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Abstract Objective Burkholderia pseudomallei, the etiological cause of melioidosis, is a soil saprophyte endemic in South-East Asia, where it constitutes a public health concern of high-priority. Melioidosis cases are sporadically identified in nonendemic areas, usually associated with travelers or import of goods from endemic regions. Due to extensive intercontinental traveling and the anticipated climate change-associated alterations of the soil bacterial flora, there is an increasing concern for inadvertent establishment of novel endemic areas, which may expand the global burden of melioidosis. Rapid diagnosis, isolation and characterization of B. pseudomallei isolates is therefore of utmost importance particularly in non-endemic locations. Data description We report the genome sequences of two novel clinical isolates (MWH2021 and MST2022) of B. pseudomallei identified in distinct acute cases of melioidosis diagnosed in two individuals arriving to Israel from India and Thailand, respectively. The data includes preliminary genetic analysis of the genomes determining their phylogenetic classification in rapport to the genomes of 131 B. pseudomallei strains documented in the NCBI database. Inspection of the genomic data revealed the presence or absence of loci encoding for several documented virulence determinants involved in the molecular pathogenesis of melioidosis. Virulence analysis in murine models of acute or chronic melioidosis established that both strains belong to the highly virulent class of B. pseudomalleii.

The diagnosis of Q fever is challenging due to nonspecific symptoms and negative standard blood culture results. Serological testing through immunofluorescence assay (IFA) is the most commonly used method for diagnosing this disease. Polymerase chain reaction (PCR) tests can also be used to detect bacterial DNA if taken at an appropriate time. Once the presence of bacteria is confirmed in a sample, an enrichment step is required before characterizing it through sequencing. Cultivating C. burnetii is challenging as it can only be isolated by inoculation into cell culture, embryonated eggs, or animals. In this article, we describe the isolation of C. burnetii from a valve specimen in Vero cells. We conducted genome sequencing and taxonomy profiling of this isolate and were able to determine its taxonomic affiliation. Furthermore, Multispacer sequence typing (MST) analysis suggests that the infection originated from a local strain of C. burnetii found around northern Israel and Lebanon. This novel strain belongs to a previously described genotype MST6, harboring the QpRS plasmid, never reported in Israel.

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms.

Abstract Objective As part of a research aiming at presenting an alternative approach for rapid determination of antimicrobial susceptibility by quantification of changes in expression levels of specific marker genes and gene sets, cultures of the virulent bacterial strain Francisella tularensis SchuS4 were grown in the presence of inhibitory/sub-inhibitory concentrations of either ciprofloxacin or doxycycline and their transcriptomic profiles were elucidated using differential expression analysis followed by functional annotation. Data description RNA sequencing was performed to identify differentially expressed genes (DEGs) in response to exposure of F. tularensis SchuS4 to either ciprofloxacin or doxycycline, the antibiotics of choice for Tularemia therapy. Accordingly, RNA samples were collected 2 h post antibiotic exposure and subjected to RNA sequence analysis. Transcriptomic quantification of RNA representing duplicated samples generated highly similar gene expression data. Exposure to sub-inhibitory concentration [0.5 x MIC (minimal inhibitory concentration)] of doxycycline or ciprofloxacin modulated the expression of 237 or 8 genes, respectively, while exposure to an inhibitory concentration (1 x MIC) resulted in the modulation of 583 or 234 genes, respectively. Amongst the genes modulated upon doxycycline exposure upregulation of 31 genes encoding for translation-functions could be distinguished, as well as downregulation of 14 genes encoding for functions involved in DNA transcription and repair. Ciprofloxacin exposure impacted differently the RNA sequence profile of the pathogen, resulting in upregulation of 27 genes encoding mainly DNA replication and repair functions, transmembrane transporters and molecular chaperons. In addition, 15 downregulated genes were involved in translation processes.

Abstract Objective As part of a research aiming at the isolation of bacteria secreting growth inhibiting compounds, cultures of Francisella tularensis were implanted in environmental samples and monitored for inhibition zones on agar. Two antibiotic-like secreting bacteria were isolated, their genomic sequence was deciphered and taxonomic profiling analysis classified them as belonging to the Pantoea genus. Data description Two bacterial isolates exhibiting growth inhibition zones to F. tularensis (LVS) were analyzed using the Oxford Nanopore Technology (ONT). Preliminary de novo assembly of the reads was performed, followed by taxonomic profiling based on Multi Locus Sequence Analysis (MLSA) and implementation of the Average Nucleotide Identity (ANI) measure. The genomic sequences resulted in the identification of two different Pantoea species, denoted EnvD and EnvH. Subsequent de novo genome assembly generated 5 and 10 contigs for EnvD and EnvH, respectively. The largest contig (4,008,183 bps and 3,740,753 bps for EnvD and EnvH, respectively), overlaps to a major extent to the chromosome of closely related Pantoea species. ANI values calculated for both isolates revealed two apparently new species of the Pantoea genus. Our study deciphered the identity of two bacteria producing antibiotic-like compounds, and the genomic sequence revealed they represent distinct Pantoea species.

Here, Israeli and Finkel et al. perform genome-wide CRISPR knockout screens to identify host factors required for the infection with SARS-CoV-2 and two additionally variants of concern, Alpha and Beta, unveiling shared and differential host pathways required by the variants, and demonstrate GATA6 is critical for SARS-CoV-2 viral entry through modulation of ACE2 expression.

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.

Severe manifestations of coronavirus disease 2019 (COVID-19) are mostly restricted to distinct groups of people who have preexisting morbidities. Most COVID-19 animal models develop a mild pathology that resolves within a relatively short period of time, reflecting the more prevalent asymptomatic-to-mild performance of the disease observed in humans. Mice are normally unaffected by SARS coronavirus-2 infection, because of the inability of the virus to bind effectively to the murine angiotensin-converting enzyme 2 (ACE2) receptor. We have previously demonstrated that induction of mild and transient pulmonary morbidity, by application of low doses of ricin, rendered CD1 mice to be susceptible to this virus, which was displayed by sustained body weight loss and mortality rates >50%. In the present study, we performed transcriptomic analyses charting the major alterations in gene expression of mice that were pre-exposed to low doses of ricin and then subjected to SARS-CoV-2 infection compared to mice that were solely exposed to ricin or infected with SARS-CoV-2. Mice intoxicated and infected with ricin and SARS-CoV-2 demonstrated a marked stimulation of essential immunity genes and biological pathways involved in the activation of natural-killer response, cell death receptors, cytotoxic T-cells, Toll-like receptor signaling and the NLRP3 inflammasome pathway. At the protein level, an induced early and transient interferon response was recorded which was subsequently suppressed. The activation of this array of genes predicts clinical manifestations that are consistent with severe COVID-19 in humans, thereby establishing the suitability of this unique animal model for the study of severe COVID-19 disease.

As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population’s immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs’ clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5′-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.

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.