RESUMEN
Tick-borne encephalitis outbreaks have been reported in Europe after consumption of raw milk products from infected animals. While molecular methods are commonly used in viral foodborne outbreak investigations due to their sensitivity, specificity and rapidity, there are very few methods to detect infectious tick-borne encephalitis virus (TBEV) in milk products for routine use/analyses. To address this gap, we developed a cell culture-based method to detect infectious TBEV in artificially contaminated raw goat milk and raw goat cheese, and evaluated the sensitivity of TBEV infectivity assays. Raw goat milk samples were spiked with TBEV to achieve inoculation levels ranging from 106 to 100 TCID50/mL, and Faisselle and Tomme cheese samples were spiked so their TBEV concentrations ranged from 9.28 × 105 to 9.28 × 101 TCID50 per 2.5g. To detect infectious TBEV, Vero cells were infected by raw goat milk. For cheese samples, after homogenisation and membrane filtration, Vero cells were infected with samples adsorbed on the filter (method A) or with samples eluted from the filter (method B). After 5 days, cytopathic effects (CPEs) were observed and TBEV replication in Vero cells was confirmed by an increase in the number of genome copies/mL that were detected in cell supernatant. Infected Vero cells exhibited CPEs for both milk and cheese samples. Infectious TBEV was detected to 103 TCID50/mL in raw milk samples and to 9.28 × 101 TCID50 from Faisselle samples using both methods A and B. For Tomme samples, method A was able to detect TBEV to 9.28 × 102 TCID50/2.5g and method B to 9.28 × 103 TCID50/2.5g. The number of positive samples detected was slightly higher with method A than with method B. To conclude, this qualitative cell culture-based method can detect infectious TBEV artificially inoculated into raw milk and cheese; it should be further evaluated during foodborne outbreak investigations to detect infectious TBEV from naturally contaminated milk and cheese.
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Queso , Virus de la Encefalitis Transmitidos por Garrapatas , Contaminación de Alimentos , Cabras , Leche , Animales , Leche/virología , Virus de la Encefalitis Transmitidos por Garrapatas/aislamiento & purificación , Células Vero , Chlorocebus aethiops , Queso/virología , Contaminación de Alimentos/análisis , Encefalitis Transmitida por Garrapatas/virología , Técnicas de Cultivo de CélulaRESUMEN
Enterovirus 71 (EV71) is recognized as a major causative agent of hand, foot, and mouth disease (HFMD), posing a significant global public health concern due to its widespread impact and resulting in a major public health issue worldwide. Despite its prevalence, current clinical therapy lacks effective antiviral agents. Fucosylated chondroitin sulfates (FCS) derived from sea cucumber exhibits a range of biological activities including potent antiviral effects. This study provides compelling evidence of the potent antiviral efficacy of FCS against EV71. To further elucidate the impact of structural variations on the anti-EV71 activity, native FCSs with diverse sulfation patterns and a varity of FCS derivatives were prepared and analyzed. Notably, this study presents the detailed structural characterization of FCSs from the sea cucumbers Holothuria scabra Jaege and Holothuria fuscopunctata. Analysis of the structure-activity relationships revealed that molecular weight, sulfated fucose branches, and sulfation pattern were all crucial factors contributing to the potent inhibitory effects of FCS against EV71. Interestingly, molecular weight emerged as the most significant structural determinant of the antiviral potency. These findings suggest the promising potential of utilizing FCS as an innovative EV71 entry inhibitor for the treatment of HFMD.
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Antivirales , Sulfatos de Condroitina , Enterovirus Humano A , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacología , Antivirales/farmacología , Antivirales/química , Animales , Enterovirus Humano A/efectos de los fármacos , Relación Estructura-Actividad , Humanos , Pepinos de Mar/química , Chlorocebus aethiops , Peso Molecular , Células VeroRESUMEN
Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic disease affecting camels and humans. The live attenuated vaccine represents a candidate human vaccine because it can induce strong immune responses in immunized hosts. The attenuated vaccine strain of the highly pathogenic virus can also be used to produce a cell-based vaccine in the BSL2 GMP facility. In this study, we evaluated the reversion potential of pathogenicity to pathogenic wild-type virus to ensure the safety of the live attenuated vaccine strain. We passaged our previously developed cold-adapted live attenuated MERS-CoV vaccine strain at 22 °C (EMC2012-CA22°C) in Vero cells at 37 °C as often as 15 times to determine the potential of pathogenicity reversion in hDPP4 (human dipeptidyl peptidase 4)-transgenic mice, K18-hDPP4. The serial passage of EMC2012-CA22°C in Vero cells at 37 °C up to 15 times did not result in pathogenicity reversion to wild-type MERS-CoV. In K18-hDPP4 mice infected with this virus, no weight loss or mortality was observed, and no virus was detected in tissues such as the lung, kidney, brain, and nasal turbinate. In addition, mice immunized with this virus produced a robust neutralizing antibody response and were fully protected from lethal challenge with wild-type MERS-CoV. The cold-adapted attenuated MERS-CoV vaccine strain (EMC2012-CA22°C) was not reverted to wild-type pathogenic virus after 15 passages in Vero cells at 37 °C.
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Frío , Coronavirus del Síndrome Respiratorio de Oriente Medio , Vacunas Atenuadas , Vacunas Virales , Animales , Chlorocebus aethiops , Células Vero , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/inmunología , Vacunas Atenuadas/inmunología , Ratones , Vacunas Virales/inmunología , Vacunas Virales/genética , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/inmunología , Ratones Transgénicos , Humanos , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Pase Seriado , Dipeptidil Peptidasa 4/genética , FemeninoRESUMEN
Chikungunya fever is a mosquito-borne disease caused by Chikungunya virus (CHIKV). Treatment of CHIKV infections is currently supportive and does not limit viral replication or symptoms of persistent chronic arthritis. Although there are multiple compounds reported as antivirals active against CHIKV in vitro, there are still no effective and safe antivirals. Thus, active research aims at the identification of new chemical structures with antiviral activity. Here, we report the screen of the Pandemic Response Box library of small molecules against a fully infectious CHIKV reporter virus. Our screening approach successfully identified previously reported CHIKV antiviral compounds within this library and further expanded potentially active hits, supporting the use of reporter-virus-based assays in high-throughput screening format as a reliable tool for antiviral drug discovery. Four molecules were identified as potential drug candidates against CHIKV: MMV1634402 (Brilacidin) and MMV102270 (Diphyllin), which were previously shown to present broad-spectrum antiviral activities, in addition to MMV1578574 (Eravacycline), and the antifungal MMV689401 (Fluopicolide), for which their antiviral potential is uncovered here.
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Antivirales , Fiebre Chikungunya , Virus Chikungunya , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas , Virus Chikungunya/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Fiebre Chikungunya/tratamiento farmacológico , Fiebre Chikungunya/virología , Humanos , Animales , Bibliotecas de Moléculas Pequeñas/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Evaluación Preclínica de Medicamentos , Replicación Viral/efectos de los fármacos , Descubrimiento de Drogas , Chlorocebus aethiops , Células VeroRESUMEN
Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.
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Animales Recién Nacidos , Infecciones por Coronavirus , Diarrea , Filogenia , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/patogenicidad , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/clasificación , Porcinos , Enfermedades de los Porcinos/virología , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Virulencia , Diarrea/virología , Diarrea/veterinaria , Glicoproteína de la Espiga del Coronavirus/genética , Genoma Viral , Mutagénesis Insercional , China , Células VeroRESUMEN
BACKGROUND: In early 2020, COVID-19 pandemic has mobilized researchers in finding new remedies including repurposing of medicinal plant products focusing on direct-acting antiviral and host-directed therapies. In this study, we performed an in vitro investigation on the standardized Marantodes pumilum extract (SKF7®) focusing on anti-SARS-CoV-2 and anti-inflammatory activities. METHODS: Anti-SARS-CoV-2 potential of the SKF7® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays. RESULTS: The SKF7® inhibited wild-type Wuhan (EC50 of 21.99 µg/mL) and omicron (EC50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC50 value of 6.31 µg/mL). The SKF7® prominently inhibited 3CL protease activity. The SKF7® inhibited the LPS induced-TLR4 response with the EC50 of 16.19 µg/mL. CONCLUSIONS: In conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7®. Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7® in affecting the COVID-19 pathogenesis are warranted.
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Antivirales , Extractos Vegetales , SARS-CoV-2 , Animales , Humanos , Antivirales/farmacología , SARS-CoV-2/efectos de los fármacos , Células Vero , Chlorocebus aethiops , Extractos Vegetales/farmacología , Células A549 , Plantas Medicinales/química , Tratamiento Farmacológico de COVID-19 , Antiinflamatorios/farmacología , Malasia , COVID-19 , Proteasas 3C de CoronavirusRESUMEN
BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) is a severe public health problem in Jiangxi province, China. Previous studies reported genetic variants of Orthohantavirus hantanense (Hantaan virus, HTNV) in rodents in this area. However, the relationship between HTNV variants and human infection needs to be confirmed. This study aimed to identify the HTNV variants in patients and to understand the clinical characteristics of HFRS caused by these variants. METHODS: Samples were collected from hospitalized suspected cases of HFRS during the acute phase. HFRS cases were confirmed using quantitative real-time RT-PCR. Peripheral blood mononuclear cells (PBMC) from patients with HFRS were inoculated into Vero-E6 cells for viral isolation. The genomic sequences of HTNV from patients were obtained by amplicon-based next-generation sequencing. A retrospective analysis was conducted on the clinical characteristics of the patients. RESULTS: HTNV RNA was detected in 53 of 183 suspected HFRS patients. Thirteen HTNVs were isolated from 32 PBMCs of HFRS cases. Whole genome sequences of 14 HTNVs were obtained, including 13 isolates in cell culture from 13 patients, and one from plasma of the fatal case which was not isolated successfully in cell culture. Genetic analysis revealed that the HTNV sequence from the 14 patients showed significant variations in nucleotide and amino acid to the HTNV strains found in other areas. Fever (100%, 53/53), thrombocytopenia (100%, 53/53), increased serum aspartate aminotransferase (100%, 53/53), and increased lactate dehydrogenase (96.2%, 51/53) were the most common characteristics. Severe acute kidney injury was observed in 13.2% (7/53) of cases. Clinical symptoms, such as pain, petechiae, and gastrointestinal or respiratory symptoms were uncommon. CONCLUSION: The HTNV genetic variants cause human infections in Jiangxi. The clinical symptoms of HFRS caused by the HTNV genetic variant during the acute phase are atypical. In addition to renal dysfunction, attention should be paid to the common liver injuries caused by these genetic variants.
Asunto(s)
Variación Genética , Fiebre Hemorrágica con Síndrome Renal , Humanos , Fiebre Hemorrágica con Síndrome Renal/virología , Fiebre Hemorrágica con Síndrome Renal/epidemiología , China/epidemiología , Masculino , Femenino , Adulto , Persona de Mediana Edad , Chlorocebus aethiops , Animales , Células Vero , Filogenia , ARN Viral/genética , Adulto Joven , Estudios Retrospectivos , Leucocitos Mononucleares/virología , Anciano , Genoma Viral , Orthohantavirus/genética , Orthohantavirus/aislamiento & purificación , Orthohantavirus/clasificación , Adolescente , Virus Hantaan/genética , Virus Hantaan/aislamiento & purificación , Virus Hantaan/clasificaciónRESUMEN
Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have shown anti-inflammatory potential in multiple inflammatory diseases. In the March 2022 issue of the Journal of Extracellular Vesicles, it was shown that EVs from human MSCs can suppress severe acute respiratory distress syndrome, coronavirus 2 (SARS-CoV-2) replication and can mitigate the production and release of infectious virions. We therefore hypothesized that MSC-EVs have an anti-viral effect in SARS-CoV-2 infection in vivo. We extended this question to ask whether also other respiratory viral infections could be treated by MSC-EVs. Adipose stem cell-derived EVs (ASC-EVs) were isolated using tangential flow filtration from conditioned media obtained from a multi-flask cell culture system. The effects of the ASC-EVs were tested in Vero E6 cells in vitro. ASC-EVs were also given i.v. to SARS-CoV-2 infected Syrian Hamsters, and H1N1 influenza virus infected mice. The ASC-EVs attenuated SARS-CoV-2 virus replication in Vero E6 cells and reduced body weight and signs of lung injury in infected Syrian hamsters. Furthermore, ASC-EVs increased the survival rate of influenza A-infected mice and attenuated signs of lung injury. In summary, this study suggests that ASC-EVs can have beneficial therapeutic effects in models of virus-infection-associated acute lung injury and may potentially be developed to treat lung injury in humans.
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Lesión Pulmonar Aguda , COVID-19 , Vesículas Extracelulares , Subtipo H1N1 del Virus de la Influenza A , Células Madre Mesenquimatosas , SARS-CoV-2 , Animales , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , SARS-CoV-2/fisiología , COVID-19/terapia , Lesión Pulmonar Aguda/terapia , Lesión Pulmonar Aguda/virología , Ratones , Células Vero , Humanos , Chlorocebus aethiops , Infecciones por Orthomyxoviridae/terapia , Replicación Viral , Mesocricetus , Modelos Animales de Enfermedad , Masculino , Gripe Humana/terapia , FemeninoRESUMEN
Rift Valley Fever Virus (RVFV) is an arbovirus that circulates among animals and can be transmitted to humans. Mosquitoes are the primary vectors that allow RVFV to spread vertically and horizontally. Egypt was exposed to frequent outbreaks with devastating economic consequences. RVFV has a high incidence of worldwide dissemination and no specific vaccine or therapy. Linear Human Cathelicidin (LL-37), is a natural antimicrobial peptide with antiviral activity against numerous viruses. In addition to immunomodulatory effects, LL-37 directly influences viral encapsulation. This study aimed to evaluate the antiviral activity of LL-37 against RVFV in vitro. The post-entry and pre-incubation of LL-37 within Vero cells were assessed in the absence and presence of RVFV. LL-37 activity was assessed using a TCID50 endpoint test, qRT-PCR, and a western blot. When genomic RVFV was quantified, it resulted in a 48% direct inactivation of the viral envelope and a 36% reduction when the virus was pre-incubated with LL-37 before infection. LL-37 decreased viral infection by 75% and protected Vero cells against RVFV infection by 47% at a 1.25 µg/ml dosage. These findings imply that LL-37 exerts antiviral efficacy against RVFV by restricting virus entrance through direct disruption of the virus envelope and indirectly by triggering an immunological response. The effect of LL-37 is time-dependent. As a result, LL-37 may provide rapid and affordable therapies for RVFV infection in Egypt, both during outbreaks and as a preventive strategy.
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Péptidos Catiónicos Antimicrobianos , Antivirales , Catelicidinas , Virus de la Fiebre del Valle del Rift , Chlorocebus aethiops , Células Vero , Animales , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Antivirales/uso terapéutico , Egipto , Humanos , Fiebre del Valle del Rift/tratamiento farmacológico , Fiebre del Valle del Rift/prevención & controlRESUMEN
This study proposes an affordable plasma device that utilizes a parallel-plate dielectric barrier discharge geometry with a metallic mesh electrode, featuring a straightforward 3D-printed design. Powered by a high-voltage supply adapted from a cosmetic plasma device, it operates on atmospheric air, eliminating the need for gas flux. Surface modification of polyethylene treated with this device was characterized and showed that the elemental composition after 15 min of plasma treatment decreased the amount of C to ~80 at% due to the insertion of O (~15 at%). Tested against Candida albicans and Staphylococcus aureus, the device achieved a reduction of over 99% in microbial load with exposure times ranging from 1 to 10 min. Simultaneously, the Vero cell viability remained consistently high, namely between 91% and 96% across exposure times. These results highlight this device's potential for the surface modification of materials and various infection-related applications, boasting affordability and facilitating effective antimicrobial interventions.
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Candida albicans , Gases em Plasma , Staphylococcus aureus , Propiedades de Superficie , Candida albicans/efectos de los fármacos , Gases em Plasma/química , Gases em Plasma/farmacología , Staphylococcus aureus/efectos de los fármacos , Animales , Células Vero , Chlorocebus aethiops , Viabilidad Microbiana/efectos de los fármacos , Polímeros/químicaRESUMEN
We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2WK521WT in VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2WK521E166V), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of Mpro, and compromised the enzymatic activity; the Ki values of recombinant MproE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to MproE166V than nirmatrelvir. SARS-CoV-2WK521WT selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and better replicated in the presence of 5h, than SARS-CoV-2WK521WT. However, no significant enzymatic or structural changes in MproA191T were observed. The replicability of SARS-CoV-2WK521E166V proved to be compromised compared to SARS-CoV-2WK521WT but predominated over SARS-CoV-2WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2's drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.
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Proteasas 3C de Coronavirus , Farmacorresistencia Viral , SARS-CoV-2 , SARS-CoV-2/efectos de los fármacos , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/química , Humanos , Chlorocebus aethiops , Animales , Farmacorresistencia Viral/genética , Células Vero , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , COVID-19/virología , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Cristalografía por Rayos X , Lactamas , Leucina , Nitrilos , ProlinaRESUMEN
Usutu (USUV), West Nile (WNV), and Zika virus (ZIKV) are neurotropic arthropod-borne viruses (arboviruses) that cause severe neurological disease in humans. However, USUV-associated neurological disease is rare, suggesting a block in entry to or infection of the brain. We determined the replication, cell tropism and neurovirulence of these arboviruses in human brain tissue using a well-characterized human fetal organotypic brain slice culture model. Furthermore, we assessed the efficacy of interferon-ß and 2'C-methyl-cytidine, a synthetic nucleoside analogue, in restricting viral replication. All three arboviruses replicated within the brain slices, with WNV reaching the highest titers, and all primarily infected neuronal cells. USUV- and WNV-infected cells exhibited a shrunken morphology, not associated with detectable cell death. Pre-treatment with interferon-ß inhibited replication of all arboviruses, while 2'C-methyl-cytidine reduced only USUV and ZIKV titers. Collectively, USUV can infect human brain tissue, showing similarities in tropism and neurovirulence as WNV and ZIKV. These data suggest that a blockade to infection of the human brain may not be the explanation for the low clinical incidence of USUV-associated neurological disease. However, USUV replicated more slowly and to lower titers than WNV, which could help to explain the reduced severity of neurological disease resulting from USUV infection.
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Encéfalo , Flavivirus , Replicación Viral , Virus del Nilo Occidental , Virus Zika , Humanos , Virus del Nilo Occidental/patogenicidad , Virus del Nilo Occidental/fisiología , Virus Zika/patogenicidad , Virus Zika/fisiología , Encéfalo/virología , Replicación Viral/efectos de los fármacos , Flavivirus/patogenicidad , Flavivirus/fisiología , Flavivirus/efectos de los fármacos , Feto/virología , Interferón beta/farmacología , Animales , Virulencia , Técnicas de Cultivo de Órganos , Tropismo Viral , Neuronas/virología , Infecciones por Flavivirus/virología , Infección por el Virus Zika/virología , Chlorocebus aethiops , Células VeroRESUMEN
Background: Toxoplasma gondii (T. gondii) is a widespread, zoonotic protozoan intracellular parasite with a complex life cycle, which can cause toxoplasmosis, a potentially serious disease. During the invasion process, T. gondii proteins first bind to the relevant host cell receptors, such as glycosaminoglycan molecule (GAG-binding motif), which is one of the main receptors for parasites or virus to infect host cells. However, research on TGME49_216510 (T. gondii Trx21), a protein from Toxoplasma gondii, is limited. Methods: Bioinformatics analysis of the Trx21 protein was performed firstly. And specific primers were then designed using the conserved domain and GAG-binding motif to amplify, express, and purify a fragment of the Trx21 protein. The purified Trx21-GST protein was used for antioxidant and cell adhesion experiments. Simultaneously, mice were immunized with Trx21-His to generate specific polyclonal antibodies for subcellular localization analysis. Results: The Trx21 protein, consisting of 774 amino acids, included a transmembrane region, three GAG-binding motifs, and a Thioredoxin-like domain. The recombinant Trx21-His protein had a molecular mass of about 31 kDa, while the Trx21-GST protein had a molecular mass of about 55 kDa, which was analyzed by SDS-PAGE and Western blot. Subcellular localization analysis by IFA revealed that Trx21 is predominantly distributed in the cytoplasm of T. gondii. Furthermore, Trx21 exhibited a protective effect on supercoiled DNA against metal-catalyzed oxidation (MCO) and demonstrated adhesion abilities to Vero cells. Conclusions: These results indicate that Trx21 plays an important role in host cell interaction and oxidative damage.
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Adhesión Celular , Proteínas Protozoarias , Tiorredoxinas , Toxoplasma , Toxoplasma/metabolismo , Toxoplasma/genética , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Animales , Ratones , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Antioxidantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Biología Computacional , Células Vero , Chlorocebus aethiops , Toxoplasmosis/parasitología , HumanosRESUMEN
SARS-CoV-2 is a highly pathogenic virus responsible for the COVID-19 disease. It belongs to the Coronaviridae family, characterized by a phospholipid envelope, which is crucial for viral entry and replication in host cells. Hypericin, a lipophilic, naturally occurring photosensitizer, was reported to effectively inactivate enveloped viruses, including SARS-CoV-2, upon light irradiation. In addition to its photodynamic activity, Hyp was found to exert an antiviral action also in the dark. This study explores the mechanical properties of heat-inactivated SARS-CoV-2 viral particles using Atomic Force Microscopy (AFM). Results reveal a flexible structure under external stress, potentially contributing to the virus pathogenicity. Although the fixation protocol causes damage to some particles, correlation with fluorescence demonstrates colocalization of partially degraded virions with their genome. The impact of hypericin on the mechanical properties of the virus was assessed and found particularly relevant in dark conditions. These preliminary results suggest that hypericin can affect the mechanical properties of the viral envelope, an effect that warrants further investigation in the context of antiviral therapies.
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Antracenos , Microscopía de Fuerza Atómica , Perileno , Fármacos Fotosensibilizantes , SARS-CoV-2 , Perileno/análogos & derivados , Perileno/farmacología , Perileno/química , Antracenos/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Virión/efectos de los fármacos , Humanos , Antivirales/farmacología , Antivirales/química , COVID-19/virología , Chlorocebus aethiops , Células Vero , Tratamiento Farmacológico de COVID-19 , AnimalesRESUMEN
A group of Niclosamide-linked isatin hybrids (Xo, X1, and X2) was created and examined using IR, 1HNMR, 13C NMR, and mass spectrometry. These hybrids' cytotoxicity, antioxidant, cell cycle analysis, and apoptosis-inducing capabilities were identified. Using the SRB assay, their cytotoxicity against the human HCT-116, MCF-7, and HEPG-2 cancer cell lines, as well as VERO (African Green Monkey Kidney), was evaluated. Compound X1 was the most effective compound. In HCT-116 cells, compound X1 produced cell cycle arrest in the G1 phase, promoted cell death, and induced apoptosis through mitochondrial membrane potential breakdown in comparison to niclosamide and the control. Niclosamide and compound X1 reduced reactive oxygen species generation and modulated the gene expression of BAX, Bcl-2, Bcl-xL, and PAR-4 in comparison to the control. Docking modeling indicated their probable binding modalities with the XIAP BIR2 domain, which selectively binds caspase-3/7, and highlighted their structural drivers of activity for further optimization investigations. Computational in silico modeling of the new hybrids revealed that they presented acceptable physicochemical values as well as drug-like characteristics, which may introduce them as drug-like candidates. The study proved that compound X1 might be a novel candidate for the development of anticancer agents as it presents antiproliferative activity mediated by apoptosis.
Asunto(s)
Antineoplásicos , Antioxidantes , Apoptosis , Proliferación Celular , Isatina , Simulación del Acoplamiento Molecular , Niclosamida , Humanos , Apoptosis/efectos de los fármacos , Isatina/farmacología , Isatina/química , Proliferación Celular/efectos de los fármacos , Antioxidantes/farmacología , Antioxidantes/química , Animales , Chlorocebus aethiops , Antineoplásicos/farmacología , Antineoplásicos/química , Células Vero , Niclosamida/farmacología , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Células HCT116 , Línea Celular Tumoral , Células MCF-7 , Células Hep G2RESUMEN
Dengue, caused by the dengue virus (DENV), is a global health threat transmitted by Aedes mosquitoes, resulting in 400 million cases annually. The disease ranges from mild to severe, with potential progression to hemorrhagic dengue. Current research is focused on natural antivirals due to challenges in vector control. This study evaluates the antiviral potential of peptides derived from the microalgae Phaeodactylum tricornutum, known for its bioactive compounds. Microalgae were cultivated under controlled conditions, followed by protein extraction and hydrolysis to produce four peptide fractions. These fractions were assessed for cytotoxicity via the MTT assay and antiviral activity against DENV serotype 2 using flow cytometry and plaque formation assays. The 10-30 kDa peptide fraction, at 150 and 300 µg/mL concentrations, demonstrated no cytotoxicity and significantly reduced the percentage of infected cells and viral titers. These findings suggest that peptides derived from Phaeodactylum tricornutum exhibit promising antiviral activity against dengue virus serotype 2, potentially contributing to developing new therapeutic approaches for dengue.
Asunto(s)
Antivirales , Virus del Dengue , Microalgas , Virus del Dengue/efectos de los fármacos , Antivirales/farmacología , Antivirales/química , Animales , Hidrolisados de Proteína/farmacología , Hidrolisados de Proteína/química , Dengue/tratamiento farmacológico , Dengue/virología , Péptidos/farmacología , Péptidos/química , Serogrupo , Chlorocebus aethiops , Humanos , Aedes/efectos de los fármacos , Células VeroRESUMEN
The COVID-19 pandemic has spread throughout the whole globe, so it is imperative that all available resources be used to treat this scourge. In reality, the development of new pharmaceuticals has mostly benefited from natural products. The widespread medicinal usage of species in the Asteraceae family is extensively researched. In this study, compounds isolated from methanolic extract of Artemisia monosperma Delile, a wild plant whose grows in Egypt's Sinai Peninsula. Three compounds, stigmasterol 3-O-ß-D-glucopyranoside 1, rhamnetin 3, and padmatin 6, were first isolated from this species. In addition, five previously reported compounds, arcapillin 2, jaceosidin 4, hispidulin 5, 7-O-methyleriodictyol 7, and eupatilin 8, were isolated. Applying molecular modelling simulations revealed two compounds, arcapillin 2 and rhamnetin 3 with the best docking interactions and energies within SARS-CoV-2 Mpro-binding site (-6.16, and -6.70 kcal mol-1, respectively). The top-docked compounds (2-3) were further evaluated for inhibitory concentrations (IC50), and half-maximal cytotoxicity (CC50) of both SARS-CoV-2 and MERS-CoV. Interestingly, arcapillin showed high antiviral activity towards SARS-CoV-2 and MERS-CoV, with IC50 values of 190.8 µg mL-1 and 16.58 µg mL-1, respectively. These findings may hold promise for further preclinical and clinical research, particularly on arcapillin itself or in collaboration with other drugs for COVID-19 treatment.
Asunto(s)
Antivirales , Artemisia , Coronavirus del Síndrome Respiratorio de Oriente Medio , Simulación del Acoplamiento Molecular , SARS-CoV-2 , Artemisia/química , Antivirales/farmacología , Antivirales/química , Antivirales/aislamiento & purificación , SARS-CoV-2/efectos de los fármacos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Humanos , Chlorocebus aethiops , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Proteasas 3C de Coronavirus/química , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/aislamiento & purificación , Células Vero , Modelos MolecularesRESUMEN
The mycobacterial F-ATP synthase is responsible for the optimal growth, metabolism and viability of Mycobacteria, establishing it as a validated target for the development of anti-TB therapeutics. Herein, we report the discovery of an N-acyl phenothiazine derivative, termed PT6, targeting the mycobacterial F-ATP synthase. PT6 is bactericidal and active against the drug sensitive, Rifampicin-resistant as well as Multidrug-resistant tuberculosis strains. Compound PT6 showed noteworthy inhibition of F-ATP synthesis, exhibiting an IC50 of 0.788 µM in M. smegmatis IMVs and was observed that it could deplete intracellular ATP levels, exhibiting an IC50 of 30 µM. PT6 displayed a high selectivity towards mycobacterial ATP synthase compared to mitochondrial ATP synthase. Compound PT6 showed a minor synergistic effect in combination with Rifampicin and Isoniazid. PT6 demonstrated null cytotoxicity as confirmed by assessing its toxicity against VERO cell lines. Further, the binding mechanism and the activity profile of PT6 were validated by employing in silico techniques such as molecular docking, Prime MM/GBSA, DFT and ADMET analysis. These results suggest that PT6 presents an attractive lead for the discovery of a novel class of mycobacterial F-ATP synthase inhibitors.
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Antituberculosos , Diseño de Fármacos , Inhibidores Enzimáticos , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis , Fenotiazinas , Fenotiazinas/farmacología , Fenotiazinas/química , Fenotiazinas/síntesis química , Antituberculosos/farmacología , Antituberculosos/síntesis química , Antituberculosos/química , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Relación Estructura-Actividad , Estructura Molecular , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Relación Dosis-Respuesta a Droga , Animales , Chlorocebus aethiops , Células Vero , Simulación del Acoplamiento Molecular , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Introduction: Oxysterol-binding protein (OSBP) is known for its crucial role in lipid transport, facilitating cholesterol exchange between the Golgi apparatus and endoplasmic reticulum membranes. Despite its established function in cellular processes, its involvement in coronavirus replication remains unclear. Methods: In this study, we investigated the role of OSBP in coronavirus replication and explored the potential of a novel OSBP-binding compound, ZJ-1, as an antiviral agent against coronaviruses, including SARS-CoV-2. We utilized a combination of biochemical and cellular assays to elucidate the interactions between OSBP and SARS-CoV-2 non-structural proteins (Nsps) and other viral proteins. Results: Our findings demonstrate that OSBP positively regulates coronavirus replication. Moreover, treatment with ZJ-1 resulted in reduced OSBP levels and exhibited potent antiviral effects against multiple coronaviruses. Through our investigation, we identified specific interactions between OSBP and SARS-CoV-2 Nsps, particularly Nsp3, Nsp4, and Nsp6, which are involved in double-membrane vesicle formation-a crucial step in viral replication. Additionally, we observed that Nsp3 a.a.1-1363, Nsp4, and Nsp6 target vesicle-associated membrane protein (VAMP)-associated protein B (VAP-B), which anchors OSBP to the ER membrane. Interestingly, the interaction between OSBP and VAP-B is disrupted by Nsp3 a.a.1-1363 and partially impaired by Nsp6. Furthermore, we identified SARS-CoV-2 orf7a, orf7b, and orf3a as additional OSBP targets, with OSBP contributing to their stabilization. Conclusion: Our study highlights the significance of OSBP in coronavirus replication and identifies it as a promising target for the development of antiviral therapies against SARS-CoV-2 and other coronaviruses. These findings underscore the potential of OSBP-targeted interventions in combating coronavirus infections.
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Antivirales , Receptores de Esteroides , SARS-CoV-2 , Proteínas no Estructurales Virales , Replicación Viral , Replicación Viral/efectos de los fármacos , Humanos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Antivirales/farmacología , Receptores de Esteroides/metabolismo , Proteínas no Estructurales Virales/metabolismo , COVID-19/virología , COVID-19/metabolismo , Chlorocebus aethiops , Células Vero , Proteínas Virales/metabolismo , Células HEK293 , Animales , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Proteínas Viroporinas/metabolismo , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Unión ProteicaRESUMEN
The terpolymers of N-vinylpyrrolidone (VP) with acrylic acid and triethylene glycol methacrylate were synthesized with more than 90% yield by radical copolymerization in ethanol from monomeric mixtures of different molar composition (98:2:2, 95:5: 2 and 98:2:5) and their monomer composition, absolute molecular masses and hydrodynamic radii in aqueous media were determined. Using the MTT test, these terpolymers were established to be low toxic for non-tumor Vero cells and HeLa tumor cells. Polymer compositions of hydrophobic dye methyl pheophorbide a (MPP) based on studied terpolymers and linear polyvinylpyrrolidone (PVP) were obtained and characterized in water solution. Quantum-chemical modeling of the MPP-copolymer structures was conducted, and the possibility of hydrogen bond formation between terpolymer units and the MPP molecule was shown. Using fluorescence microscopy, the accumulation and distribution of polymer particles in non-tumor (FetMSC) and tumor (HeLa) cells was studied, and an increase in the accumulation of MPP with both types of particles was found.