Dynamic bidirectional regulation of NLRC3 and gammaherpesviruses during viral latency in B lymphocytes.

While most NOD-like receptors (NLRs) are predominately expressed by innate immune cells, NLRC3, an inhibitory NLR of immune signaling, exhibits the highest expression in lymphocytes. The role of NLRC3 or any NLRs in B lymphocytes is completely unknown. Gammaherpesviruses, including human Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68), establish latent infection in B lymphocytes, which requires elevated NF-κB. This study shows that during latent EBV infection of human B cells, viral-encoded latent membrane protein 1 (LMP1) decreases NLRC3 transcript. LMP1-induced-NF-κB activation suppresses the promoter activity of NLRC3 via p65 binding to the promoter. Conversely, NLRC3 inhibits NF-κB activation by promoting the degradation of LMP1 in a proteasome-dependent manner. In vivo, MHV-68 infection reduces Nlrc3 transcripts in splenocytes, and Nlrc3-deficient mice show greater viral latency than controls. These results reveal a bidirectional regulatory circuit in B lymphocytes, where viral latent protein LMP1 reduces NLRC3 expression, while NLRC3 disrupts gammaherpesvirus latency, which is an important step for tumorigenesis.

Pipette-free field-deployable molecular diagnostic kit for bimodal visual detection of infectious RNA viruses.

Here, we developed a field-deployable molecular diagnostic kit for the detection of RNA viruses that operates in a pipette-free manner. The kit is composed of acrylic sticks, PCR tubes, and palm-sized three-dimensional(3D)-printed heaters operated by batteries. The kit performs RNA extraction, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP), and visual detection in one kit. An acrylic stick was engraved with one shallow and one deep cylindrical chamber at each end for the insertion of an FTA card and ethidium homodimer-1 (EthD-1), respectively, to perform RNA extraction/purification and bimodal visual detection of the target amplicons. First, an intercalation of EthD-1 into the target DNA initially produces fluorescence upon UV illumination. Next, the addition of a strong oxidant, in this case sodium (meta) periodate (NaIO4 ), produces intense aggregates in the presence of EthD-1-intercalated DNA, realized by electrostatic interaction. In the absence of the target amplicon, no fluorescence or aggregates are observed. Using this kit, two major infectious viruses-severe fever with thrombocytopenia syndrome virus (SFTSV) and severe acute respiratory syndrome coronavirus (SARS-CoV-2)-were successfully detected in 1 h, and the limits of detection (LOD) were approximately 1 virus µL-1 for SFTSV and 103 copies µL-1 for SARS-CoV-2 RNA. The introduced kit is portable, end-user-friendly, and can be operated in a pipette-free manner, paving the way for simple and convenient virus detection in resource-limited settings.

A DNA vaccine against GII.4 human norovirus VP1 induces blocking antibody production and T cell responses.

Human noroviruses (HuNoVs) are highly contagious and a leading cause of epidemics of acute gastroenteritis worldwide. Among the various HuNoV genotypes, GII.4 is the most prevalent cause of outbreaks. However, no vaccines have been approved for HuNoVs to date. DNA vaccines are proposed to serve as an ideal platform against HuNoV since they can be easily produced and customized to express target proteins. In this study, we constructed a CMV/R vector expressing a major structural protein, VP1, of GII.4 HuNoV (CMV/R-GII.4 HuNoV VP1). Transfection of CMV/R-GII.4 HuNoV VP1 into human embryonic kidney 293T (HEK293T) cells resulted in successful expression of VP1 proteins in vitro. Intramuscular or intradermal immunization of mice with the CMV/R-GII.4 HuNoV VP1 construct elicited the production of blocking antibodies and activation of T cell responses against GII.4 HuNoV VP1. Our collective data support the utility of CMV/R-GII.4 HuNoV VP1 as a promising DNA vaccine candidate against GII.4 HuNoV.

A study on the fabrication of metal microneedle array electrodes for ECG detection based on low melting point Bi-In-Sn alloys.

This study describes the fabrication and characteristics of microneedle array electrodes (MAEs) using Bismuth-Indium-Tin (Bi-In-Sn) alloys. The MAEs consist of 57 pyramid-shaped needles measuring 340 µm wide and 800 µm high. The fabrication process involved micromolding the alloys in a vacuum environment. Physical tests demonstrated that Bi-In-Sn MAEs have good mechanical strength, indicating their suitability for successful skin penetration. The electrode-skin interface impedance test confirmed that Bi-In-Sn MAEs successfully penetrated the skin. Impedance measurements revealed the importance of insulating the microneedle electrodes for optimal electrical performance, and a UV-curable Polyurethane Acrylate coating was applied to enhance insulation. Electrocardiogram measurements using the Bi-In-Sn MAEs demonstrated performance comparable to that of traditional Ag/AgCl electrodes, which shows promise for accurate data collection. Overall, the study demonstrates successful, minimally-invasive skin insertion, improved electrical insulation, and potential applications of Bi-In-Sn microneedle array. These findings contribute to advancements in microneedle technology for biomedical applications.

A CRISPR-Cas12a-Based Diagnostic Method for Japanese Encephalitis Virus Genotypes I, III, and V.

The Japanese encephalitis virus (JEV) is prevalent in Asian countries, including Korea, Japan, China, Vietnam, and India. JEV is transmitted to humans by Culex mosquitoes. Despite extensive research efforts, no approved antiviral agents are currently available, although JE can be prevented by vaccination. DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a newly emerging CRISPR-Cas12a-based molecular diagnostic method combined with isothermal nucleic acid amplification. In this study, DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) was effectively utilized for JEV diagnosis and detected down to 10 RNA copies for JEV genotype I (GI) and 1 × 102 copies for both GIII and GV, achieving similar sensitivity to RT-PCR while displaying no cross-reaction with other viruses. A one-tube, one-temperature format of DETECTR was further developed, and its efficiency compared with that of conventional DETECTR.

Kaempferol Interferes with Varicella-Zoster Virus Replication in Human Foreskin Fibroblasts.

Kaempferol, a natural flavonoid abundantly found in plants, is known to have pharmacological properties, such as anti-inflammatory and anti-cancer effects. In this study, we investigated the antiviral effects of kaempferol against a varicella-zoster virus (VZV) clinical isolate in vitro. We found that kaempferol significantly inhibited VZV replication without exhibiting cytotoxicity. Kaempferol exerted its antiviral effect at a similar stage of the VZV life cycle as acyclovir, which inhibits VZV DNA replication. Taken together, our results suggest that kaempferol inhibits VZV infection by blocking the DNA replication stage in the viral life cycle.

A CRISPR-Cas12a-based diagnostic method for multiple genotypes of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) infection is commonly reported in countries of Northeast Asia including China, Japan and South Korea. The majority of the SFTS patients are elderly and the average fatality rate is more than 10%. A rapid and sensitive diagnostic method to monitor and prevent SFTSV transmission remains an urgent clinical challenge. In this study, we developed a molecular diagnostic technique for detection of SFTSV using the CRISPR-Cas12a system combined with reverse transcription recombinase polymerase amplification (RT-RPA). Using this method, we successfully diagnosed SFTSV infections with the reaction time of 50 min from blood plasma without cross-reactivity to other viruses, supporting its application for rapid and sensitive diagnosis of SFTS.

Discovery and Photoisomerization of New Pyrrolosesquiterpenoids Glaciapyrroles D and E, from Deep-Sea Sediment Streptomyces sp.

Two new pyrrolosesquiterpenes, glaciapyrroles D (1) and E (2) were discovered along with the previously reported glaciapyrrole A (3) from Streptomyces sp. GGS53 strain isolated from deep-sea sediment. This study elucidated the planar structures of 1 and 2 using nuclear magnetic resonance (NMR), mass spectrometry (MS), ultraviolet (UV), and infrared (IR) spectroscopic data. The absolute configurations of the glaciapyrroles were determined by Mosher's method, circular dichroism spectroscopy, and X-ray crystallography. Under 366 nm UV irradiation, the glaciapyrroles were systematically converted to the corresponding photoglaciapyrroles (4-6) via photoisomerization, resulting in the diversification of the glaciapyrrole family compounds. The transformation of the glaciapyrrole Z to E isomers occurred in a 1:1 ratio, based on virtual validation of the photoisomerization of these olefinic compounds by 1H-NMR spectroscopy and liquid chromatography/mass spectrometry (LC/MS) analysis. Finally, when encapsulated in poly(lactic-co-glycolic acid) nanoparticles, glaciapyrrole E and photoglaciapyrrole E displayed significant inhibitory activity against influenza A virus. This is the first report of antiviral effects from glaciapyrrole family compounds, whose biological functions have only been subjected to limited studies so far.

Coordinated regulation of interferon and inflammasome signaling pathways by SARS-CoV-2 proteins.

Type I and III interferons (IFNs) and the nucleotide-binding domain (NBD) leucine-rich repeat (LRR)-containing receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome play pivotal roles in the pathogenesis of SARS-CoV-2. While optimal IFN and inflammasome responses are essential for limiting SARS-CoV-2 infection, aberrant activation of these innate immune responses is associated with COVID-19 pathogenesis. In this review, we focus our discussion on recent findings on SARS-CoV-2-induced type I and III IFNs and NLRP3 inflammasome responses and the viral proteins regulating these mechanisms.

Antiviral Activities of Ethyl Pheophorbides a and b Isolated from Aster pseudoglehnii against Influenza Viruses.

Screening of the antiviral and virucidal activities of ethanol extracts from plants endemic to the Republic of Korea revealed the inhibitory activity of a 70% ethanol extract of the whole plant of A. pseudoglehnii (APE) against influenza virus infection. Two chlorophyll derivatives, ethyl pheophorbides a and b, isolated as active components of APE, exerted virucidal effects with no evident cytotoxicity. These compounds were effective only under conditions of direct incubation with the virus, and exerted no effects on the influenza A virus (IAV) surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Interestingly, virucidal activities of ethyl pheophorbides a and b were observed against enveloped but not non-enveloped viruses, suggesting that these compounds act by affecting the integrity of the viral membrane and reducing infectivity.

Microfluidic electrical cell lysis for high-throughput and continuous production of cell-free varicella-zoster virus.

Varicella-zoster virus (VZV), the causative agent of varicella and herpes zoster, is highly cell-associated and spreads via cell-to-cell contact in tissue culture. The lack of cell-free VZV hampers studies on VZV biology as well as antiviral and vaccine development. In the present study, a poly(methylmethacrylate) microfluidic device integrated with arrays of microelectrode was fabricated to continuously electrolyse VZV-infected cells to produce cell-free viruses. By designing multiple constrictions and microelectrode arrays, a high electric field is focused on the constricted region of the microchannel to disrupt large numbers of virus-infected cells with high-throughput on a microfluidic platform. Plaque assay and scanning electron microscopy were conducted to quantify and characterize cell-free VZV produced using the microfluidic continuous-flow electrical cell lysis device. The process of microfluidic electrical cell lysis followed by subsequent filtration and virus concentration process yielded a 1.4-2.1 × 104 plaque-forming units (PFUs) per mL of cell-free VZV from 7.0 × 106 VZV-infected human foreskin fibroblasts (HFF) cells. The high electric field formed inside a microfluidic channel combined with the continuous-flow of virus-infected cells within the microchannel enabled the rapid and efficient production of high-titer cell-free virus in large quantities with relatively low input of the voltage.

Specific Detection of Influenza A and B Viruses by CRISPR-Cas12a-Based Assay.

A rapid and accurate on-site diagnostic test for pathogens including influenza viruses is critical for preventing the spread of infectious diseases. Two types of influenza virus, A and B cause seasonal flu epidemics, whereas type A can cause influenza pandemics. To specifically detect influenza A (IAV) and B (IBV) viruses, we developed a clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system-based assay. By coupling reverse transcription recombinase polymerase amplification (RT-RPA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP), a CRISPR-Cas12a DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) detected IAV and IBV titers as low as 1 × 100 plaque forming units (PFUs) per reaction without exhibiting cross-reactivity. Only 75 to 85 min were required to detect IAV and IBV, depending on isothermal nucleic acid amplification methods, and results were verified using a lateral flow strip assay that does not require additional analytic equipment. Taken together, our findings establish RT-RPA and RT-LAMP-coupled DETECTR-based diagnostic tests for rapid, specific and high-sensitivity detection of IAV and IBV using fluorescence and lateral flow assays. The diagnostic test developed in this study can be used to distinguish IAV and IBV infections, a capability that is necessary for monitoring and preventing the spread of influenza epidemics and pandemics.

SARS-CoV-2 Nonstructural Proteins 1 and 13 Suppress Caspase-1 and the NLRP3 Inflammasome Activation.

Viral infection-induced activation of inflammasome complexes has both positive and negative effects on the host. Proper activation of inflammasome complexes induces down-stream effector mechanisms that inhibit viral replication and promote viral clearance, whereas dysregulated activation has detrimental effects on the host. Coronaviruses, including SARS-CoV and MERS-CoV, encode viroporins that activate the NLRP3 inflammasome, and the severity of coronavirus disease is associated with the inflammasome activation. Although the NLRP3 inflammasome activation is implicated in the pathogenesis of coronaviruses, these viruses must evade inflammasome-mediated antiviral immune responses to establish primary replication. Screening of a complementary DNA (cDNA) library encoding 28 SARS-CoV-2 open reading frames (ORFs) showed that two nonstructural proteins (NSPs), NSP1 and NSP13, inhibited caspase-1-mediated IL-1ß activation. NSP1 amino acid residues involved in host translation shutoff and NSP13 domains responsible for helicase activity were associated with caspase-1 inhibition. In THP-1 cells, both NSP1 and NSP13 significantly reduced NLRP3-inflammasome-induced caspase-1 activity and IL-1ß secretion. These findings indicate that SARS-CoV-2 NSP1 and NSP13 are potent antagonists of the NLRP3 inflammasome.

Antiviral Activities of Quercetin and Isoquercitrin Against Human Herpesviruses.

We previously reported that the ethyl acetate (EtOAc) fraction of a 70% ethanol extract of Elaeocarpus sylvestris (ESE) inhibits varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) replication in vitro. PGG (1,2,3,4,6-penta-O-galloyl-ß-D-glucose) is a major chemical constituent of the EtOAc fraction of ESE that inhibits VZV but not HCMV replication. In this study, we comprehensively screened the chemical compounds identified in the EtOAc fraction of ESE for potential antiviral properties. Among the examined compounds, quercetin and isoquercitrin displayed potent antiviral activities against both VZV and HCMV with no significant cytotoxic effects. Both compounds strongly suppressed the expression of VZV and HCMV immediate-early (IE) genes. Our collective results indicated that, in addition to PGG, quercetin and isoquercitrin are bioactive compounds in the EtOAc fraction of ESE that effectively inhibit human herpesvirus replication.

Human cytomegalovirus IE86 protein aa 136-289 mediates STING degradation and blocks the cGAS-STING pathway.

We previously reported that human cytomegalovirus (HCMV) 86 kDa immediate-early 2 gene product (IE86) promotes proteasome-dependent degradation of STING. In the present study, we determined the specific residues of IE86 responsible for STING degradation using a STING-firefly luciferase fusion protein expression system for quantitative meas-urement of STING protein levels. IE86 amino acids (aa) 136-289 were sufficient to promote STING degradation and further induced down-regulation of 2'3'-cyclic GMP-AMP (cGAMP)-mediated IFN-ß promoter activation. Interestingly, transactivation domains (TAD) of the IE86 protein located at the N- and C-termini were required for down-regulation of Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon ß (IFN-ß) (TRIF)-mediated IFN-ß-and p65/RelA-induced NF-κB-dependent promoter activation while amino acids (aa) 136-289 had no significant effects. Our collective data suggest that the IE86 protein utilizes the aa 136-289 region to promote STING degradation and inhibit the cGAS-STING pathway.

Spicatoside A derived from Liriope platyphylla root ethanol extract inhibits hepatitis E virus genotype 3 replication in vitro.

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans worldwide. Although hepatitis E is self-limiting without chronic infection development, HEV infection often leads to severe liver diseases causing high mortality in pregnant women in addition to chronic hepatitis and cirrhosis in immunosuppressed patients. In this study, we investigated the effect of a Liriope platyphylla ethanol extract (LPE) on HEV replication. Interestingly, LPE suppressed replication of the genotype 3 HEV replicon. Sequential solvent fractionation revealed that the ethyl acetate (EA) fraction of LPE exerts the most potent inhibitory effects. With the aid of activity-guided fractionation and multi-step column chromatography, spicatoside A was subsequently isolated in the EA fraction of LPE and specifically shown to exert inhibitory effects on replication of the genotype 3 HEV replicon. In addition, spicatoside A interfered with replication of the HEV genotype 3 strain 47832c and expression of HEV ORF2 capsid proteins. Our findings clearly support the potential utility of spicatoside A as an effective anti-HEV agent.

Anti-varicella-zoster virus activity of cephalotaxine esters in vitro.

Harringtonine (HT) and homoharringtonine (HHT), alkaloid esters isolated from the genus Cephalotaxus, exhibit antitumor activity. A semisynthetic HHT has been approved for treatment of chronic myelogenous leukemia. In addition to antileukemic activity, HT and HHT are reported to possess potent antiviral activity. In this study, we investigated the effects of HT and HHT on replication of varicella-zoster virus (VZV) in vitro. HT and HHT, but not their biologically inactive parental alkaloid cephalotaxine (CET), significantly inhibited replication of recombinant VZV-pOka luciferase. Furthermore, HT and HHT, but not CET, strongly induced down-regulation of VZV lytic genes and exerted potent antiviral effects against a VZV clinical isolate. The collective data support the utility of HT and HHT as effective antiviral candidates for treatment of VZV-associated diseases.

Analysis of Anticancer Activity and Chemical Sensitization Effects of Dendropanax morbifera and Commersonia bartramia Extracts.

BACKGROUND/AIM: Dendropanax morbifera (DM) and Commersonia bartramia (CB) are possible candidates for immunotherapy. In this study, the cytotoxicity and chemical sensitization of DM and CB extracts on gynecologic and colon cancers were evaluated. MATERIALS AND METHODS: The malignant cell lines were cultured and analyzed for cytotoxicity and chemical sensitization. A mouse model was also constructed to make the condition similar to in vivo. Reverse transcription-polymerase chain reaction was conducted to determine alterations in drug-resistant genes. RESULTS: The extracts from DM and CB showed specific cytotoxicity to malignant cell lines. DM increased chemical sensitivity to cervical and ovarian cancer, while CB showed improved sensitization to endometrial cancer. The effects of the extracts were confirmed using a mouse model. The extracts induced differences in the expression levels of a number of genes related to drug resistance. CONCLUSION: DM and CB extracts could be novel agents for immunotherapy and chemical sensitization in gynecologic and colon cancers.

Corrigendum: Human Cytomegalovirus IE2 86 kDa Protein Induces STING Degradation and Inhibits cGAMP-Mediated IFN-ß Induction.

[This corrects the article on p. 1854 in vol. 8, PMID: 29018427.].

An ethanol extract of Lysimachia mauritiana exhibits inhibitory activity against hepatitis E virus genotype 3 replication.

Hepatitis E virus (HEV) is an etiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. HEV can cause fulminant hepatic failure with high mortality rates in pregnant women, and genotype 3 is reported to trigger chronic hepatitis in immunocompromised individuals worldwide. Screening of plant extracts for compounds with potential anti-HEV effects led to the identification of a 70% ethanol extract of Lysimachia mauritiana (LME) that interferes with replication of the swine HEV genotype 3 replicon. Furthermore, LME significantly inhibited replication of HEV genotype 3 and expression of HEV ORF2 in infected cells without exerting cytotoxic effects. Collectively, our findings demonstrate the potential utility of LME in the development of novel antiviral drugs against HEV infection.