Radicicol Inhibits Chikungunya Virus Replication by Targeting Nonstructural Protein 2.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes a debilitating febrile illness characterized by persistent muscle and joint pain. The widespread distribution of transmission-competent vectors, Aedes species mosquitoes, indicates the potential risk of large-scale epidemics with high attack rates that can severely impact public health globally. Despite this, currently, there are no antivirals available for the treatment of CHIKV infections. Thus, we aimed to identify potential drug candidates by screening a chemical library using a cytopathic effect-based high-throughput screening assay. As a result, we identified radicicol, a heat shock protein 90 (Hsp90) inhibitor that effectively suppressed CHIKV replication by blocking the synthesis of both positive- and negative-strand viral RNA as well as expression of viral proteins. Interestingly, selection for viral drug-resistant variants and mutational studies revealed nonstructural protein 2 (nsP2) as a putative molecular target of radicicol. Moreover, coimmunoprecipitation and in silico modeling analyses determined that G641D mutation in the methyltransferase (MT)-like domain of nsP2 is essential for its interaction with cytoplasmic Hsp90ß chaperone. Our findings collectively support the potential application of radicicol as an anti-CHIKV agent. The detailed study of the underlying mechanism of action further contributes to our understanding of virus-host interactions for novel therapeutics against CHIKV infection.

A Pan-Dengue Virus Reverse Transcription-Insulated Isothermal PCR Assay Intended for Point-of-Need Diagnosis of Dengue Virus Infection by Use of the POCKIT Nucleic Acid Analyzer.

Dengue virus (DENV) infection is considered a major public health problem in developing tropical countries where the virus is endemic and continues to cause major disease outbreaks every year. Here, we describe the development of a novel, inexpensive, and user-friendly diagnostic assay based on a reverse transcription-insulated isothermal PCR (RT-iiPCR) method for the detection of all four serotypes of DENV in clinical samples. The diagnostic performance of the newly established pan-DENV RT-iiPCR assay targeting a conserved 3' untranslated region of the viral genome was evaluated. The limit of detection with a 95% confidence was estimated to be 10 copies of in vitro-transcribed (IVT) RNA. Sensitivity analysis using RNA prepared from 10-fold serial dilutions of tissue culture fluid containing DENVs suggested that the RT-iiPCR assay was comparable to the multiplex real-time quantitative RT-PCR (qRT-PCR) assay for DENV-1, -3, and -4 detection but 10-fold less sensitive for DENV-2 detection. Subsequently, plasma collected from patients suspected of dengue virus infection (n = 220) and individuals not suspected of dengue virus infection (n = 45) were tested by the RT-iiPCR and compared to original test results using a DENV NS1 antigen rapid test and the qRT-PCR. The diagnostic agreement of the pan-DENV RT-iiPCR, NS1 antigen rapid test, and qRT-PCR tests was 93.9%, 84.5%, and 97.4%, respectively, compared to the composite reference results. This new RT-iiPCR assay along with the portable POCKIT nucleic acid analyzer could provide a highly reliable, sensitive, and specific point-of-need diagnostic assay for the diagnosis of DENV in clinics and hospitals in developing countries.

Discovery and synthesis of 1,2,4-oxadiazole derivatives as novel inhibitors of Zika, dengue, Japanese encephalitis, and classical swine fever virus infections.

Zika virus (ZIKV), an arbovirus of the Flaviviridae family, has emerged as a significant public health concern owing to its association with congenital abnormalities and severe neurological sequelae. Thus, there is an urgent need to develop effective therapeutic approaches to efficiently treat ZIKV infections. This study used phenotypic screening to identify a series of 1,2,4-oxadiazole derivatives that possess antiviral activity against ZIKV infection. Subsequently, 28 new derivatives were designed, synthesized, and evaluated for this purpose. Among these compounds, 4-(5-phenyl-1,2,4-oxadiazol-3-yl)-N-(pyridin-3-ylmethyl)aniline (5d) had potent antiviral activity against ZIKV infections. Furthermore, a structure-activity relationship analysis indicated that a benzyl substitution on the aniline nitrogen of this compound improved potency while augmenting its drug-like properties. In addition, 5d exhibited antiviral activity against various viruses of Flaviviridae family of worldwide public health importance, such as dengue, Japanese encephalitis and classical swine fever viruses, indicating its potential as a lead compound for generating 1,2,4-oxadiazole derivatives with broad-spectrum anti-flaviviral properties.

Development of Artificial Intelligence to Support Needle Electromyography Diagnostic Analysis.

OBJECTIVES: This study proposes a method for classifying three types of resting membrane potential signals obtained as images through diagnostic needle electromyography (EMG) using TensorFlow-Slim and Python to implement an artificial-intelligence-based image recognition scheme. METHODS: Waveform images of an abnormal resting membrane potential generated by diagnostic needle EMG were classified into three types-positive sharp waves (PSW), fibrillations (Fibs), and Others-using the TensorFlow-Slim image classification model library. A total of 4,015 raw waveform data instances were reviewed, with 8,576 waveform images subsequently collected for training. Images were learned repeatedly through a convolutional neural network. Each selected waveform image was classified into one of the aforementioned categories according to the learned results. RESULTS: The classification model, Inception v4, was used to divide waveform images into three categories (accuracy = 93.8%, precision = 99.5%, recall = 90.8%). This was done by applying the pretrained Inception v4 model to a fine-tuning method. The image recognition model was created for training using various types of image-based medical data. CONCLUSIONS: The TensorFlow-Slim library can be used to train and recognize image data, such as EMG waveforms, through simple coding rather than by applying TensorFlow. It is expected that a convolutional neural network can be applied to image data such as the waveforms of electrophysiological signals in a body based on this study.

Neutralization of Acidic Intracellular Vesicles by Niclosamide Inhibits Multiple Steps of the Dengue Virus Life Cycle In Vitro.

Dengue fever is one of the most important mosquito-borne viral infections in large parts of tropical and subtropical countries and is a significant public health concern and socioeconomic burden. There is an urgent need to develop antivirals that can effectively reduce dengue virus (DENV) replication and decrease viral load. Niclosamide, an antiparasitic drug approved for human use, has been recently identified as an effective antiviral agent against a number of pH-dependent viruses, including flaviviruses. Here, we reveal that neutralization of low-pH intracellular compartments by niclosamide affects multiple steps of the DENV infectious cycle. Specifically, niclosamide-induced endosomal neutralization not only prevents viral RNA replication but also affects the maturation of DENV particles, rendering them non-infectious. We found that niclosamide-induced endosomal neutralization prevented E glycoprotein conformational changes on the virion surface of flaviviruses, resulting in the release of non-infectious immature virus particles with uncleaved pr peptide from host cells. Collectively, our findings support the potential application of niclosamide as an antiviral agent against flavivirus infection and highlight a previously uncharacterized mechanism of action of the drug.

Evaluation and Clinical Validation of Two Field-Deployable Reverse Transcription-Insulated Isothermal PCR Assays for the Detection of the Middle East Respiratory Syndrome-Coronavirus.

Middle East respiratory syndrome (MERS) is an emerging zoonotic viral respiratory disease that was first identified in Saudi Arabia in 2012. In 2015, the largest MERS outbreak outside of the Middle East region occurred in the Republic of Korea. The rapid nosocomial transmission of MERS-coronavirus (MERS-CoV) in Korean health care settings highlighted the importance and urgent need for a rapid and reliable on-site diagnostic assay to implement effective control and preventive measures. Here, the evaluation and validation of two newly developed reverse transcription-insulated isothermal PCR (RT-iiPCR) methods targeting the ORF1a and upE genes of MERS-CoV are described. Compared with World Health Organization-recommended singleplex real-time quantitative RT-PCR (RT-qPCR) assays, both RT-iiPCR assays had comparable analytical sensitivity for the detection of MERS-CoV RNA in tissue culture fluid and in sputum samples spiked with infectious virus. Furthermore, clinical evaluation was performed with sputum samples collected from subjects with acute and chronic respiratory illnesses, including patients infected with MERS-CoV. The overall agreement values between the two RT-iiPCR assays and the reference RT-qPCR assays were 98.06% (95% CI, 94.43%-100%; κ = 0.96) and 99.03% (95% CI, 95.88%-100%; κ = 0.99) for ORF1a and upE assays, respectively. The ORF1a and upE MERS-CoV RT-iiPCR assays coupled with a field-deployable system provide a platform for a highly sensitive and specific on-site tool for diagnosis of MERS-CoV infections.