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Controlling the progression of contagious diseases is crucial for public health management, emphasizing the importance of early viral infection diagnosis. In response, lateral flow assays (LFAs) have been successfully utilized in point-of-care (POC) testing, emerging as a viable alternative to more traditional diagnostic methods. Recent advancements in virus detection have primarily leveraged methods such as reverse transcription-polymerase chain reaction (RT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and the enzyme-linked immunosorbent assay (ELISA). Despite their proven effectiveness, these conventional techniques are often expensive, require specialized expertise, and consume a significant amount of time. In contrast, LFAs utilize nanomaterial-based optical sensing technologies, including colorimetric, fluorescence, and surface-enhanced Raman scattering (SERS), offering quick, straightforward analyses with minimal training and infrastructure requirements for detecting viral proteins in biological samples. This review describes the composition and mechanism of and recent advancements in LFAs for viral protein detection, categorizing them into colorimetric, fluorescent, and SERS-based techniques. Despite significant progress, developing a simple, stable, highly sensitive, and selective LFA system remains a formidable challenge. Nevertheless, an advanced LFA system promises not only to enhance clinical diagnostics but also to extend its utility to environmental monitoring and beyond, demonstrating its potential to revolutionize both healthcare and environmental safety.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro . We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease., Competing Interests: The authors declare no conflict of interest.

In influenza A virus-infected cells, newly synthesized viral neuraminidases (NAs) transiently localize at the host cell Golgi due to glycosylation, before their expression on the cell surface. It remains unproven whether Golgi-localized intracellular NAs exhibit sialidase activity. We have developed a sialidase imaging probe, [2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenyl]-α-D-N-acetylneuraminic acid (BTP9-Neu5Ac). This probe is designed to be cleaved by sialidase activity, resulting in the release of a hydrophobic fluorescent compound, 2-(benzothiazol-2-yl)-5-(non-1-yn-1-yl) phenol (BTP9). BTP9-Neu5Ac makes the location of sialidase activity visually detectable by the BTP9 fluorescence that results from the action of sialidase activity. In this study, we established a protocol to visualize the sialidase activity of intracellular NA at the Golgi of influenza A virus-infected cells using BTP9-Neu5Ac. Furthermore, we employed this fluorescence imaging protocol to elucidate the intracellular inhibition of laninamivir octanoate, an anti-influenza drug. At approximately 7 h after infection, newly synthesized viral NAs localized at the Golgi. Using our developed protocol, we successfully histochemically stained the sialidase activity of intracellular viral NAs localized at the Golgi. Importantly, we observed that laninamivir octanoate effectively inhibited the intracellular viral NA, in contrast to drugs like zanamivir or laninamivir. Our study establishes a visualization protocol for intracellular viral NA sialidase activity and visualizes the inhibitory effect of laninamivir octanoate on Golgi-localized intracellular viral NA in infected cells., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: T. T., O. T., K. I., and A. M. are inventors of a Japanese patent (patent registration JP6493964) on a chemical structure of BTP9-Neu5Ac. This patent was implicated by institutions, University of Shizuoka and Josho Gakuen. The other authors declare that they have no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Extracellular vesicles are surrounded by a phospholipid bilayer, carrying various active biomolecules and participating in many physiological and pathological processes, including infectious ones., Objective: To research the role of exosomes in intercellular interactions in the pathogenesis of various types of lung damage in fatal cases of COVID-19., Material and Methods: We conducted a clinical and morphological analysis of 118 fatal cases caused by coronavirus infection in Moscow. We selected 32 cases with morphological signs of various types of lung lesions for immunohistochemical reaction (IHC) with antibodies against tetraspanin proteins (CD63, CD81), which are involved in the assembly of exosomes, as well as with antibodies against viral proteins: nucleocapsid and spike protein. We determined the main producing cells of extracellular vesicles and cells containing viral proteins, carried out their comparison and quantitative analysis., Results: IHC reaction with antibodies against CD63 showed cytoplasmic granular uniform and subapical staining of cells, as well as granular extracellular staining. We determined similar staining using antibodies against viral proteins. Extracellular vesicles were found in the same cells as viral proteins. The main producing cells of vesicles and cells containing viral proteins were found to be macrophages, type II pneumocytes, and endothelial cells., Conclusion: Taking into account the results of the literature, the localization of viral proteins and extracellular vesicles in the same cells indicates the key role of vesicles in the pathogenesis of various forms of lung damage by the SARS-CoV-2 virus, in the dissemination of the pathogen in the organism, which leads to interaction with the adaptive immune system and the formation of immunity.

Importance: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family., Competing Interests: The authors declare no conflict of interest.

Influenza A virus (IAV) infection relies on the action of the hemagglutinin (HA) and neuraminidase (NA) membrane proteins. The HA ligands anchor the IAV virion to the cell's surface by binding the sialic acid (SA) present on the host's receptors while NA is an enzyme capable of cleaving the SA from the extracellular environment. It is believed that the activity of NA ligands increases the motility of the virions favoring the propagation of the infection. In this work, we develop a numerical framework to study the dynamics of a virion moving across the cell surface for timescales much bigger than the typical ligand-receptor reaction times. We find that the rates controlling the ligand-receptor reactions and the maximal distance at which a pair of ligand-receptor molecules can interact greatly affect the motility of the virions. We also report on how different ways of organizing the two types of ligands on the virions' surface result in different types of motion that we rationalize using general principles. In particular, we show how the emerging motility of the virion is less sensitive to the rate controlling the enzymatic activity when NA ligands are clustered.

Filoviruses are filamentous enveloped viruses belonging to the family Filoviridae, in the order Mononegavirales. Some filovirus members, such as Ebola virus and Marburg virus, cause severe hemorrhagic fever in humans and non-human primates. The filovirus ribonucleoprotein complex, called the nucleocapsid, forms a double-layered helical structure in which a non-segmented, single-stranded, negative-sense RNA genome is encapsidated by the nucleoprotein (NP), viral protein 35 (VP35), VP24, VP30 and RNA-dependent RNA polymerase (L). The inner layer consists of the helical NP-RNA complex, acting as a scaffold for the binding of VP35 and VP24 that constitute the outer layer. Recent structural studies using cryo-electron microscopy have advanced our understanding of the molecular mechanism of filovirus nucleocapsid formation. Here, we review the key characteristics of the Ebola virus and Marburg virus nucleocapsid structures, highlighting the similarities and differences between the two viruses. In particular, we focus on the structure of the helical NP-RNA complex, the RNA binding mechanism and the NP-NP interactions in the helix. The structural analyses reveal a possible mechanism of nucleocapsid assembly and provide potential targets for the anti-filovirus drug design., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Human cytomegalovirus (HCMV) is a major cause of illness in immunocompromised individuals. The HCMV lytic cycle contributes to the clinical manifestations of infection. The lytic cycle occurs over ∼96 h in diverse cell types and consists of viral DNA (vDNA) genome replication and temporally distinct expression of hundreds of viral proteins. Given its complexity, understanding this elaborate system can be facilitated by the introduction of mechanistic computational modeling of temporal relationships. Therefore, we developed a multiplicity of infection (MOI)-dependent mechanistic computational model that simulates vDNA kinetics and late lytic replication based on in-house experimental data. The predictive capabilities were established by comparison to post hoc experimental data. Computational analysis of combinatorial regulatory mechanisms suggests increasing rates of protein degradation in association with increasing vDNA levels. The model framework also allows expansion to account for additional mechanisms regulating the processes. Simulating vDNA kinetics and the late lytic cycle for a wide range of MOIs yielded several unique observations. These include the presence of saturation behavior at high MOIs, inefficient replication at low MOIs, and a precise range of MOIs in which virus is maximized within a cell type, being 0.382 IU to 0.688 IU per fibroblast. The predicted saturation kinetics at high MOIs are likely related to the physical limitations of cellular machinery, while inefficient replication at low MOIs may indicate a minimum input material required to facilitate infection. In summary, we have developed and demonstrated the utility of a data-driven and expandable computational model simulating lytic HCMV infection.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality., (© 2021 Janssen Vaccines & Prevention B.V. Electrophoresis published by Wiley-VCH GmbH.)

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies., Competing Interests: The authors have declared that no competing interests exist.

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66-85.84%) and 95.39% (95% confidence interval: 94.32-96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods., (© 2022. The Author(s).)

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies., Competing Interests: AH, KT, MU, JA, LE, FE No competing interests declared, DF, TM, JV, RP, SF, SH, IB, AB, LC employed by Waters Corporation, MR, RY, MP, TP, LA employed by SISCAPA Assay Technologies, (© 2021, Hober et al.)

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21-99.98) and specificity (100%, 95% CI 96.31-100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation., (© 2021. The Author(s).)

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.

The viral protein containers that encapsulate a virus' genetic material are repurposed as virus-like particles in a host of nanotechnology applications, including cargo delivery, storage, catalysis, and vaccination. These viral architectures have evolved to sit on the knife's edge between stability, to provide adequate protection for their genetic cargoes, and instability, to enable their efficient and timely release in the host cell environment upon environmental cues. By introducing a percolation theory for viral capsids, we demonstrate that the geometric characteristics of a viral capsid in terms of its subunit layout and intersubunit interaction network are key for its disassembly behavior. A comparative analysis of all alternative homogeneously tiled capsid structures of the same stoichiometry identifies evolutionary drivers favoring specific viral geometries in nature and offers a guide for virus-like particle design in nanotechnology.

The last step in influenza virus replication involves the assembly of viral components on the infected cell's plasma membrane followed by budding of intact virus from the host cell surface. Because viral neuraminidase and hemagglutinin are both inserted into the host cell's membrane during this process, influenza virus-infected cells are distinguished from uninfected cells by the presence of viral neuraminidase and hemagglutinin on their cell surfaces. In an effort to exploit this difference in cell surface markers for development of diagnostic and therapeutic agents, we have modified an influenza neuraminidase inhibitor, zanamivir, for targeting of attached imaging and therapeutic agents selectively to influenza viruses and virus-infected cells. We have designed here a zanamivir-conjugated rhodamine dye that allows visual monitoring of binding, internalization, and intracellular trafficking of the fluorescence-labeled neuraminidase in virus-infected cells. We also synthesize a zanamivir- 99m Tc radioimaging conjugate that permits whole body imaging of the virus's biodistribution and abundance in infected mice. Finally, we create both a zanamivir-targeted cytotoxic drug (i.e., zanamivir-tubulysin B) and a viral neuraminidase-targeted CAR T cell and demonstrate that they are both able to kill viral neuraminidase-expressing cells without damaging healthy cells. Taken together, these data suggest that the influenza virus neuraminidase inhibitor, zanamivir, can be exploited to improve the diagnosis, imaging, and treatment of influenza virus infections.

Hunters are at a higher risk for exposure to zoonotic pathogens due to their close interactions with wildlife and arthropod vectors. In this study, high throughput sequencing was used to explore the viromes of two tick species, Amblyomma dissimile and Haemaphysalis juxtakochi, removed from hunted wildlife in Trinidad and Tobago. We identified sequences from 3 new viral species, from the viral families Orthomyxoviridae, Chuviridae and Tetraviridae in A. dissimile., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mycoplasma pneumoniae is the prevalent cause of acquired respiratory infections around the globe. A multi-epitope vaccine (MEV) must be developed to combat infections of M. pneumoniae because there is no specific disease-modifying treatment or vaccination is present. The objective of this research is to design a vaccine that targets M. pneumoniae top five highly antigenic proteins using a combination of immunological techniques and molecular docking. T-cell (HTL & CTL), B-cell, and IFN-γ of target proteins were forecasted and highly conservative epitopes were chosen for further study. For designing of final vaccine, 4LBL, 7CTL, and 5HTL epitopes were joined by linkers of KK, AAY, and GPGPG. The N-end of the vaccine was linked to an adjuvant (Cholera enterotoxin subunit B) with a linker named EAAAK to enhance immunogenicity. After the addition of adjuvants and linkers, the size of the construct was 395 amino acids. The epitopes of IFN-γ and B-cells illustrate that the model construct is optimized for cell-mediated immune or humoral responses. To ensure that the final design is safer and immunogenic, properties like non-allergens, antigenicity, and various physicochemical properties were evaluated. Molecular docking of the vaccine with the toll-like receptor 4 (TLR4) was conducted to check the compatibility of the vaccine with the receptor. Besides, in-silico cloning was utilized for validation of the credibility and proper expression of the vaccine. Furthermore, to confirm that the multi-epitope vaccine created is protective and immunogenic, this research requires experimental validation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4 + T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic ( n  = 18) and antiretroviral therapy (ART)-treated subjects ( n  = 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. IMPORTANCE Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus , order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae , which is available at ictv.global/report/portogloboviridae.

Aptamers are short single-stranded DNA or RNA oligonucleotides selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have been demonstrated to bind various targets from small-molecule to cells or even tissues in the way of antibodies. Thus, they are called chemical antibodies. We summarize and evaluate recent developments in aptamer-based sensors (for short aptasensors) for virus detection in this review. These aptasensors are mainly classified into optical and electronic aptasensors based on the type of transducer. Nowadays, the smartphone has become the most widely used mobile device with billions of users worldwide. Considering the ongoing COVID-19 outbreak, smartphone-based aptasensors for a portable and point-of-care test (POCT) of COVID-19 detection will be of great importance in the future., (© 2021 Wiley-VCH GmbH.)

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (R h ) measured by online DLS with the R h values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R g /R h ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.

Cyprinid herpesvirus 2 (CyHV-2), a member of the genus Cyprinivirus in the family Alloherpesviridae, has attracted worldwide attention because it causes severe disease and high mortality in crucian carp and goldfish. In this study, we focus on mRNA, protein and viral miRNA expression profiles in C. auratus gibelio caudal fin (GiCF) cells infected with CyHV-2, using high-throughput sequence techniques and TMT-labelled analyses. The results revealed that 156 virus genes were differentially expressed during the infection. Among these differentially expressed genes, 7 viral genes were significantly up-regulated and 28 were significantly down-regulated at 96 hpi (hours post-infection) vs 48 hpi. Besides, a total of 78 viral proteins, including a large number of membrane proteins and capsid proteins associated with the viral assembly, were successfully detected by using proteome analysis. Furthermore, a total of 225,143,474 raw reads were generated from cDNA library of CyHV-2-infected GiCF cells using high-throughput sequencing technology. Following annotation and secondary structure prediction, 10 viral miRNAs were found as significantly modulated in CyHV-2-infected GiCF cells (2 down-regulated and 8 up-regulated). Finally, the CyHV-2 genes (orf19, orf23, orf118, orf121, orf127) targeted by the viral miRNA CyHV-2-KT-635 identified in this study, were predicted and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the regulation of CyHV-2-KT-635 on orf121 protein expression was verified by western blotting assay. Taken together, this study provides a valuable basis for further research on the expression of virus genes during CyHV-2 replication and the molecular mechanisms by which miRNA may regulate CyHV-2 virus., (© 2021 John Wiley & Sons Ltd.)

A major part of the analysis of parallel reaction monitoring (PRM) data is the comparison of observed fragment ion intensities to a library spectrum. Classically, these libraries are generated by data-dependent acquisition (DDA). Here, we test Prosit, a published deep neural network algorithm, for its applicability in predicting spectral libraries for PRM. For this purpose, we targeted 1529 precursors derived from synthetic viral peptides and analyzed the data with Prosit and DDA-derived libraries. Viral peptides were chosen as an example, because virology is an area where in silico library generation could significantly improve PRM assay design. With both libraries a total of 1174 precursors were identified. Notably, compared to the DDA-derived library, we could identify 101 more precursors by using the Prosit-derived library. Additionally, we show that Prosit can be applied to predict tandem mass spectra of synthetic viral peptides with different collision energies. Finally, we used a spectral library predicted by Prosit and a DDA library to identify SARS-CoV-2 peptides from a simulated oropharyngeal swab demonstrating that both libraries are suited for peptide identification by PRM. Summarized, Prosit-derived viral spectral libraries predicted in silico can be used for PRM data analysis, making DDA analysis for library generation partially redundant in the future., (© 2021 The Authors. Proteomics published by Wiley-VCH GmbH.)

Porcine circovirus type 3 (PCV3) has been reported in many countries such as USA, China, Korea and many European countries during 2015-2018. The six PCV3 strains named IH, SJ, N5, N10, N13 and N62 were detected out of 220 samples by PCR methods while the prevalence our study was conducted in 2017 to 2018. The six detected strains were hard to genotype with reference viruses due to their diverse phylogenetic relationship. PCV3 capsid, ORF3 and replicase protein coding genes were reassembled at the nucleotide sequence level, then 16 new reassembled PCV3 sequences were generated. Based on the maximum likelihood mapping analysis of 303 PCV3 sequences a model with a combination of replicase, ORF3 and capsid protein coding genes was selected as the most appropriate target for genotyping, which provided the best support for the clade classification into three genotypes and several subtypes (genotype 1, genotype 2; subtype: a and b, genotype 3; subtype a, b, c, d, e, f, g, h). This study, the IH_Korea_2017 and N62_Korea_2018 strains belong to genogroup 3 (subtype a) the SJ_Korea_2017 strain genogroup 3 (subtype g) and the N5, N10, N13 Korea_ 2018 strains genogroup 3 (subtype f), respectively. In conclusion, this study may provide insights to classification of PCV3 genotypes around the world., (© 2020 The Authors Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

The graphene revolution, which has taken place during the last 15 years, has represented a paradigm shift for science. The extraordinary properties possessed by this unique material have paved the road to a number of applications in materials science, optoelectronics, energy, and sensing. Graphene-related materials (GRMs) are now produced in large scale and have found niche applications also in the biomedical technologies, defining new standards for drug delivery and biosensing. Such advances position GRMs as novel tools to fight against the current COVID-19 and future pandemics. In this regard, GRMs can play a major role in sensing, as an active component in antiviral surfaces or in virucidal formulations. Herein, the most promising strategies reported in the literature on the use of GRM-based materials against the COVID-19 pandemic and other types of viruses are showcased, with a strong focus on the impact of functionalization, deposition techniques, and integration into devices and surface coatings., (© 2021 Wiley-VCH GmbH.)

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Background & Aims: During treatment of chronic HBV infections, loss or seroconversion of the HBV surface antigen (HBsAg) is considered a functional cure. HBsAg consists of the large (LHBs), middle (MHBs), and small surface protein (SHBs) and their relative proportions correlate strongly with disease stage. Our aim was to assess the association between HBsAg composition and functional cure during treatment., Methods: A total of 83 patients were retrospectively analyzed. HBsAg loss was achieved by 17/64 patients during nucleos(t)ide analogue (NA) treatment and 3/19 patients following treatment with pegylated interferon-alfa2a (PEG-IFN) for 48 weeks. Sixty-three patients without HBsAg loss were matched as controls. LHBs, MHBs and SHBs were quantified in sera collected before and during treatment., Results: Before treatment, median MHBs levels were significantly lower in patients with subsequent HBsAg loss than in those without (p = 0.005). During treatment, MHBs and LHBs proportions showed a fast decline in patients with HBsAg loss, but not in patients with HBV e antigen seroconversion only or patients without serologic response. MHBs became undetectable by month 6 of NA treatment in all patients with HBsAg loss, which occurred on average 12.8 ± 8.7 (0-52) months before loss of total HBsAg. Receiver-operating characteristic analyses revealed that the proportion of MHBs was the best early predictor of HBsAg loss before NA treatment (AUC = 0.726, p = 0.019). In patients achieving HBsAg loss with PEG-IFN, the proportions of MHBs and LHBs showed similar kinetics., Conclusion: Quantification of HBsAg proteins shows promise as a novel tool to predict early treatment response. These assessments may help optimize individual antiviral treatment, increasing the rates of functional cure in chronically HBV-infected patients., Lay Summary: The hepatitis B surface antigen (HBsAg) is a key serum marker for viral replication. Loss of HBsAg is considered stable remission, which can be achieved with antiviral treatments. We have investigated whether the ratios of the different components of HBsAg, namely the large (LHBs) and medium (MHBs) HBsAg during different treatments are associated with the occurrence of HBsAg loss. We found that LHBs and MHBs decrease earlier than total HBsAg before HBsAg loss and we propose LHBs and MHBs as promising novel biomarker candidates for predicting cure of HBV infection., Competing Interests: Conflict of interest There are no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Supramolecular coordination complexes (SCCs) have emerged as anticancer agents. Tracking the movement of these metallic anticancer agents plays an important role in the field of biomedicines. Herein, we describe a method for tracking the movement of a rhomboidal Pt(II) metallacycle agent using the quantum dots encapsidation in vitro self-assembly system of viral proteins. When incubated with living Vero cells, self-assembly of hybrid viral nanoparticles were employed for simultaneous cell imaging and visual transmission of the Pt(II) metallacycle agent. Considering these results, we believe that the multifunctional biomaterials consisting of a supramolecular coordination complex and quantum dots provide a new alternative for probing of the delivery of Pt(II) metallacycle drugs.

Proteomics offers a wide collection of methodologies to study biological systems at the finest granularity. Faced with COVID-19, the most worrying pandemic in a century, proteomics researchers have made significant progress in understanding how the causative virus hijacks the host's cellular machinery and multiplies exponentially, how the disease can be diagnosed, and how it develops, as well as its severity predicted. Numerous cellular targets of potential interest for the development of new antiviral drugs have been documented. Here, the most striking results obtained in the proteomics field over this first semester of the pandemic are presented. The molecular machinery of SARS-CoV-2 is much more complex than initially believed, as many post-translational modifications can occur, leading to a myriad of proteoforms and a broad heterogeneity of viral particles. The interplay of protein-protein interactions, protein abundances, and post-translational modifications has yet to be fully documented to provide a full picture of this intriguing but lethal biological threat. Proteomics has the potential to provide rapid detection of the SARS-CoV-2 virus by mass spectrometry proteotyping, and to further increase the knowledge of severe respiratory syndrome COVID-19 and its long-term health consequences., (© 2020 Wiley-VCH GmbH.)

Vaccines against infectious diseases are urgently needed. Therefore, modern analytical method development should be as efficient as possible to speed up vaccine development. The objectives of the study were to identify critical method parameters (CMPs) and to establish a set of steps to efficiently develop and validate a CE-SDS method for vaccine protein analysis based on a commercially available gel buffer. The CMPs were obtained from reviewing the literature and testing the effects of gel buffer dilution. A four-step approach, including two multivariate DoE (design of experiments) steps, was proposed, based on CMPs and was verified by CE-SDS method development for: (i) the determination of influenza group 1 mini-hemagglutinin glycoprotein; and (ii) the determination of polio virus particle proteins from an inactivated polio vaccine (IPV). The CMPs for sample preparation were incubation temperature(s) and time(s), pH, and reagent(s) concentration(s), and the detection wavelength. The effects of gel buffer dilution revealed the CMPs for CE-SDS separation to be the effective length, the gel buffer concentration, and the capillary temperature. The four-step approach based on the CMPs was efficient for the development of the two CE methods. A four-step approach to efficiently develop capillary gel electrophoresis methods for viral vaccine protein analysis was successfully established., (© 2020 The Authors. Electrophoresis published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Duck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh., Aim: This study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV., Methods: A total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank., Results: Of the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity., Conclusion: Based on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry., Competing Interests: The authors declare that there is no conflict of interest.

The pandemic causing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally infected more than 50 million people and ∼1.2 million have succumbed to this deadly pathogen. With the vaccine trials still in clinical phases, mitigation of Coronavirus Disease 2019 (COVID-19) relies primarily on robust virus detection methods and subsequent quarantine measures. Hence, the importance of rapid, affordable and reproducible virus testing will serve the need to identify and treat infected subjects in a timely manner. Based on the type of diagnostic assay, the primary targets are viral genome (RNA) and encoded proteins. Currently, COVID-19 detection is performed using various molecular platforms as well as serodiagnostics that exhibit approximately 71% sensitivity. These methods encounter several limitations including sensitivity, specificity, availability of skilled expertise and instrument access. Saliva-based COVID-19 diagnostics are emerging as a superior alternative to nasal swabs because of the ease of sample collection, no interaction during sampling, and high viral titers during early stages of infection. In addition, SARS-CoV-2 is detected in the environment as aerosols associated with suspended particulate matter. Designing virus detection strategies in diverse samples will allow timely monitoring of virus spread in humans and its persistence in the environment. With the passage of time, advanced technologies are overcoming limitations associated with detection. Enhanced sensitivity and specificity of next-generation diagnostics are key features enabling improved prognostic care. In this comprehensive review, we analyze currently adopted advanced technologies and their concurrent use in the development of diagnostics for SARS-CoV-2 detection.

Chronic hepatitis B virus (HBV) infection has been a serious public health burden worldwide. Current anti-HBV therapies could not eliminate HBV ultimately. Considering the characteristics of HBV, it is impossible to be entirely cured based on current therapies. Therefore, it is urgently needed to develop novel therapeutic agents with new mechanism of action. The dihydroquinolizinone (DHQ) derivatives exhibited potent anti-HBV activity by decreasing HBV DNA and HBsAg level in an obscure mechanism of action. In this study, we have optimized the DHQ scaffold, developed the photoaffinity probe, with which to identify potential binding proteins., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The natural MERS-CoV infection in dromedary camels is understudied. Recent experimental studies showed no obvious clinical signs in the infected dromedary camels. Aim: To study the pathological changes associated with natural MERS-CoV infection in dromedary camels. Methods: Tissues from three MERS-CoV positive animals as well as two negative animals were collected and examined for the presence of pathological changes. The screening of the animals was carried out first by the rapid agglutination test and then confirmed by the RT-PCR. The selected animals ranged from six to twelve months in age. The sensitivity of the latter technique was much higher in the detection of MERS-CoV than the Rapid test (14 out of 75 animals positive or 18% versus 31 out of 75 positive or 41%). Results: MERS-CoV induced marked desquamation of the respiratory epithelium accompanied by lamina propria and submucosal mononuclear cells infiltration, epithelial hyperplasia in the respiratory tract, and interstitial pneumonia. Ciliary cell loss was seen in the trachea and turbinate. In addition, degeneration of glomerular capillaries with the complete destruction of glomerular tufts that were replaced with fibrinous exudate in renal corpuscles in the renal cortex were noticed. Expression of the MERS-CoV-S1 and MERS-CoV-N proteins was revealed in respiratory tract, and kidneys. Conclusion: To our knowledge, this is the first study describing the pathological changes of MERS-CoV infection in dromedary camels under natural conditions. In contrast to experimental infection in case of spontaneous infection interstitial pneumonea is evident at least in some affected animals.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

We present here the results of the analysis of the complete genome sequence of a lytic bacteriophage, vB_ButM_GuL6, which is the first virus isolated from Buttiauxella. Electron microscopy revealed that vB_ButM_GuL6 belongs to the family Myoviridae, order Caudovirales. The genome of vB_ButM_GuL6 is a linear, circularly permuted 178,039-bp dsDNA molecule with a GC content of 43.4%. It has been predicted to contain 282 protein-coding genes and two tRNA genes, tRNA-Met and tRNA-Gly. Using bioinformatics approaches, 99 (36%) of the vB_ButM_GuL6 genes were assigned a putative function. Genome-wide comparisons and phylogenetic analysis indicated that vB_ButM_GuL6 represents a new species of the subfamily Tevenvirinae and is most closely related to Escherichia virus RB43. These phages, together with Cronobacter phages Miller, CfP1, and IME-CF2, likely form a new genus within the subfamily Tevenvirinae.

At the time of writing, FIND has listed four CE-marked SARSCoV-2 antigen tests. We evaluated the recently CE-approved rapid POCT SD-Biosensor for SARS-CoV-2 nucleoprotein detection in nasopharyngeal secretions from 330 patients admitted to the Emergency Room for a suspect of COVID-19 and travelers returning home from high risk countries. Sensitivity, specificity, accuracy, negative and predictive values were consistent with the use of the test to mass-screening for SARS-CoV-2 surveillance., (Copyright © 2020. Published by Elsevier B.V.)

Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. In addition, the potential adverse effects of CAR T cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a PET reporter gene for imaging of T-cell trafficking in patients with brain tumor. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir. Here we report the molecular engineering, imaging, and ganciclovir-mediated destruction of B7H3 CAR T cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for ganciclovir. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[ 18 F]-fluoro-3-[hydroxymethyl]butyl)guanine ([ 18 F]FHBG) of B7H3-sr39tk CAR T cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T cells confirmed complete tumor ablation with intraperitoneal ganciclovir administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities. SIGNIFICANCE: This study showcases the only genetically engineered system capable of serving the dual role both as an effective PET imaging reporter and as a suicide switch for CAR T cells., (©2020 American Association for Cancer Research.)

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.

Infection by SARS-CoV-2 commonly begins in the nasopharynx, and the cytologic and molecular correlates are not characterized. Fifty-eight cytologic preps (20 oral and 38 from the nasopharynx) were obtained from ten patients and analyzed in a blinded fashion for SARS-CoV-2 spike and envelope protein by immunohistochemistry and viral RNA by in situ hybridization. qRTPCR identified three positive cases and seven controls; the three cases reported mild symptoms that resolved in 2-3 days. Blinded analyses confirmed the presence of the SARS-CoV-2 spike and envelope proteins and viral RNA in the three cases and viral absence in the seven controls. A signal for the positive cases was evident in each nasopharyngeal and none of the oral samples. Viral RNA/proteins localized exclusively to glandular cells and was present in high copy number. Blinded analysis of the cytology documented that the glandular cells infected by SARS-CoV-2 showed marked degeneration with ciliocytophthoria; viral inclusions were not evident. Co-expression analysis showed viral infected cells had increased apoptosis, marked by strong expression of activated caspase 3. Weekly serial testing of two of the cases showed persistence of productive viral infection for up to 2 weeks after symptom onset. It is concluded that the target cell of SARS-CoV-2 in the head and neck region is the glandular cell of the nasal passages, that viral infection is lytic and associated with high copy number that facilitates viral spread. The method outlines a simple, rapid test for productive SARS-CoV-2 based on immunohistochemistry or in situ hybridization of the glandular cells from the nasopharynx., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to report., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In recent years, the H9N2 influenzavirus has been circulating widely in poultry farms causing extensive damage. The hemagglutinin (HA) genes of the two virus isolates of H9N2 subtype in specific pathogen-free chickens were studied to determine the shedding rate in the host’s oropharyngeal and cloacal routes and their genetic relationship. The sequence analysis and phylogenetic study of the samples were performed by comparing each isolate with other H9N2 isolates in the gene bank. In the present study, the chickens were inoculated with low pathogenic avian influenza virus (LPAIV) (A/Chicken/Iran/ZMT-101/1998 [H9N2]) through the intranasal route. Oropharyngeal and cloacal swabs were collected from the chickens within 1-10 days after inoculation. The rate of viral shedding was measured within the previous 10 days by the real-time reverse transcriptase polymerase chain reaction molecular technique. No clinical symptoms were observed during the experiment in the chickens. The results obtained from this technique showed that the main route of shedding for LPAIV was oropharyngeal areas (p <0.05). Both isolates had a similar proteolytic R-S-S-R sequence at the cleavage site of the HA gene and contained glutamine (Q) amino acid at position 226 of the HA receptor-binding site, indicating that these isolates were nonpathogenic. Phylogenetic analysis demonstrated that both isolates belonged to the Eurasian clade. The comparison of these isolates with other isolates in the gene bank showed that they had the greatest similarity with the isolates in clade 1 and the least homology with the isolates in clade 4., (Copyright © 2020, Archives of Razi Institute. Published by Kowsar.)

Sacbrood virus (SBV) is one of the most damaging viruses in honey bee colonies. Genetic differences among sacbrood viruses detected in honey bees in different locales have been reported in previous studies. The aim of this study was to construct phylogenetic trees based on the structural polyprotein and non-structural RNA dependent RNA polymerase gene regions and to make a molecular characterization of the Tur/Bur/Sac01 and Tur/Bur/Sac02 strains identified in Apis mellifera in Turkey. As a result of the study, the tree based on the structural polyprotein region separated into four lineages: Tur/Bur/Sac01 and Tur/Bur/Sac02 were in the same branch as the Turkish sacbrood virus strains identified in previous studies and formed the Turkish clade. Strains isolated from adjacent geographical areas were in the same clade in this tree. The phylogenetic tree based on the non-structural RNA dependent RNA polymerase gene region divides into two main branches, reflecting host affiliation: Apis cerana and A. mellifera. Strains formed clusters based on their geographic distribution and host affiliation. The Tur/Bur/Sac01 and Tur/Bur/Sac02 strains formed a separate cluster among the European strains. Sacbrood viruses from Turkey were genetically different from SBV strains detected in other countries and in A. cerana., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection of SARS-CoV-2 facilitates early intervention and prevents the disease spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, and visual SARS-CoV-2 detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop the AIOD-CRISPR assay and detect the nucleic acids with a sensitivity of few copies. We validate the assay by using COVID-19 clinical swab samples and obtain consistent results with RT-PCR assay. Furthermore, a low-cost hand warmer (~$0.3) is used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care. Thus, our method has the significant potential to provide a rapid, sensitive, one-pot point-of-care assay for SARS-CoV-2.

The world is on the cusp of a post-antibiotic era, but researchers and medical doctors have found a way forward-by looking back at how infections were treated before the advent of antibiotics, namely using phage therapy. Although bacteriophages (phages) continue to lack drug approval in Western medicine, an increasing number of patients are being treated on an expanded-access emergency investigational new drug basis. To streamline the production of high-quality and clinically safe phage preparations, we developed a systematic procedure for medicinal phage isolation, liter-scale cultivation, concentration and purification. The 16- to 21-day procedure described in this protocol uses a combination of modified classic techniques, modern membrane filtration processes and no organic solvents to yield on average 23 mL of 10 11 plaque-forming units (PFUs) per milliliter for Pseudomonas, Klebsiella, and Serratia phages tested. Thus, a single production run can produce up to 64,000 treatment doses at 10 9 PFUs, which would be sufficient for most expanded-access phage therapy cases and potentially for clinical phase I/II applications. The protocol focuses on removing endotoxins early by conducting multiple low-speed centrifugations, microfiltration, and cross-flow ultrafiltration, which reduced endotoxins by up to 10 6 -fold in phage preparations. Implementation of a standardized phage cultivation and purification across research laboratories participating in phage production for expanded-access phage therapy might be pivotal to reintroduce phage therapy to Western medicine.