Search

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 108 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. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03214-w.

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., Here, using the K18-hACE2 transgenic mice model, the authors report the in vivo efficacy of a fully human neutralizing antibody against SARS-CoV-2 and show that when administered before or up to 3 days post infection, treated mice do not exhibit disease symptoms while 80% of control animals succumb to the infection.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

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., Here, the authors generate a replication-competent VSV based vaccine expressing SARS-CoV-2 spike protein and show protection in the hamster model with one dose. Analysis of the antibody response in mice shows induction of neutralizing antibodies and suggests a desirable Th1-biased response to the vaccine.

BriLife

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.

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. Nonclinical safety, immunogenicity and efficacy studies were conducted in 4 animal species, using multiple dose levels (up to 108 PFU/animal) and various dosing regimens. There were no treatment related mortalities in any study, or any noticeable clinical signs. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings gave cause for safety concern in any of the studies. There was no viral shedding in urine, nor viral RNA detected in whole blood or serum samples 7 days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccine immune response gave rise to neutralizing antibodies, cellular immune response, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph node. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive IFNAR KO mice. Vaccine virus replication and distribution in K18 hACE2 transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The rVSV-ΔG-SARS-CoV-2-S vaccine was well tolerated locally and systemically and elicited an effective immunogenic response up to the highest dose tested, supporting further clinical development.

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in vivo neutralization of SARS-CoV-2 is not yet clear. Delineating the role this process plays in antibody-mediated protection will have a great impact on the design of such therapeutics. Here, the Fc of two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, targeting distinct domains of the spike, was engineered to abrogate their Fc-dependent functions. The protective activity of these antibodies was tested against lethal SARS-CoV-2 infections in K18-hACE2 transgenic mice, both before or two days post-exposure in comparison to their original, Fc-active antibodies. Antibody treatment with both Fc-variants similarly rescued the mice from death, reduced viral load and prevented signs of morbidity. In addition, surviving animals developed a significant endogenous immune response towards the virus. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in antibody-mediated protection, which should aid in future design of effective antibody-based therapies.

SummaryA wide range of SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) were reported to date, most of which target the spike glycoprotein and in particular its receptor binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit. The therapeutic implementation of these antibodies has been recently challenged by emerging SARS-CoV-2 variants that harbor extensively mutated spike versions. Consequently, the re-assessment of mAbs, previously reported to neutralize the original early-version of the virus, is of high priority.Four previously selected mAbs targeting non-overlapping epitopes, were evaluated for their binding potency to RBD versions harboring individual mutations at spike positions 417, 439, 453, 477, 484 and 501. Mutations at these positions represent the prevailing worldwide distributed modifications of the RBD, previously reported to mediate escape from antibody neutralization. Additionally, the in vitro neutralization potencies of the four RBD-specific mAbs, as well as two NTD-specific mAbs, were evaluated against two frequent SARS-CoV-2 variants of concern (VOCs): (i) the B.1.1.7 variant, emerged in the UK and (ii) the B.1.351 variant, emerged in South Africa. Variant B.1.351 was previously suggested to escape many therapeutic mAbs, including those authorized for clinical use. The possible impact of RBD mutations on recognition by mAbs is addressed by comparative structural modelling. Finally, we demonstrate the therapeutic potential of three selected mAbs by treatment of K18-hACE2 transgenic mice two days post infection with each of the virus strains.Our results clearly indicate that despite the accumulation of spike mutations, some neutralizing mAbs preserve their potency against SARS-CoV-2. In particular, the highly potent MD65 and BL6 mAbs are shown to retain their ability to bind the prevalent novel viral mutations and to effectively protect against B.1.1.7 and B.1.351 variants of high clinical concern.

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.

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.

We announce the genome sequences of two strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated in Israel, one imported by a traveler who returned from Japan and the second strain collected from a patient infected by a traveler returning from Italy. The sequences obtained are valuable as early manifestations for future follow-up of the local spread of the virus in Israel.

The COVID-19 pandemic caused by SARS-CoV-2 that emerged in December 2019 in China resulted in over 7.8 million infections and over 430,000 deaths worldwide, imposing an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we generated a replication competent recombinant VSV-ΔG-spike vaccine, in which the glycoprotein of VSV was replaced by the spike protein of the SARS-CoV-2. In vitro characterization of the recombinant VSV-ΔG-spike indicated 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 was implemented. We show that vaccination of hamsters with recombinant VSV-ΔG-spike results in rapid and potent induction of neutralizing antibodies against SARS-CoV-2. Importantly, single-dose vaccination was able to protect hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss of the immunized hamsters compared to unvaccinated hamsters. Furthermore, whereas lungs of infected hamsters displayed extensive tissue damage and high viral titers, immunized hamsters’ lungs showed only minor lung pathology, and no viral load. Taken together, we suggest recombinant VSV-ΔG-spike as a safe, efficacious and protective vaccine against SARS-CoV-2 infection.

Highlights • SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies, using different buffers, support the elimination of the extraction step (direct approaches). • We obtained similar limit of detection levels of SARS-CoV-2 when analyzing laboratory (non-clinical) samples using viral RNA extraction prior to RT-qPCR assay and direct approaches. • However, in clinical oro-nasopharyngeal SARS-CoV-2 swabs there was an advantage for the extraction procedure. • Although being previously reported to facilitate viral detection, the tested buffers severely compromised the level of detection of clinical samples. • A direct no-buffer approach might be an alternative, in times of need, since it identified more than 60% of positive clinical specimens., SARS-CoV-2 genetic identification is based on viral RNA extraction prior to RT-qPCR assay, however recent studies support the elimination of the extraction step. Herein, we assessed the RNA extraction necessity, by comparing RT-qPCR efficacy in several direct approaches vs. the gold standard RNA extraction, in detection of SARS-CoV-2 from laboratory samples as well as clinical Oro-nasopharyngeal SARS-CoV-2 swabs. Our findings show advantage for the extraction procedure, however a direct no-buffer approach might be an alternative, since it identified more than 60% of positive clinical specimens.

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection., Here, Noy-Porat, Makdasi et al. report the isolation of a panel of neutralizing mAbs selected against SARS-CoV-2 receptor-binding domain (RBD) from a phage display library constructed based on patient samples collected in the acute phase of the disease, which show efficient neutralizing activities against authentic virus in vitro.

Ricin, a plant-derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl-agarose (LA) beads. (b) Tryptic digestion. (c) LC-MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM-based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP-II toxins, by applying multiplex format.

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.

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.

Poxvirus detection assays are based on morphology, viral antigens and specific nucleic acids, none of which indicates virus viability or infectious capacity. Determination of virus viability is achieved by propagation in cell cultures and subsequent analysis by the mentioned methods, a process that takes days. Thus, presented here the development of a new assay, named PILA (Poxvirus Infection Luciferase Assay), for rapid detection of infectious poxviruses which is a cell-based reporter assay. The assay is composed of two steps: (i) Transfection of cells with a poxvirus specific reporter vector which consists of the early 7.5-kDa-STR promoter, regulating the expression of luciferase gene; (ii) Infection with a poxvirus containing sample. Luciferase activity measured post infection, indicates the presence of infectious poxvirus in the sample. The assay can detect quantities as low as 100 PFU of VACV, six hours post infection. Orthopox virus universality was confirmed by detection of various Orthopoxviruses, and specificity was verified by using pox-specific neutralizing antibodies. The PILA is specific, rapid, simple, and suitable for detecting viable virus. The assay can be utilized for applications such as poxvirus titration, neutralizing assay and drug discovery. The assay was adjusted for live detection assay by using GFP as reporting gene.

Leptospirosis is re-emerging in developed countries as a travel-related infection. In this nationwide study of travel-related leptospirosis in Israel, all cases diagnosed at the Central Reference Laboratory for Leptospirosis, during 2002–2008 were retrospectively reviewed and only travel-related cases were included. During the study years, 20 (42%) of 48 leptospirosis cases in Israel were travel-related. Exposure occurred in Southeast Asia in 15 (75%) of 20 cases. The estimated yearly incidence of travel-related leptospirosis was 1.78/100,000 travelers compared with an incidence of endemic cases of 0.06/100,000 inhabitants (risk ratio = 29.6, 95% confidence interval = 16.7–52.4). Most patients (89%) were infected during water-related activities. Severe disease was present in 10 (55%) of 18 patients; 7 of them were presumptively infected with the Icterohaemorrhagiae serogroup. Thus, travel-related leptospirosis is becoming increasingly important in the epidemiology of leptospirosis in Israel. Leptospirosis should be suspected in any traveler with undifferentiated febrile illness, especially when water exposure is reported.

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG-q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGalphaB. anthracis and q-dots655-IgGalphaY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 10(3) bacteria per ml in the sample.

During recent months, the Centers for Disease Control and Prevention (CDC) announced the occurrence of three major biosafety incidents, raising serious concern about biosafety and biosecurity guideline implementation in the most prestigious agencies in the United States: the CDC, the National Institutes of Health (NIH) and the Federal Drug Administration (FDA). These lapses included: a) the mishandling of Bacillus anthracis spores potentially exposing dozens of employees to anthrax; b) the shipment of low pathogenic influenza virus unknowingly cross-contaminated with a highly pathogenic strain; and c) an inventory lapse of hundreds of samples of biological agents, including six vials of variola virus kept in a cold storage room for decades, unnoticed. In this review we present the published data on these events, report the CDC inquiry's main findings, and discuss the key lessons to be learnt to ensure safer scientific practice in biomedical and microbiological service and research laboratories.

Leptospirosis is considered an important reemerging infectious disease worldwide. The standard and most widespread method for the diagnosis of leptospirosis is the microscopic agglutination test (MAT). This test is laborious and time-consuming, and the interpretation of the results is subjective. In the present work we describe an application of flow cytometry (FCM) as a tool for the serological diagnosis of leptospirosis. The analysis is based on the sensitivity of FCM to the size and shape of the bacteria analyzed by measurement of light scatter parameters: forward scatter (FSC) and side scatter (SSC). The addition of positive serum to an infecting leptospiral serovar results in a shift of the light scatter parameter to a different location with higher FSC and SSC values, indicating the formation of leptospiral aggregates. By using immunofluorescent staining, we have shown that the large particles formed are the agglutinated leptospires. Quantification of the agglutination process has been achieved by calculating an agglutination factor ( A f ), based on changes in the light scatter parameters measured by FCM. A f enables us to determine the specificity of the serological reaction of the patient serum with each leptospiral serovar. In this work, 27 serum samples from 18 leptospirosis patients were tested by both the MAT and the FCM techniques, in which each serum sample was tested against 13 serovars. Twenty-six human serum samples derived from patients with a variety of other defined illnesses were used as negative controls and enabled us to define the A f threshold value as

Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.

The epidemiology and clinical features of 46 cases of human leptospirosis diagnosed in Israel between 1986 and 1999 were analysed. The median patient age was 37.5 years (range, 16-85 years), and the male/female ratio was 43/3. The most common serogroup found was Leptospira icterohaemorrhagiae. The disease was associated with jaundice in 71% of cases, acute renal failure in 62%, rhabdomyolysis in 52%, pancytopenia in 28%, respiratory failure in 14% and disseminated intravascular coagulation in 5%. Leptospirosis occurs sporadically throughout the year, peaking during the summer months. A shift occurred from predominantly agriculture-related serogroups in the 1970s to urban-related serogroups during the study period reported, with Leptospira icterohaemorrhagiae being the dominant serogroup.

In this paper we will describe a new developed contribution of fluorescence nano-crystal (q-dots) as a fluorescence label for detecting pathogenic bacteria by flow cytometry (FCM) and the use of nano-magnetic particles to improve bacterial sorting by Flow cytometry cell sorting (FACS).FCM or FACS systems are based upon single cell detection by light scatter and Immunofluorescence labeling signals. The common FACS systems are based upon single or dual excitation as excitation source both for light scatter parameters and for several fluorescence detectors. Hence, for multi-labeling detection, there is a need for fluorophores with broad excitation wave length and sharp emission bands. Moreover, such fluorophores should be with high fluorescence efficiency, stable, and available for bio-molecules conjugation. Q-dots benefit from practical features which meet those -criteria. We will describe the use of q-dots as fluorescence labels for specific conjugates against Bacillus anthracis spores and Yersinia pestis bacteria, which enable the specific detection of the different species. A specific and sensitive multiplex analysis procedure for both pathogens was achieved, with high sensitivity down to 10(3) bacteria per ml in the sample.Sorting bacteria by FACS has a tremendous advantage for sensitive and selective analysis and sorting of sub-populations. However it has always been a difficult task due to the fact that bacteria are small particles (usually 1-3 μm). For such small particles, light scatter signal is on the threshold level, and many positive events may be lost. Here we will present the development of a procedure for sorting of the gram negative bacteria Y. pestis from environment samples. We will show that the application of nano-magnetic particles, as a tool for the immunomagnetic labeling and separation of the bacteria, enables fast sorting in high and low bacterial concentration down to 10 (5) cfu/ml. The nano-metric physical size of the immunospecific labeling particles disguises them from the FACS detectors; hence the bacterial population becomes the major population as opposed to being "rare events population" when using standard micro-magnetic beads for pre-enrichment.The procedure of separation and collection of bacteria enables sensitive detection and characterization methods of bacteria from complex samples.

In this paper we will describe a new developed contribution of fluorescence nano-crystal (q-dots) as a fluorescence label for detecting pathogenic bacteria by flow cytometry (FCM) and the use of nano-magnetic particles to improve bacterial sorting by Flow cytometry cell sorting (FACS).

Aim: Combination of immunomagnetic separation (IMS) and lateral flow device (LFD) assays for the development of a sensitive, rapid, on-site methodology that enables concentration and detection of Bacillus anthracis spores in complex samples. Methods and Results: The data presents the development of an optimized, 30 min, IMS assay, with about 95% capture of B. anthracis spores from different dairy products (n = 38). No cross reactivity was detected with typical milk flora and some closely related Bacilli. To enable direct application of the IMS captured spores on the LFD, spores were eluted from the bead–spore complex utilizing 95% (v/v) formamide-10 mmol l−1 EDTA for 30 s in a microwave oven. Detached spores were analysed on LFD enabling detection within 10 min. The combined IMS–LFD methodology (40 min) demonstrates a 60-fold improvement in sensitivity, relative to samples that were applied directly on the LFD without the IMS concentrating step. Conclusions: The IMS–LFD method is a powerful platform, combining rapidity, specificity and efficiency for concentrating and detecting B. anthracis from water and milk contaminated samples. Significant and Impact of the Study: The combination of IMS and LFD enhances the sensitivity and flexibility of B. anthracis spore detection from complex samples. This method can potentially be extended to other toxins and micro-organisms in a variety of matrices.

Mediterranean spotted fever (MSF) usually occurs as sporadic cases. We report five clusters of MSF in Israel. Each cluster consisted of two to three patients. In two clusters, one patient died while the other recovered. In the other three clusters the patients presented with a benign course of the disease. The diagnosis of MSF in the fatal cases was confirmed by nested-polymerase chain reaction (PCR) tests performed on samples obtained from internal organs. Rickettsial DNA was also found in a tick obtained from a dog owned by one of the patients. MSF was diagnosed in the recovered patients by serology. The diagnosis of MSF fever in one family member should raise the awareness to the possibility of other cases in the vicinity.

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.

A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GalphaRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.

Leptospirosis is a zoonotic disease that occurs worldwide, found predominantly in agricultural workers, port workers and dairy workers.To investigate the risk of disease transmission to dairy workers following an outbreak in 1999 of Leptospirosis hardjo in the dairy herds of two kibbutzim in southern Israel.A seroepidemiologic survey of all the dairy workers from these two kibbutzim was conducted, including individual interview and examination. Data were collected on the presence of clinical symptoms of leptospirosis during the previous month. One month later the medical personnel on the two kibbutzim were contacted in order to determine if any worker had subsequently developed clinical signs or symptoms of leptospirosis. All dairy workers had blood drawn for serology. Those workers whose initial serology had been borderline for leptospirosis had a repeated serology test between 2 and 4 weeks later. Doxycycline was given prophylactically to all dairy workers on one kibbutz only.Either with or without chemoprophylaxis, no dairy workers exposed to herds infected with Leptospira hardjo showed evidence of seroconversion or disease. This indicated a low risk of transmission of this serovar from cows to dairy workers.Since human illness with leptospirae can cause illness associated with significant morbidity, we recommend that physicians make an informed decision regarding doxycycline prophylaxsis for dairy workers exposed to cattle herds infected with Leptospira hardjo.