Overview of the immunological mechanisms in hepatitis B virus reactivation: Implications for disease progression and management strategies.

Hepatitis B virus (HBV) reactivation is a clinically significant challenge in disease management. This review explores the immunological mechanisms underlying HBV reactivation, emphasizing disease progression and management. It delves into host immune responses and reactivation's delicate balance, spanning innate and adaptive immunity. Viral factors' disruption of this balance, as are interactions between viral antigens, immune cells, cytokine networks, and immune checkpoint pathways, are examined. Notably, the roles of T cells, natural killer cells, and antigen-presenting cells are discussed, highlighting their influence on disease progression. HBV reactivation's impact on disease severity, hepatic flares, liver fibrosis progression, and hepatocellular carcinoma is detailed. Management strategies, including anti-viral and immunomodulatory approaches, are critically analyzed. The role of prophylactic anti-viral therapy during immunosuppressive treatments is explored alongside novel immunotherapeutic interventions to restore immune control and prevent reactivation. In conclusion, this comprehensive review furnishes a holistic view of the immunological mechanisms that propel HBV reactivation. With a dedicated focus on understanding its implications for disease progression and the prospects of efficient management strategies, this article contributes significantly to the knowledge base. The more profound insights into the intricate interactions between viral elements and the immune system will inform evidence-based approaches, ultimately enhancing disease management and elevating patient outcomes. The dynamic landscape of management strategies is critically scrutinized, spanning anti-viral and immunomodulatory approaches. The role of prophylactic anti-viral therapy in preventing reactivation during immunosuppressive treatments and the potential of innovative immunotherapeutic interventions to restore immune control and proactively deter reactivation.

The Potential Role of Autophagy in Progression of Liver Fibrosis in Chronic Hepatitis B Patients Receiving Antiviral Treatment: A Brief Report.

Despite antiviral treatment, some patients with chronic hepatitis B (CHB) progress to cirrhosis. Enhancement of autophagy was implicated in the proliferation of hepatitis B in hepatocytes. This study aimed to evaluate the potential role of autophagy in the progression of liver fibrosis in patients receiving antiviral treatments and having completely inhibited viral replication. This descriptive-analytical study was designed and conducted in 2020 at Mottahhari Hepatitis Clinic affiliated with Shiraz University of Medical Science (Shiraz, Iran). Patients who were on anti-hepatitis B nucleotide treatments for at least two years, and those who were not cirrhotic at baseline but later progressed to cirrhosis were identified to be included in the case group. Besides, for the control group, patients on the nucleotide regimens who did not have cirrhosis at baseline or during follow-up were randomly selected. Ultimately, 16 cases and 14 controls were included in the study. Data were analyzed using SPSS software, and P<0.05 was considered statistically significant. Serum Beclin-1 and LC3 levels were compared between the two groups using enzyme-linked immunosorbent assays. The t test was used to assess the statistical differences between the case and control groups. Beclin-1 level was significantly higher in cirrhosis patients than the control group (1283±244 vs. 1063±257, P=0.024). However, there was no statistical difference between the level of LC3 in the cirrhotic group (168±31) and the control group (150±16) (P=0.065). Autophagy may have a role in the progression of cirrhosis in patients with CHB. Future larger prospective studies are required to determine the effect of blocking on the progression of liver disease in this population A preprint of this study was published at https://www.researchsquare.com/article/rs-1435490/v1.pdf.

Aloperine-Type Alkaloids with Antiviral and Antifungal Activities from the Seeds of Sophora alopecuroides L.

As a continuous flow investigation of novel pesticides from natural quinolizidine alkaloids, the chemical compositions of the seeds of Sophora alopecuroides were thoroughly researched. Fifteen new aloperine-type alkaloids (1-15) as well as six known aloperine-type alkaloids (16-21) were obtained from the extract of S. alopecuroides. The structures of 1-21 were confirmed via HRESIMS, NMR, UV, IR, ECD calculations, and X-ray diffraction. The antiviral activities of 1-21 against tobacco mosaic virus (TMV) were detected following the improved method of half-leaf. Compared with ningnanmycin (protective: 69.7% and curative: 64.3%), 15 exhibited excellent protective (71.7%) and curative (64.6%) activities against TMV. Further biological studies illustrated that 15 significantly inhibited the transcription of the TMV-CP gene and increased the activities of polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL). The antifungal activities of 1-21 against Phytophythora capsica, Botrytis cinerea, Alternaria alternata, and Gibberella zeae were screened according to a mycelial inhibition test. Compound 13 displayed excellent antifungal activity against B. cinerea (EC50: 7.38 µg/mL). Moreover, in vitro antifungal mechanism studies displayed that 13 causes accumulation of reactive oxygen species and finally leads to mycelia cell membrane damage and cell death in vitro.

Recent Progress in Human Milk Oligosaccharides and Its Antiviral Efficacy.

Gastrointestinal (GI)-associated viruses, including rotavirus (RV), norovirus (NV), and enterovirus, usually invade host cells, transmit, and mutate their genetic information, resulting in influenza-like symptoms, acute gastroenteritis, encephalitis, or even death. The unique structures of human milk oligosaccharides (HMOs) enable them to shape the gut microbial diversity and endogenous immune system of human infants. Growing evidence suggests that HMOs can enhance host resistance to GI-associated viruses but without a systematic summary to review the mechanism. The present review examines the lactose- and neutral-core HMOs and their antiviral effects in the host. The potential negative impacts of enterovirus 71 (EV-A71) and other GI viruses on children are extensive and include neurological sequelae, neurodevelopmental retardation, and cognitive decline. However, the differences in the binding affinity of HMOs for GI viruses are vast. Hence, elucidating the mechanisms and positive effects of HMOs against different viruses may facilitate the development of novel HMO derived oligosaccharides.

Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity.

The human respiratory syncytial virus (hRSV) and the human metapneumovirus (hMPV) are important human respiratory pathogens from the Pneumoviridae family. Both are responsible for severe respiratory tract infections in infants, young children, elderly individuals, adults with chronic medical conditions, and immunocompromised patients. Despite their large impact on human health, vaccines for hRSV were only recently introduced, and only limited treatment options exist. Here we show that Ginkgolic acid (GA), a natural compound from the extract of Ginkgo biloba, with known antiviral properties for several viruses, efficiently inhibits these viruses' infectivity and spread in cultures in a dose-dependent manner. We demonstrate that the drug specifically affects the entry step during the early stages on the viruses' life cycle with no effect on post-entry and late stage events, including viral gene transcription, genome replication, assembly and particles release. We provide evidence that GA acts as an efficient antiviral for members of the Pneumoviridae family and has the potential to be used to treat acute infections.

SARS-CoV-2 resistance to monoclonal antibodies and small-molecule drugs.

Over four years have passed since the beginning of the COVID-19 pandemic. The scientific response has been rapid and effective, with many therapeutic monoclonal antibodies and small molecules developed for clinical use. However, given the ability for viruses to become resistant to antivirals, it is perhaps no surprise that the field has identified resistance to nearly all of these compounds. Here, we provide a comprehensive review of the resistance profile for each of these therapeutics. We hope that this resource provides an atlas for mutations to be aware of for each agent, particularly as a springboard for considerations for the next generation of antivirals. Finally, we discuss the outlook and thoughts for moving forward in how we continue to manage this, and the next, pandemic.

Structure elucidation and antiviral activity of a cold water-extracted mannogalactofucan Ts1-1A from Trametes sanguinea against human cytomegalovirus in vitro.

In this study, we purified a partially acetylated heteropolysaccharide (Ts1-1A) from the fruit bodies of Trametes sanguinea Lloyd through cold water extraction and serial chromatographic separation. The purified polysaccharide Ts1-1A (12.8 kDa) was characterized as a branched mannogalactofucan with a backbone of alternately connected 1,3-linked α-Fucp and 1,6-linked α-Galp, which was partially substituted by non-reducing end units of ß-Manp at O-2 and O-3 positions of 1,6-linked α-Galp. Ts1-1A showed pronounced anti-human cytomegalovirus activity at the concentration of 200 and 500 µg/mL in systematical assessments including morphological changes, western blotting, qPCR, indirect immunofluorescence and tissue culture infective dose assays. Moreover, Ts1-1A exerted its antiviral activity at two distinct stages of viral proliferation manifesting as significantly inhibiting viral protein (IE1/2 and p52) expression and reducing viral gene (UL123, UL44 and UL32) replication in the HCMV-infected WI-38 cells. At viral attachment stage, Ts1-1A interacted with HCMV and prevented HCMV from attaching to its host cells. While at early phase of viral replication stage, Ts1-1A suppressed HCMV replication by downregulating NQO1 and HO-1 proteins related to oxidative stress as an antioxidant. To sum up, Ts1-1A is a promising anti-HCMV agent which could be developed for HCMV infection prevention and therapy.

Inflammatory Consequences: Hepatitis C Virus-Induced Inflammasome Activation and Pyroptosis.

Hepatitis C virus (HCV), despite the availability of effective direct-acting antivirals (DAAs) that clear the virus from >95% of individuals treated, continues to cause significant health care burden due to disease progression that can lead to fibrosis, cirrhosis, and/or hepatocellular carcinoma. The fact that some people who are treated with DAAs still go on to develop worsening liver disease warrants further study into the immunopathogenesis of HCV. Many viral infections, including HCV, have been associated with activation of the inflammasome/pyroptosis pathway. This inflammatory cell death pathway ultimately results in cell lysis and release of inflammatory cytokines, IL-18 and IL-1ß. This review will report on studies that investigated HCV and inflammasome activation/pyroptosis. This includes clinical in vivo data showing elevated pyroptosis-associated cytokines in the blood of individuals living with HCV, studies of genetic associations of pyroptosis-related genes and development of liver disease, and in vitro studies aimed at understanding the mechanism of pyroptosis induced by HCV. Finally, we discuss major gaps in understanding and outstanding questions that remain in the field of HCV-induced pyroptosis.

Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid.

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

CNS Viral Infections-What to Consider for Improving Drug Treatment: A Plea for Using Mathematical Modeling Approaches.

Neurotropic viruses may cause meningitis, myelitis, encephalitis, or meningoencephalitis. These inflammatory conditions of the central nervous system (CNS) may have serious and devastating consequences if not treated adequately. In this review, we first summarize how neurotropic viruses can enter the CNS by (1) crossing the blood-brain barrier or blood-cerebrospinal fluid barrier; (2) invading the nose via the olfactory route; or (3) invading the peripheral nervous system. Neurotropic viruses may then enter the intracellular space of brain cells via endocytosis and/or membrane fusion. Antiviral drugs are currently used for different viral CNS infections, even though their use and dosing regimens within the CNS, with the exception of acyclovir, are minimally supported by clinical evidence. We therefore provide considerations to optimize drug treatment(s) for these neurotropic viruses. Antiviral drugs should cross the blood-brain barrier/blood cerebrospinal fluid barrier and pass the brain cellular membrane to inhibit these viruses inside the brain cells. Some antiviral drugs may also require intracellular conversion into their active metabolite(s). This illustrates the need to better understand these mechanisms because these processes dictate drug exposure within the CNS that ultimately determine the success of antiviral drugs for CNS infections. Finally, we discuss mathematical model-based approaches for optimizing antiviral treatments. Thereby emphasizing the potential of CNS physiologically based pharmacokinetic models because direct measurement of brain intracellular exposure in living humans faces ethical restrictions. Existing physiologically based pharmacokinetic models combined with in vitro pharmacokinetic/pharmacodynamic information can be used to predict drug exposure and evaluate efficacy of antiviral drugs within the CNS, to ultimately optimize the treatments of CNS viral infections.

Metformin Inhibits Zika Virus Infection in Trophoblast Cell Line.

Zika virus (ZIKV) infections have been associated with severe clinical outcomes, which may include neurological manifestations, especially in newborns with intrauterine infection. However, licensed vaccines and specific antiviral agents are not yet available. Therefore, a safe and low-cost therapy is required, especially for pregnant women. In this regard, metformin, an FDA-approved drug used to treat gestational diabetes, has previously exhibited an anti-ZIKA effect in vitro in HUVEC cells by activating AMPK. In this study, we evaluated metformin treatment during ZIKV infection in vitro in a JEG3-permissive trophoblast cell line. Our results demonstrate that metformin affects viral replication and protein synthesis and reverses cytoskeletal changes promoted by ZIKV infection. In addition, it reduces lipid droplet formation, which is associated with lipogenic activation of infection. Taken together, our results indicate that metformin has potential as an antiviral agent against ZIKV infection in vitro in trophoblast cells.

In vitro suppression of porcine epidemic diarrhea virus by Panax notoginseng saponins: assessing antiviral potential.

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality in neonatal suckling piglets, leading to significant economic losses to the swine industry. Panax notoginseng saponins (PNS) are bioactive extracts derived from the P. notoginseng plant. In this study, we investigated the anti-PEDV effect of PNS by employing various methodologies to assess their impact on PEDV in Vero cells. Using a CCK-8 (Cell Counting Kit-8) assay, we found that PNS had no significant cytotoxicity below the concentration of 128 µg/mL in Vero cells. Using immunofluorescence assays (IFAs), an enzyme-linked immunosorbent assay (ELISA), and plaque formation assays, we observed a dose-dependent inhibition of PEDV infection by PNS within 24-48 hours postinfection. PNS exerts its anti-PEDV activity specifically at the genome replication stage, and mRNA-seq analysis demonstrated that treatment with PNS resulted in increased expression of various genes, including IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), CFH (complement factor H), IGSF10 (immunoglobulin superfamily member 10), ID2 (inhibitor of DNA binding 2), SPP1 (secreted phosphoprotein 1), PLCB4 (phospholipase C beta 4), and FABP4 (fatty acid binding protein 4), but it resulted in decreased expression of IL1A (interleukin 1 alpha), TNFRSF19 (TNF receptor superfamily member 19), CDH8 (cadherin 8), DDIT3 (DNA damage inducible transcript 3), GADD45A (growth arrest and DNA damage inducible alpha), PTPRG (protein tyrosine phosphatase receptor type G), PCK2 (phosphoenolpyruvate carboxykinase 2), and ADGRA2 (adhesion G protein-coupled receptor A2). This study provides insights into the potential mechanisms underlying the antiviral effects of PNS. Taken together, the results suggest that the PNS might effectively regulate the defense response to the virus and have potential to be used in antiviral therapies.

The metabolic footprint of Vero E6 cells highlights the key metabolic routes associated with SARS-CoV-2 infection and response to drug combinations.

SARS-CoV-2 burdens healthcare systems worldwide, yet specific drug-based treatments are still unavailable. Understanding the effects of SARS-CoV-2 on host molecular pathways is critical for providing full descriptions and optimizing therapeutic targets. The present study used Nuclear Magnetic Resonance-based metabolic footprinting to characterize the secreted cellular metabolite levels (exometabolomes) of Vero E6 cells in response to SARS-CoV-2 infection and to two candidate drugs (Remdesivir, RDV, and Azithromycin, AZI), either alone or in combination. SARS-CoV-2 infection appears to force VE6 cells to have increased glucose concentrations from extra-cellular medium and altered energetic metabolism. RDV and AZI, either alone or in combination, can modify the glycolic-gluconeogenesis pathway in the host cell, thus impairing the mitochondrial oxidative damage caused by the SARS-CoV-2 in the primary phase. RDV treatment appears to be associated with a metabolic shift toward the TCA cycle. Our findings reveal a metabolic reprogramming produced by studied pharmacological treatments that protects host cells against virus-induced metabolic damage, with an emphasis on the glycolytic-gluconeogenetic pathway. These findings may help researchers better understand the relevant biological mechanisms involved in viral infection, as well as the creation of mechanistic hypotheses for such candidate drugs, thereby opening up new possibilities for SARS-CoV-2 pharmacological therapy.

Non-Thermal Plasma Reduces HSV-1 Infection of and Replication in HaCaT Keratinocytes In Vitro.

Herpes simplex virus type 1 (HSV-1) is a lifelong pathogen characterized by asymptomatic latent infection in the trigeminal ganglia (TG), with periodic outbreaks of cold sores caused by virus reactivation in the TG and subsequent replication in the oral mucosa. While antiviral therapies can provide relief from cold sores, they are unable to eliminate HSV-1. We provide experimental results that highlight non-thermal plasma (NTP) as a new alternative therapy for HSV-1 infection that would resolve cold sores faster and reduce the establishment of latent infection in the TG. Additionally, this study is the first to explore the use of NTP as a therapy that can both treat and prevent human viral infections. The antiviral effect of NTP was investigated using an in vitro model of HSV-1 epithelial infection that involved the application of NTP from two separate devices to cell-free HSV-1, HSV-1-infected cells, and uninfected cells. It was found that NTP reduced the infectivity of cell-free HSV-1, reduced viral replication in HSV-1-infected cells, and diminished the susceptibility of uninfected cells to HSV-1 infection. This triad of antiviral mechanisms of action suggests the potential of NTP as a therapeutic agent effective against HSV-1 infection.

Treatment of highly virulent mammarenavirus infections-status quo and future directions.

INTRODUCTION: Mammarenaviruses are negative-sense bisegmented enveloped RNA viruses that are endemic in Africa, the Americas, and Europe. Several are highly virulent, causing acute human diseases associated with high case fatality rates, and are considered to be significant with respect to public health impact or bioterrorism threat. AREAS COVERED: This review summarizes the status quo of treatment development, starting with drugs that are in advanced stages of evaluation in early clinical trials, followed by promising candidate medical countermeasures emerging from bench analyses and investigational animal research. EXPERT OPINION: Specific therapeutic treatments for diseases caused by mammarenaviruses remain limited to the off-label use of ribavirin and transfusion of convalescent sera. Progress in identifying novel candidate medical countermeasures against mammarenavirus infection has been slow in part because of the biosafety and biosecurity requirements. However, novel methodologies and tools have enabled increasingly efficient high-throughput molecular screens of regulatory-agency-approved small-molecule drugs and led to the identification of several compounds that could be repurposed for the treatment of infection with several mammarenaviruses. Unfortunately, most of them have not yet been evaluated in vivo. The most promising treatment under development is a monoclonal antibody cocktail that is protective against multiple lineages of the Lassa virus in nonhuman primate disease models.

Review of COVID-19 Therapeutics by Mechanism: From Discovery to Approval.

The global research and pharmaceutical community rapidly mobilized to develop treatments for coronavirus disease 2019 (COVID-19). Existing treatments have been repurposed and new drugs have emerged. Here we summarize mechanisms and clinical trials of COVID-19 therapeutics approved or in development. Two reviewers, working independently, reviewed published data for approved COVID-19 vaccines and drugs, as well as developmental pipelines, using databases from the following organizations: United States Food and Drug Administration (US-FDA), European Medicines Agency (EMA), Japanese Pharmaceutical and Medical Devices Agency (PMDA), and ClinicalTrials.gov. In all, 387 drugs were found for initial review. After removing unrelated trials and drugs, 66 drugs were selected, including 17 approved drugs and 49 drugs under development. These drugs were classified into six categories: 1) drugs targeting the viral life cycle 2) Anti-severe acute respiratory syndrome coronavirus 2 Monoclonal Antibodies, 3) immunomodulators, 4) anti-coagulants, 5) COVID-19-induced neuropathy drugs, and 6) other therapeutics. Among the 49 drugs under development are the following: 6 drugs targeting the viral life cycle, 12 immunosuppression drugs, 2 immunostimulants, 2 HIF-PHD targeting drugs, 3 GM-CSF targeting drugs, 5 anti-coagulants, 2 COVID-19-induced neuropathy drugs, and 17 others. This review provides insight into mechanisms of action, properties, and indications for COVID-19 medications.

In silico study of alkaloids with quercetin nucleus for inhibition of SARS-CoV-2 protease and receptor cell protease.

Covid-19 disease caused by the deadly SARS-CoV-2 virus is a serious and threatening global health issue declared by the WHO as an epidemic. Researchers are studying the design and discovery of drugs to inhibit the SARS-CoV-2 virus due to its high mortality rate. The main Covid-19 virus protease (Mpro) and human transmembrane protease, serine 2 (TMPRSS2) are attractive targets for the study of antiviral drugs against SARS-2 coronavirus. Increasing consumption of herbal medicines in the community and a serious approach to these drugs have increased the demand for effective herbal substances. Alkaloids are one of the most important active ingredients in medicinal plants that have wide applications in the pharmaceutical industry. In this study, seven alkaloid ligands with Quercetin nucleus for the inhibition of Mpro and TMPRSS2 were studied using computational drug design including molecular docking and molecular dynamics simulation (MD). Auto Dock software was used to evaluate molecular binding energy. Three ligands with the most negative docking score were selected to be entered into the MD simulation procedure. To evaluate the protein conformational changes induced by tested ligands and calculate the binding energy between the ligands and target proteins, GROMACS software based on AMBER03 force field was used. The MD results showed that Phyllospadine and Dracocephin-A form stable complexes with Mpro and TMPRSS2. Prolinalin-A indicated an acceptable inhibitory effect on Mpro, whereas it resulted in some structural instability of TMPRSS2. The total binding energies between three ligands, Prolinalin-A, Phyllospadine and Dracocephin-A and two proteins MPro and TMRPSS2 are (-111.235 ± 15.877, - 75.422 ± 11.140), (-107.033 ± 9.072, -84.939 ± 10.155) and (-102.941 ± 9.477, - 92.451 ± 10.539), respectively. Since the binding energies are at a minimum, this indicates confirmation of the proper binding of the ligands to the proteins. Regardless of some Prolinalin-A-induced TMPRSS2 conformational changes, it may properly bind to TMPRSS2 binding site due to its acceptable binding energy. Therefore, these three ligands can be promising candidates for the development of drugs to treat infections caused by the SARS-CoV-2 virus.

Viral deubiquitinating proteases and the promising strategies of their inhibition.

Several viruses are now known to code for deubiquitinating proteases in their genomes. Ubiquitination is an essential post-translational modification of cellular substrates involved in many processes in the cell, including in innate immune signalling. This post-translational modification is regulated by the ubiquitin conjugation machinery, as well as various host deubiquitinating enzymes. The conjugation of ubiquitin chains to several innate immune related factors is often needed to induce downstream signalling, shaping the antiviral response. Viral deubiquitinating proteins, besides often having a primary function in the viral replication cycle by cleaving the viral polyprotein, are also able to cleave ubiquitin chains from such host substrates, in that way exerting a function in innate immune evasion. The presence of viral deubiquitinating enzymes has been firmly established for numerous animal-infecting viruses, such as some well-researched and clinically important nidoviruses, and their presence has now been confirmed in several plant viruses as well. Viral proteases in general have long been highlighted as promising drug targets, with a current focus on small molecule inhibitors. In this review, we will discuss the range of viral deubiquitinating proteases known to date, summarise the various avenues explored to inhibit such proteases and discuss novel strategies and models intended to inhibit and study these specific viral enzymes.

Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, 27 inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.

Antiviral activity of adenoviral vector expressing human interferon lambda-4 against influenza virus.

Interferon lambda (IFNλ), classified as a type III IFN, is a representative cytokine that plays an important role in innate immunity along with type I IFN. IFNλ can elicit antiviral states by inducing peculiar sets of IFN-stimulated genes (ISGs). In this study, an adenoviral vector expression system with a tetracycline operator system was used to express human IFNλ4 in cells and mice. The formation of recombinant adenovirus (rAd-huIFNλ4) was confirmed using immunohistochemistry assays and transmission electron microscopy. Its purity was verified by quantifying host cell DNA and host cell proteins, as well as by confirming the absence of the replication-competent adenovirus. The transduction of rAd-huIFNλ4 induced ISGs and inhibited four subtypes of the influenza virus in both mouse-derived (LA-4) and human-derived cells (A549). The antiviral state was confirmed in BALB/c mice following intranasal inoculation with 109 PFU of rAd-huIFNλ4, which led to the inhibition of four subtypes of the influenza virus in mouse lungs, with reduced inflammatory lesions. These results imply that human IFNλ4 could induce antiviral status by modulating ISG expression in mice.