ABSTRACT
BACKGROUND: The largest West African monkeypox outbreak began September 2017, in Nigeria. Four individuals traveling from Nigeria to the United Kingdom (n = 2), Israel (n = 1), and Singapore (n = 1) became the first human monkeypox cases exported from Africa, and a related nosocomial transmission event in the United Kingdom became the first confirmed human-to-human monkeypox transmission event outside of Africa. METHODS: Epidemiological and molecular data for exported and Nigerian cases were analyzed jointly to better understand the exportations in the temporal and geographic context of the outbreak. RESULTS: Isolates from all travelers and a Bayelsa case shared a most recent common ancestor and traveled to Bayelsa, Delta, or Rivers states. Genetic variation for this cluster was lower than would be expected from a random sampling of genomes from this outbreak, but data did not support direct links between travelers. CONCLUSIONS: Monophyly of exportation cases and the Bayelsa sample, along with the intermediate levels of genetic variation, suggest a small pool of related isolates is the likely source for the exported infections. This may be the result of the level of genetic variation present in monkeypox isolates circulating within the contiguous region of Bayelsa, Delta, and Rivers states, or another more restricted, yet unidentified source pool.
Subject(s)
Monkeypox virus , Mpox (monkeypox) , Disease Outbreaks , Humans , Mpox (monkeypox)/epidemiology , Monkeypox virus/genetics , Nigeria/epidemiology , United KingdomABSTRACT
Exposure to sulfur mustard (SM) may result in severe ocular injuries. While some of the eyes show a clinical resolution of the injury (defined as clinically non-impaired), part of the eyes develop irreversible late ocular pathologies (defined as clinically impaired) that may lead to corneal blindness. Understanding the pathological mechanisms underlying the development of the late pathology may lead to improved treatment options. Therefore, this study aimed to investigate the mRNA expression profiles of corneas from clinically impaired, clinically non-impaired and naïve eyes. Rabbit eyes were exposed to SM vapor and a clinical follow-up was carried out up to 4 weeks using a slit lamp microscope. At this time point, corneal tissues from clinically impaired, clinically non-impaired and naïve eyes were processed for RNA sequencing (RNA-seq) and differential expression analyses. The differential expression profiles were further subjected to pathway enrichment analysis using Ingenuity Pathway Analysis (IPA). Real-time PCR was used for RNA-seq validation. The late pathology developed in 54%-80% of the eyes following ocular exposure to SM, clinically manifested by inflammation, corneal opacity and neovascularization. RNA-seq results showed significant differences in mRNA levels of hundreds of genes between clinically impaired, clinically non-impaired and naïve corneas. Pathway enrichment analysis showed common pathways that were activated in all of the exposed eyes, such as Th1 and Th2 activation pathway, in addition to pathways that were activated only in the clinically impaired eyes compared to the clinically non-impaired eyes, such as IL-6 and ERK5 signaling. Corneal mRNA expression profiles for the clinically impaired, clinically non-impaired and naïve eyes generated a comprehensive database that revealed new factors and pathways, which for the first time were shown to be involved in SM-induced late pathology. Our data may contribute to the research on both the pathological mechanisms that are involved in the development of the late pathology and the protective pathways that are activated in the clinically non-impaired eyes and may point out towards novel therapeutic strategies for this severe ocular injury.
Subject(s)
Chemical Warfare Agents/adverse effects , Corneal Neovascularization , Corneal Opacity , Mustard Gas/adverse effects , RNA, Messenger/metabolism , Animals , Cornea , Corneal Neovascularization/chemically induced , Corneal Neovascularization/metabolism , Corneal Opacity/chemically induced , Corneal Opacity/metabolism , Disease Models, Animal , RabbitsABSTRACT
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.
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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.
Subject(s)
Burkholderia pseudomallei , Genome, Bacterial , Melioidosis , Phylogeny , Burkholderia pseudomallei/genetics , Burkholderia pseudomallei/isolation & purification , Burkholderia pseudomallei/pathogenicity , Melioidosis/microbiology , Melioidosis/epidemiology , Thailand/epidemiology , Humans , Genome, Bacterial/genetics , India , Animals , Israel/epidemiology , Virulence/genetics , Mice , Whole Genome SequencingABSTRACT
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.
Subject(s)
Doxycycline , Francisella tularensis , Doxycycline/pharmacology , Francisella tularensis/genetics , Ciprofloxacin/pharmacology , Transcriptome/genetics , Anti-Bacterial Agents/pharmacology , RNAABSTRACT
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.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Humans , Mice , COVID-19/prevention & control , SARS-CoV-2/genetics , Spike Glycoprotein, CoronavirusABSTRACT
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.
Subject(s)
Pantoea , Anti-Bacterial Agents/pharmacology , Israel , Pantoea/genetics , Phylogeny , Sequence Analysis, DNA , Whole Genome SequencingABSTRACT
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.
Subject(s)
Mpox (monkeypox) , Nanopore Sequencing , Orthopoxvirus , Smallpox , Variola virus , Diagnosis, Differential , Humans , Mpox (monkeypox)/epidemiology , Monkeypox virus/genetics , Smallpox/diagnosis , Variola virus/geneticsABSTRACT
The global spread of SARS-CoV-2 led to major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitate the development of novel therapeutics. Here, employing a genome-scale CRISPR screen, we provide a comprehensive data-set of cellular factors that are exploited by wild type SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. We identified several host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination, Heparan sulfate biogenesis and host phosphatidylglycerol biosynthesis. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential proviral gene for all variants inspected. We show that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals shows elevated levels of GATA6, suggesting a role in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and inhibition of viral infectivity. Overall, we show GATA6 may represent a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , GATA6 Transcription Factor/genetics , Humans , Peptidyl-Dipeptidase A/metabolism , Proviruses/genetics , SARS-CoV-2/geneticsABSTRACT
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.
ABSTRACT
During evolution, organisms have gained functional complexity mainly by modifying and improving existing functioning systems rather than creating new ones ab initio. Here we explore the interplay between two processes which during evolution have had major roles in the acquisition of new functions: gene duplication and protein domain rearrangements. We consider four possible evolutionary scenarios: gene families that have undergone none of these event types; only gene duplication; only domain rearrangement, or both events. We characterize each of the four evolutionary scenarios by functional attributes. Our analysis of ten fungal genomes indicates that at least for the fungi clade, species significantly appear to gain complexity by gene duplication accompanied by the expansion of existing domain architectures via rearrangements. We show that paralogs gaining new domain architectures via duplication tend to adopt new functions compared to paralogs that preserve their domain architectures. We conclude that evolution of protein families through gene duplication and domain rearrangement is correlated with their functional properties. We suggest that in general, new functions are acquired via the integration of gene duplication and domain rearrangements rather than each process acting independently.
Subject(s)
Biological Evolution , Fungal Proteins/genetics , Fungal Proteins/metabolism , Fungi/genetics , Gene Duplication , Proteome/chemistry , Proteome/genetics , Databases, Genetic , Fungal Proteins/chemistry , Fungi/chemistry , Gene Order , Genes, Fungal/genetics , Proteome/metabolismABSTRACT
We report the genome sequences and the identification of genetic variations in eight clinical samples of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Samples were collected from nasopharyngeal swabs of symptomatic and asymptomatic individuals from five care homes for elderly and infirm persons in Israel. The sequences obtained are valuable, as they carry a newly reported nonsynonymous substitution located within the nucleoprotein open reading frame.
ABSTRACT
Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing lethal infection. The various phases of pneumonic plague are yet to be fully understood. A well-established way to address the pathology of infectious diseases in general, and pneumonic plague in particular, is to conduct concomitant transcriptomic analysis of the bacteria and the host. The analysis of dual RNA by RNA sequencing technology is challenging, due the difficulties of extracting bacterial RNA, which is overwhelmingly outnumbered by the host RNA, especially at the critical early time points post-infection (prior to 48 h). Here, we describe a novel technique that employed the infusion of an RNA preserving reagent (RNAlater) into the lungs of the animals, through the trachea, under deep anesthesia. This method enabled the isolation of stable dual mRNA from the lungs of mice infected with Y. pestis, as early as 24 h post-infection. The RNA was used for transcriptomic analysis, which provided a comprehensive gene expression profile of both the host and the pathogen.
ABSTRACT
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.
Subject(s)
Bleomycin/toxicity , COVID-19/pathology , Lung Injury , Ricin/toxicity , Animals , Chlorocebus aethiops , Comorbidity , Disease Models, Animal , Female , Lung Injury/chemically induced , Lung Injury/virology , Mice , Vero Cells , Virus Attachment , Virus Internalization/drug effectsABSTRACT
Francisella tularensis is a highly infectious intracellular bacterium representing the causative agent of tularemia, a severe disease which requires prompt antibacterial intervention for mitigating its potential high mortality. Inhaled bacteria interact with lung cells belonging to various subpopulations, including those of the epithelium. As of today, the host epithelial response to inhalational infection with F. tularensis is poorly understood. Here, we announce host transcriptome data sets which systematically address the human epithelial response to F. tularensis at different time points postinfection.
ABSTRACT
We report the whole-genome sequence of a monkeypox virus strain isolated in Israel. The strain was isolated in 2018 from a patient travelling back from West Africa. The virus was fully sequenced on the Illumina MiSeq and Oxford Nanopore Technologies MinION platforms.
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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.
ABSTRACT
High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis, F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5-5.9 orders of magnitude, in samples with bacteria spiked-in at 103-105 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8-9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.
ABSTRACT
The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA-disrupted strains following H2O2-induced oxidative stress.