RESUMO
In this study, we demonstrated the antiviral efficacy of hesperetin against multiple poxviruses, including buffalopox virus (BPXV), vaccinia virus (VACV), and lumpy skin disease virus (LSDV). The time-of-addition and virus step-specific assays indicated that hesperetin reduces the levels of viral DNA, mRNA, and proteins in the target cells. Further, by immunoprecipitation (IP) of the viral RNA from BPXV-infected Vero cells and a cell-free RNA-IP assay, we demonstrated that hesperetin-induced reduction in BPXV protein synthesis is also consistent with diminished interaction between eukaryotic translation initiation factor eIF4E and the 5' cap of viral mRNA. Molecular docking and MD simulation studies were also consistent with the binding of hesperetin to the cap-binding pocket of eIF4E, adopting a conformation similar to m7GTP binding. Furthermore, in a BPXV egg infection model, hesperetin was shown to suppress the development of pock lesions on the chorioallantoic membrane and associated mortality in the chicken embryos. Most importantly, long-term culture of BPXV in the presence of hesperetin did not induce the generation of drug-resistant viral mutants. In conclusion, we, for the first time, demonstrated the antiviral activity of hesperetin against multiple poxviruses, besides providing some insights into its potential mechanisms of action.
Assuntos
Fator de Iniciação 4E em Eucariotos , Hesperidina , Vaccinia virus , Animais , Bovinos , Chlorocebus aethiops , Embrião de Galinha , Células Vero , Simulação de Acoplamento Molecular , Vaccinia virus/genética , Antivirais/farmacologia , RNA Mensageiro , Replicação ViralRESUMO
Bordetella bronchiseptica is a well-known Gram-negative bacterial pathogen causing a plethora of diseases in different animals. Although its infection has been reported from pigs and dogs in India, no report of B. bronchiseptica from horses is described. We report for the first time, isolation, identification and characterization of strains of B. bronchiseptica from respiratory infection in horses from different states in India. The antimicrobial susceptibility testing showed resistance to penicillins, ceftazidime, and chloramphanicol. The virulence capability of the strains was confirmed by sequencing genes such as adenylate cyclase toxin (cyaA), bordetella virulence gene (bvgA) and by PCR detection of flagellin gene (fla). We demonstrate the involvement of B. bronchiseptica strains in respiratory tract infection in horses in India.
RESUMO
Micro RNAs (miRNAs) have been implicated in the regulation of maturation, proliferation, differentiation, and activation of immune cells. In this study, we demonstrated that miR-29a antagonizes IFN-γ production at early times post-LSDV infection in cattle. miR-29a was predicted to target upstream IFN-γ regulators, and its inhibition resulted in enhanced IFN-γ production in sensitized peripheral blood mononuclear cells (PBMCs). Further, stimulation of PBMCs with LSDV antigen exhibited lower levels of miR-29a, concomitant with a potent cell-mediated immune response (CMI), characterized by an increase in LSDV-specific CD8+ T cell counts and enhanced levels of IFN-γ, which eventually facilitated virus clearance. In addition, a few immunocompromised cattle (developed secondary LSDV infection at ~ 6 months) that failed to mount a potent cell-mediated immune response, were shown to maintain higher miR-29a levels. Furthermore, as compared to the sensitized crossbred cattle, PBMCs from sensitized Rathi (a native Indian breed) animals exhibited lower levels of miR-29a along with an increase in CD8+ T cell counts and enhanced levels of IFN-γ. Finally, we analysed that a ≥ 60% decrease in miR-29a expression levels in the PBMCs of sensitized cattle correlated with a potent CMI response. In conclusion, miR-29a expression is involved in antagonizing the IFN-γ response in LSDV-infected cattle and may serve as a novel biomarker for the acute phase of LSDV infection, as well as predicting the functionality of T cells in sensitized cattle. In addition, Rathi cattle mount a more potent CMI response against LSDV than crossbred cattle.
Assuntos
Doenças dos Bovinos , Vírus da Doença Nodular Cutânea , MicroRNAs , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/genética , Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Vírus da Doença Nodular Cutânea/genética , MicroRNAs/genética , Reação em Cadeia da Polimerase , BiomarcadoresRESUMO
Countries in the Indian subcontinent are currently facing a deadly epidemic of lumpy skin disease (LSD). LSD is primarily a disease of cattle. Buffaloes may sometimes develop mild illness, however, other domestic animals are considered resistant to LSD. We confirmed the LSDV infection in camels as evidenced by skin nodules on the body surface of the affected camels, isolation of LSD virus (LSDV) and amplification of LSDV-specific gene segments from the skin nodules (PCR), nucleotide sequencing of the viral genome and, demonstration of anti-LSDV antibodies in serum. Phylogenetic analysis based on nucleotide sequencing of ORF011, ORF012 and ORF036 revealed that the virus (LSDV/Camel/India/2022/Bikaner) is related to the historical NI-2490/Kenya/KSGP-like field strains which are predominantly circulating in the Indian subcontinent. This is the first report wherein LSDV has been to infect camels.
Assuntos
Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Animais , Bovinos , Vírus da Doença Nodular Cutânea/genética , Doença Nodular Cutânea/epidemiologia , Camelus , Filogenia , Búfalos , Nucleotídeos , Surtos de Doenças/veterináriaRESUMO
Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50th LSDV passage in Vero cells, named as Lumpi-ProVacInd) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.
Assuntos
Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Vacinas Virais , Animais , Bovinos , Feminino , Chlorocebus aethiops , Doença Nodular Cutânea/prevenção & controle , Doença Nodular Cutânea/epidemiologia , Vírus da Doença Nodular Cutânea/genética , Vacinas Atenuadas/efeitos adversos , Células Vero , Vacinas Virais/administração & dosagemRESUMO
Emetine is a FDA-approved drug for the treatment of amebiasis. Previously we demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. In this study, we evaluated the in vitro antiviral efficacy of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and found it to be a low nanomolar (nM) inhibitor. Interestingly, emetine exhibited protective efficacy against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment led to a decrease in viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding. In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 mRNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation of protein translation). Further, molecular docking and molecular dynamics simulation studies suggested that emetine may bind to the cap-binding pocket of eIF4E, in a similar conformation as m7-GTP binds. Additionally, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathway for its effective replication in the target cells. Collectively our results suggest that further detailed evaluation of emetine as a potential treatment for COVID-19 may be warranted.
Assuntos
Antivirais , Emetina , Vírus da Bronquite Infecciosa/efeitos dos fármacos , RNA Viral/metabolismo , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Embrião de Galinha , Chlorocebus aethiops , Infecções por Coronavirus/tratamento farmacológico , Emetina/farmacologia , Emetina/uso terapêutico , Fator de Iniciação 4E em Eucariotos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais , Células VeroRESUMO
Wound healing in horses is complicated by the excessive growth of granulation tissue, commonly known as proud flesh and is similar to keloids in human beings. At present, there is no satisfactory treatment for proud flesh in horses. In this study, we, for the first time, demonstrated that leaf extract of Aerva javanica suppresses excessive growth of granulation tissue in horses. Many plant flavonoids are claimed to have antiproliferative properties. Kaempferol is a natural flavonoid containing 3-hydroxy flavone backbone found in many plants in its aglycone form and attached with various sugars. Ecdysteroids are steroidal analogs of invertebrate steroidal hormones found in plants. Both flavonoids and ecdysteroids accumulate more in plants during abiotic stress. We hypothesized that Aerva javanica may have high levels of ecdysteroids and kaempferols for surviving in stressful conditions of desert. Those kaempferols may suppress the growth of granulation tissue by their antiangiogenesis property. Ecdysteroids may control the larvae of habronema if associated with proud flesh. Extract was prepared using solvent-based fractionation and silica gel column flash chromatography. Application of the leaf extract in horses suppressed growth of granulation tissue along with restoration of normal skin function. Various purification steps and mass spectrometry were used to identify the active components in the study.
Assuntos
Amaranthaceae , Doenças dos Cavalos , Queloide , Animais , Tecido de Granulação , Cavalos , Queloide/veterinária , Extratos Vegetais/farmacologia , CicatrizaçãoRESUMO
The present study was conducted with the hypothesis that addition of cholesterol to the extender would stabilize the sperm membranes by increasing the cholesterol-to-phospholipid (C:P) ratio and would result in an improved post-thaw semen quality and reduce oxidative stress in the jack (Martina franca) semen. Forty-eight ejaculates from six jacks were collected and analyzed for the present study. The freshly collected semen sample of each jack stallion was divided into five equal fractions after addition of the primary extender without cholesterol-loaded cyclodextrin (CLC) (C) and with 1, 1.5, 2, and 3 mg/mL CLC to obtain 120 × 106 sperm/mL spermatozoa concentration. The semen was cryopreserved using customized freezing protocols. Evaluation of seminal parameters, the C:P ratio, and the oxidative status of jack spermatozoa was analyzed at all stages of cryopreservation. The oxidative status in the jack semen was evaluated by measuring malondialdehyde, glutathione and total antioxidant capacity levels. The results indicated that the mean percent values for various seminal quality parameters and the oxidative parameters were found to be significantly higher (P < .05) in CLC-treated groups with the highest values for 2 mg of CLC/120 × 106 spermatozoa. In conclusion, the present study revealed that the supplementation of CLC before cryopreservation has significantly reduced the oxidative stress and also increased the C:P ratio during semen cryopreservation process. Furthermore, a reduction in lipid peroxidation levels, reduced damage to the sperm plasma and acrosome membranes and improvement in the post-thaw sperm integrity as well as stability were recorded.
Assuntos
Ciclodextrinas , Preservação do Sêmen , Animais , Colesterol , Criopreservação/veterinária , Crioprotetores/farmacologia , Ciclodextrinas/farmacologia , Suplementos Nutricionais , Cavalos , Masculino , Estresse Oxidativo , Fosfolipídeos , Análise do Sêmen/veterinária , Preservação do Sêmen/veterinária , Motilidade dos Espermatozoides , EspermatozoidesRESUMO
We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.
Assuntos
Antivirais/farmacologia , Apigenina/farmacologia , Farmacorresistência Viral , Vaccinia virus/efeitos dos fármacos , Animais , Embrião de Galinha/virologia , Galinhas , Chlorocebus aethiops , DNA Viral/genética , DNA Polimerase Dirigida por DNA , Humanos , Vaccinia virus/enzimologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Equine influenza is a contagious viral disease that affects all members of the family Equidae, i.e., horses, donkeys and mules. The authors describe the pattern of equine influenza outbreaks in a number of states of India from July 2008 to June 2009. The disease was first reported in June 2008 in Katra (Jammu and Kashmir) and spread to ten other states within a year. All outbreaks of equine influenza in the various states were confirmed by laboratory investigations (virus isolation and/or serological confirmation based on haemagglutination inhibition [HI] assays of paired samples) before declaring them as equine influenza virus-affected state(s). The virus (H3N8) was reported from various locations in the country including Katra, Mysore (Karnataka), Ahmedabad (Gujarat), Gopeshwar and Uttarkashi (Uttarakhand) and was isolated in 9- to 11-day-old embryonated chicken eggs. The virus was confirmed as H3N8 by HI assays with standard serum and amplification of full-length haemagglutinin and neuraminidase genes by reverse transcriptase-polymerase chain reaction. Serum samples (n = 4 740) of equines from 13 states in India screened by HI revealed 1074 (22.65%) samples as being positive for antibodies to equine influenza virus (H3N8).