Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCR sample solution.

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

Differential RNA editing landscapes in host cell versus the SARS-CoV-2 genome.

The SARS-CoV-2 pandemic was defined by the emergence of new variants formed through virus mutation originating from random errors not corrected by viral proofreading and/or the host antiviral response introducing mutations into the viral genome. While sequencing information hints at cellular RNA editing pathways playing a role in viral evolution, here, we use an in vitro human cell infection model to assess RNA mutation types in two SARS-CoV-2 strains representing the original and the alpha variants. The variants showed both different cellular responses and mutation patterns with alpha showing higher mutation frequency with most substitutions observed being C-U, indicating an important role for apolipoprotein B mRNA editing catalytic polypeptide-like editing. Knockdown of select APOBEC3s through RNAi increased virus production in the original virus, but not in alpha. Overall, these data suggest a deaminase-independent anti-viral function of APOBECs in SARS-CoV-2 while the C-U editing itself might function to enhance genetic diversity enabling evolutionary adaptation.

One for all-human kidney Caki-1 cells are highly susceptible to infection with corona- and other respiratory viruses.

In vitro investigations of host-virus interactions are reliant on suitable cell and tissue culture models. Results are only as good as the model they are generated in. However, choosing cell models for in vitro work often depends on availability and previous use alone. Despite the vast increase in coronavirus research over the past few years, scientists are still heavily reliant on: non-human, highly heterogeneous or not fully differentiated, or naturally unsusceptible cells requiring overexpression of receptors and other accessory factors. Complex primary or stem cell models are highly representative of human tissues but are expensive and time-consuming to develop and maintain with limited suitability for high-throughput experiments.Using tissue-specific expression patterns, we identified human kidney cells as an ideal target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and broader coronavirus infection. We show the use of the well-characterized human kidney cell line Caki-1 for infection with three human coronaviruses (hCoVs): Betacoronaviruses SARS-CoV-2 and Middle Eastern respiratory syndrome coronavirus and Alphacoronavirus hCoV 229E. Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cell lines for the cultivation of the respective virus. Antibody staining against SARS-CoV-2 N protein shows comparable replication rates. A panel of 26 custom antibodies shows the location of SARS-CoV-2 proteins during replication using immunocytochemistry. In addition, Caki-1 cells were found to be susceptible to two other human respiratory viruses, influenza A virus and respiratory syncytial virus, making them an ideal model for cross-comparison for a broad range of respiratory viruses. IMPORTANCE Cell lines remain the backbone of virus research, but results are only as good as their originating model. Despite increased research into human coronaviruses following the COVID-19 pandemic, researchers continue to rely on suboptimal cell line models of: non-human origin, incomplete differentiation, or lacking active interferon responses. We identified the human kidney Caki-1 cell line as a potential target for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This cell line could be shown to be infectable with a wide range of coronaviruses including common cold virus hCoV-229E, epidemic virus MERS-CoV, and SARS-CoV-2 as well as other important respiratory viruses influenza A virus and respiratory syncytial virus. We could show the localization of 26 SARS-CoV-2 proteins in Caki-1 cells during natural replication and the cells are competent of forming a cellular immune response. Together, this makes Caki-1 cells a unique tool for cross-virus comparison in one cell line.

Self-derived peptides from the SARS-CoV-2 spike glycoprotein disrupting shaping and stability of the homotrimer unit.

The structural spike (S) protein from the SARS-CoV-2 ß-coronavirus is shown to make different pre- and post-fusion conformations within its homotrimer unit. To support the ongoing novel vaccine design and development strategies, we report the structure-based design approach to develop self-derived S peptides. A dataset of crucial regions from the S protein were transformed into linear motifs that could act as the blockers or stabilizers for the S protein homotrimer unit. Among these distinct S peptides, the pep02 (537-QQFGRDIAD-545) and pep07 (821-RDLICAQKFNGLTVLPPLLTDE-842) were found making stable folded binding with the S protein (550-750 and 950-1050 regions). Upon inserting SARS-CoV-2 S variants in the peptide destabilized the complexed S protein structure, resulting an allosteric effect in different functional regions of the protein. Particularly, the molecular dynamics revealed that A544D mutation in the pep02 peptide induced instability for the complexed S protein, whereas the N943K variant from pep09 exhibited an opposite behavior. An increased protein-peptide binding affinity and the stable structural folding were observed in mutated systems, compared to that of the wild type systems. The presence of mutation has induced an "up" active conformation of the spike (RBD) domain, responsible for interacting the host cell receptor. Among the lower affinity peptide datasets (e.g., pep01), the S1 and S2 subunit in the protein formed an "open" conformation, whereas with higher affinity peptides (e.g., pep07) these domains gained a "closed" conformation. These findings propose that our designed self-derived S peptides could replace a single S protein monomer, blocking the homotrimer formation or inducing stability.

Direct Lysis RT-qPCR of SARS-CoV-2 in Cell Culture Supernatant Allows for Fast and Accurate Quantification.

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

Highly Conserved Homotrimer Cavity Formed by the SARS-CoV-2 Spike Glycoprotein: A Novel Binding Site.

An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an "up" and "down" state. In addition, the trimer cavity may function as a "bouncing spring" that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses.

Meta-analysis of coefficient alpha for scores on the Narcissistic Personality Inventory.

The Narcissistic Personality Inventory (NPI) has greatly facilitated the scientific study of trait narcissism. However, there is great variability in the reported reliability of scores on the NPI. This study meta-analyzes coefficient alpha for scores on the NPI and its sub-scales (e.g. entitlement) with transformed alphas weighted by the inverse of the variance of alpha. Three coders evaluated 1213 individual studies for possible inclusion and determined that 1122 independent samples were suitable for coding on 12 different characteristics of the sample, scale, and study. A fourth author cross-coded 15 percent of these samples resulting in 85 percent overall agreement. In the independent samples, comprised of 195,038 self-reports, the expected population coefficient alpha for the NPI was .82. The population value for alpha on the various sub-scales ranged from .48 for narcissistic self-sufficiency to .76 for narcissistic leadership/authority. Because significant heterogeneity existed in coded study alphas for the overall NPI, moderator tests and an explanatory model were also conducted and reported. It was found that longer scales, the use of a Likert response scale as opposed to the original forced choice response format, higher mean scores and larger standard deviations on the scale, as well as the use of samples with a larger percentage of female respondents were all positively related to the expected population alpha for scores on the overall NPI. These results will likely aid researchers who are concerned with the reliability of scores on the NPI in their research on non-clinical subjects.

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus, Magnaporthe grisea.

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.