Development of an immunofluorescence assay for detection of SARS-CoV-2.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis in 2019. Currently, the main method for identification of SARS-CoV-2 is a reverse transcription (RT)-PCR assay designed to detect viral RNA in oropharyngeal (OP) or nasopharyngeal (NP) samples. While the PCR assay is considered highly specific and sensitive, this method cannot determine the infectivity of the sample, which may assist in evaluation of virus transmissibility from patients and breaking transmission chains. Thus, cell-culture-based approaches such as cytopathic effect (CPE) assays are routinely employed for the identification of infectious viruses in NP/OP samples. Despite their high sensitivity, CPE assays take several days and require additional diagnostic tests in order to verify the identity of the pathogen. We have therefore developed a rapid immunofluorescence assay (IFA) for the specific detection of SARS-CoV-2 in NP/OP samples following cell culture infection. Initially, IFA was carried out on Vero E6 cultures infected with SARS-CoV-2 at defined concentrations, and infection was monitored at different time points. This test was able to yield positive signals in cultures infected with 10 pfu/ml at 12 hours postinfection (PI). Increasing the incubation time to 24 hours reduced the detectable infective dose to 1 pfu/ml. These IFA signals occur before the development of CPE. When compared to the CPE test, IFA has the advantages of specificity, rapid detection, and sensitivity, as demonstrated in this work.

Nanodissection of Selected Viral Particles by Scanning Transmission Electron Microscopy/Focused Ion Beam for Genetic Identification.

This study presents the development of a new correlative workflow to bridge the gap between electron microscopy imaging and genetic analysis of viruses. The workflow enables the assignment of genetic information to a specific biological entity by harnessing the nanodissection capability of focused ion beam (FIB). This correlative workflow is based on scanning transmission electron microscopy (STEM) and FIB followed by a polymerase chain reaction (PCR). For this purpose, we studied the tomato brown rugose fruit virus (ToBRFV) and the adenovirus that have significant impacts on plant integrity and human health, respectively. STEM imaging was used for the identification and localization of virus particles on a transmission electron microscopy (TEM) grid followed by FIB milling of the desired region of interest. The final-milled product was subjected to genetic analysis by the PCR. The results prove that the FIB-milling process maintains the integrity of the genetic material as confirmed by the PCR. We demonstrate the identification of RNA and DNA viruses extracted from a few micrometers of an FIB-milled TEM grid. This workflow enables the genetic analysis of specifically imaged viral particles directly from heterogeneous clinical samples. In addition to viral diagnostics, the ability to isolate and to genetically identify specific submicrometer structures may prove valuable in additional fields, including subcellular organelle and granule research.

An upstream open reading frame (uORF) signals for cellular localization of the virulence factor implicated in pregnancy associated malaria.

Plasmodium falciparum, the causative agent of the deadliest form of human malaria, alternates expression of variable antigens, encoded by members of a multi-copy gene family named var. In var2csa, the var gene implicated in pregnancy-associated malaria, translational repression is regulated by a unique upstream open reading frame (uORF) found only in its 5' UTR. Here, we report that this translated uORF significantly alters both transcription and posttranslational protein trafficking. The parasite can alter a protein's destination without any modifications to the protein itself, but instead by an element within the 5' UTR of the transcript. This uORF-dependent localization was confirmed by single molecule STORM imaging, followed by fusion of the uORF to a reporter gene which changes its cellular localization from cytoplasmic to ER-associated. These data point towards a novel regulatory role of uORF in protein trafficking, with important implications for the pathology of pregnancy-associated malaria.

Diagnosis of Imported Monkeypox, Israel, 2018.

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.

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1.

A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories.

The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed "viral factories," where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as "production lines" of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed "viral factories," are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles.

Virus-host interactions: insights from the replication cycle of the large Paramecium bursaria chlorella virus.

The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membrane-containing sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans and mice, proceeds through a bacteriophage-like infection pathway.

Enhanced killing of cervical cancer cells by combinations of methyl jasmonate with cisplatin, X or alpha radiation.

Current therapies for treatment of advanced cervical cancer involve the use of cisplatin, often in combination with radiotherapy. These treatments do not lead to a high survival rate and furthermore, serious side effects are dose-limiting factors. Methyl jasmonate (MJ) was recently identified as potent and selective cytotoxic agent towards cervical cancer cells. In the present study we evaluated the effectiveness of combined treatments of MJ with cisplatin or X-irradiation on a variety of cervical cancer cells including SiHa, CaSki, HeLa and C33A. Cytotoxicity of alpha particles, emitted from (224)Ra atoms, was also evaluated as a single agent and in combination with MJ. Cooperation between MJ and cisplatin in reducing cell viability (XTT assays) and survival (clonogenicity assays) was exhibited towards several cancer cell lines at a range of combination doses. MJ effectively cooperated also with X-ray irradiation, significantly lowering the radiation doses required to inhibit cell survival (ID50) of all tested cells lines. We show for the first time, that alpha irradiation selectively reduced cell viability and survival of cervical cancer cells. Lower doses of α irradiation were required as compared to X-irradiation to inhibit cell survival. Cooperation with MJ was demonstrated in part of the cancer cell lines. In conclusion, our studies point to α irradiation and MJ, novel anticancer agents, as potent candidates for treatment of cervical cancer, in single agent regiments and in combination. MJ can be added also to conventional X-ray and cisplatin therapies to increase their cytotoxic effect while lowering the effective dose.

Insights from the Infection Cycle of VSV-ΔG-Spike Virus.

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

Iron-Modified Blood Culture Media Allow for the Rapid Diagnosis and Isolation of the Slow-Growing Pathogen Francisella tularensis.

The life-threatening disease tularemia is caused by Francisella tularensis, an intracellular Gram-negative bacterial pathogen. Due to the high mortality rates of the disease, as well as the low respiratory infectious dose, F. tularensis is categorized as a Tier 1 bioterror agent. The identification and isolation from clinical blood cultures of F. tularensis are complicated by its slow growth. Iron was shown to be one of the limiting nutrients required for F. tularensis metabolism and growth. Bacterial growth was shown to be restricted or enhanced in the absence or addition of iron. In this study, we tested the beneficial effect of enhanced iron concentrations on expediting F. tularensis blood culture diagnostics. Accordingly, bacterial growth rates in blood cultures with or without Fe2+ supplementation were evaluated. Growth quantification by direct CFU counts demonstrated significant improvement of growth rates of up to 6 orders of magnitude in Fe2+-supplemented media compared to the corresponding nonmodified cultures. Fe2+ supplementation significantly shortened incubation periods for successful diagnosis and isolation of F. tularensis by up to 92 h. This was achieved in a variety of blood culture types in spite of a low initial bacterial inoculum representative of low levels of bacteremia. These improvements were demonstrated with culture of either Francisella tularensis subsp. tularensis or subsp. holarctica in all examined commercial blood culture types routinely used in a clinical setup. Finally, essential downstream identification assays, such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), immunofluorescence, or antibiotic susceptibility tests, were not affected in the presence of Fe2+. To conclude, supplementing blood cultures with Fe2+ enables a significant shortening of incubation times for F. tularensis diagnosis, without affecting subsequent identification or isolation assays. IMPORTANCE In this study, we evaluated bacterial growth rates of Francisella tularensis strains in iron (Fe)-enriched blood cultures as a means of improving and accelerating bacterial growth. The shortening of the culturing time should facilitate rapid pathogen detection and isolation, positively impacting clinical diagnosis and enabling prompt onset of efficient therapy.