Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging vi

Sars-cov-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy

New viruses in idiopathic human diarrhea cases, the Netherlands

Emerging viral infections can be identified by using a viral metagenomics approach for clinical human material. Diarrhea samples of patients with unexplained gastroenteritis from the Netherlands were analyzed by using viral metagenomics. Novel circular DNA viruses, bufaviruses, and genogroup III picobirnaviruses were identified. These data expand our knowledge of the human virome

Metagenomic survey for viruses in Western Arctic caribou, Alaska, through iterative assembly of taxonomic units

Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-)emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive

Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models

Detection of circovirus in Foxes with Meningoencephalitis, United Kingdom, 2009–2013

A fox circovirus was identified in serum samples from foxes with unexplained neurologic signs by using viral metagenomics. Fox circovirus nucleic acid was localized in histological lesions of the cerebrum by in situ hybridization. Viruses from the family Circoviridae may have neurologic tropism more commonly than previously anticipated

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorg

Development of a liposomal delivery system for temperature-triggered release of a tumor targeting agent, Ln(III)-DOTA-phenylboronate

Liposomes, capable of temperature-triggered content release at the site of interest, can be of great importance for imaging and therapy of tumors. The delivery of imaging agents or therapeutics can be improved by application of liposomes with a gel-to-liquid phase-transition temperature suitable for mild hyperthermia (41-43 degrees C), and by prolonging their circulation time by incorporation of lipids containing polyethyleneglycol moieties. Still, the rapid wash out of the delivered material from the tumor tissue is a major obstacle for both imaging and therapy. In this study, we developed an optimized temperature sensitive liposomal system to be used with mild hyperthermia: highly stable at physiological temperature and with a sharp transition of the bilayer at 41.5 degrees C, with subsequent rapid release of entrapped compounds such as calcein or tumor cell-targeting contrast agents. Intravital microscopy on calcein/rhodamine containing liposomes was applied to demonstrate the applicability of this system in vivo. The calcein loaded liposomes were injected iv into nude mice with a human BLM melanoma tumor implanted in a dorsal skin-fold window chamber. Arrival of the liposomes at the tumor site and content release after temperature increase were monitored. The results demonstrated not only accumulation of the liposomes at the tumor site, but also a massive release of calcein after increase of the temperature to 41 degrees C. The versatility of the thermosensitive liposomes was further demonstrated by encapsulation of a tumor cell-targeting DOTA-phenylboronate conjugate and its release at elevated temperatures. The DOTA ligand in this system is able to chelate a variety of metals suitable for both diagnostic and therapeutic applications, whereas the phenylboronate function is able to target specifically to tumor cells through a covalent binding with sialic acid moieties over-expressed on their surface upon heat-triggered release from the liposomal carrier. (C) 2010 Elsevier Ltd. All rights reserved