Search

Abstract Background In an event of a smallpox outbreak in humans, the window for efficacious treatment by vaccination with vaccinia viruses (VACV) is believed to be limited to the first few days post-exposure (p.e.). We recently demonstrated in a mouse model for human smallpox, that active immunization 2–3 days p.e. with either VACV-Lister or modified VACV Ankara (MVA) vaccines, can rescue animals from lethal challenge of ectromelia virus (ECTV), the causative agent of mousepox. The present study was carried out in order to determine whether a single dose of the anti-viral cidofovir (CDV), administered at different times and doses p.e. either alone or in conjunction with active vaccination, can rescue ECTV infected mice. Methods Animals were infected intranasally with ECTV, treated on different days with various single CDV doses and monitored for morbidity, mortality and humoral response. In addition, in order to determine the influence of CDV on the immune response following vaccination, both the "clinical take”, IFN-gamma and IgG Ab levels in the serum were evaluated as well as the ability of the mice to withstand a lethal challenge of ECTV. Finally the efficacy of a combined treatment regime of CDV and vaccination p.e. was determined. Results A single p.e. CDV treatment is sufficient for protection depending on the initiation time and dose (2.5 – 100 mg/kg) of treatment. Solid protection was achieved by a low dose (5 mg/kg) CDV treatment even if given at day 6 p.e., approximately 4 days before death of the control infected untreated mice (mean time to death (MTTD) 10.2). At the same time point complete protection was achieved by single treatment with higher doses of CDV (25 or 100 mg/kg). Irrespective of treatment dose, all surviving animals developed a protective immune response even when the CDV treatment was initiated one day p.e.. After seven days post treatment with the highest dose (100 mg/kg), virus was still detected in some organs (e.g. lung and liver) yet all animals survived, suggesting that efficacious single CDV treatment requires a potent immune system. The combination of CDV and vaccination provided no additional protection over CDV alone. Yet, combining CDV and vaccination maintained vaccination efficacy. Conclusions Altogether, our data substantiate the feasibility of single post-exposure antiviral treatment to face orthopoxvirus infection.

Abstract Background Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. Results We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Conclusion Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

The emergence of rapidly spreading variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a major challenge to vaccines' protective efficacy. Intramuscular (IM) vaccine administration induces short-lived immunity but does not prevent infection and transmission. New vaccination strategies are needed to extend the longevity of vaccine protection, induce mucosal and systemic immunity and prevent viral transmission. The intranasal (IN) administration of the VSV-ΔG-spike vaccine candidate directly to mucosal surfaces yielded superior mucosal and systemic immunity at lower vaccine doses. Compared to IM vaccination in the K18-hACE2 model, IN vaccination preferentially induced mucosal IgA and T-cells, reduced the viral load at the site of infection, and ameliorated disease-associated brain gene expression. IN vaccination was protective even one year after administration. As most of the world population has been vaccinated by IM injection, we demonstrate the potential of a heterologous IM + IN vaccination regimen to induce mucosal immunity while maintaining systemic immunity. Furthermore, the IM + IN regimen prevented virus transmission in a golden Syrian hamster co-caging model. Taken together, we show that IN vaccination with VSV-ΔG-spike, either as a homologous IN + IN regimen or as a boost following IM vaccination, has a favorable potential over IM vaccination in inducing efficient mucosal immunity, long-term protection and preventing virus transmission.

The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses., (© 2024. The Author(s).)

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Following viral infection, T-cells are crucial for an effective immune response to intracellular pathogens, including respiratory viruses. During the COVID-19 pandemic, diverse assays were required in pre-clinical trials to evaluate the immune response following vaccination against SARS-CoV-2 and assess the response following exposure to the virus. To assess the nature and potency of the cellular response to infection or vaccination, a reliable and specific activity assay was needed. A cellular activity assay based on the presentation of short peptides (epitopes) allows the identification of T cell epitopes displayed on different alleles of the MHC, shedding light on the strength of the immune response towards antigens and aiding in antigen design for vaccination. In this report, we describe two approaches for scanning T cell epitopes on the surface glycoprotein of the SARS-CoV-2 (spike), which is utilized for attachment and entry and serves as an antigen in many vaccine candidates. We demonstrate that epitope scanning is feasible using peptide libraries or computational scanning combined with a cellular activity assay. Our scans identified four CD8 T cell epitopes, including one novel undescribed epitope. These epitopes enabled us to establish a reliable T-cell response assay, which was examined and used in various experimental mouse models for SARS-CoV-2 infection and vaccination. These approaches could potentially aid in future antigen design for vaccination and establish cellular activity assays against uncharacterized antigens of emerging pathogens.

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

Electrical doping of metal halide perovskites (MPHs) is a key step towards the use of this efficient and cost-effective semiconductor class in modern electronics. In this work, we demonstrate n-type doping of methylammonium lead iodide (CH 3 NH 3 PbI 3 ) by the post-fabrication introduction of Sm 2+ . The ionic radius of the latter is similar to that of Pb 2+ and can replace it without altering the perovskite crystal lattice. It is demonstrated that once incorporated, Sm 2+ can act as a dopant by undergoing oxidation to Sm 3+ . This results in the release of a negative charge that n-dopes the material, resulting in an increase of conductivity of almost 3 orders of magnitude. Unlike substitution doping with heterovalent ions, furtive dopants do not require counterions to maintain charge neutrality with respect to the ions they replace and are thus more likely to be incorporated into the crystalline structure. The incorporation of the dopant throughout the material is evidenced by XPS and ToF-SIMS, while the XRD pattern shows no phase separation at low and medium doping concentrations. A shift of the Fermi level towards a conduction energy of 0.52 eV confirms the doping to be n-type with a charge carrier density, calculated using the Mott-Schottky method, estimated to be nearly 10 17 cm -3 for the most conductive samples. Variable-temperature conductivity experiments show that the dopant is only partially ionized at room temperature due to dopant freeze-out.

Since the emergence of the original SARS-CoV-2, several variants were described, raising questions as to the ability of recently developed vaccine platforms to induce immunity and provide protection against these variants. Here, we utilized the K18-hACE2 mouse model to show that VSV-ΔG-spike vaccination provides protection against several SARS-CoV-2 variants: alpha, beta, gamma, and delta. We show an overall robust immune response, regardless of variant identity, leading to reduction in viral load in target organs, prevention of morbidity and mortality, as well as prevention of severe brain immune response, which follows infection with various variants. Additionally, we provide a comprehensive comparison of the brain transcriptomic profile in response to infection with different variants of SARS-CoV-2 and show how vaccination prevents these disease manifestations. Taken together, these results highlight the robust VSV-ΔG-spike protective response against diverse SARS-CoV-2 variants, as well as its promising potential against newly arising variants.

The current monkeypox virus global spread and lack of data regarding clinical specimens' infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value (Cq ≥ 35, ≤ 4,300 DNA copies/mL), corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox.

BriLife ® , a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (10 6 , 10 7 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of the COVID-19 pandemic. More than 274 million individuals have suffered from COVID-19 and over five million people have died from this disease so far. Therefore, there is an urgent need for therapeutic drugs. Repurposing FDA approved drugs should be favored since evaluation of safety and efficacy of de-novo drug design are both costly and time consuming. We report that imatinib, an Abl tyrosine kinase inhibitor, robustly decreases SARS-CoV-2 infection and uncover a mechanism of action. We show that imatinib inhibits the infection of SARS-CoV-2 and its surrogate lentivector pseudotype. In latter, imatinib inhibited both routes of viral entry, endocytosis and membrane-fusion. We utilized a system to quantify in real-time cell-cell membrane fusion mediated by the SARS-CoV-2 surface protein, Spike, and its receptor, hACE2, to demonstrate that imatinib inhibits this process in an Abl1 and Abl2 independent manner. Furthermore, cellular thermal shift assay revealed a direct imatinib-Spike interaction that affects Spike susceptibility to trypsin digest. Collectively, our data suggest that imatinib inhibits Spike mediated viral entry by an off-target mechanism. These findings mark imatinib as a promising therapeutic drug in inhibiting the early steps of SARS-CoV-2 infection., (© 2022. The Author(s).)

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 10 8 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife ® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants' mutations. We show that human sera from BriLife ® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife ® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife ® -acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a severe global pandemic. Mice models are essential to investigate infection pathology, antiviral drugs, and vaccine development. However, wild-type mice lack the human angiotensin-converting enzyme 2 (hACE2) that mediates SARS-CoV-2 entry into human cells and consequently are not susceptible to SARS-CoV-2 infection. hACE2 transgenic mice could provide an efficient COVID-19 model, but are not always readily available, and practically restricted to specific strains. Therefore, there is a dearth of additional mouse models for SARS-CoV-2 infection. We applied lentiviral vectors to generate hACE2 expression in interferon receptor knock-out (IFNAR1 -/- ) mice. Lenti-hACE2 transduction supported SARS-CoV-2 replication in vivo, simulating mild acute lung disease. Gene expression analysis revealed two modes of immune responses to SARS-CoV-2 infection: one in response to the exposure of mouse lungs to SARS-CoV-2 particles in the absence of productive viral replication, and the second in response to productive SARS-CoV-2 infection. Our results infer that immune response to immunogenic elements on incoming virus or in productively infected cells stimulate diverse immune effectors, even in absence of type I IFN signaling. Our findings should contribute to a better understanding of the immune response triggered by SARS-CoV-2 and to further elucidate COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients., Competing Interests: The authors have declared that no competing interests exist.

Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed., (© 2021 Vitner et al.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic., (© 2021. The Author(s).)

The COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models included the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of human COVID-19. A group activity of animals in their home cage continuously monitored by the HCMS100 (Home cage Monitoring System 100) was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters rechallenged with SARS-CoV-2 exhibited minor reduction in group activity compared to naive hamsters. To evaluate the rVSV-ΔG-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor the group activity of hamsters in their home cage. A single-dose rVSV-ΔG-spike vaccination of the immunized group showed a faster recovery than the nonimmunized infected hamsters, substantiating the efficacy of rVSV-ΔG-spike vaccine. HCMS100 offers nonintrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Melamed, Politi, Grauer, Achdout, Aftalion, Gur, Tamir, Yahalom-Ronen, Maimon, Yitzhak, Weiss, Rosner, Erez, Yitzhaki, Shapira, Paran, Mamroud, Vagima and Israely.)

Mice are normally unaffected by SARS coronavirus 2 (SARS-CoV-2) infection since the virus does not bind effectively to the murine version of the angiotensin-converting enzyme 2 (ACE2) receptor molecule. Here, we report that induced mild pulmonary morbidities rendered SARS-CoV-2-refractive CD-1 mice susceptible to this virus. Specifically, SARS-CoV-2 infection after application of low doses of the acute lung injury stimulants bleomycin or ricin caused severe disease in CD-1 mice, manifested by sustained body weight loss and mortality rates greater than 50%. Further studies revealed markedly higher levels of viral RNA in the lungs, heart, and serum of low-dose ricin-pretreated mice compared with non-pretreated mice. Furthermore, lung extracts prepared 2-3 days after viral infection contained subgenomic mRNA and virus particles capable of replication only when derived from the pretreated mice. The deleterious effects of SARS-CoV-2 infection were effectively alleviated by passive transfer of polyclonal or monoclonal antibodies generated against the SARS-CoV-2 receptor binding domain (RBD). Thus, viral cell entry in the sensitized mice seems to depend on viral RBD binding, albeit by a mechanism other than the canonical ACE2-mediated uptake route. This unique mode of viral entry, observed over a mildly injured tissue background, may contribute to the exacerbation of coronavirus disease 2019 (COVID-19) pathologies in patients with preexisting morbidities.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), exhibits high levels of mortality and morbidity and has dramatic consequences on human life, sociality and global economy. Neutralizing antibodies constitute a highly promising approach for treating and preventing infection by this novel pathogen. In the present study, we characterize and further evaluate the recently identified human monoclonal MD65 antibody for its ability to provide protection against a lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice. Eighty percent of the untreated mice succumbed 6-9 days post-infection, while administration of the MD65 antibody as late as 3 days after exposure rescued all infected animals. In addition, the efficiency of the treatment is supported by prevention of morbidity and ablation of the load of infective virions in the lungs of treated animals. The data demonstrate the therapeutic value of human monoclonal antibodies as a life-saving treatment for severe COVID-19 infection.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and  alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2.

Objectives: Environmental surfaces have been suggested as likely contributors in the transmission of COVID-19. This study assessed the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminating surfaces and objects in two hospital isolation units and a quarantine hotel., Methods: SARS-CoV-2 virus stability and infectivity on non-porous surfaces was tested under controlled laboratory conditions. Surface and air sampling were conducted at two COVID-19 isolation units and in a quarantine hotel. Viral RNA was detected by RT-PCR and infectivity was assessed by VERO E6 CPE test., Results: In laboratory-controlled conditions, SARS-CoV-2 gradually lost its infectivity completely by day 4 at ambient temperature, and the decay rate of viral viability on surfaces directly correlated with increase in temperature. Viral RNA was detected in 29/55 surface samples (52.7%) and 16/42 surface samples (38%) from the surroundings of symptomatic COVID-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild COVID-19 patients. None of the surface and air samples from the three sites (0/97) were found to contain infectious titres of SARS-Cov-2 on tissue culture assay., Conclusions: Despite prolonged viability of SARS-CoV-2 under laboratory-controlled conditions, uncultivable viral contamination of inanimate surfaces might suggest low feasibility for indirect fomite transmission., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Routine methods for virus detection in clinical specimens rely on a variety of sensitive methods, such as genetic, cell culture and immuno-based assays. It is imperative that the detection assays would be reliable, reproducible, sensitive and rapid. Isolation of viruses from clinical samples is crucial for deeper virus identification and analysis. Here we introduce a rapid cell-based assay for isolation and detection of viruses. As a proof of concept several model viruses including West Nile Virus (WNV), Modified Vaccinia Ankara (MVA) and Adenovirus were chosen. Suspended Vero cells were employed to capture the viruses following specific antibody labeling which enables their detection by flow cytometry and immuno-fluorescence microscopy assays. Using flow cytometry, a dose response analysis was performed in which 3.6e4 pfu/mL and 1e6 pfu/mL of MVA and WNV could be detected within two hours, respectively. When spiked to commercial pooled human serum, detection sensitivity was slightly reduced to 3e6 pfu/mL for WNV, but remained essentially the same for MVA. In conclusion, the study demonstrates a robust and rapid methodology for virus detection using flow cytometry and fluorescence microscopy. We propose that this proof of concept may prove useful in identifying future pathogens.

Large numbers of rodents are often used in the study of disease progression and in the evaluation of its potential treatments. To avoid subjective observation and to minimize home cage interference, we developed a computerized home cage monitoring system (HCMS100) based on a standard cage rack adapted with a single laser beam and a detector mounted on each cage, enabling to monitor mice movements based on laser beam interruptions. This retrofit system provided continuous and uninterrupted monitoring of spontaneous movement of a group of mice in a home cage. Validity was evaluated using disease state induced by LPS modelling bacterial infection and by influenza virus. RESULTS: Spontaneous activity of different number of mice (2-8) per cage showed the expected circadian rhythm with increased activity during the night, and its extent dependent on the number of mice in the cage. Females and males show similar circadian rhythm. Intranasal LPS administration and pulmonary infection with live influenza virus resulted in major reduction of mice activity along disease progression. Increase in activity over time was a good indicator of the recovery process from both LPS exposure and the flu infection. CONCLUSIONS: HCMS100 was shown to be a reliable, inexpensive, easy to use system that requires no changes in the common housing of various experimental animals (mice, hamsters, rats etc.). With minimal intervention, HCMS100 provides a continuous record of group activity with clear pattern of circadian rhythm, allowing long term recording of home cage activity even in restricted access environments., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Provisional Patent pending US62977333 for the HCMS100 was filed by the Israel Institute for Biological Research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We report a case of monkeypox in a man who returned from Nigeria to Israel in 2018. Virus was detected in pustule swabs by transmission electron microscopy and PCR and confirmed by immunofluorescence assay, tissue culture, and ELISA. The West Africa monkeypox outbreak calls for increased awareness by public health authorities worldwide.

Viral infections of the central nervous system (CNS) lead to a broad range of pathologies. CNS infections with Orthopox viruses have been mainly documented as an adverse reaction to smallpox vaccination with vaccinia virus. To date, there is insufficient data regarding the mechanisms underlying pathological viral replication or viral clearance. Therefore, informed risk assessment of vaccine adverse reactions or outcome prediction is limited. This work applied a model of viral infection of the CNS, comparing neurovirulent with attenuated strains. We followed various parameters along the disease and correlated viral load, morbidity, and mortality with tissue integrity, innate and adaptive immune response and functionality of the blood⁻brain barrier. Combining these data with whole brain RNA-seq analysis performed at different time points indicated that neurovirulence is associated with host immune silencing followed by induction of tissue damage-specific pathways. In contrast, brain infection with attenuated strains resulted in rapid and robust induction of innate and adaptive protective immunity, followed by viral clearance and recovery. This study significantly improves our understanding of the mechanisms and processes determining the consequence of viral CNS infection and highlights potential biomarkers associated with such outcomes., Competing Interests: The authors declare no conflict of interest.

Eczema vaccinatum is a severe and occasionally lethal complication of smallpox vaccine, characterized by systemic viral dissemination, distant from the initial inoculation site of the vaccine. A major risk factor for eczema vaccinatum is a background of atopic dermatitis, a chronic, common allergic, relapsing disorder, manifested by dry and inflamed skin, itchy rash, Th2 biased immune response and hypersensitivity to various antigens. Unlike the severe manifestations of eczema vaccinatum in humans, current models present only mild symptoms that limits examination of potential therapeutics for eczema vaccinatum. The atopic dermatitis and eczema vaccinatum models we present here, are the first to simulate the severity of the diseases in humans. Indeed, dermatitic mice display persistent severe dermatitis, characterized by dry and inflamed skin with barrier dysfunction, epidermal hyperplasia and significant elevation of serum IgE. By exposing atopic dermatitis mice to ectromelia virus, we generated eczema vaccinatum that mimic the human disease better than known eczema vaccinatum models. Similarly to humans, eczematous mice displayed enlarged and disseminated skin lesions, which correlated with elevated viral load. Cidofovir and antiviral antibodies conferred protection even when treatment started at a late eczematous stage. Moreover, we are the first to demonstrate that despite a severe background of atopic dermatitis, modified vaccinia Ankara virus (MVA) vaccination protects against lethal ectromelia virus exposure. We finally show that protection by MVA vaccination is dependent on CD4 + T cells and is associated with significant activation of CD8 + cytotoxic T cells and induction of humoral immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Background: Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium., Results: We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection., Conclusion: Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

Objective: To evaluate the potential protective affects of Epo on left ventricular (LV) function and remodeling after acute myocardial infarction (MI)., Methods: Epo was injected into the peritoneum of male Wistar rats (250 g) during 6 weeks post induction of MI. Rats were divided into five groups: MI treated with single high dose (MT1, 5,000 U/kg, n=10), single high dose (5,000 U/kg) and repeated high doses (MTHi, 1,000 U/kg twice a week; n=8), or single high dose (5,000 U/kg) and repeated low doses (MTLo, 750 U/kg once a week, n=10), MI non-treated (MNT, n=10), sham (S, n=5). Echocardiography was performed 3.6+/-1.5 days and 43.7+/-2.3 days post MI. Collagen deposition and infarct size were measured on histological sections using computerized image analysis. Apoptosis was assessed by ApopTag staining., Results: Baseline fractional shortening (FS) was similar between groups. Six weeks after MI the FS of MTLo (26.9%) was significantly higher compared to MNT (17.8%), MT1 (19.5%) and MTH (22.3%) (p=0.01). However, remodeling indices (end diastolic and end systolic areas, LV circumference) did not improve in the Epo groups, and even worsened in the MTHi group. There was significantly less collagen staining in non-infarct areas in MT1 and MTHi groups compared to MNT and MTLo (0.38+/-0.3%, 0.49+/-0.34%, vs 0.89+/-0.41%, 0.95+/-0.33%, respectively, p<0.001). The number of ApopTag positive nucleus was significantly higher in the MNT group compared to the MT1, MTHi, MTLo groups (14.4+/-8, 7.6+/-4, 5.8+/-7, 4.8+/-5, respectively, p=0.01 for trend)., Conclusion: Repeated low doses of Epo after MI improved LV function, but the role of Epo on remodeling is not clear. It did not reduce left ventricular indices, but reduces fibrosis and apoptosis. High Epo doses reduced LV function and aggravated remodeling.

The extent measurement error on CT colonography influences polyp categorisation according to established management guidelines is studied using twenty-eight observers of varying experience to classify polyps seen at CT colonography as either 'medium' (maximal diameter 6-9 mm) or 'large' (maximal diameter 10 mm or larger). Comparison was then made with the reference diameter obtained in each patient via colonoscopy. The Bland-Altman method was used to assess agreement between observer measurements and colonoscopy, and differences in measurement and categorisation was assessed using Kruskal-Wallis and Chi-squared test statistics respectively. Observer measurements on average underestimated the diameter of polyps when compared to the reference value, by approximately 2-3 mm, irrespective of observer experience. Ninety-five percent limits of agreement were relatively wide for all observer groups, and had sufficient span to encompass different size categories for polyps. There were 167 polyp observations and 135 (81%) were correctly categorised. Of the 32 observations that were miscategorised, 5 (16%) were overestimations and 27 (84%) were underestimations (i.e. large polyps misclassified as medium). Caution should be exercised for polyps whose colonographic diameter is below but close to the 1-cm boundary threshold in order to avoid potential miscategorisation of advanced adenomas.

Our purpose was to assess the effect of reader experience, fatigue, and scan findings on interpretation time for CT colonography. Nine radiologists (experienced in CT colonography); nine radiologists and ten technicians (both groups trained using 50 validated examinations) read 40 cases (50% abnormal) under controlled conditions. Individual interpretation times for each case were recorded, and differences between groups determined. Multi-level linear regression was used to investigate effect of scan category (normal or abnormal) and observer fatigue on interpretation times. Experienced radiologists (mean time 10.9 min, SD 5.2) reported significantly faster than less experienced radiologists and technicians; odds ratios of reporting times 1.4 (CI 1.1, 1.8) and 1.6 (1.3, 2.0), respectively (P

Prenatal ultrasonographic diagnosis of urethral valves is now common. Treatment of the urethral obstruction has sometimes to be done in low weight newborn babies. In these babies, the stripping of valves using a Fogarty catheter provides an helpful way of treatment, easier than endoscopy. The technique is presented here. The results of this procedure in 10 neonates with a follow up of 3 to 22 months are reported. The urethral obstruction is easily cured in most patients without using a previous diversion. This procedure is indicated in premature or hypotrophic newborns when the urethral size is too small to allow a safe endoscopic section of the valves. We did not observe secondary stricture. Follow up is too short to allow a correct investigation of the continence.

In August and September 1977 a discrete cluster of 27 serologically or pathologically confirmed cases of Legionnaires' disease, plus six highly presumptive cases were identified in the area of Kingsport, Tennessee. Three patients died. Most patients manifested severe pneumonia and fever; no mild or asymptomatic disease forms were recognized despite intensive case-finding efforts. Illness was epidemiologically associated with residing, visiting, or working in one geographic area of Kingsport, residence there being the factor most strongly associated. Although the attack rate for area residents was 0.64%, the randomly determined prevalence of serologic reactors was 5.2%, which is not significantly different from that in a nonimplicated control neighborhood. The epidemic did not correlate temporally with any identified environmental or demographic event. No source of the bacterium was found either by a detailed case-control study of area associations or by bacterial isolation from sentinel guinea pigs or environmental specimens. There was no evidence of person-to-person spread.

Thirty-nine cases of Legionnaires' disease in a 16-month period were identified in visitors to and residents of Bloomington, Indiana. Thirty-five patients had spent at least one night at the Indiana Memorial Union in the 2 weeks before becoming ill. Five of 32 sporadic cases nationwide between 1 January and 31 March 1978 were retrospectively shown to be in persons who had recently visited the Union. The risk of acquiring Legionnaires' disease as a Union visitor was at least 17 times greater than that for Bloomington residents 20 years or older. Employees who had worked at the Union 5 years or longer were more likely to be seropositive than workers in other Bloomington hotels. Legionnaires' disease bacterium was isolated from five environmental sites in Bloomington. A cooling tower may have been involved in disease spread, but it was not the only source. Hypochlorite solution was added to cooling tower water as a precautionary measure; however, one case was confirmed in a man with Union exposure 9 days after hypochlorite treatment had begun.

We analyzed 24 environmental samples collected in or near the Indiana Memorial Union, where an epidemic of Legionnaires' disease occurred in early 1978. We conducted fluorescent antibody analyses and culture on F-G and charcoal yeast extract agars of each sample directly; splenic tissue of guinea pigs inoculated with the sample; and yolk sacs from embryonated eggs inoculated with splenic tissue of guinea pigs injected with the sample. Legionnaires' disease (LD) bacterium was isolated from seven of the 24 samples: one water sample from the air-conditioner cooling tower of the Union; three water samples from a stream near the Union; and three mud samples from the same stream. The LD bacterium strains were of three different serotypes. These findings indicate that LD bacteria may be widespread in nature.

One hundred patients with sporadic, community-acquired, serologically confirmed Legionnaires' disease were matched with control subjects known by the patients (acquaintance controls) and control subjects chosen from among patients with negative serodiagnostic tests for Legionnaires' disease (clinical controls). Each clinical control subject was also matched with an acquaintance control of his own. Legionnaires' disease patients had smoked more cigarettes, consumed more alcohol, and were more likely to have resided near excavation sites than acquaintance or clinical control subjects. Parallel differences between clinical controls and their acquaintances were not seen. Legionnaires' disease patients had traveled away from home for more time during the 2 weeks before onset of illness than had their acquaintances. The difference was of greater magnitude than that between clinical control subjects and their acquaintances. Legionnaires' disease patients were more likely to have resided near construction sites than clinical controls, and there were more construction workers among patients than among clinical control subjects.