Post-treatment Assay Research Articles

Enhanced synergistic antiviral effects of thermally expanded graphite and copper oxide nanosheets in the form of a novel nanocomposite against herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) is responsible for a wide range of human infections, including skin and mucosal ulcers, encephalitis, and keratitis. The gold standard for treating HSV-1 infections is acyclovir. However, the use of this drug is associated with several limitations such as toxic reactions and the development of drug-resistant strains. So, there is an urgent need to discover and develop novel and effective agents against this virus. For the first time, this study aimed to investigate the antiviral effects of the Thermally Expanded Graphite (TEG)-copper oxide (CuO) nanocomposite against HSV-1 and compare results with its constituent components. After microwave (MW)-assisted synthesis of TEG and CuO nanosheets as well as MW-CuO/TEG nanocomposite and characterization of all these nanomaterials, an MTT assay was used to determine their cytotoxicity. The quantitative real-time PCR was then used to investigate the effects of these nanomaterials on viral load. Three-hour incubation of HSV-1 with TEG nanosheets (500 μg/mL), MW-CuO nanosheets (15 μg/mL), and MW-CuO/TEG nanocomposite (35 μg/mL) resulted in a decrease in viral load with an inhibition rate of 31.4 %, 49.2 %, and 74.4 %, respectively. The results from the post-treatment assay also showed that TEG nanosheets (600 μg/mL), MW-CuO nanosheets (15 μg/mL), and MW-CuO/TEG nanocomposite (10 μg/mL) led to a remarkable decrease in viral load with an inhibition rate of 56.9 %, 63 %, and 99.9 %, respectively. The combination of TEG and MW-CuO nanosheets together and the formation of a nanocomposite structure display strong synergy in their ability to inhibit HSV-1 infection. MW-CuO/TEG nanocomposites can be considered a suitable candidate for the treatment of HSV-1 infection.

In Vitro Anti-Rotaviral Activity of Bavachin Isolated from Psoralea corylifolia L. (Fabaceae).

Rotavirus is the main causative agent of viral gastroenteritis among young animals worldwide. Currently, no clinically approved or effective antiviral drugs are available to combat rotavirus infections. Herein, we evaluated the anti-rotaviral activities of extracts and bavachin isolated from Psoralea corylifolia L. (Fabaceae) (P. corylifolia) against the bovine rotavirus G8P[7] and porcine rotavirus G5P[7] in vitro. Two assay strategies were performed: (1) a virucidal assay to reduce viral infectivity by virus neutralization and (2) a post-treatment assay to assess viral replication suppression. The results from the virucidal assay showed that the extracts and bavachin did not exert anti-rotaviral activities. In the follow-up analysis after treatment, bavachin exhibited robust antiviral efficacy, with 50% effective concentration (EC50) values of 10.6 μM (selectivity index [SI] = 2.38) against bovine rotavirus G8P[7] and 13.0 μM (SI = 1.94) against porcine rotavirus G5P[7]. Bavachin strongly suppressed viral RNA synthesis in the early (6 h) and late stages (18 h) after rotaviral infection. These findings strongly suggest that bavachin may have hindered the virions by effectively inhibiting the early stages of the virus replication cycle after rotaviral infection. Furthermore, confocal imaging showed that bavachin suppressed viral protein synthesis, notably that of the rotaviral protein (VP6). These results suggest that bavachin has strong antiviral activity against rotaviruses, inhibits viral replication, and is a candidate natural therapeutic drug targeting rotaviral infection. The utilization of bavachin isolated from P. corylifolia may contribute to decreased mortality rates, lower medication expenses, and enhanced economic viability in domestic farms.

Evaluation of cytotoxicity and antiviral activity of Kyllinga nemoralis leaves and stems methanolic extract

This present study aimed to investigate the potential stems and leaves methanolic extract of Kyllinga nemoralis as an antiviral agent against Herpes Simplex Virus Type 1 (HSV-1). The leaves and stems of K. nemoralis methanolic extract were assessed in the cytotoxicity test using the MTT assays. The plaque reduction assay was evaluated in the post-treatment assay, pre-treatment assay, virucidal assay, time-addition test, and time-removal test. Cytotoxicity screening of stems and leaves of K. nemoralis against Vero cells shows that the cytotoxicity concentration of 50% of cell viability (CC50) values was 0.75 mg/mL, which indicates that K. nemoralis is non-cytotoxic and safe. The antiviral assay provides a Selective Index (SI) value (CC50/EC50) to determine the good SI for antiviral activity. In post-treatment, the SI value for this extract against HSV-1 was 8.33. In pre-treatment, the SI value for this extract was 21.43. In the virucidal test, the SI value for this extract was 24.19 mg/mL. In the time addition assay, the plaque inhibition reached 43% when the extract was added at 2 h.p.i, and it dropped to 15% at 10 h.p.i. In the time removal assay, the plaque inhibition reached 50% after the extract was removed at 2 h.p.i., and it increased to 87% at 24 h.p.i. This study showed that K. nemoralis has potential as a drug used to fight herpes infections.

Antiviral Activity and Molecular Dynamics Simulation of Hops Compounds against Oropouche Virus (Peribunyaviridae).

The Oropouche virus (OROV) is a member of the family Peribunyaviridae (order Bunyavirales) and the cause of a dengue-like febrile illness transmitted mainly by biting midges and mosquitoes. In this study, we aimed to explore acylphloroglucinols and xanthohumol from hops (Humulus lupulus L.) as a promising alternative for antiviral therapies. The evaluation of the inhibitory potential of hops compounds on the viral cycle of OROV was performed through two complementary approaches. The first approach applies cell-based assay post-inoculation experiments to explore the inhibitory potential on the latest steps of the viral cycle, such as genome translation, replication, virion assembly, and virion release from the cells. The second part covers in silico methods evaluating the ability of those compounds to inhibit the activity of the endonuclease domain, which is essential for transcription, binding, and cleaving RNA. In conclusion, the beta acids showed strongest inhibitory potential in post-treatment assay (EC50 = 26.7 µg/mL). Xanthohumol had the highest affinity for OROV endonuclease followed by colupulone and cohumulone. This result contrasts with that observed for docking and MM/PBSA analysis, where cohumulone was found to have a higher affinity. Finally, among the three tested ligands, Lys92 and Arg33 exhibited the highest affinity with the protein.

In vitro antiviral activity of abietane diterpenoids isolated from Torreya nucifera against rotavirus infection

Although vaccines have been developed for rotavirus infections, there is currently no effective therapeutic treatment. Therefore, this study aimed to evaluate the efficacy of 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera (T. nucifera) against bovine G8P[7] and porcine G5P[7] rotaviruses using two different assay strategies: 1) reduction of viral infectivity by neutralizing the virus (virucidal assay) and 2) inhibition of viral replication after infection (post-treatment assay). In the post-treatment assay, compounds 1 and 2 exhibited strong anti-rotavirus activity, with 50% effective concentration values of 24.7 μM (selectivity index; SI= 2.52) and 23.2 μM (SI= 1.75) against bovine G8P[7], 21.1 μM (SI= 2.95) and 22.6 μM (SI= 1.80) against porcine G5P[7], respectively. During viral replication, the two compounds demonstrated stronger inhibition of viral RNA synthesis in the late stages (18 h) than in the early stages (6 h). Compounds 1 and 2 also inhibited the synthesis of viral proteins such as VP6, as determined by immunofluorescence assay. Thus, it appears that compounds 1 and 2 isolated from T. nucifera possess strong antiviral activity against rotaviruses, inhibit viral replication, and may be developed into potential plant-derived therapeutic candidates against rotavirus infection.

Abietane Diterpenoids Isolated from Torreya nucifera Disrupt Replication of Influenza Virus by Blocking the Phosphatidylinositol-3-Kinase (PI3K)-Akt and ERK Signaling Pathway.

Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera exhibited strong anti-influenza activity, with 50% inhibitory concentration values of 13.6 and 18.3 μM against H1N1, 12.8 and 10.8 μM against H9N2, and 29.2 μM (only compound 2) against H3N2 in the post-treatment assay, respectively. During the viral replication stages, the two compounds demonstrated stronger inhibition of viral RNA and protein in the late stages (12-18 h) than in the early stages (3-6 h). Moreover, both compounds inhibited PI3K-Akt signaling, which participates in viral replication during the later stages of infection. The ERK signaling pathway is also related to viral replication and was substantially inhibited by the two compounds. In particular, the inhibition of PI3K-Akt signaling by these compounds inhibited viral replication by sabotaging influenza ribonucleoprotein nucleus-to-cytoplasm export. These data indicate that compounds 1 and 2 could potentially reduce viral RNA and viral protein levels by inhibiting the PI3K-Akt signaling pathway. Our results suggest that abietane diterpenoids isolated from T. nucifera may be potent antiviral candidates for new influenza therapies.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aβ1-42). We found two compounds that were selective for TTR and did not disrupt Aβ-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Anti-Herpes Simplex Virus Type-1 Activity of Kyllinga nemoralis Roots Aqueous Extract

Human Herpesvirus 1 known as herpes simplex virus type 1 (HSV-1) is belonged to the Herpesviridae family, Alphaherpesvirinae subfamily, and Simplexvirus genus. Kyllinga nemoralis, known as whitehead spike sedge, white kyllinga, white globose spike, or poverty grass, is a monocotyledonous flowering graminoid similar to grasses and rushes. Acyclovir (ACV) did not eradicate HSV-1 infection as the drug only focuses on inhibiting the production of new viral genomes by interfering with viral DNA synthesis. This study aimed to identify the cytotoxic concentration (CC50) of K. nemoralis roots aqueous extract and evaluate its antiviral activities against HSV-1. 
 
 Cytotoxicity assay involved the incubation of Vero cells with 10 concentrations of extract ranging between 0.002 mg/mL and 1.000 mg/mL. MTT solution was added and absorbance was measured at 540 nm [1]. The CC50 of extract was determined from the graph of cell viability (%) against extract concentrations. Antiviral assays of post-treatment, pre-treatment and virucidal tests determined the mode of antiviral activity. The 50 % effective concentration (EC50) of extract was derived from the graph of plaque inhibition (%) against concentrations. Selectivity index (SI) was evaluated as the ratio of CC50 to EC50 [2]. 
 
 
 Post-treatment assay: Cells were inoculated with HSV-1 for 2 hours. Extract with (2% DMEM and 1% MCS) was added into cells and incubated for 48 hours. 
 Pre-treatment assay: Cells were treated with extract in 5% DMEM for 24 hours and inoculated with HSV-1 for 1 hour 30 minutes. Then, cells were overlaid with (2% DMEM and 1% MCS) and incubated for 48 hours. 
 Virucidal assay: HSV-1 was incubated with extract for 30 minutes. Then, virus-extract suspension was inoculated into cells and overlaid with 2% DMEM and 1% MCS for 48 hours. 
 
 
 After 48 hours incubation, cells were stained with crystal violet solution and incubated at room temperature for 30 minutes. The plaques were counted and percentage of plaque inhibition (%) was calculated. 
 The CC50 revealed aqueous extract concentration was able to kill at 50% of cell population. Post-treatment assay showed that 0.062 mg/mL of the extract inhibited intracellular viral replication. Pre-treatment study indicated that 0.02 mg/mL of extract produced protective effects towards cells. Finally, virucidal assay demonstrated that 0.222 mg/mL of extract initiated extracellular interaction with virus in inhibiting infection [3] as shown in Table 1. 
 The cytotoxicity study showed that CC50, 0.72 mg/mL was toxic on cells. Therefore, lower concentrations between 0.016 mg/mL to 0.5 mg/mL, that were least or non-toxic to the cells were utilized in the subsequent antiviral screenings [4]. The extract showed good inhibition of intracellular virus replication with an EC50 of 0.062 mg/mL and SI more than 10 which is 11.61. Similarly, in pre-treatment investigation, the extract produced excellent antiviral activity with an EC50 of 0.02 mg/mL and corresponding SI of 36.00. This finding highlight that the extract is most effective when administered as pre-treatment. Antiviral activity exhibited by roots extract probably results from the inhibition of host cell glycoprotein receptors or due to prevention of binding between HSV-1 virions and host cells [5]. Virucidal assay revealed a weak extracellular interaction between HSV-1 and extract. It was supported by the low SI, 3.24 as shown in Figure 1.1. The finding suggests that 30 minutes of virus-extract exposure could be insufficient for roots extract to show an effective inhibition of virus [2]. In conclusion, the present study revealed that K. nemoralis roots aqueous extract inhibits in-vitro productive HSV-1 infection through three different modes that are effective prevention of intracellular viral replication, excellent interaction with host cell surface to inhibit HSV-1 infection and moderate direct destruction the virus particles.

Cytotoxicity and Antiviral Activity of Kyllinga nemoralis Methanolic Extract

Herpes Simplex Virus (HSV) is a double-stranded DNA virus in the Herpesviridae family. The most prevalent in humans is Human Simplex Virus Type 1 (HSV-1). It can reach the brain without causing the symptoms [1]. Currently, the most antiviral drug used is Acyclovir (ACV); nucleotide analogues target DNA polymerase inhibiting viral replication. However, ACV drug-resistant occurs, especially in immunocompromised patients [2]. Therefore, alternate therapy for patients with established resistance was required. Plant extract is commonly used as antiviral agent. Kyllinga nemoralis is known as White Spike Head had antiviral, antibacterial, antibleeding and anti-poisoning properties [3]. It contains antiviral sources such as the highest flavonoid and flavonol concentrations [4].
 
 The objective of this experimental study was to investigate the potential of K. nemoralis methanolic extract as antiviral agent against HSV-1. The root of K. nemoralis methanolic plant extract, Vero Cells and HSV-1 were used in this study. The Cytotoxic Concentration (CC50) of the K. nemoralis methanolic extract against Vero Cells and the antiviral activity of K. nemoralis methanolic extract against HSV-1 by using post-treatment, pre-treatment and virucidal assay were performed in this study.
 
 Results showed that the percentage of cell population versus concentration of root of K. nemoralis methanolic extract (figure 1). The CC50 of this extract was determined at 0.91 mg/mL. This showed that the root of K. nemoralis methanolic extract was considered not toxic to the cells as the CC50 of extract showed more than 4 µg/mL [5].
 
 The results indicate that the root of K. nemoralis methanolic extract was more effective in protecting the Vero cells. In pre-treatment, the SI value of the extract was 8.27. This finding shows that the root of K. nemoralis methanolic extract had a moderate ability to bind to the Vero cells and inhibited the binding of HSV-1 to the cell surface of the Vero cells. In a virucidal test, the root extract towards HSV-1 showed the SI value was 8.125. This data suggests that the root of the extract was moderately inactivates the extracellular component of HSV-1.
 In conclusion, the findings of this study show that K. nemoralis methanolic extract was non-toxic to the Vero cells and has the potential to be an antiviral agent against HSV-1.

Vathasura Kudineer, an Andrographis based polyherbal formulation exhibits immunomodulation and inhibits chikungunya virus (CHIKV) under invitro conditions

Ethnopharmacological relevanceChikungunya disease (CHIKD) is caused by the alphavirus, chikungunya virus (CHIKV) and is characterized by acute fever and joint inflammation; the inflammation continues even after clearance of the virus from the system, persisting for several months to years. Currently, there are no modern medicines/vaccines available for its treatment and use of over-the-counter anti-inflammatory generic medicines to relieve symptoms is generally practiced. In India, Indian traditional medicines hold a lot of promise to treat this infection and are routinely used during outbreaks. Aim of the studyIn the present study, we characterized the phytochemical and physicochemical properties of aqueous and ethanol extracts of the Vathasura Kudineer (VSK), a Andrographis based Siddha polyherbal formulation. Additionally, we evaluated its immunomodulatory and antiviral potential using an in vitro system. Materials and methodsAqueous and ethanolic extracts of VSK were prepared and their physico and phytochemical properties were obtained by biochemical and biophysical assays, HPTLC and FTIR. The aqueous extracts of VSK and several of its ingredients were evaluated for their cytotoxicity in Vero cells and using the maximum non-toxic concentration (MNTC), were processed further for evaluating their ability to inhibit CHIKV infection in Vero cells. We performed the co-treatment assay with ethanol extract of VSK and several of its ingredients to assess the antiviral activity against chikungunya virus on Vero cells and through pre-treatment assay (anti-adhesive effect), co-incubation assay (virucidal effect) and post-treatment assay (post-entry effect) were evaluated. Further, we tested the aqueous extract of VSK along with some of its ingredients for their immunomodulatory properties. We performed antioxidant and anti-inflammatory assays using LPS-simulated RAW 264.7 cells. For antioxidant capacity of extracts, we performed extra-cellular ABTS radical scavenging activity and intra-cellular effects on ROS generation and SOD activity. We assessed the effect on most important inflammatory mediators like Nitric oxide (NO) and Prostaglandin E2 (PGE2) and pro-inflammatory cytokines like interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα). ResultsWe provided the fingerprint of the phytochemicals of both ethanol and aqueous extracts of VSK that can be used for identification. We observed that ethanol extract was able to inhibit CHIKV infection at MNTC with 48 h of treatment on Vero cells. Its ingredient VSKI-As (Anethum sowa) found to be most effective to show virucidal effect while VSKI-Cs (Clerodendrum serratum) and VSKI-Pn (Pipper nigrum) found to be effective in post-entry effect. VSK was able to show ABTS radical scavenging activity, reduce ROS generation, inhibit the inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (IL-1β and TNFα) production in LPS-stimulated RAW 264.7 cells. ConclusionsWe provided the evidence that VSK has both immunomodulatory as well as antiviral potential. It shows virucidal as well as post-entry effects on chikungunya virus. VSK can inhibit pro-inflammatory cytokines, IL-1β and TNFα production by suppressing the inflammatory mediators, NO and PGE2.

Naoxintong capsule limits myocardial infarct expansion by inhibiting platelet activation through the ERK5 pathway

BackgroundIn the clinic, Naoxintong capsule (NXT) has been applied in two level prevention of ischemic disease. However, its mechanism of action requires further study. PurposeThis study investigated whether NXT could affect platelet function and activation under ischemic pathological conditions. Materials and methodsWistar rats were divided into six groups, sham, saline, NXT (250, 500, 1000 mg/kg), and aspirin group (10 mg/kg). For the pre-treatment assays, MI model was established after pre-administration of saline, NXT-L, NXT-M, NXT-H, and aspirin respectively for 14 days, and after surgery, there were no continuous treatments. For the post-treatment assay, rats were orally administered for 3 days after MI. FeCl3-induced thrombosis model was applied to determine the thrombus wet weight. Bleeding time was used to assess the ability of the platelets to develop a hemostatic plug. ResultsNXT decreased infarct size, decreased LDH, CK, and CK-MB values, and improved cardiac function. NXT inhibited platelets activation through reducing CD62P-positive platelets and inhibited infarct expansion by decreasing the number of CD45-positive cells and the amount of MMP9 secreted into the heart tissue. Mechanistically, NXT inhibited platelets activation through decreasing ROS levels, decreasing ERK5 phosphorylation, and increasing RAC1 phosphorylation in MI rats. Pre-treatment with NXT decreased thrombus formation and had normal bleeding times. ConclusionNXT showed obviously preventive effects, which was associated with negative control of platelet activation. The above results provide a basis for clinically expanding application of NXT.

In vitro Evaluation of Anti-Chikungunya Virus Activities of Tualang Honey.

Chikungunya is an infection caused by chikungunya virus (CHIKV). Although chikungunya has affected many countries in recent times, specific treatment or licensed vaccine are nonexistent. In this study the potential antiviral properties of Tualang honey against in vitro CHIKV infection was evaluated. Cytotoxic test was performed using the XTT Cell Viability assay to determine maximum non-toxic dose (MNTD) in Vero cells. Using plaque assay, the potential antiviral activities of Tualang honey at various non-toxic concentrations and treatment regimens were evaluated. Tualang honey demonstrated virucidal effect with maximum inhibition CHIKV observed was 99.71% (p< 0.05). Tualang honey also had a prophylactic property by conferring protection to Vero cells during pre-treatment assay, resulting in up to 98.22% reduction of CHIKV replication under certain treatment regimen. Furthermore, Tualang honey exhibited anti-viral activities, with as much as 94.87% inhibition following post-treatment assay of Tualang honey in CHIKV-infected Vero cells. Additionally, Tualang honey also affected viral entry up to 82.21% after 48 hours of infection. These results suggest that Tualang honey has wide anti-CHIKV activities in Vero cells and exerts its effect through different mechanisms although these need to be further validated in other cells or model of CHIKV infection.

Bio Efficacy Assay of Laccase Isolated and Characterized from Trichoderma viride in Biodegradation of Low Density Polyethylene (LDPE) and Textile Industrial Effluent Dyes

This study is based on the biosynthesis of laccase enzyme from fungi Trichoderma viride and its exploitation in biodegradation of polyethylene using Laccase mediator system (LMS -Laccase + 1-HBT) in Low Density Polyethylene (LDPE) and in biodegradation of textile industrial effluent dyes. In different parts of our lifespan we have seen the numerous types of plastics are getting degraded by various methods, still the average time to completely degrade a plastic bottle is at least 450 years.Similarly industry produces over 3.6 thousand individual textile dyes today and utilizes more than 8000 toxic chemicals in numerous methods for textile manufacture comprising dyeing and printing. As polyethylene and textile industrial effluent dyes are causing severe hazardous effect on environment and health issues in all kind of living organisms, it is necessary to degrade plastics and textile industrial effluent dyes in rapid way. Recently researchers have come up with an idea of degrading plastic and textile industrial effluent dyes with the help of microorganisms and enzymes much faster than normal rate. The Laccase enzyme extracted was tested for its optimum temperature and pH. Lowry’s method is used for protein estimation. A control and sample LDPE was subjected to LMS. The tensile strength and elongation of the sample was less than that of the control after 5 days of treatment. This study showed that laccase together with 1-HBT helps to biodegrade polyethylene. The purified laccase enzyme was used for the pretreatment assay and post treatment assay. The Laccase degrades certain reactive dyes like Congo red, Acid Red, Methylene Blue, Brilliant Blue, Metallic Blue and Black. Thus recommends the application of laccase in textile dye colour removal (bioremediation).

Antiviral Activity of Fritillaria thunbergii Extract against Human Influenza Virus H1N1 (PR8) In Vitro, In Ovo and In Vivo.

Influenza viruses cause respiratory diseases in humans and animals with high morbidity and mortality rates. Conventional anti-influenza drugs are reported to exert side effects and newly emerging viral strains tend to develop resistance to these commonly used agents. Fritillaria thunbergii (FT) is traditionally used as an expectorant for controlling airway inflammatory disorders. Here, we evaluated the therapeutic effects of FT extracts against influenza virus type A (H1N1) infection in vitro, in ovo, and in vivo. In the post-treatment assay, FT extracts showed high CC50 (7,500 µg/ml), indicating low toxicity, and exerted moderate antiviral effects compared to oseltamivir (SI 50.6 vs. 222) in vitro. Antiviral activity tests in ovo revealed strong inhibitory effects of both FT extract and oseltamivir against H1N1 replication in embryonated eggs. Notably, at a treatment concentration of 150 mg/kg, only half the group administered oseltamivir survived whereas the FT group showed 100% survival, clearly demonstrating the low toxicity of FT extracts. Consistent with these findings, FT-administered mice showed a higher survival rate with lower body weight reduction relative to the oseltamivir group upon treatment 24 h after viral infection. Our collective results suggest that FT extracts exert antiviral effects against influenza H1N1 virus without inducing toxicity in vitro, in ovo or in vivo, thereby supporting the potential utility of FT extract as a novel candidate therapeutic drug or supplement against influenza.

Antiviral Properties of Orthosiphon stamineus Aqueous Extract in Herpes Simplex Virus Type 1 Infected Cells

A study was conducted to investigate the antiviral activity of aqueous extracts from Orthosiphon stamineus (OS). Extraction was done using different parts of OS. The whole plant except root (WPOS), leaves (LOS) and flowers (FOS) of OS were extracted using aqueous extraction method. Cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Plaque reduction assays were carried out to evaluate the antiviral activity of OS extract against herpes simplex virus type 1 (HSV-1). These include post-treatment, pre-treatment and virucidal assays. High antiviral activity was observed in post-treatment and virucidal assay with 100% reduction of HSV-1 plaque at 0.39 mg/mL in LOS, FOS and WPOS. In pre-treatment assay, 79%, 84% and 97% plaque reduction using the same concentration was observed in FOS, LOS and WPOS, respectively. In conclusion, this study showed that OS aqueous extract has promising potential to be explored as anti-HSV-1 agent regardless of the mode of treatment.

Antiviral effects and mechanisms of Yinhuapinggan granule against H1N1 influenza virus infection in RAW264.7 cells.

Yinhuapinggan granule (YHPG), a modified prescription based on Ma-Huang-Tang (MHT), is used in traditional Chinese medicine (TCM) to treat influenza, cough, and viral pneumonia. In this study, we investigated the antiviral effects of YHPG by means of pre-, post-, and co-treatment, and its underlying mechanisms on regulating the levels of inflammatory-related cytokines, modulating the mRNA expressions of interferon-stimulated genes in influenza virus-infected murine macrophage cells (RAW264.7), and evaluating the protein expressions of key effectors in the Type I IFN and pattern recognition receptor (PRRs) signaling pathways. The results showed that YHPG markedly inhibited influenza virus (IFV) replication in pre-, post- and co-treatment assay, especially in post-treatment assay. Antiviral mechanisms studies revealed that YHPG (500 and 250μg/mL) significantly up-regulated levels of IFN-β, IFN-stimulated genes (Mx-1, ISG-15 and ISG-56) compared with the IFV control group, while the levels of IL-6 and TNF-α were significantly down-regulated. Furthermore, western blot analysis results revealed that the protein expressions of the phosphorylated forms of TBK1, IRF3, ERK1/2, P38 MAPK and NF-κB p65 were significantly down-regulated in RAW264.7 cells with the YHPG (500 and 250μg/mL) treatment, while the expression of the phosphorylated form of STAT1 was significantly enhanced. Based on these results, YHPG had antiviral effects in IFV-infected RAW264.7 cells, which might be associated with regulation of the inflammatory cytokines production, evaluation of the levels of IFN-stimulated genes, and modulation of the protein expressions of key effectors in the Type I IFN and PRRs signaling pathways.

Ethanolic Extract of Melia Fructus Has Anti-influenza A Virus Activity by Affecting Viral Entry and Viral RNA Polymerase.

Meliae Fructus (MF) is the dried ripe fruit of Melia toosendan Siebold et Zuccarini, Meliaceae family. MF is widely used in traditional medicine to treat inflammation and helminthic infection and has anti-bacterial, anti-oxidant, anti-cancer, anti-inflammatory, and analgesic activities. However, potential anti-influenza properties of MF have yet to be investigated. We determined whether an ethanolic extract of MF (EMF) has anti-viral activity via an EMF pre-, co-, and post-treatment assay, using the Influenza A/PR/8/34 and H3N2 virus on Madin-Darby canine kidney (MDCK) cells. The EMF had anti-influenza virus activity in pre- and co-treated cells in a dose-dependent manner, but not in post-treated cell. EMF inhibited the activity of hemagglutinin (HA) and neuraminidase (NA) of influenza virus. EMF inhibited viral HA, nucleoprotein (NP), matrix protein 2 (M2), non-structural protein 1 (NS1), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) mRNA synthesis at 5 h post infection (hpi), however, the levels of PA, PB1, and PB2 mRNA were increased in pre- and co-EMF treated cells compared with control virus-infected and EMF post-treated cells at 18 hpi. The level of M2 protein expression was also decreased upon pre- and co-treatment with EMF. The PA protein was accumulated and localized in not only the nucleus but also the cytoplasm of virus-infected MDCK cells at 18 hpi. Pre-EMF treatment inhibited the expression of pAKT, which is induced by influenza virus infection, at the stage of virus entry. We also found that treatment of EMF up-regulated the antiviral protein Mx1, which may play a partial role in inhibiting influenza virus infection in pre- and co-EMF treated MDCK cells. In summary, these results strongly suggested that an ethanolic extract of Meliae Fructus inhibited influenza A virus infection by affecting viral entry, PA proteins of the RNA polymerase complex, and Mx1 induction and may be a potential and novel anti-influenza agent.

Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto).

Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2h, followed by challenges with LPS for 3 and 18h. Post-treatment assay was done by initial treatment of the cells with LPS for 18h, followed by the spores for 3 and 6h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.

CtDNA dynamics: a novel indicator to track resistance in metastatic breast cancer treated with anti-HER2 therapy.

BackgroundMost studies utilizing circulating tumor DNA (ctDNA) to monitor disease interrogated only one or a few genes and failed to develop workable criteria to inform clinical practice. We evaluated the feasibility of detecting resistance to anti-HER2 therapy by serial gene-panel ctDNA sequencing.ResultsPrimary therapeutic resistance was identified in 6 out of 14 patients with events of progressive disease. For this subset comparison of pre- and post-treatment ctDNA assay results revealed that HER2 amplification concurred with disease progression (4/6, 66.7%). Mutations in TP53 (3/6, 50.0%) and genes implicated in the PI3K/mTOR pathway (3/6, 50.0%) were also dominant markers of resistance. Together, resistance to HER2 blockade should be indicated during treatment if any of the following situations applies: 1) recurrence or persistence of HER2 amplification in the blood; 2) emergence or ≥20% increase in the fraction of mutations in any of these resistance-related genes including TP53/PIK3CA/MTOR/PTEN. Compared with CT scans, dynamic ctDNA profiling utilizing pre-defined criteria was sensitive in identifying drug resistance (sensitivity 85.7%, specificity 55.0%), with a concordance rate up to 82.1%. Besides, the ctDNA criteria had a discriminating role in the prognosis of HER2-positive metastatic breast cancer.Methods52 plasma samples were prospectively collected from 18 patients with HER2-positive metastatic breast cancer who were treated with an oral anti-HER1/HER2 tyrosine kinase inhibitor (ClinicalTrials.gov NCT01937689). ctDNA was assayed by gene-panel target-capture next-generation sequencing.ConclusionsLongitudinal gene-panel ctDNA sequencing could be exploited to determine resistance and guide the precise administration of anti-HER2 targeted therapy in the metastatic setting.

Assessment of Anti-Influenza Activity and Hemagglutination Inhibition of Plumbago indica and Allium sativum Extracts.

Background:Human influenza is a seasonal disease associated with significant morbidity and mortality. Anti-flu ayurvedic/herbal medicines have played a significant role in fighting the virus pandemic. Plumbagin and allicin are commonly used ingredients in many therapeutic remedies, either alone or in conjunction with other natural substances. Evidence suggests that these extracts are associated with a variety of pharmacological activities.Objective:To evaluate anti-influenza activity from Plumbago indica and Allium sativum extract against Influenza A (H1N1)pdm09.Materials and Methods:Different extraction procedures were used to isolate the active ingredient in the solvent system, and quantitative HPLTC confirms the presence of plumbagin and allicin. The cytotoxicity was carried out on Madin-Darby Canine kidney cells, and the 50% cytotoxic concentration (CC50) values were below 20 mg/mL for both plant extracts. To assess the anti-influenza activity, two assays were employed, simultaneous and posttreatment assay.Results:A. sativum methanolic and ethanolic extracts showed only 14% reduction in hemagglutination in contrast to P. indica which exhibited 100% reduction in both simultaneous and posttreatment assay at concentrations of 10 mg/mL, 5 mg/mL, and 1 mg/mL.Conclusions:Our results suggest that P. indica extracts are good candidates for anti-influenza therapy and should be used in medical treatment after further research.SUMMARY The search for natural antiviral compounds from plants is a promising approach in the development of new therapeutic agents. In the past century, several scientific efforts have been directed toward identifying phytochemicals capable of inhibiting virus. Knowledge of ethnopharmacology can lead to new bioactive plant compounds suitable for drug discovery and development. Macromolecular docking studies provides most detailed possible view of drug-receptor interaction where the structure of drug is designed based on its fit to three dimensional structures of receptor site rather than by analogy to other active structures or random leads. Our previous studies indicate that Allicin sand Plumbagin could be used as the potent multi drug targets against the Neuraminidase, Hemagglutinin and M2 protein channel of influenza A (H1N1) pdm09. This in-vittro study has shown that P. indica L. and A. sativum extracts can inhibit influenza A (H1N1)pdm09 virus by inhibiting viral nucleoprotein synthesis and polymerase activity.