CoV-2 variants including WA1/2020 strains, a B.1.1.7 isolate, and chimeric strains with South African (B.1.351) or Brazilian (B.1.1.28) spike genes. Although some individual mAbs showed reduced or abrogated neutralizing activity against B.1.351 and B.1.1.28 viruses with E484K spike protein mutations in cell culture, low prophylactic doses of mAb combinations protected against infection in K18-hACE2 transgenic mice, 129S2 immunocompetent mice, and hamsters without emergence of resistance. Two exceptions were mAb LY-CoV555 monotherapy which lost all protective activity in vivo, and AbbVie 2B04/47D11, which showed partial loss of activity. When administered after infection as therapy, higher doses of mAb cocktails protected in vivo against viruses displaying a B.1.351 spike gene. Thus, many, but not all, of the antibody products with Emergency Use Authorization should retain substantial efficacy against the prevailing SARS-CoV-2 variant strains.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality1,2. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here we show that SARS-CoV-2 infects human CD4+T helper cells, but not CD8+T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.

Center rot of onion, caused by

Although neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of most COVID-19 vaccines and being developed as therapeutics, escape mutations could compromise such countermeasures. To define the immune-mediated mutational landscape in S protein, we used a VSV-eGFP-SARS-CoV-2-S chimeric virus and 19 neutralizing monoclonal antibodies (mAbs) against the receptor binding domain (RBD) to generate 48 escape mutants. These variants were mapped onto the RBD structure and evaluated for cross-resistance by convalescent human plasma. Although each mAb had unique resistance profiles, many shared residues within an epitope, as several variants were resistant to multiple mAbs. Remarkably, we identified mutants that escaped neutralization by convalescent human sera, suggesting that some humans induce a narrow repertoire of neutralizing antibodies. By comparing the antibody-mediated mutational landscape in S protein with sequence variation in circulating SARS-CoV-2 strains, we identified single amino acid substitutions that could attenuate neutralizing immune responses in some humans. Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGM Biopharmaceuticals, and on the Scientific Advisory Board of Moderna and Immunome. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions. The Ellebedy laboratory has received unrelated funding support in sponsored research agreements from Emergent BioSolutions and funding support in sponsored research agreement from Abbvie to further develop 2B04 and 2H04 as therapeutic mAbs. A.H.E. and Washington University have filed a patent application that includes the SARS-CoV-2 antibodies 2B04 and 2H04 for potential commercial development. S.P.J.W. and Z.H.L have filed a disclosure with Washington University for VSV-SARS-CoV-2 mutants to characterize antibody panels. S. P. J. W has received unrelated funding support in sponsored research agreements with Vir Biotechnology and Abbvie. Ethical Approval: The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance number A3381-01).

Center rot, caused by Pantoea ananatis, has been one of the most important bacterial diseases of onion leading to considerable economic losses. Symptoms can be expressed in the onion foliage and bulb, with the pathogen moving from the infected leaves to bulb scales. However, little is known regarding which growth stage the plant is most susceptible to bulb infection and if there are differences in susceptibility to bulb infection among sweet onion cultivars. In this study, five cultivars of sweet onion (Pirate, Sweet Harvest, 1518, Granex YPRR, and 1407) were inoculated by clipping the tips of onion foliage and depositing 1 ml of 1 × 108 CFU/ml of P. ananatis suspension into the central leaf cavity. The inoculations were done at three growth stages (first leaf senescence, bulb initiation, and bulb swelling). Center rot incidence was assessed for precured and cured onion bulbs. In addition, total bulb incidence of center rot for each cultivar inoculated at three growth stages were also calculated. Total bulb center rot incidence was significantly higher for Granex YPRR (84%) compared with other cultivars. Also, cultivars 1518 (49%) and 1407 (33%) had significantly lower incidence of bulb infection compared with other tested cultivars. Onions were significantly more susceptible to bulb infection when inoculated during first leaf senescence (62%) as compared with bulb initiation (37%) and bulb swelling (31%) stages in precured bulbs (P = 0.041). Significantly higher incidence of center rot was observed for bulbs whose foliage were inoculated during first leaf senescence stage (64%) compared with bulb initiation (55%) and bulb swelling (52%) stages (P = 0.048). Interactions between onion cultivar and inoculation stage on center rot bulb incidence were not significant (P ≥ 0.218), when evaluated at different assessment periods. However, different cultivars displayed significant variability in susceptibility to bulb infection. The outcomes of this study may have implications in devising management strategies aimed at protecting most susceptible onion growth stages against P. ananatis.

Piperazate (Piz) is a nonproteinogenic amino acid noted for its unusual N-N bond motif. Piz is a proline mimic that imparts conformational rigidity to peptides. Consequently, piperazyl molecules are often bioactive and desirable for therapeutic exploration. The in vitro characterization of Kutzneria enzymes KtzI and KtzT recently led to a biosynthetic pathway for Piz. However, Piz anabolism in vivo has remained completely uncharacterized. Herein, we describe the systematic interrogation of actinobacterial Piz metabolism using a combination of bioinformatics, genetics, and select biochemistry. Following studies in Streptomyces flaveolus, Streptomyces lividans, and several environmental Streptomyces isolates, our data suggest that KtzI-type enzymes are conditionally dispensable for Piz production. We also demonstrate the feasibility of Piz monomer production using engineered actinobacteria for the first time. Finally, we show that some actinobacteria employ fused KtzI-KtzT chimeric enzymes to produce Piz. Our findings have implications for future piperazyl drug discovery, pathway engineering, and fine chemical bioproduction.

Neutralizing antibodies against the SARS-CoV-2 spike (S) protein are a goal of COVID-19 vaccines and have received emergency use authorization as therapeutics. However, viral escape mutants could compromise efficacy. To define immune-selected mutations in the S protein, we exposed a VSV-eGFP-SARS-CoV-2-S chimeric virus, in which the VSV glycoprotein is replaced with the S protein, to 19 neutralizing monoclonal antibodies (mAbs) against the receptor-binding domain (RBD) and generated 50 different escape mutants. Each mAb had a unique resistance profile, although many shared residues within an epitope of the RBD. Some variants (e.g., S477N) were resistant to neutralization by multiple mAbs, whereas others (e.g., E484K) escaped neutralization by convalescent sera. Additionally, sequential selection identified mutants that escape neutralization by antibody cocktails. Comparing these antibody-mediated mutations with sequence variation in circulating SARS-CoV-2 revealed substitutions that may attenuate neutralizing immune responses in some humans and thus warrant further investigation., Graphical abstract, Highlights • Generation of SARS-CoV-2 spike mutants that escape antibody neutralization • Mutant S477N is resistant to neutralization by multiple monoclonal antibodies • Mutant E484K is less sensitive to neutralization by convalescent human sera • Sequential selection identifies mutants that escape neutralization by antibody cocktails, Liu et al. isolated 50 escape mutants to 19 monoclonal antibodies targeting the SARS-CoV-2 spike using a VSV-SARS2 chimera. Some mutants (S477N) resist neutralization by multiple mAbs, and others (E484K) are less sensitive to neutralization by convalescent sera. Several mutants exist in clinical SARS2-CoV-2 isolates and thus warrant further study.