AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication

We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted

Stiffness, pain, and joint counts in chronic chikungunya disease: relevance to disability and quality of life.

Introduction/Objectives: To characterize the importance of musculoskeletal stiffness in a cohort of chikungunya patients with chronic joint symptoms. Method: Eighty-two patients were followed up 3 years after chikungunya infection. Tender and swollen joint counts, a pain intensity scale, Health Assessment Questionnaire-Disability Index (HAQ-DI), and the EuroQol EQ-5D quality of life instrument were completed. A musculoskeletal stiffness questionnaire provided scores for overall stiffness and its components: stiffness severity, physical impact, and psychosocial impact. Results: Patients had a mean age 51 ± 14 years. Sixty-seven patients were still experiencing chronic arthralgia. Musculoskeletal stiffness was reported by 43/67 patients with arthralgia and 3/15 patients without arthralgia. A physical impact of stiffness was reported by 87% patients and psychosocial impact by 71% patients. Mean tender joint count in patients reporting arthralgia was 6 ± 7, mean pain intensity 65 ± 20 out of 100, mean HAQ-DI was 0.54 ± 0.52, and mean EQ-VAS global health perception was 68 ± 62 out of 100. Stiffness severity was correlated with tender joint counts (ρ = 0.46) and pain intensity (ρ = 0.40). All three measures were equally well correlated with the EuroQol-VAS global health perception. Pain and tender joints were better correlated with the HAQ-DI (ρ = 0.68 and ρ = 0.63), but stiffness was more strongly correlated with several quality of life domains, including mobility. Swollen joints were a poor predictor of outcomes. Conclusions: Musculoskeletal stiffness following chikungunya infection is distinct from arthralgia. It does not always occur in the same patients or with a corresponding intensity. Joint pain and stiffness may be independently associated with disability and quality of life assessments

Exploring inactivation of SARS-CoV-2, MERS-CoV, Ebola, Lassa, and Nipah viruses on N95 and KN95 respirator material using photoactivated methylene blue to enable reuse

BACKGROUND: The COVID-19 pandemic resulted in a worldwide shortage of N95 respirators, prompting the development of decontamination methods to enable limited reuse. Countries lacking reliable supply chains would also benefit from the ability to safely reuse PPE. Methylene blue (MB) is a light-activated dye with demonstrated antimicrobial activity used to sterilize blood plasma. Decontamination of respirators using photoactivated MB requires no specialized equipment, making it attractive for use in the field during outbreaks. METHODS: We examined decontamination of N95 and KN95 respirators using photoactivated MB and 3 variants of SARS-CoV-2, the virus that causes COVID-19; and 4 World Health Organization priority pathogens: Ebola virus, Middle East respiratory syndrome coronavirus, Nipah virus, and Lassa virus. Virus inactivation by pretreating respirator material was also tested. RESULTS: Photoactivated MB inactivated all tested viruses on respirator material, albeit with varying efficiency. Virus applied to respirator material pre-treated with MB was also inactivated, thus MB pretreatment may potentially protect respirator wearers from virus exposure in real-time. CONCLUSIONS: These results demonstrate that photoactivated MB represents a cost-effective, rapid, and widely deployable method to decontaminate N95 respirators for reuse during supply shortages

Development of an accessible and scalable qPCR assay for Monkeypox virus detection

During the 2022 monkeypox (MPX) outbreak, testing has been limited and results delayed, allowing ongoing transmission. Gold-standard qPCR diagnostics are difficult to obtain. This research adapted the June 2022 CDC MPX qPCR assay for broad implementation. Validated using MPX stocks in a matrix with multiple sample types, MPX was detected with Cq values of 17.46 to 35.59 and titer equivalents 8.01 × 106 to 2.45 × 100 PFU/mL. The detection limit was 3.59 PFU/mL. Sensitivity and specificity were both 100%. This qPCR assay can be quickly and broadly implemented in research and public health labs to increase diagnostic capacity amid the growing MPX outbreak

Effects of rIL2/anti-IL2 antibody complex on chikungunya virus-induced chronic arthritis in a mouse model

Abstract Chikungunya virus (CHIKV) is characterized by disabling joint pain that can cause persistent arthritis in approximately one-fourth of patients. Currently, no standard treatments are available for chronic CHIKV arthritis. Our preliminary data suggest that decreases in interleukin-2 (IL2) levels and regulatory T cell (Treg) function may play a role in CHIKV arthritis pathogenesis. Low-dose IL2-based therapies for autoimmune diseases have been shown to up-regulate Tregs, and complexing IL2 with anti-IL2 antibodies can prolong the half-life of IL2. A mouse model for post-CHIKV arthritis was used to test the effects of recombinant IL2 (rIL2), an anti-IL2 monoclonal antibody (mAb), and the complex on tarsal joint inflammation, peripheral IL2 levels, Tregs, CD4 + effector T cells (Teff), and histological disease scoring. The complex treatment resulted in the highest levels of IL2 and Tregs, but also increased Teffs, and therefore did not significantly reduce inflammation or disease scores. However, the antibody group, which had moderately increased levels of IL2 and activated Tregs, resulted in a decreased average disease score. These results suggest the rIL2/anti-IL2 complex stimulates both Tregs and Teffs in post-CHIKV arthritis, while the anti-IL2 mAb increases IL2 availability enough to shift the immune environment towards a tolerogenic one

A mouse model for studying post-acute arthritis of chikungunya

Chikungunya virus (CHIKV) was introduced to the Americas in 2013, causing two million infections across over thirty countries. CHIKV causes a chronic debilitating arthritis in one fourth of infected individuals and currently evidence-based targeted therapies for the treatment of CHIKV arthritis are lacking. Multiple mouse models of chikungunya have been developed to study acute CHIKV infection. In humans, post-CHIKV arthritis may persist for months to years after viremia from a CHIKV infection has resolved. Therefore, the development of a mouse model of post-acute arthritis of chikungunya may facilitate the study of potential novel therapeutics for this arthritis. In this article we describe the development of a wild-type immunocompetent C57BL/6 mouse model for post-acute arthritis of chikungunya, including a histologic inflammation scoring system, as well as suggestions for how this mouse model may be used to examine the efficacy of novel therapies for CHIKV arthritis

L1000CDS2: LINCS L1000 characteristic direction signatures search engine

The library of integrated network-based cellular signatures (LINCS) L1000 data set currently comprises of over a million gene expression profiles of chemically perturbed human cell lines. Through unique several intrinsic and extrinsic benchmarking schemes, we demonstrate that processing the L1000 data with the characteristic direction (CD) method significantly improves signal to noise compared with the MODZ method currently used to compute L1000 signatures. The CD processed L1000 signatures are served through a state-of-the-art web-based search engine application called L1000CDS2. The L1000CDS2 search engine provides prioritization of thousands of small-molecule signatures, and their pairwise combinations, predicted to either mimic or reverse an input gene expression signature using two methods. The L1000CDS2 search engine also predicts drug targets for all the small molecules profiled by the L1000 assay that we processed. Targets are predicted by computing the cosine similarity between the L1000 small-molecule signatures and a large collection of signatures extracted from the gene expression omnibus (GEO) for single-gene perturbations in mammalian cells. We applied L1000CDS2 to prioritize small molecules that are predicted to reverse expression in 670 disease signatures also extracted from GEO, and prioritized small molecules that can mimic expression of 22 endogenous ligand signatures profiled by the L1000 assay. As a case study, to further demonstrate the utility of L1000CDS2, we collected expression signatures from human cells infected with Ebola virus at 30, 60 and 120 min. Querying these signatures with L1000CDS2 we identified kenpaullone, a GSK3B/CDK2 inhibitor that we show, in subsequent experiments, has a dose-dependent efficacy in inhibiting Ebola infection in vitro without causing cellular toxicity in human cell lines. In summary, the L1000CDS2 tool can be applied in many biological and biomedical settings, while improving the extraction of knowledge from the LINCS L1000 resource

AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication

We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α—dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12—stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286–2302, 2016. © 2016 Wiley Periodicals, Inc.Fil: Booth, Laurence. Virginia Commonwealth University; Estados UnidosFil: Roberts, Jane L.. Virginia Commonwealth University; Estados UnidosFil: Ecroyd, Heath. University of Wollongong; AustraliaFil: Tritsch, Sarah R.. United States Army Medical Research Institute of Infectious Diseases; Estados UnidosFil: Bavari, Sina. United States Army Medical Research Institute of Infectious Diseases; Estados UnidosFil: Reid, St. Patrick. United States Army Medical Research Institute of Infectious Diseases; Estados UnidosFil: Proniuk, Stefan. Arno Therapeutics; Estados UnidosFil: Zukiwski, Alexander. Arno Therapeutics; Estados UnidosFil: Jacob, Abraham. University of Arizona; Estados UnidosFil: Sepúlveda, Claudia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Giovannoni, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Garcia, Cybele. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Damonte, Elsa Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: González Gallego, Javier. Universidad de León; EspañaFil: Tuñón, María J.. Universidad de León; EspañaFil: Dent, Paul. Virginia Commonwealth University; Estados Unido