A definitive diagnosis of HIV typically requires a positive nucleic acid test. Limited access to these tests means that initiation of anti-HIV therapy is delayed or does not occur in a significant part of the world. While rapid antigen tests are more broadly available, these are insufficient for diagnosis on their own. To address the challenge of improving access to HIV testing, we have developed a passive, paper-based microfluidic sample preparation device we term the QuickDraw. We demonstrate that QuickDraw efficiently processes HIV-containing finger stick-quantities of whole blood to yield purified viral RNA. The output of the QuickDraw is then used as input for a colorimetric reverse transcriptase - loop-mediated isothermal amplification (RT-LAMP) assay. Coupled with sample preparation conducted with the QuickDraw, the assay demonstrated a limit of detection of 1000 copies per mL. QuickDraw simplifies viral nucleic acid sample preparation and detection by dramatically reducing the amount of equipment needed, suggesting it could be suitable for deployment in clinical and low-resource settings. By decentralizing nucleic acid testing, the QuickDraw platform has the potential to expand access to nucleic acid diagnostics in low-middle income countries (LMICs), while also supporting the UNAIDS goals for HIV detection, leading to wider access to treatment and reduced community transmission. It is also a significant step towards the goal of a simple-to-use nucleic acid-based HIV self-test.

Cancer cells often have defects in antiviral pathways, making them susceptible to oncolytic viruses like vesicular stomatitis virus (VSV). However, some cancer cells resist viral infection through the constitutive expression of interferon-stimulated genes. This study examined whether NF-κB activation and NF-κB-dependent antiviral signaling contribute to resistance to VSV infection in the PC3 cell line, derived from an aggressive metastatic prostate cancer (PrCa) tumor. We found that NF-κB localized to the nucleus in VSV-infected PC3 cells, but not in the VSV-susceptible LNCaP PrCa cell line. Analysis of the upstream NF-κB inhibitor IκB-α revealed higher levels of both total and phosphorylated IκB-α in PC3 cells compared to LNCaP cells, indicating constitutive activation of the NF-κB pathway via an IκB-α-dependent mechanism. Notably, VSV infection did not alter IκB-α phosphorylation in PC3 cells, suggesting that VSV may amplify NF-κB signaling through an IκB-α–independent pathway. Furthermore, PC3 cells displayed elevated levels of the NF-κB p65 protein subunit compared to LNCaP cells, with its phosphorylated form significantly increased upon VSV infection. These results from phosphorylation assays confirm that multiple steps in the NF-κB pathway are differentially activated in PC3 and LNCaP cells. Finally, the expression of several NF-κB-dependent cytokines and proinflammatory genes, including IL12 and IL6, was upregulated following VSV infection in PC3 cells, as compared to LNCaP cells. Collectively, these findings suggest that enhanced NF-κB signaling may underlie the resistance of PC3 cells to VSV oncolysis, potentially offering new insights into therapeutic strategies targeting NF-κB in resistant prostate cancers.

The therapeutic efficacy of oncolytic viruses is often limited by the presence of tumor cells that resist virus-mediated killing. Here, we investigated the molecular mechanisms underlying resistance to Vesicular Stomatitis Virus (VSV) in PC3 cells, an aggressive metastatic prostate cancer (PrCa) cell line, using the VSV-sensitive LNCaP cell line as a comparator. RNA sequencing revealed that, relative to untreated cells, VSV-infected PC3 cells upregulated both pro-apoptotic genes, including BIM, PUMA, and NOXA, and anti-apoptotic and antiviral genes, including A20 and RIG-I. In addition, genes associated with antiviral and pro-survival pathways, including NFκB and PI3K-Akt signaling, were more highly expressed in PC3 cells than in LNCaP cells. At baseline, PC3 cells also exhibited elevated expression of multiple pro-survival genes, including BCL-xL, MCL1, and CK2, compared with LNCaP cells. Complementary proteomic analyses identified enhanced activation of NFκB, PI3K-Akt, and MSK1 signaling in VSV-infected PC3 cells relative to infected LNCaP cells. Furthermore, pharmacological inhibition of BCL-2 family proteins or NFκB signaling restored sensitivity to VSV-induced cell death in PC3 cells. Collectively, these findings identify NFκB-centered pro-survival signaling networks as key contributors to the resistant phenotype of PC3 cells and suggest that combining oncolytic virotherapy with targeted inhibitors may improve therapeutic efficacy in resistant prostate cancers.

INTRODUCTION:The ability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to evade antiviral immune signaling in the airway contributes to the severity of coronavirus disease 2019 (COVID-19). Additionally, COVID-19 is influenced by age and has more severe presentations in older individuals. Hence we investigate the role of innate immune signaling as a function of lung development and age. METHOD:We investigated the transcriptome of the airway epithelium using pediatric and adult lung tissue samples from the LungMAP Human Tissue Core Biorepository. Specifically, lung lobes were digested and cultured into a biomimetic model of the airway epithelium on an air-liquid interface. Cells were then infected with SARS-CoV-2 and subjected to single-cell RNA sequencing. The data was analyzed using Seurat, scType, Monacle and tools to infer cell-cell communication. RESULTS:The clustering analysis identified following six cell populations: club cells, proliferating epithelial cells, multiciliated precursor cells, ionocytes, and two biologically distinct clusters of ciliated cells (FOXJ1high and FOXJ1low). Interestingly, the two ciliated cell clusters showed different infection rates and enrichment of processes involved in ciliary biogenesis and function; we observed a cell-type-specific suppression of innate immunity in infected cells from the FOXJ1low subset. We also identified a significant number of genes that were differentially expressed in lung cells derived from children as compared to adults, suggesting the differential pathogenesis of SARS-CoV-2 infection in children versus adults. CONCLUSION:This work reveals age dependent differences in the lung epithelial cell response to SARS-CoV-2 infection. The results can be used to identify drug targets to modulate molecular signaling cascades that mediate an innate immune response.

Influenza viruses cause mild to severe lower respiratory infections, sometimes resulting in hospitalization and death. Vaccination remains the primary prophylactic strategy. Live attenuated influenza vaccines (LAIVs) efficiently induce antiviral immune responses and contain temperature-sensitive and cold-adapted mutations that render them safe. These mutations are principally located in the PB1 and PB2 subunits of the viral RNA polymerase, but the mechanism by which they attenuate the virus is unclear. We introduced the PB1 and PB2 mutations from two LAIV backbones, A/Ann Arbor/6/1960 H2N2 (AA) and A/Leningrad/134/17/1957 H2N2 (Len), into the model influenza strain A/Puerto Rico/8/1934 H1N1 (PR8). In contrast to the wild-type (WT) PR8 polymerase, the two “PR8-LAIV” polymerase complexes demonstrated maximal activity at cold temperatures (30–32 °C) and greatly reduced activity at elevated temperatures (>37 °C). To further understand the impact of the LAIV mutations, we infected MDCK cells with WT and mutated PR8 viruses that contain the Len and AA LAIV mutations in PB1 and PB2. The PR8-LAIV mutant viruses exhibited a selective, temperature-dependent defect in the replicase activity of the viral RNA polymerase relative to WT PR8, while also demonstrating a temperature-dependent enhancement in the transcriptional activity of the enzyme. In addition, the PR8-LAIV mutant viruses produced similar levels of viral proteins to WT PR8 at 37 °C, but greatly (2–3 log10) reduced levels of infectious viral progeny. Collectively, these data show that LAIV mutations selectively alter influenza viral RNA polymerase function, favoring transcription over genome synthesis at 37 °C, thereby preserving viral antigen production while also contributing to viral attenuation.

Background: Vaccination against SARS-CoV-2 has been pivotal in controlling the COVID-19 pandemic. However, understanding vaccine-induced immunity in immunocompromised individuals remains critical, particularly how prior exposure to other coronaviruses modulates immune responses. The influence of previous infections with endemic human coronaviruses (HCoVs), such as OC43, on SARS-CoV-2 immunity is not fully understood. This study evaluates antibody responses to COVID-19 vaccination in hemodialysis patients (HD), transplant recipients (TR), and healthy controls (CO), accounting for prior SARS-CoV-2 infection and baseline human coronavirus (HCoV) reactivity. Methods: We obtained longitudinal antibody measurements from 70 subjects (CO: n = 33; HD: n = 13; TR: n = 24) and assessed antibody kinetics across multiple post-vaccination time points using multivariate linear mixed modeling (MLMM). Results: Limited but measurable cross-reactivity was observed between SARS-CoV-2 and endemic HCoVs, particularly the β-coronavirus OC43. Pre-existing immunity in healthy individuals modestly enhanced vaccine-induced anti-spike (S) IgG responses, supported by post-vaccination increases in SARS-CoV-2 IgG. Prior SARS-CoV-2 infection significantly influenced anti-S and nucleocapsid (N) IgG responses but had limited impact on endemic HCoVs responses. Vaccine type and immune status significantly affected antibody kinetics. mRNA vaccination (BNT162b2) elicited stronger and more durable SARS-CoV-2 anti-S IgG responses than the inactivated CoronaVac vaccine, especially in immunocompetent individuals. Immunocompromised groups showed delayed or attenuated responses, with modest anti-S IgG cross-reactive boosting. Elevated anti-N IgG in CoronaVac recipients raised questions about its origin—infection or vaccine effects. MLMM identified key immunological and clinical predictors of antibody responses, emphasizing the critical role of host immune history. Conclusions: These findings highlight a constrained but meaningful role for HCoV cross-reactivity in SARS-CoV-2 immunity and vaccine responsiveness, underscore the need for infection markers unaffected by vaccination, and support development of broadly protective pan-coronavirus vaccines and tailored strategies for at-risk populations.

[RETRACTED] Coronavirus disease (COVID-19) is an infectious disease caused by the SARS coronavirus 2 (SARS-CoV-2) virus. Direct assessment, detection, and quantitative analysis using high throughput methods like single-cell RNA sequencing (scRNAseq) is imperative to understanding the host response to SARS-CoV-2. One barrier to studying SARS-CoV-2 in the laboratory setting is the requirement to process virus-infected cell cultures, and potentially infectious materials derived therefrom, under Biosafety Level 3 (BSL-3) containment. However, BSL3 laboratory facilities are rare and only a subset of these facilities are outfitted with the equipment needed to perform high-throughput molecular assays. Here, we describe a method for preparing non-hazardous RNA samples from SARS-CoV-2 infected cells, that enables scRNAseq analyses to be conducted safely in a BSL2 facility – thereby making molecular assays of SARS-CoV-2 cells accessible to a much larger community of researchers. (The last step in this version contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.)

SARS-CoV-2 E and 3a proteins are important for the assembly, budding, and release of viral particles. These two transmembrane proteins have been implicated in forming channels in the membrane that allow the transport of ions to favor viral replication. During an active infection, both proteins generally localize to the endoplasmic reticulum (ER), ER-Golgi intermediate compartment (ERGIC), and the Golgi where viral assembly occurs. The ER and Golgi are critical for the proper packaging and trafficking of cellular proteins along the secretory pathways which determine a protein’s final destination inside or outside of the cell. The SARS-CoV-2 virus primarily infects epithelial cells that are highly secretory in nature such as those in the lung and gut. Here we quantified the distribution of SARS-CoV-2 E and 3a proteins along the secretory pathways in a human intestinal epithelial cell line. We used NaturePatternMatch to demonstrate that epitope-tagged E and 3a proteins expressed alone via transient transfection have a similar immunoreactivity pattern as E and 3a proteins expressed by wild-type viral infection. While E and 3a proteins localized with all selected cellular markers to varying degrees, 3a protein displayed a higher correlation coefficient with the Golgi, early/late endosome, lysosome, and plasma membrane when compared to E protein. This work is the first to provide quantification of the subcellular distribution of E and 3a proteins along the multiple components of the secretory pathway and serves as a basis to develop models for examining how E and 3a alter proteostasis within these structures and affect their function.

Anticipated emotions are the feelings one expects if a hypothetical future event were to occur, whereas anticipatory emotions are those one experiences right now while imagining the event.There has been little direct comparison of these two forms of future-oriented emotion, and authors have typically focused on positive emotions (e.g., pleasure).Besides, their sensitivity to depressive symptoms-which may help to explain motivational problems in depression-has only recently been investigated (e.g., Anderson et al., 2023;Gamble et al., 2021).The present study (conducted September-November 2022) used innovative picture-and-text vignettes depicting everyday positive and negative future events, to which participants rated their anticipated and anticipatory responses on separate dimensions of valence (i.e., how positive or negative) and arousal (i.e., emotional intensity).Based on prior literature, anticipatory emotions were expected to be correlated with, yet weaker than, anticipated emotions, reflecting a conceptualization of anticipatory emotions as a "foretaste" of the affective response one expects in the future.We also predicted that high depressive symptoms would coincide with diminished emotion ratings overall and specifically for anticipatory emotions (tightly coupled with event expectations; Carrera et al., 2012).Results largely supported these preregistered predictions, yet anticipatory emotions (positive and negative) were only weaker in more highly depressed participants.Depressive symptoms may therefore affect how one currently feels about future possibilities without altering one's expectations of how such events would actually feel.Implications and future research objectives arising from this are discussed.

Background: The global COVID-19 pandemic has resulted in approximately 7 million deaths and a historic vaccination effort, with over 13.6 billion doses administered. Despite this, understanding of immune responses in vulnerable populations, such as transplant recipients (TR) and hemodialysis patients (HD), remains limited, especially outside the US and Europe. Methods: To address this gap, we analyzed blood samples and deidentified data from the Instituto Nacional de Coordinación de Trasplante (INCORT) in The Dominican Republic, measuring antibody levels to SARS-CoV-2 post-infection and vaccination with BNT162b2 (Pfizer-BioNTech) and Sinovac-CoronaVac (Sinovac) in TR, HD, and healthy controls (CO). Using a fluorescent multiplex assay (mPlex-CoV) and mixed-effects modeling, we assessed variations in anti-S, anti-RBD, and anti-N IgG antibodies. Results: The results indicate that the CO group experienced an early peak in anti-S and anti-RBD antibodies, followed by stabilization. In contrast, the TR and HD groups showed a slower, gradual increase in antibodies. Despite fluctuations in the HD group, both the TR and HD groups maintained high anti-S and anti-RBD IgG levels, indicating a back-boosting effect from vaccination. However, elevated anti-N IgG levels in the TR and HD groups suggest potential reinfections. Additionally, prior SARS-CoV-2 infection led to higher anti-S IgG levels, with BNT162b2 associated with higher anti-S IgG and CoronaVac associated with higher anti-N IgG levels. Conclusion: These findings highlight the variability in antibody responses and the need for targeted public health strategies to diverse immunological profiles.

ABSTRACT COVID-19 can cause neurological symptoms such as fever, dizziness, and nausea. However, such neurological symptoms of SARS-CoV-2 infection have been hardly assessed in mouse models. In this study, we infected two commonly used wild-type mouse lines (C57BL/6J and 129/SvEv) and a 129S calcitonin gene-related peptide (αCGRP) null-line with mouse-adapted SARS-CoV-2 and demonstrated neurological signs including fever, dizziness, and nausea. We then evaluated whether a CGRP receptor antagonist, olcegepant, a “gepant” antagonist used in migraine treatment, could mitigate acute neuroinflammatory and neurological signs of SARS-COV-2 infection. First, we determined whether CGRP receptor antagonism provided protection from permanent weight loss in older (>18 m) C57BL/6J and 129/SvEv mice. We also observed acute fever, dizziness, and nausea in all older mice, regardless of treatment. In both wild-type mouse lines, CGRP antagonism reduced acute interleukin 6 (IL-6) levels with virtually no IL-6 release in mice lacking αCGRP. These findings suggest that migraine inhibitors such as those blocking CGRP receptor signaling protect against acute IL-6 release and subsequent inflammatory events after SARS-CoV-2 infection, which may have repercussions for related pandemic or endemic coronavirus outbreaks. IMPORTANCE Coronavirus disease (COVID-19) can cause neurological symptoms such as fever, headache, dizziness, and nausea. However, such neurological symptoms of severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infection have been hardly assessed in mouse models. In this study, we first infected two commonly used wild-type mouse lines (C57BL/6J and 129S) with mouse-adapted SARS-CoV-2 and demonstrated neurological symptoms including fever and nausea. Furthermore, we showed that the migraine treatment drug olcegepant could reduce long-term weight loss and IL-6 release associated with SARS-CoV-2 infection. These findings suggest that a migraine blocker can be protective for at least some acute SARS-CoV-2 infection signs and raise the possibility that it may also impact long-term outcomes.

Previous research demonstrating that positive episodic simulation enhances future expectancies has relied on explicit expectancy measures. The current study investigated the effects of episodic simulation on implicit expectancies. Using the Future Thinking Implicit Relational Assessment Procedure (FT-IRAP), participants made true/false decisions to indicate whether or not they expected positive/negative outcomes after adopting orientations consistent or inconsistent with an optimistic disposition. The outcome measure, DIRAP, was based on response time differences between consistent and inconsistent blocks. Participants then engaged in either positive simulation training, in which they imagined positive future events, or a neutral visualisation task before repeating the FT-IRAP twice following 10-minute intervals. Positive simulation training increased DIRAP scores for don't-expect-negative trials-boosting participants' readiness to affirm that negative events were unlikely to happen to them. Although findings did not generalise across all trial types, they show potential for positive simulation training to enhance implicit future expectancies.

An academic partnership between a university and an upstate New York school serving students with intellectual and developmental disabilities established a research-driven, weekly onsite asymptomatic COVID-19 testing protocol supplemented by symptomatic testing. The research team, including school leadership, met at least weekly to address implementation issues, impacts of changing pandemic and regulatory requirements, and interpretation and dissemination of findings. The dynamic partnership resulted in 380 participants, more than 16 000 polymerase chain reaction tests, and additional research studies. ( Am J Public Health. 2024;114(S5):S384–S387. https://doi.org/10.2105/AJPH.2024.307687 )

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic associated with morbidity and mortality in humans. Although disease severity correlates with immune dysregulation, the cellular mechanisms of inflammation and pathogenesis of COVID-19 remain relatively poorly understood. Here, we used mouse-adapted SARS-CoV-2 strain MA10 to investigate the role of adaptive immune cells in disease. We found that while infected wild-type mice lost ~10% weight by 3 to 4 days postinfection, rag −/− mice lacking B and T lymphocytes did not lose weight. Infected lungs at peak weight loss revealed lower pathology scores, fewer neutrophils, and lower interleukin-6 and tumor necrosis factor–α in rag −/− mice. Mice lacking αβ T cells also had less severe weight loss, but adoptive transfer of T and B cells into rag −/− mice did not significantly change the response. Collectively, these findings suggest that while adaptive immune cells are important for clearing SARS-CoV-2 infection, this comes at the expense of increased inflammation and pathology.

[This corrects the article DOI: 10.1371/journal.pone.0291840.].

An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.

Vesicular Stomatitis Virus (VSV) has emerged as a promising candidate for various clinical applications, including vaccine development, virus pseudotyping, and gene delivery. Its broad host range, ease of propagation, and lack of pre-existing immunity in humans make it ideal for therapeutic use. VSV’s potential as an oncolytic virus has garnered attention; however, resistance to VSV-mediated oncolysis has been observed in some cell lines and tumor types, limiting its effectiveness. This review provides a detailed analysis of recent advances in VSV-based oncolysis, focusing on resistance mechanisms such as sustained type-I IFN signaling, upregulation of ISGs, immune cell activation, the tumor microenvironment (TME), and tumor-intrinsic factors. Strategies to overcome resistance include enhancing viral oncoselectivity, inhibiting IFN responses, modulating the TME, and combining VSV with chemotherapies, radiation, and immune checkpoint inhibitors. Several VSV-based phase I/II clinical trials show promise; however, addressing resistance and developing novel strategies to enhance therapeutic efficacy are essential for realizing the full potential of VSV oncolytic virotherapy. Future research should focus on patient-specific approaches, as tumor heterogeneity implies varying resistance mechanisms. Personalized treatments tailored to tumor molecular profiles, along with identifying biomarkers predictive of resistance to VSV oncolysis, will enhance patient selection and enable more effective, individualized VSV-based therapies.

Two experiments investigated the role of visual imagery in prospective memory (PM). In experiment 1, 140 participants completed a general knowledge quiz which included a PM task of writing a letter "X" next to any questions that referred to space. Participants either visualised themselves performing this task, verbalised an implementation intention about the task, did both, or did neither. Performance on the PM task was enhanced in both conditions involving visual imagery but not by implementation intentions alone. In experiment 2, 120 participants imagined themselves writing a letter "X" next to questions about space, or in a bizarre imagery condition imagined themselves drawing an alien next to those questions. Relative to the control condition, PM was significantly enhanced when participants imagined writing a letter "X" next to the target questions, but not by the bizarre imagery task. The findings indicate that the robust effects of imagery observed in retrospective memory also extend to PM.