Rapid detection of human adenovirus subgroup B using recombinase polymerase amplification assay.

Human adenovirus subgroup B (HAdV B) is one of the major pathogens of human respiratory virus infections, which has considerable transmission and morbidity in a variety of populations. Therefore, rapid and specific detection of HAdV B in clinical samples is essential for diagnosis. This study aimed to develop a product for rapid nucleic acid detection of HAdV B using recombinase polymerase amplification assay (RPA) and validate the performance of this method by using clinical samples. Results showed that this method achieved a lower limit of detection (LOD) of 10 copies/µL and had no cross-reactivity with other adenovirus subgroups or respiratory pathogens. In addition to high sensitivity, it can be completed within 30 min at 40 °C. There is no need to perform nucleic acid extraction on clinical samples. Taking qPCR as the gold standard, the RPA assay possessed a high concordance (Cohen's kappa, 0.896; 95% CI 0.808-0.984; P < 0.001), with a sensitivity of 87.80% and a specificity of 100.00%. The RPA assay developed in this study provided a simple and highly specific method, making it an important tool for rapid adenovirus nucleic acid detection and facilitating large-scale population screening in resource-limited settings.

Tiratricol inhibits yellow fever virus replication through targeting viral RNA-dependent RNA polymerase of NS5.

Yellow fever virus (YFV) infection is a major public concern that threatens a large population in South America and Africa. No specific antiviral drugs are available for treating yellow fever. Here, we report that tiratricol (triiodothyroacetic acid, TRIAC), a clinically approved drug used to treat thyroid hormone resistance syndrome (THRS), is a potent YFV inhibitor both in host cells and in animal models.An in vitro study demonstrates that TRIAC remarkably suppresses viral RNA synthesis and protein expression in a dose-dependent manner in human hepatoma cell lines (Huh-7) with an EC50 value of 2.07 µM and a CC50 value of 385.77 µM respectively. The surface plasmon resonance assay and molecular docking analysis indicate that TRIAC hinders viral replication by binding to the RNA-dependent RNA polymerase (RdRp) domain of viral nonstructural protein NS5, probably through interacting with the active sites of RdRp.The inhibitory effect of TRIAC in vivo is also confirmed in 3-week old C57BL/6 mice challenged with YFV infection, from which the survival of the mice as well as lesions and infection in their tissues and serum issignificantly promoted following oral administration of TRIAC (0.2 mg/kg/day). Additionally, TRIAC shows a broad-spectrum antiviral activity against multiple flaviviruses such as TBEV, WNV,ZIKV, andJEV in vitro. Our data demonstrate that the TH analogue TRIAC is an effective anti-YFV compound and may act as a potential therapeutic candidate for the treatment of YFV infection if its clinical importance is determined in patients in future.

Identification of tyrphostin AG879 and A9 inhibiting replication of chikungunya virus by screening of a kinase inhibitor library.

Chikungunya virus (CHIKV) is a globally public health threat. There are currently no medications available to treat CHIKV infection. High-throughput screening of 419 kinase inhibitors was performed based on the cytopathic effect method, and six kinase inhibitors with reduced cytopathic effects, including tyrphostin AG879 (AG879), tyrphostin 9 (A9), sorafenib, sorafenib tosylate, regorafenib, and TAK-632, were identified. The anti-CHIKV activities of two receptor tyrosine kinase inhibitors, AG879 and A9, that have not been previously reported, were selected for further evaluation. The results indicated that 50% cytotoxic concentration (CC50) of AG879 and A9 in Vero cells were greater than 30 µM and 6.50 µM, respectively and 50% effective concentration (EC50) were 0.84 µM and 0.36 µM, respectively. The time-of-addition and time-of-removal assays illustrated that both AG879 and A9 function in the middle stage of CHIKV life cycle. Further, AG879 and A9 do not affect viral attachment; however, they inhibit viral RNA replication, and exhibit antiviral activity against CHIKV Eastern/Central/South African and Asian strains, Ross River virus and Sindbis virus in vitro.

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6-mediated endocytic pathway.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

Recent Advances in Antivirals for Japanese Encephalitis Virus.

Culex mosquitoes are the primary vectors of the Japanese encephalitis virus (JEV). Since its discovery in 1935, Japanese encephalitis (JE), caused by JEV, has posed a significant threat to human health. Despite the widespread implementation of several JEV vaccines, the transmission chain of JEV in the natural ecosystem has not changed, and the vector of transmission cannot be eradicated. Therefore, JEV is still the focus of attention for flaviviruses. At present, there is no clinically specific drug for JE treatment. JEV infection is a complex interaction between the virus and the host cell, which is the focus of drug design and development. An overview of antivirals that target JEV elements and host factors is presented in this review. In addition, drugs that balance antiviral effects and host protection by regulating innate immunity, inflammation, apoptosis, or necrosis are reviewed to treat JE effectively.

Multi-omic and comparative analyses revealed monocyte-derived alpha-defensin-1 correlated with COVID-19 severity and inhibited SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological pathogen of coronavirus disease 2019 (COVID-19), a highly contagious disease, spreading quickly and threatening global public health. The symptoms of COVID-19 vary from mild reactions to severe respiratory distress or even fatal outcomes probably due to the different status of host immunity against the virus. Here in the study, we unveiled plasma proteomic signatures and transcriptional patterns of peripheral blood mononuclear cells (PBMCs) using blood samples of 10 COVID-19 patients with different severity. Through systemic analysis, α-defensin-1 (DEFA1) was identified to be elevated in both plasma and PBMCs, and correlated with disease severity and stages. In vitro study demonstrated that DEFA1 was secreted from immunocytes and suppressed SARS-CoV-2 infection of both original and mutated strains with dose dependency. By using sequencing data, we discovered that DEFA1 was activated in monocytes through NF-κB signaling pathway after infection, and secreted into circulation to perturb SARS-CoV-2 infection by interfering protein kinase C expression. It worked mainly during virus replication instead of entry in host cells. Together, the anti-SARS-CoV-2 mechanism of DEFA1 has unveiled a corner of how innate immunity is against SARS-CoV-2 and explored its clinical potential in disease prognosis and therapeutic intervention.

Recent Advancements in Mosquito-Borne Flavivirus Vaccine Development.

Lately, the global incidence of flavivirus infection has been increasing dramatically and presents formidable challenges for public health systems around the world. Most clinically significant flaviviruses are mosquito-borne, such as the four serotypes of dengue virus, Zika virus, West Nile virus, Japanese encephalitis virus and yellow fever virus. Until now, no effective antiflaviviral drugs are available to fight flaviviral infection; thus, a highly immunogenic vaccine would be the most effective weapon to control the diseases. In recent years, flavivirus vaccine research has made major breakthroughs with several vaccine candidates showing encouraging results in preclinical and clinical trials. This review summarizes the current advancement, safety, efficacy, advantages and disadvantages of vaccines against mosquito-borne flaviviruses posing significant threats to human health.

Virus-host Interactions in Early Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus that can cause severe viral encephalitis. Initial interactions between JEV and host cells are required for productive viral infection and initiation of the viral life cycle. The elucidation of these interactions is critical, not only to understand the pathogenesis of JEV infection, but also to design efficient antiviral strategies. In this review, we outline the known viral and cellular components involved in JEV entry into host cells, with a particular focus on the initial virus-host cell interaction on the cell surface and the downstream early events such as endocytosis, membrane fusion, and viral genome release.

mTOR signaling regulates Zika virus replication bidirectionally through autophagy and protein translation.

Zika virus (ZIKV) reemerged in 2016 and attracted much more attention worldwide. To date, the limited knowledge of ZIKV interactions with host cells in the early stages of infection impedes the prevention of viral epidemics and the treatment of ZIKV disease. The mammalian target of rapamycin (mTOR) signaling pathway plays an essential role in the regulation of autophagy and protein synthesis during multiple viral infections. This study aimed to investigate the functional role of mTOR signaling in ZIKV replication in human umbilical vein endothelial cells. Immunoblotting demonstrated that ZIKV infection inhibited mTORC1 signaling, enhancing autophagy but obstructing protein translation. Drugs or siRNA for interfering with mTOR signaling molecules were utilized to demonstrate that AKT/TSC2/mTORC1 signaling was involved in ZIKV infection and that autophagy promoted ZIKV production, but viral protein expression was regulated by mTORC1 signaling. Moreover, confocal microscopy indicated a robust correlation between autophagy and viral RNA transcription. This study clarifies the dual functions of mTOR signaling during ZIKV infection and provides theoretical support for developing potential anti-ZIKV drugs based on mTOR signaling molecules and deeper insights to better understand the mechanism between ZIKV and host cells.

Research Advances on the Role of Lipids in the Life Cycle of Human Coronaviruses.

Coronaviruses (CoVs) are emerging pathogens with a significant potential to cause life-threatening harm to human health. Since the beginning of the 21st century, three highly pathogenic and transmissible human CoVs have emerged, triggering epidemics and posing major threats to global public health. CoVs are enveloped viruses encased in a lipid bilayer. As fundamental components of cells, lipids can play an integral role in many physiological processes, which have been reported to play important roles in the life cycle of CoVs, including viral entry, uncoating, replication, assembly, and release. Therefore, research on the role of lipids in the CoV life cycle can provide a basis for a better understanding of the infection mechanism of CoVs and provide lipid targets for the development of new antiviral strategies. In this review, research advances on the role of lipids in different stages of viral infection and the possible targets of lipids that interfere with the viral life cycle are discussed.

Zika virus infection triggers lipophagy by stimulating the AMPK-ULK1 signaling in human hepatoma cells.

Zika virus (ZIKV) is a globally transmitted mosquito-borne pathogen, and no effective treatment or vaccine is available yet. Lipophagy, a selective autophagy targeting lipid droplets (LDs), is an emerging subject in cellular lipid metabolism and energy homeostasis. However, the regulatory mechanism of lipid metabolism and the role of lipophagy in Zika virus infection remain largely unknown. Here, we demonstrated that ZIKV induced lipophagy by activating unc-51-like kinase 1 (ULK1) through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) in Huh7 cells. Upon ZIKV infection, the average size and triglyceride content of LDs significantly decreased. Moreover, ZIKV infection significantly increased lysosomal biosynthesis and LD-lysosome fusion. The activities of AMPK at Thr-172 and ULK1 at Ser-556 were increased in ZIKV-infected cells and closely correlated with lipophagy induction. Silencing of AMPK expression inhibited ZIKV infection, autophagy induction, and LD-lysosome fusion and decreased the triglyceride content of the cells. The activities of mammalian target of rapamycin (mTOR) at Ser-2448 and ULK1 at Ser-757 were suppressed independently of AMPK during ZIKV infection. Therefore, ZIKV infection triggers AMPK-mediated lipophagy, and the LD-related lipid metabolism during ZIKV infection is mainly regulated via the AMPK-ULK1 signaling pathway.

Lipid Droplets and Their Participation in Zika Virus Infection.

Lipid droplets (LDs) are highly conserved and dynamic intracellular organelles. Their functions are not limited to serving as neutral lipid reservoirs; they also participate in non-energy storage functions, such as cell lipid metabolism, protection from cell stresses, maintaining protein homeostasis, and regulating nuclear function. During a Zika virus (ZIKV) infection, the viruses hijack the LDs to provide energy and lipid sources for viral replication. The co-localization of ZIKV capsid (C) protein with LDs supports its role as a virus replication platform and a key compartment for promoting the generation of progeny virus particles. However, in view of the multiple functions of LDs, their role in ZIKV infection needs further elucidation. Here, we review the basic mechanism of LD biogenesis and biological functions and discuss how ZIKV infection utilizes these effects of LDs to facilitate virus replication, along with the future application strategy of developing new antiviral drugs based on the interaction of ZIKV with LDs.

High-Throughput Screening of FDA-Approved Drug Library Reveals Ixazomib Is a Broad-Spectrum Antiviral Agent against Arboviruses.

The emergence of significant arboviruses and their spillover transmission to humans represent a major threat to global public health. No approved drugs are available for the treatment of significant arboviruses in circulation today. The repurposing of clinically approved drugs is one of the most rapid and promising strategies in the identification of effective treatments for diseases caused by arboviruses. Here, we screened small-molecule compounds with anti-tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus activity from 2580 FDA-approved drugs. In total, 60 compounds showed antiviral efficacy against all four of the arboviruses in Huh7 cells. Among these compounds, ixazomib and ixazomib citrate (inhibitors of 20S proteasome ß5) exerted antiviral effects at a low-micromolar concentration. The time-of-drug-addition assay suggested that ixazomib and ixazomib citrate disturbed multiple processes in viruses' life cycles. Furthermore, ixazomib and ixazomib citrate potently inhibited chikungunya virus replication and relieved virus-induced footpad swelling in a mouse model. These results offer critical information which supports the role of ixazomib as a broad-spectrum agent against arboviruses.

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

The West Nile virus (WNV) is a member of the flavivirus and is known to cause encephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors, ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.

Mosquito-Borne Flaviviruses and Current Therapeutic Advances.

Mosquito-borne flavivirus infections affect approximately 400 million people worldwide each year and are global threats to public health. The common diseases caused by such flaviviruses include West Nile, yellow fever, dengue, Zika infection and Japanese encephalitis, which may result in severe symptoms and disorders of multiple organs or even fatal outcomes. Till now, no specific antiviral agents are commercially available for the treatment of the diseases. Numerous strategies have been adopted to develop novel and promising inhibitors against mosquito-borne flaviviruses, including drugs targeting the critical viral components or essential host factors during infection. Research advances in antiflaviviral therapy might optimize and widen the treatment options for flavivirus infection. This review summarizes the current developmental progresses and involved molecular mechanisms of antiviral agents against mosquito-borne flaviviruses.

Antiviral efficacy of selective estrogen receptor modulators against SARS-CoV-2 infection in vitro and in vivo reveals bazedoxifene acetate as an entry inhibitor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh member of the coronavirus family that can infect humans. Recently, more contagious and pathogenic variants of SARS-CoV-2 have been continuously emerging. Clinical candidates with high efficacy and ready availability are still in urgent need. To identify potent anti-SARS-CoV-2 repurposing drugs, we evaluated the antiviral efficacy of 18 selective estrogen receptor modulators (SERMs) against SARS-CoV-2 infection. Six SERMs exhibited excellent anti-SARS-CoV-2 effects in Vero E6 cells and three human cell lines. Clomifene citrate, tamoxifen, toremifene citrate, and bazedoxifene acetate reduced the weight loss of hamsters challenged with SARS-CoV-2, and reduced hamster pulmonary viral load and interleukin-6 expression when assayed at 4 days postinfection. In particular, bazedoxifene acetate was identified to act on the penetration stage of the postattachment step via altering cholesterol distribution and endosome acidification. And, bazedoxifene acetate inhibited pseudoviruses infection of original SARS-CoV-2, Delta variant, Omicron variant, and SARS-CoV. These results offer critical information supporting bazedoxifene acetate as a promising agent against coronaviruses.

Hepatitis C Virus Infection Cycle-Specific MicroRNA Profiling Reveals Stage-Specific miR-4423-3p Targets RIG-I to Facilitate Infection.

Hepatitis C virus (HCV) infection is one of the main causes of chronic liver diseases, the disorders of which involve multiple pathological processes and elements including host factors such as non-coding small RNAs. Although several genes have been reported to be correlated with HCV infection, the potential regulatory network has not been deciphered clearly. By small RNA sequencing, we clarified the expression profile of microRNAs (miRNAs) in HCV-infected Huh7 and Huh7.5.1 cells and identified 6 dysregulated miRNAs with the same expression trend and 32 dysregulated miRNAs with different expression trends during different stages of HCV life cycle. By looking into each infection stage, we found that 6 miRNAs were entry stage specific, 4 miRNAs were replication stage specific, and 1 miRNA was related to the transmission stage. Moreover, due to the fact that Huh7.5.1 cells have a retinoic acid-inducible gene 1 (RIG-I) mutation which causes reduced production of interferons (IFNs), we here focused on the miRNAs of different trends to decipher the RIG-I/IFN specific miRNAs. Among them, miR-4423-3p showed a significant promotive effect on HCV infection by suppressing RIG-I/IFN pathway through direct binding to RIG-I mRNA. Together, the results displayed novel insights into the miRNA regulatory networks in HCV infection and progression, thus providing a prosperous perspective into the establishment of novel therapeutic and diagnostic targets of the disease.

Entry Inhibitors of Hepatitis C Virus.

Hepatitis C virus (HCV) infection affects approximately 1% of the world's population and is a major cause of chronic liver diseases. Although antiviral therapy consisting of direct-acting antivirals (DAAs) can cure the majority of HCV patients, it is still limited by viral resistances, drug-drug interactions, and high costs. Moreover, the role of DAAs in the prevention of occurrences of graft reinfection in HCV patients who receive liver transplantations is still under comprehensive clinical investigation, bringing the risk of recipient reinfection. HCV entry is composed of initial non-specific attachment and binding, post-binding interactions with essential host factors, internalization, and virion-cell membrane fusion to release viral RNA to cytosol. Thus, a number of novel and promising targets from either virion or cellular factors of these processes become optimal interfering elements for antiviral therapy, eliminating viral infection at the very beginning. Therefore, entry inhibitors can be supplemented into the future treatment regimens to optimize and widen the prevention and therapeutics of HCV infection. This chapter introduces the basic HCV entry processes and summarizes molecular mechanisms and research status of the current antiviral agents targeting HCV entry in preclinical and clinical study.