Reduced Virus Titers Research Articles

Deciphering the antiviral nature of endophytic Bacillus spp. against groundnut bud necrosis virus in cowpea and tomato.

Tomato, the important vegetable crop, is severely affected by Orthotospovirus arachinecrosis which impacts heavy economic losses. The application of insecticide to manage viral diseases is not an environmentally safe approach. In view of these issues, we investigated the antiviral efficacy of 21 bacterial endophytes against GBNV in local lesion host (Cowpea-VBN3). Based on the reduction in lesion number and virus titer as estimated through both DAC ELISA and qPCR in cowpea, the bacterial endophytes viz., Bacillus licheniformis Soya1, Bacillus tequilensis NBL6, and Bacillus velezensis VB7 were selected and further tested in tomato. The study revealed the well-defined antiviral efficacy of these endophytes against GBNV. The percentage of disease incidence ranged from 16 to 24% in endophyte-treated tomato plants compared with untreated plants (88%). In addition, symptom severity was reduced, and the application of endophytes also in promotion of the growth compared with untreated control. DAC ELISA revealed that the tomato plants treated with bacterial endophytes challenged with GBNV showed reduction in the virus titer (0.26-0.39 @ OD 405 nm) at different days of interval after inoculation (0, 5, and 10 days) compared with untreated control (3.475 @ OD 405 nm). Additionally, reduction in the viral copy number in bacterial endophyte-treated plants was evident by real-time PCR. Furthermore, tomato plants bacterized with endophytes depicted significant correlation and reduction in viral load and disease incidence as revealed by the principal-component biplot analysis. Thus, the application of bacterial endophytes has a potential role in reducing the disease incidence, severity, and titer value of GBNV, which will be the promising management approach in future to mitigate the virus infection in tomato plants.

ISKNV Triggers AMPK/mTOR-Mediated Autophagy Signaling through Oxidative Stress, Inducing Antioxidant Enzyme Expression and Enhancing Viral Replication in GF-1 Cells.

Infectious spleen and kidney necrosis virus (ISKNV) infections can induce the process of host cellular autophagy but have rarely been identified within the molecular autophagy signaling pathway. In the present study, we demonstrated that ISKNV induces ROS-mediated oxidative stress signals for the induction of 5'AMP-activated protein kinase/mechanistic target of rapamycin kinase (AMPK/mTOR)-mediated autophagy and upregulation of host antioxidant enzymes in fish GF-1 cells. We also examined ISKNV-induced oxidative stress, finding that reactive oxidative species (ROS) increased by 1.5-fold and 2.5-fold from day 2 to day 3, respectively, as assessed by the H2DCFDA assay for tracing hydrogen peroxide (H2O2), which was blocked by NAC treatment in fish GF-1 cells. Furthermore, ISKNV infection was shown to trigger oxidative stress/Nrf2 signaling from day 1 to day 3; this event was then correlated with the upregulation of antioxidant enzymes such as Cu/ZnSOD and MnSOD and was blocked by the antioxidant NAC. Using an MDC assay, TEM analysis and autophagy marker LC3-II/I ratio, we found that ROS stress can regulate autophagosome formation within the induction of autophagy, which was inhibited by NAC treatment in GF-1 cells. Through signal analysis, we found that AMPK/mTOR flux was modulated through inhibition of mTOR and activation of AMPK, indicating phosphorylation levels of mTOR Ser 2448 and AMPK Thr 172 from day 1 to day 3; however, this process was reversed by NAC treatment, which also caused a reduction in virus titer (TCID50%) of up to 1000 times by day 3 in GF-1 cells. Thus, ISKNV-induced oxidative stress signaling is blocked by antioxidant NAC, which can also either suppress mTOR/AMPK autophagic signals or reduce viral replication. These findings may provide the basis for the creation of DNA control and treatment strategies.

Persistent Neurological Deficits in Mouse PASC Reveal Antiviral Drug Limitations.

Post-Acute Sequelae of COVID-19 (PASC) encompasses persistent neurological symptoms, including olfactory and autonomic dysfunction. Here, we report chronic neurological dysfunction in mice infected with a virulent mouse-adapted SARS-CoV-2 that does not infect the brain. Long after recovery from nasal infection, we observed loss of tyrosine hydroxylase (TH) expression in olfactory bulb glomeruli and neurotransmitter levels in the substantia nigra (SN) persisted. Vulnerability of dopaminergic neurons in these brain areas was accompanied by increased levels of proinflammatory cytokines and neurobehavioral changes. RNAseq analysis unveiled persistent microglia activation, as found in human neurodegenerative diseases. Early treatment with antivirals (nirmatrelvir and molnupiravir) reduced virus titers and lung inflammation but failed to prevent neurological abnormalities, as observed in patients. Together these results show that chronic deficiencies in neuronal function in SARS-CoV-2-infected mice are not directly linked to ongoing olfactory epithelium dysfunction. Rather, they bear similarity with neurodegenerative disease, the vulnerability of which is exacerbated by chronic inflammation.

Betacyanins from red pitahaya (Hylocereus polyrhizus) exhibit antiviral response against influenza A virus

Seasonal influenza affects millions of lives worldwide, with the influenza A virus (IAV) responsible for pandemics and annual epidemics, causing the most severe illnesses resulting in patient hospitalizations or death. With IAV threatening the next global influenza pandemic, it is a race against time to search for antiviral drugs. Betacyanins are unique nitrogen-containing and water-soluble reddish-violet pigments that have been reported to possess antiviral properties against the dengue virus. This study aimed to examine the antiviral effect of betacyanins from red pitahaya (Hylocereus polyrhizus) on IAV-infected lung epithelial A549 cells. HPLC and LC-MS analysis of extracted betacyanin showed four betacyanins in the betacyanin fraction: phyllocactin, hylocerenin, betanin, and isobetanin. Cytotoxicity assay showed that betacyanin fractions were not cytotoxic to A549 cells at concentrations below 100 μg/mL. Betacyanin fraction concentrations of 12.5, 25.0, and 50.0 μg/mL prevented the formation of viral cytopathic effect and reduced virus titer in IAV-infected cells up to 72 h. A downregulation of protein and mRNA nucleoprotein expression levels was observed after treatment with 25.0 and 50.0 μg/mL of betacyanin fraction after 24 h, thereby providing evidence for the antiviral activity of betacyanin from red pitahaya against IAV in vitro.

A Novel Recombinant Modified Vaccinia Ankara Virus expressing Interleukin-13 Receptor α2 Antigen for Potential Cancer Immunotherapy.

Genetically altered recombinant poxviruses hold great therapeutic promise in animal models of cancer. Poxviruses can induce effective cellmediated immune responses against tumor-associated antigens. Preventive and therapeutic vaccination with a DNA vaccine expressing IL-13Rα2 can mediate partial regression of established tumors in vivo, indicating that host immune responses against IL-13Rα2 need further augmentation. The aim of the study is developing a recombinant modified vaccinia Ankara (MVA) expressing IL-13Rα2 (rMVA-IL13Rα2) virus and study in vitro infectivity and efficacy against IL-13Rα2 positive cell lines. We constructed a recombinant MVA expressing IL-13Rα2 and a green fluorescent protein (GFP) reporter gene. Purified virus titration by infection of target cells and immunostaining using anti-vaccinia and anti-IL-13Rα2 antibodies was used to confirm the identity and purity of the rMVA-IL13Rα2. Western Blot analysis confirmed the presence of IL-13Rα2 protein (~52 kDa). Flow cytometric analysis of IL-13Rα2 negative T98G glioma cells when infected with rMVA-IL13Rα2 virus demonstrated cell-surface expression of IL-13Rα2, indicating the infectivity of the recombinant virus. Incubation of T98G-IL13Rα2 cells with varying concentrations (0.1-100 ng/ml) of interleukin-13 fused to truncated Pseudomonas exotoxin (IL13-PE) resulted in depletion of GFP+ fluorescence in T98G-IL13Rα2 cells. IL13-PE (10-1000 ng/ml) at higher concentrations also inhibited the protein synthesis in T98G-IL13Rα2 cells compared to cells infected with the control pLW44-MVA virus. IL13- PE treatment of rMVA-IL13Rα2 infected chicken embryonic fibroblast and DF-1 cell line reduced virus titer compared to untreated cells. rMVA-IL13Rα2 virus can successfully infect mammalian cells to express IL-13Rα2 in a biologically active form on the surface of infected cells. To evaluate the efficacy of rMVA-IL13Rα2, immunization studies are planned in murine tumor models.

Virological evaluation of natural and modified attapulgite against porcine epidemic diarrhoea virus

BackgroundThe Porcine Epidemic Diarrhea Virus (PEDV) has caused significant economic losses in the global swine industry. As a potential drug for treating diarrhea, the antiviral properties of attapulgite deserve further study.MethodsIn this study, various methods such as RT-qPCR, Western blot, viral titer assay, Cytopathic Effect, immunofluorescence analysis and transmission electron microscopy were used to detect the antiviral activity of attapulgite and to assess its inhibitory effect on PEDV.ResultsWhen exposed to the same amount of virus, there was a significant decrease in the expression of the S protein, resulting in a viral titer reduction from 10-5.613 TCID50/mL to 10-2.90 TCID50/mL, which represents a decrease of approximately 102.6 folds. Results of cytopathic effect and indirect immunofluorescence also indicate a notable decrease in viral infectivity after attapulgite treatment. Additionally, it was observed that modified materials after acidification had weaker antiviral efficacy compared to powdered samples that underwent ultrasonic disintegration, which showed the strongest antiviral effects.ConclusionAs a result, Attapulgite powders can trap and adsorb viruses to inhibit PEDV in vitro, leading to loss of viral infectivity. This study provides new materials for the development of novel disinfectants and antiviral additives.

Exploring the Antiviral Potential of Esters of Cinnamic Acids with Quercetin.

Severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) has infected more than 762 million people to date and has caused approximately 7 million deaths all around the world, involving more than 187 countries. Although currently available vaccines show high efficacy in preventing severe respiratory complications in infected patients, the high number of mutations in the S proteins of the current variants is responsible for the high level of immune evasion and transmissibility of the virus and the reduced effectiveness of acquired immunity. In this scenario, the development of safe and effective drugs of synthetic or natural origin to suppress viral replication and treat acute forms of COVID-19 remains a valid therapeutic challenge. Given the successful history of flavonoids-based drug discovery, we developed esters of substituted cinnamic acids with quercetin to evaluate their in vitro activity against a broad spectrum of Coronaviruses. Interestingly, two derivatives, the 3,4-methylenedioxy 6 and the ester of acid 7, have proved to be effective in reducing OC43-induced cytopathogenicity, showing interesting EC50s profiles. The ester of synaptic acid 7 in particular, which is not endowed with relevant cytotoxicity under any of the tested conditions, turned out to be active against OC43 and SARS-CoV-2, showing a promising EC50. Therefore, said compound was selected as the lead object of further analysis. When tested in a yield reduction, assay 7 produced a significant dose-dependent reduction in viral titer. However, the compound was not virucidal, as exposure to high concentrations of it did not affect viral infectivity, nor did it affect hCoV-OC43 penetration into pre-treated host cells. Additional studies on the action mechanism have suggested that our derivative may inhibit viral endocytosis by reducing viral attachment to host cells.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Immune-enhancing activity of compound polysaccharide on the inactivated influenza vaccine

Traditional Chinese medicine polysaccharides have numerous biological activities with broad applications in the biomedical industries. However, a clear understanding of the pharmacological activities of compound polysaccharides with multi-component structures remain challenging. This study aimed to investigate the immune boosting effect of compound polysaccharides on the influenza vaccine and assess the preliminary structure-activity relationship. The compound polysaccharide (CP) was isolated from the combined Chinese herbs lentinan, pachymaran and tremellan, and purified by gradient ethanol precipitation to obtain its subcomponents of CP-20, CP-40, CP-60, and CP-80 with decreasing molecular weights. These polysaccharides were mainly composed of glucans with different linkage patterns, including α-(1 → 3)-glucan, α-(1 → 4)-glucan and β-(1 → 6)-glucan. A significant improvement was observed in the survival of mice vaccinated with inactivated (IAV) vaccine and the isolated polysaccharides as adjuvants. A reduction in the pulmonary virus titer and weight loss were also observed. Moreover, CP-40 and CP-60, as well as the original CP, significantly enhanced the serum anti-IAV antibody titers and interleukin IL-2, IL-5, and IL-6 concentrations. These preliminary results indicate the immune boosting effect of the compound polysaccharides is highly relevant to the specific structural properties of the subcomponent, and CP-40 is worthy of further exploration as a glycan adjuvant for the IAV vaccine.

A combination of nirmatrelvir and ombitasvir boosts inhibition of SARS-CoV-2 replication

Antiviral therapeutics are highly effective countermeasures for the treatment of coronavirus disease 2019 (COVID-19). However, development of resistance to antivirals undermines their effectiveness. Combining multiple antivirals during patient treatment has the potential to overcome the evolutionary selective pressure towards antiviral resistance, as well as provide a more robust and efficacious treatment option. The current evidence for effective antiviral combinations to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication is limited. Here, we demonstrate a combination of nirmatrelvir with ombitasvir, to jointly bring about potent inhibition of SARS-CoV-2 replication. We developed an in vitro 384- well plate cytopathic effect assay for the evaluation of antiviral combinations against Calu-3 cells infected with SARS-CoV-2 and found, that a combination of ombitasvir and nirmatrelvir was synergistic; thereby decreasing the nirmatrelvir IC50 by approx. 16-fold. The increased potency of the nirmatrelvir-ombitasvir combination, over nirmatrelvir alone afforded a greater than 3 log10 reduction in viral titre, which is sufficient to fully prevent the detection of progeny SARS-CoV-2 viral particles at 48 h post infection. The mechanism of this potentiated effect was shown to be, in-part, due to joint inhibition of the 3-chymotrypsin-like protease via a positive allosteric modulation mechanism.

A study on influence of wettability on antiviral coating using polyethylene glycol (PEG) and acrylic binder

Abstract One of the biggest problems facing medical science today is preventing viral outbreaks, which highlights the significance of research initiatives aimed at creating antimicrobial coatings for a range of products, including textiles, medical devices, and public spaces. In this study, we aimed to determine the possible antiviral effects of polyethylene glycol (PEG) coating on feline coronavirus (FCoV). The PEG coatings were synthesized by a simple mixing method with a water-based acrylic binder in different weight percentages (3, 5, 10, 15, 20, and 25 wt%). The Spearman–Karber technique was used to calculate the viral titers, which were then expressed as the tissue culture infectious dose at 50 % CPE (TCID50/ml). 20 wt% PEG could result in a 3 log10 reduction in virus titer with an inhibition rate of approximately 99.9 % against FCoV. The increment of PEG weight percent from 0 to 25 wt% decreases the hardness and glass transition temperature of the coatings from 38.1 to 5.5 HV and 15.45 to −15.48 °C. Apart from that, the wettability analysis has revealed that PEG coating is hydrophilic with water contact angle (WCA) of around 75 ± 0.5°–85 ± 0.5°. Adding 25 wt% of PEG makes the coating to be superhydrophilic with WCA of 39.85 ± 0.5°.

A comprehensive approach for evaluating the virucidal performance of domestic laundry detergents under practical conditions.

We aimed to develop a method to assess the virucidal performance of domestic laundry in a lab-scale washing machine (Rotawash) based on EN 17658. For method development, virus recovery was investigated after drying on cotton carriers for three test viruses murine norovirus (MNV), modified vaccinia virus Ankara (MVA), and bovine coronavirus (BCoV), followed by washing simulations in flasks and Rotawash. MNV and MVA demonstrated sufficient recovery from carriers after drying and washing (up to 40°C and 60min). BCoV exhibited lower recovery, indicating less relevance as a test virus. Rotawash efficacy tests conducted with MNV, a resistant, non-enveloped virus, showed limited efficacy of a bleach-free detergent, aligning with results from a domestic washing machine. Rotawash washes achieved higher reductions in infectious virus titers than suspension tests, indicating the role of washing mechanics in virus removal. This study established a practical method to test the virucidal efficacy of laundry detergents in Rotawash, simulating domestic washing.

Ivermectin antiviral activity against Varicellovirus bovinealpha 1: assessment of intracellular drug accumulation in virus-infected cells.

Varicellovirus bovinealpha 1 (formerly bovine alphaherpesvirus type 1, BoAHV-1) is associated with several syndromes in cattle, including respiratory disease and is one of the main agents involved in the bovine respiratory disease complex (BRDC). Its infectious cycle is characterized by latent infections with sporadic virus reactivation and transmission. Although the acute disease can be prevented by the use of vaccines, specific therapeutic measures are not available. Ivermectin (IVM) is a semi-synthetic avermectin with a broad-spectrum antiparasitic activity, which has previously shown to have potential as an antiviral drug. In this study, IVM antiviral activity against BoAHV-1 was characterized in two cell lines (MDBK [Madin Darby bovine kidney] and BT [bovine turbinate]), including the measurement of intracellular drug accumulation within virus-infected cells. IVM antiviral activity was assessed at three different drug concentrations (1.25, 2.5 and 5µM) after incubation for 24, 48 and 72h. Slight cytotoxicity was only observed with 5µM IVM. Even the lowest IVM dose was able to induce a significant reduction in virus titers in both cell lines. These findings indicate that the antiviral effects of IVM were evident in our experimental model within the range of concentrations achievable through therapeutic in vivo administration. Consequently, additional in vivo trials are necessary to validate the potential utility of these results in effectively managing BoAHV-1 in infected cattle.

Complement Suppresses the Initial Type 1 Interferon Response to Ocular Herpes Simplex Virus Type 1 Infection in Mice.

The complement system (CS) contributes to the initial containment of viral and bacterial pathogens and clearance of dying cells in circulation. We previously reported mice deficient in complement component 3 (C3KO mice) were more sensitive than wild-type (WT) mice to ocular HSV-1 infection, as measured by a reduction in cumulative survival and elevated viral titers in the nervous system but not the cornea between days three and seven post infection (pi). The present study was undertaken to determine if complement deficiency impacted virus replication and associated changes in inflammation at earlier time points in the cornea. C3KO mice were found to possess significantly (p < 0.05) less infectious virus in the cornea at 24 h pi that corresponded with a decrease in HSV-1 lytic gene expression at 12 and 24 h pi compared to WT animals. Flow cytometry acquisition found no differences in the myeloid cell populations residing in the cornea including total macrophage and neutrophil populations at 24 h pi with minimal infiltrating cell populations detected at the 12 h pi time point. Analysis of cytokine and chemokine content in the cornea measured at 12 and 24 h pi revealed that only CCL3 (MIP-1α) was found to be different between WT and C3KO mice with >2-fold increased levels (p < 0.05, ANOVA and Tukey's post hoc t-test) in the cornea of WT mice at 12 h pi. C3KO mouse resistance to HSV-1 infection at the early time points correlated with a significant increase in type I interferon (IFN) gene expression including IFN-α1 and IFN-β and downstream effector genes including tetherin and RNase L (p < 0.05, Mann-Whitney rank order test). These results suggest early activation of the CS interferes with the induction of the type I IFN response and leads to a transient increase in virus replication following corneal HSV-1 infection.

Screening and Stability Evaluation of Freeze-Dried Protective Agents for a Live Recombinant Pseudorabies Virus Vaccine.

Infection of pigs with the pseudorabies virus (PRV) causes significant economic losses in the pig industry. Immunization with live vaccines is a crucial aspect in the prevention of pseudorabies in swine. The TK/gE/gI/11k/28k deleted pseudorabies vaccine is a promising alternative for the eradication of epidemic pseudorabies mutant strains. This study optimized the lyophilization of a heat-resistant PRV vaccine to enhance the quality of a live vaccine against the recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k-. The A4 freeze-dried protective formulation against PRV was developed by comparing the reduction in virus titer after lyophilization and after seven days of storage at 37 °C. The formulation contains 1% gelatin, 5% trehalose, 0.5% poly-vinylpyrimidine (PVP), 0.5% thiourea, and 1% sorbitol. The A4 freeze-dried vaccine demonstrated superior protection and thermal stability. It experienced a freeze-dried loss of 0.31 Lg post-freeze-drying and a heat loss of 0.42 Lg after being stored at a temperature of 37 °C for 7 consecutive days. The A4 freeze-dried vaccine was characterized through XRD, FTIR, and SEM analyses, which showed that it possessed an amorphous structure with a consistent porous interior. The trehalose component of the vaccine formed stable hydrogen bonds with the virus. Long-term and accelerated stability studies were also conducted. The A4 vaccine maintained viral titer losses of less than 1.0 Lg when exposed to 25 °C for 90 days, 37 °C for 28 days, and 45 °C for 7 days. The A4 vaccine had a titer loss of 0.3 Lg after storage at 2-8 °C for 24 months, and a predicted shelf life of 6.61 years at 2-8 °C using the Arrhenius equation. The A4 freeze-dried vaccine elicited no side effects when used to immunize piglets and produced specific antibodies. This study provides theoretical references and technical support to improve the thermal stability of recombinant PRV rHN1201TK-/gE-/gI-/11k-/28k- vaccines.

Methotrimeprazine is a neuroprotective antiviral in JEV infection via adaptive ER stress and autophagy

Japanese encephalitis virus (JEV) pathogenesis is driven by a combination of neuronal death and neuroinflammation. We tested 42 FDA-approved drugs that were shown to induce autophagy for antiviral effects. Four drugs were tested in the JE mouse model based on in vitro protective effects on neuronal cell death, inhibition of viral replication, and anti-inflammatory effects. The antipsychotic phenothiazines Methotrimeprazine (MTP) & Trifluoperazine showed a significant survival benefit with reduced virus titers in the brain, prevention of BBB breach, and inhibition of neuroinflammation. Both drugs were potent mTOR-independent autophagy flux inducers. MTP inhibited SERCA channel functioning, and induced an adaptive ER stress response in diverse cell types. Pharmacological rescue of ER stress blocked autophagy and antiviral effect. MTP did not alter translation of viral RNA, but exerted autophagy-dependent antiviral effect by inhibiting JEV replication complexes. Drug-induced autophagy resulted in reduced NLRP3 protein levels, and attenuation of inflammatory cytokine/chemokine release from infected microglial cells. Our study suggests that MTP exerts a combined antiviral and anti-inflammatory effect in JEV infection, and has therapeutic potential for JE treatment.

Procedures for the Detection of Wolbachia-Conferred Antiviral Protection in Drosophila melanogaster.

Spread of Wolbachia infections in host populations may be enhanced by Wolbachia-conferred protection from viral pathogens. Wolbachia-infected Drosophila melanogaster survive the pathogenic effects of positive-sense single-stranded RNA virus infections at a higher rate than the flies without Wolbachia. The protection can occur with or without detectable reduction in virus titer. For the comparisons to be meaningful, Wolbachia-harboring and Wolbachia-free insects need to be genetically matched, and original populations of gut microbiota need to be restored after the removal of Wolbachia using antibiotics. Here, I describe the procedures needed to detect Wolbachia-conferred antiviral protection against Drosophila C virus measured as the difference in survival and viral titer between flies with and without Wolbachia.

Enhanced in vivo antiviral activity against pseudorabies virus through transforming gallic acid into graphene quantum dots with stimulation of interferon-related immune responses.

With the urgent need for antiviral agents, antiviral materials with high biocompatibility and antiviral effects have attracted a lot of attention. In this study, gallic acid, a natural polyphenolic compound, was transformed into biocompatible graphene quantum dots (GAGQDs) which exhibit enhanced antiviral activity against pseudorabies virus (PRV). The as-prepared GAGQDs inhibit PRV proliferation with a 104-fold reduction in viral titers. Investigation of the antiviral mechanism revealed that GAGQDs inhibit the adsorption, invasion and replication of PRV infection. Treatment with GAGQDs regulates the expression levels of interferon-related antiviral proteins, including mitochondrial antiviral-signaling protein (MAVS), signal transducer and activator of transcription 1 (STAT1) and 2',5'-oligoadenylate synthetase 1 (OAS1), suggesting that GAGQDs can stimulate innate antiviral immune responses, resulting in enhanced antiviral effects. More importantly, GAGQD treatments alleviate clinical symptoms and reduce mortality in PRV-infected mice. Our results reveal the enhanced therapeutic effects of GAGQDs against PRV infection in vitro and in vivo, suggesting the potential of GAGQDs as a promising novel antiviral agent.

Antiviral effect of alkaloids-free Ephedra Herb extract on respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children. Despite decades of efforts, no effective therapies are available. We recently reported that extracts of Ephedra Herb and Cinnamon Bark interacted with the G attachment protein of RSV to inhibit infectivity. The present in vitro study aimed to investigate the antiviral effect of ephedrine alkaloids-free Ephedra Herb extract (EFE), which is characterized by free of harmful effects of ephedrine alkaloids in Ephedra Herb, on experimental RSV infection. Infection of RSV into A549 cells simultaneously with EFE resulted the significant reduction of RSV RNA, viral protein, and viral titers after the incubation of the cells. We found that RSV attachment to the cell surface was inhibited both in the presence of EFE and when RSV particles were pre-treated with EFE. We also found that EFE specifically interacted with the central conserved domain of RSV G protein by surface plasmon resonance, demonstrating that specific binding of G protein to the cellular receptor was inhibited by EFE. Another mechanism was found in which a higher concentration of EFE inhibited the viral load immediately after the viral entry into host cells, suggesting the inhibition of viral RNA replication. These results demonstrate that EFE worked against RSV infection through multiple antiviral mechanisms, a unique feature of this crude drug extract.

Identification and Characterization of the Stimulator of Interferon Genes (STING) in Chinese Giant Salamander Andrias davidianus

The stimulator of interferon genes (STING) is a central and multifaceted mediator in innate immunity, and plays vital roles in defending against pathogen infection. In this study, we identified and functionally characterized the STING homolog from the Chinese giant salamander Andrias davidianus (AdSTING) for the first time. The open reading frame of AdSTING encodes a 362 amino acid protein with a predicted molecular mass of 41.6 kDa, which shares 31.1–46.7% of its sequence identity with STING homologs in other vertebrates. Structural analysis revealed that AdSTING possesses four predicted transmembrane domains (TMs) at the N-terminal, and a C-terminal domain (CTD) featuring a dimerization domain (DD), a c-di-GMP-binding domain (CBD), and a short C-terminal tail (CTT). Tissue distribution analysis showed that AdSTING mRNA was ubiquitously expressed in all examined tissues, with abundant expression in muscles, intestine, and thymus. During Andrias davidianus ranavirus (ADRV) infection, significant up-regulation of AdSTING expression was observed in the thymus, spleen, and kidney. Upon different stimuli in vitro, the expression of AdSTING was significantly induced by ADRV infection or polyinosin-polycytidylic acid (poly I:C) stimulation, but no obvious changes were observed during lipopolysaccharide (LPS) stimulation. Subcellular localization analysis revealed that AdSTING mainly localized in the cytoplasm in the Chinese giant salamander thymus cell line (GSTC) and co-localized with the endoplasmic reticulum (ER). Luciferase reporter assays confirmed the ability of AdSTING to activate the interferon-stimulated response element (ISRE) and interferon (IFN) promoter. Furthermore, overexpression of AdSTING effectively decreased ADRV infection, as evidenced by the reduction of virus titers and viral gene expression. Collectively, our findings underscore the pivotal role of AdSTING in the antiviral innate immunity of the Chinese giant salamander, offering insights into the functional evolution of STING in amphibians.