Single Virus Research Articles

Quantum Dots: A Promising Fluorescent Label for Probing Virus Trafficking.

Recent research has highlighted the immense potential of the quantum dot (QD)-based single-virus tracking (SVT) technique in virology. In these experiments, the infection behaviors of single viruses or viral components, labeled with QDs, could be tracked on time scales of milliseconds to hours in host cells. The trajectories of individual viruses are reconstructed with nanometer accuracy, and the underlying dynamic information on virus infection can be extracted to uncover the infection mechanisms of viruses. Therefore, QD-based single-virus tracking (QSVT) is an exquisitely selective and powerful approach to investigating how viruses are internalized in host cells dynamically to release their genome for viral replication and assembly that ensure the completion of viral life cycles.QDs are better candidates than organic dyes and fluorescent proteins for virus labeling and subsequent SVT due to the following considerations: (i) the high brightness of QDs makes it possible to label a virus with sufficient brightness using very few QDs or even just one QD; (ii) the extraordinary photostability of QDs allows one to track the infection process long term and quantify low probability events; (iii) the color-tunable emission property of QDs ensures multicolor labeling of various components of a virus simultaneously; and (iv) the abundant surface ligands of QDs facilitate the conjugation of a virus with a variety of labeling strategies. Therefore, the photoproperties of QDs make it possible to perform multicolor long-term SVT experiments quantitatively. Nowadays, the QD-based SVT (QSVT) technique has made prodigious achievements in unraveling the entry, trafficking, and uncoating mechanisms of viruses. This fascinating technique can provide spatiotemporal dynamic information on the viral journey in unprecedented detail and has revolutionized our understanding of virus infection.In this Account, we first introduce the advantages and the limitations of conventional SVT in virological research and the unique features of QDs as labels in the SVT field. We subsequently focus on the principles and related methods of QSVT and the current state of QD chemistry and QD-based virus labeling that resolves many issues associated with the tracking of individual viruses in live cells. Then we emphasize some new findings by this technique in the study of infection mechanisms. Finally, we will provide our insights into future challenges on this topic. With this Account, we hope to further stimulate the development of QSVT with a combined effort from different disciplines and, more importantly, to accelerate the applications of QSVT in virological research.

A Psychosocial Impact of Covid-19 Pandemic on Frontline Workers and General People in Kathmandu Valley

A new variant of the corona virus family found in the Wuhan city market of the China causing serious harm to human being. After the WHO decided COVID-19 as a pandemic situation, everyone has started to measure the prevention from infectious disease according to WHO guidelines. It includes social distancing, isolation, quarantine, lockdown, sanitation and masking respectively. During the time researcher has observed the difficulties of cultivating new normal to people in Nepal. People have perceived the single corona virus differently; common populations and frontline workers have different perception towards to corona virus. Researcher started to measures the psychosocial impact of COVID-19 pandemic into frontline workers and general populations in Kathmandu valley. The total number of sample unit for this research is 82; it includes 52 general populations and 30 frontline workers.

Satellite Subgenomic Particles Are Key Regulators of Adeno-Associated Virus Life Cycle.

Historically, adeno-associated virus (AAV)-defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double-stranded DNA genome in a “snapback” configuration. The 5′- snapback genomes (SBGs) include the P5 promoters and partial rep gene sequences. The 3′-SBGs contains the capsid region. The molecular configuration of 5′-SBGs theoretically may allow double-stranded RNA transcription in their dimer configuration. Our studies demonstrated that 5-SBG regulated AAV rep expression and improved AAV packaging. In contrast, 3′-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5′-SBGs and 3′-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5′-SBGs and 3′-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of a viral genome and encodes additional essential functions.

Novel universal primer-pentaplex PCR assay based on chimeric primers for simultaneous detection of five common pig viruses associated with diarrhea

Viral pathogens associated with diarrhea in pigs include porcine circovirus 2 (PCV2), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (RVA) and C (RVC) among others. In this study, a novel universal primer-based pentaplex PCR (UP-M-PCR) assay was developed for simultaneous detection and differentiation of these five viruses. The assay uses a short-cycle multiplex amplification by chimeric primers (CP), which are virus specific, with a tail added at the 5′ end of the universal primer (UP), followed by universal amplification using UPs and a regular cycle amplification. Five universal primers with CPs (UP1-5) were designed and evaluated in an UP-based single PCR (UP–S-PCR). All five UPs were found to work efficiently and UP2 exhibited the best performance. After system optimizations, the analytical sensitivity of the UP-M-PCR, using plasmids containing the specific viral target fragments, was 5 copies/reaction for each of the five viruses irrespective of presence of a single or multiple viruses in the reaction. No cross-reaction was observed with other non-target viruses. When 273 fecal samples from clinically healthy pigs were tested, the assay sensitivity was 90.9–100%, the specificity was 98.0–100%, and the agreement rate with the UP-S-PCR was 98.5–99.6% with a Kappa value being 0.95–0.98. In summary, the UP-M-PCR developed here is a rapid and highly sensitive and specific detection method that can be used to demonstrate mixed infections in pigs with diarrhea.

Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

Enteric viruses seize their immunomodulatory niche

Enteric eukaryotic viruses are increasingly recognized as non-pathogenic, immunomodulatory participants in the healthy intestinal microbiota. In this issue of Cell Host & Microbe, Dallari etal. (2021) use a single virus infection approach to reveal both generalized viral immune responses and numerous unique, virus-specific response profiles.

Electronic Spiking Protein-Based COVID Sensors

Viruses are ultimate nanotechnology objects with cleverly designed capsid proteins to bind with cell membranes and exquisite DNA/RNA machinery to invade cells and replicate themselves using cells' machinery. Here I will discuss an electronic virus sensor that uses aptamers designed to bind with the COVID-19 spiking proteins S1 and S2 to capture the virus inside a quantum mechanical tunneling gap device. The current through the gap, that is sized to match the COVID-19 diameter of 125 nm, is monitored to detect the presence of the virus. The gap impedance also changes and can be monitored. The sensor works with small amount of saliva deposited over its active area. Saliva is a complex fluid composed of 99.49% water, enzymes, human cells, bacteria, opiorphin, haptocorrin, alfa-amylase, antibacterial compounds, mucus, and electrolytes (sodium, potassium, calcium, etc.). It also contains 60 nm -100 nm exosomes. Except for human cells, bacteria and exosomes, all other fresh saliva components have small molecules (mucus can produce large particles in old saliva). Exosomes and any other nanoparticles inside the fresh saliva can potentially produce false positive results. False negative results, on the other hand, are produced when the viruses in the saliva do not bind with the sensor aptamers. Filtering is used to exclude large (>200 nm) particles. Ultrasonication as well as electric field biasing/excitation techniques are used to ensure virus-aptamer encounter and binding. Exosomes' sizes make them difficult to exclude using filtering. Currently I am developing a technique based on dielectrophoretic responses of exosomes and viruses to preferentially trap the COVID-19 and repel the exosomes. The sensor's limit of detection is close to a single virus owing to its transduction technique of quantum mechanical tunneling current. I will discuss COVID-19 electronic sensor performance and its performance along with its fabrication and applications in this talk.

SOI-FET Snsors for Virus Detection

The recent outbreak of coronavirus disease caused by the respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need to develop fast and highly sensitive analytical tools and diagnostic devices to detect and study of the fundamental properties viruses and their nucleic acid and protein components [1]. Silicon-on-insulator field-effect transistors (SOI-FETs) based sensors provide the versatile platform for direct detection of biological species as excellent electrical signal converters. These devices have demonstrated applications for label-free, ultra-sensitive, and selective real-time detection of a wide range of biological species, including nucleic acids, proteins and viruses in either single-element or multiplexed formats [2-5]. Dielectrophoresis (DEP) and electro-hydrodynamic techniques are well known as the methods of selection and delivery of analytes to overcome limitations of diffusive transport to sensor elements without additional processing or labeling steps [6].The aim of this study was to investigate the features of the behavior of the viruses when they are indicated by SOI-FET sensors with DEF-control. For this, SOI-FET sensors with lateral DEP-electrodes and multichannel sensors, for comparison, were used. Top-down technology with using optical lithography was applied for sensor fabrication. Devices manufacturing details are described elsewhere [3]. As an analyte, we used nuclear polyhedrosis viruses (NPVs), coronavirus virus-like particles (CVP) and suspension of specific antibodies to the virus created in the Federal research center of Virology and biotechnology "Vector" of Rospotrebnadzor.The results showed that the sensors used in the study provide the subatomolar level of virus detection. DEP-concentration of viruses increase the response of the sensors by factors of 2 to 9 (compared to the response of sensors without DEP control) and allows the false positives can be eliminated. NPVs lose their mobility with prolonged exposure to an alternating field in the sub-MHz range. The DEP-concentration of CVP leads to the formation of crystal-like structures (Fig.1).This study was supported by grant no. 18-29-02091 of the Russian Foundation for Basic Research. Sample preparation of biological materials was done within the framework of the State Task of Rospotrebnadzor.Referencies: WHO Director-General's opening remarks at the media briefing on COVID-19 – 23 April 2021 23 апреля 2021 г.https://www.who.int/director-general/speeches/detail/who-director-general-s-opening-remarks-at-the-media-briefing-on-covid-19-23-april-2021.Patolsky F., Zheng G., Hayden O., Lakadamyali M., Zhuang X., Lieber C. M. Electrical detection of single viruses // Proc. Natl. Acad. Sci. 2004, v. 101. p. 14017-14022.O. V. Naumova, V. M. Generalov, E. G. Zaitseva, A. V. Latyshev, A. L. Aseev, S. A. Pyankov, I. V. Kolosov, G. G. Ananko, A. P. Agafonov, E. V. Gavrilova, R. A. Maksyutov, and A. S. Safatov. Biosensors Based on Soi Nanowire Transistors for Biomedicine and Virusology. Russian Microelectronics, 2021, v. 50, N3, p. 137–145.Ivanov Y.D., Pleshakova T.O., Kozlov A.F., Malsagova K.A., Krohin N.V., Shumyantseva V.V., Shumov I.D., Popov V.P., Naumova O.V., Fomin B.I., Nasimov D.A., Aseev A.L., Archakov A.I. SOI nanowire for the high-sensitive detection of HBsAg and a-fetoprotein. Lab Chip. 2012, v. 12, p. 5104-5111.Dmitrienko E., Naumova O., Fomin B., Kupryushkin M., Volkova A., Amirkhanov N., Semenov D., Pyshnaya I., Pyshnyi D. Surface modification of SOI FET sensors for label-free and specific detection of short RNA analyte. Nanomedicine 2016, v. 11, N 16, p. 2073-2082.Lee S., Roh S.M., Lee E., Park Y., Lee B.C., Kwon Y., Kim H.J., Kim J. Applications of converged various forces for detection of biomolecules and novelty of dielectrophoretic force in the applications(Review). Sensors, 2020, v.20, p.3242. Fig.1 – Optical image of sensor with lateral DEP electrodes after CVP detection. In the left - CVP organized in crystal-like structures under DEP-concentration, in the right (green dots on dark field) - viruses with luminescent labels. S - source, D - drain. G1, G2 - lateral DEP electrodes. Figure 1

Bacteriophages as surrogates for the study of viral dispersion in open air.

The SARS-CoV-2 pandemic has revived the debate about the routes of virus transmission and their likelihoods. It is of utmost importance to assess the risks of contamination of susceptible people by infectious individuals and to evaluate the level of SARS-CoV-2 and other respiratory viruses transmission in the community. Most countries have imposed non-pharmaceutical measures to contain SARS-CoV-2 transmission, including physical distancing and mask wearing. Here we evaluated the spreading of viruses in open air using harmless Escherichia coli bacteriophages as a surrogate. Phages were sprayed towards Petri dishes seeded with bacteria at different lengths and angles. Our results showed that the transmission rate decreased exponentially with distance. The highest recorded transmission rate was 9times 10^{-6} PFU/plate when phages were sprayed from a 1 m distance, suggesting that the probability of transmission of a single virus at a 1 m distance is 1:100,000.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00203-021-02382-8.

Preclinical evaluation of an engineered oncolytic herpes simplex virus for pediatric osteosarcoma.

10040 Background: Osteosarcoma is the most common primary bone tumor in children. For those with relapsed or metastatic disease, the five-year survival rate is approximately 20%, and survivors often suffer from long-term disability from current therapies. The high morbidity and mortality for these patients highlight a great need for improved therapies. One such novel therapeutic approach is oncolytic herpes simplex virus (oHSV) immunovirotherapy. We previously demonstrated that M002, an engineered oHSV that contains deletions of the neurovirulence gene preventing infection of normal cells, effectively infects and kills neuroblastoma and rhabdomyosarcoma. Currently, similar oHSVs are being evaluated in early phase clinical trials for children and adults with relapsed or refractory brain tumors. To date, there has been limited investigation of oncolytic virotherapy in osteosarcoma. Thus, we sought to examine the ability of oHSV, M002, to infect and kill osteosarcoma cells in vitro. Methods: We evaluated two long-term passaged human osteosarcoma cell lines, U2-OS and MG-63. Flow cytometry was used to assess baseline expression of oHSV viral entry-mediated receptors (CD111, CD112, syndecan, HVEM). Single and multi-step viral recovery experiments measured virus infectivity and replication. Cells were infected with increasing multiplicity of infection (MOI) of M002, and cell viability was measured 72 hours post-infection via alamarBlue assay. Results: Both MG-63 and U2-OS cells expressed HSV entry molecules (Table) including high levels of the primary HSV entry molecule CD111. Single step virus recovery experiments in MG-63 cells infected at a MOI of 10 plaque-forming units (PFU)/cell demonstrated a 3 log-fold increase in virus titer from 12 to 24 hours post-infection. For multi-step experiments, MG-63 cells were infected with a MOI of 0.1 PFU/cell; viral replication significantly increased from 1.1x103 PFU at 6 hours post-infection to 3.8x1010 PFU at 72 hours post-infection. M002 successfully decreased osteosarcoma viability with a lethal dose in 50% of cells (LD50)of 2.82 and0.67 PFU/cell for MG-63 and U2-OS cells, respectively. Notably, at a virus MOI of 5 PFU/cell, viability was decreased by 64% ± 0.1% (p<0.001 vs control) in MG-63 cells and 96% ± 0.1% (p<0.001 vs control) in U2-OS cells. Conclusions: MG-63 and U2-OS osteosarcoma cells express high levels of HSV entry receptors. Virus recovery experiments demonstrated the ability of M002 to infect cells and replicate over time. The viability of osteosarcoma cells significantly decreased following infection with M002. These data suggest M002 may be a promising novel therapeutic option for patients with osteosarcoma and warrant further investigation for translation to the clinical setting.[Table: see text]

Incidence of enterovirus in patients with acute gastroenteritis.

Enteroviruses (EV) have been linked to lymphocytic meningitis and exanthems, but they may also be involved in acute gastroenteritis (AGE), a condition whose aetiological agent often remains unidentified. In this work 1214 samples from individuals with AGE were studied with the aim of establishing the incidence of EV. The samples were collected between September and December in three different years and subjected to real-time genomic amplification in order to determine the viral load (VL). Of the 1214 samples studied, infection by a single virus was found in 328 cases (27%) and coinfection in 69 (5.7%). While adenoviruses (AdV) were the most frequent (14.8% of total), EV were present in 126 (10.4%) of the individuals tested. Of the 126 EV-positive samples, this virus was found as a single infection and coinfection in 76 (6.3%) and 50 (4.1%) cases, respectively. VL for EV was 5.58±1.51 log copies/ml (range 3.73-9.69) in the former and 6.27±1.75 (range 3.73-10.5) (p=0.02) in the latter. EV were identified in 97 children under 5 (16.9%) and in 29 (4.5%) patients over 5. Patients less than 5 years showed a higher VL that those more than 5 years age [6.08±1.57 (range 3.82-9.69) vs. 5.07±1.53 (range 3.73-10.58); (p=0.002)]. There was a high incidence of EV in AGE patients, and they were more frequent in those under 5, where they were found to replicate more efficiently. These results therefore indicate that testing for EV should be included in the diagnosis of AGE.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane.

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.

Coinfections with Respiratory Pathogens among COVID-19 Patients in Korea.

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in upper and lower respiratory specimens and coinfection with other respiratory pathogens in patients with coronavirus disease 2019 (COVID-19) was investigated. Study subjects (N = 342) were retrospectively enrolled after being confirmed as SARS-CoV-2 positive, and their nasopharyngeal swab (NPS), oropharyngeal swab (OPS), and sputum specimens were restored for SARS-CoV-2 retesting and respiratory pathogen detection. The majority of the subjects (96.5%, N = 330) were confirmed as SARS-CoV-2 positive using NPS/OPS specimens. Among the COVID-19 patients (N = 342), 7.9% (N = 27) and 0.9% (N = 3) were coinfected with respiratory viruses and Mycoplasma pneumoniae, respectively, yielding an 8.8% (N = 30) overall respiratory pathogen coinfection rate. Of the respiratory virus coinfection cases (N = 27), 92.6% (N = 25) were coinfected with a single respiratory virus and 7.4% (N = 2) with two viruses (metapneumovirus/adenovirus and rhinovirus/bocavirus). No triple coinfections of other respiratory viruses or bacteria with SARS-CoV-2 were detected. Respiratory viruses coinfected in the patients with COVID-19 were as follows: rhinovirus (N = 7, 2.1%), respiratory syncytial virus A and B (N = 6, 1.8%), non-SARS-CoV-2 coronaviruses (229E, NL63, and OC43, N = 5, 1.5%), metapneumovirus (N = 4, 1.2%), influenza A (N = 3, 0.9%), adenovirus (N = 3, 0.9%), and bocavirus (N = 1, 0.3%). In conclusion, the diagnostic value of utilizing NPS/OPS specimens is excellent, and, as the first report in Korea, coinfection with respiratory pathogens was detected at a rate of 8.8% in patients with COVID-19.

A Simulation of the Use of High Throughput Sequencing as Pre-Screening Assay to Enhance the Surveillance of Citrus Viruses and Viroids in the EPPO Region

Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected. In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described.

Is viral coinfection a risk factor for severe lower respiratory tract infection? A retrospective observational study.

ObjectiveTo determine whether viral coinfection is a risk for severe lower respiratory tract infection (LRTI).Working HypothesisChildren with viral coinfection had a higher risk for admission to the intensive care unit (ICU) than those with a single virus infection.Study DesignRetrospective, observational study for 10 years.Patient‐Subject SelectionChildren between 1 and 60 months of age hospitalized with LRTI.

The Human Bone Marrow Is Host to the DNAs of Several Viruses.

The long-term impact of viruses residing in the human bone marrow (BM) remains unexplored. However, chronic inflammatory processes driven by single or multiple viruses could significantly alter hematopoiesis and immune function. We performed a systematic analysis of the DNAs of 38 viruses in the BM. We detected, by quantitative PCRs and next-generation sequencing, viral DNA in 88.9% of the samples, up to five viruses in one individual. Included were, among others, several herpesviruses, hepatitis B virus, Merkel cell polyomavirus and, unprecedentedly, human papillomavirus 31. Given the reactivation and/or oncogenic potential of these viruses, their repercussion on hematopoietic and malignant disorders calls for careful examination. Furthermore, the implications of persistent infections on the engraftment, regenerative capacity, and outcomes of bone marrow transplantation deserve in-depth evaluation.

Anti-TMV Activities of Pantoea agglomerans Lipopolysaccharides in vitro

Today there are no antiviral drugs of chemical nature that can completely cure virus-infected plants. The fact that their effect is limited to minimizing the pathogenic effect of viruses motivates many researchers to look for alternatives. In recent years it has been shown that lipopolysaccharides (LPS) of some bacteria, in particular representatives of the Pseudomonas genus were active against Tobacco mosaic virus (TMV). Therefore, we were interested in the additional study of LPS of phytopathogenic bacteria Pantoea agglomerans as a possible drug acting as antiviral agent. The aim of current study was to evaluate the antiviral activities of LPS obtained from phytopathogenic bacteria P. agglomerans against TMV in vitro. Methods. The antiviral activity of LPS preparations was investigated in vitro and assessed according to the inhibition percentage towards the number of local lesions in Datura stramonium leaves. P. agglomerans LPS was isolated from dry bacterial mass by phenol-water method. LPS mild acid degradation allowed to separate O-specific polysaccharide (OPS) and lipid A, which structures were identified by us earlier. The analysis of TMV and LPS interactions was carried out using a JEM 1400 transmission electron microscope (Jeol, Japan) at an accelerating voltage of 80 kV. Results. The most active were LPS preparations from P. agglomerans P324 and 8488. In vitro inhibitory efficacies of TMV infection by these LPS preparations was 59 and 60% respectively. LPS preparations of P. agglomerans 7969, 7604 and 9637, on the contrary, were inactive. Comparative analysis of the antiviral activity of LPS structural components of two P. agglomerans P324 and 7604 strains showed that the greatest inhibitory effect on the infectivity of TMV was exhibited by P. agglomerans P324 lipid A, the antiviral activity of which practically did not differ from the activity of the LPS molecule (it was lower by 7%). At the same time, the inhibitory effect of P. agglomerans 7604 core oligosaccharide (OG-core) against TMV was slightly higher compared to the effect of the whole LPS molecule. It can be assumed that the OG-core stimulated the defense mechanisms of plants and prevented the development of viral infection. Electron microscopic dates have shown that P. agglomerans P324 LPS at the concentration of 1 mg/ml influenced on freely located virions in the control causing “sticking” thus forming dense clusters, complexes or “bundles” of the virus. The individual structural components of P. agglomerans P324 LPS (lipid A and OG-core) did not have the same effect as a whole molecule. Conclusions. The study of the antiviral activity of LPS in the model system TMV – Datura stramonium L. plants showed that the most active were LPS preparations of only two strains of P. agglomerans (P324 and 8488) while the other seven strains were inactive. Individual structural components: lipid A from P. agglomerans P324 and OG-core from P. agglomerans 7604 decreased the infectivity of TMV by 7 and 15% higher than the initial LPS molecule. According to electron microscopy data the virions sticked together forming the dense clusters in case of the direct LPS-virus contacting in vitro whereas in the control it was observed just a single free virus particles. A more detailed study of the effect of individual structural components will help to understand the regularities of the LPS structure effect on TMV infectivity.

Constricted Apertures for Dynamic Trapping and Micro-/Nanoscale Discrimination Based on Recapture Kinetics.

Sensing via analyte passage through a constricted aperture is a powerful and robust technology which is being utilized broadly, from DNA sequencing to single virus and cell characterization. Micro- and nanoscale structures typically translocate a constricted aperture, or pore, using electrophoretic force. In the present work, we explore the advances in metrology which can be achieved through rapid directional switching of hydrodynamic forces. Interestingly, multipass measurements of microscale and nanoscale structures achieve cell discrimination. We explore this cell-discrimination phenomenon as well as other features of hydrodynamic focusing such as dynamic trapping and discrete interval sensing.

Information Encoded by the Flavivirus Genomes beyond the Nucleotide Sequence.

The genus Flavivirus comprises numerous, small, single positive-stranded RNA viruses, many of which are important human pathogens. To store all the information required for their successful propagation, flaviviruses use discrete structural genomic RNA elements to code for functional information by the establishment of dynamic networks of long-range RNA–RNA interactions that promote specific folding. These structural elements behave as true cis-acting, non-coding RNAs (ncRNAs) and have essential regulatory roles in the viral cycle. These include the control of the formation of subgenomic RNAs, known as sfRNAs, via the prevention of the complete degradation of the RNA genome. These sfRNAs are important in ensuring viral fitness. This work summarizes our current knowledge of the functions performed by the genome conformations and the role of RNA–RNA interactions in these functions. It also reviews the role of RNA structure in the production of sfRNAs across the genus Flavivirus, and their existence in related viruses.

Rescue of Infectious Sindbis Virus by Yeast Spheroplast-Mammalian Cell Fusion.

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.