RESUMEN
Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.
Asunto(s)
Neuraminidasa , Infecciones por Rotavirus , Rotavirus , Replicación Viral , Animales , Neuraminidasa/metabolismo , Neuraminidasa/genética , Rotavirus/efectos de los fármacos , Rotavirus/fisiología , Porcinos , Replicación Viral/efectos de los fármacos , Línea Celular , Células Epiteliales/virología , Células Epiteliales/microbiología , Acoplamiento Viral/efectos de los fármacos , Ácido N-Acetilneuramínico/metabolismo , Ácido N-Acetilneuramínico/farmacología , Antivirales/farmacología , Haplorrinos , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/microbiologíaRESUMEN
Rotavirus (RV) is the main pathogen that causes severe diarrhea in infants and children under 5 years of age. No specific antiviral therapies or licensed anti-rotavirus drugs are available. It is crucial to develop effective and low-toxicity anti-rotavirus small-molecule drugs that act on novel host targets. In this study, a new anti-rotavirus compound was selected by ELISA, and cell activity was detected from 453 small-molecule compounds. The anti-RV effects and underlying mechanisms of the screened compounds were explored. In vitro experimental results showed that the small-molecule compound ML241 has a good effect on inhibiting rotavirus proliferation and has low cytotoxicity during the virus adsorption, cell entry, and replication stages. In addition to its in vitro effects, ML241 also exerted anti-RV effects in a suckling mouse model. Transcriptome sequencing was performed after adding ML241 to cells infected with RV. The results showed that ML241 inhibited the phosphorylation of ERK1/2 in the MAPK signaling pathway, thereby inhibiting IκBα, activating the NF-κB signaling pathway, and playing an anti-RV role. These results provide an experimental basis for specific anti-RV small-molecule compounds or compound combinations, which is beneficial for the development of anti-RV drugs.
Asunto(s)
Antivirales , Infecciones por Rotavirus , Rotavirus , Replicación Viral , Rotavirus/efectos de los fármacos , Rotavirus/fisiología , Animales , Ratones , Infecciones por Rotavirus/tratamiento farmacológico , Infecciones por Rotavirus/virología , Replicación Viral/efectos de los fármacos , Humanos , Antivirales/farmacología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación , Ratones Endogámicos BALB C , Línea Celular , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Los rotavirus del grupo A (RVA) constituyen la principal causa de diarrea grave y mortalidad infantil. La porción variable de los anticuerpos de cadena pesada derivados de camélidos presentan una amplia capacidad de unión antigénica (reconocen sitios antigénicos no accesibles a los anticuerpos tradicionales, con elevada afinidad) tienen bajos costos de producción y resultan ideales para las terapias orales. A la fecha, se desarrollaron 2 pares de nanoanticuerpos VHH contra RVA: ARP1-ARP3 y 2KD1-3B2. En este trabajo, exploramos el potencial de ambos grupos de nanoanticuerpos como estrategias de prevención complementarias a la vacunación y como una opción de tratamiento frente a la diarrea asociada a RVA en poblaciones de riesgo. Ambos pares de nanoanticuerpos fueron expresados en diferentes sistemas de producción y mostraron amplia capacidad neutralizante contra diversas cepas de RVA in vitro. También fueron usados en el modelo de ratón lactante, en el que evidenciaron distintos grados de éxito en la prevención o el tratamiento de la diarrea inducida por RVA. Es interesante destacar que la mitigación de los síntomas también se logró con ARP1 liofilizado y conservado, por lo que podría ser utilizado en áreas donde es difícil mantener la cadena de frío. Asimismo, 3B2 fue testeado en una prueba preclínica utilizando como modelo al cerdo gnotobiótico, al cual confirió completa protección contra la diarrea inducida por RVA. ARP1 fue usado en la primera prueba clínica de nanoanticuerpos VHH contra RVA, donde redujo significativamente las deposiciones en pacientes pediátricos con diarrea positivos para RVA, sin evidenciar ninguna reacción adversa
Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects
Asunto(s)
Rotavirus/efectos de los fármacos , Diarrea/prevención & control , Anticuerpos de Dominio Único/uso terapéutico , Anticuerpos/uso terapéutico , Prevención Primaria/tendencias , Mortalidad del Niño , Anticuerpos de Dominio Único/administración & dosificaciónRESUMEN
Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.
Asunto(s)
Humanos , Animales , Adenoviridae/efectos de los fármacos , Péptidos Catiónicos Antimicrobianos/farmacología , Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Rotavirus/efectos de los fármacos , Línea Celular , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.
Asunto(s)
Animales , Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismo , Rotavirus/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Chlorocebus aethiops , Nocodazol/farmacología , Ensayo de Radioinmunoprecipitación , Rotavirus/efectos de los fármacos , Rotavirus/genéticaRESUMEN
Isoprinosine (IPS) is a synthetic drug whose antiviral effect on rotavirus replication in vitro has been characterized in terms of the decrease in metachromasia after acridine orange staining. The present study describes the effect of IPS on the synthesis of viral RNA in vitro. MA-104 cell cultures infected with simian rotavirus strain SA-11 were incubated with zero, 250, 500 and 1,000 microg/ml IPS and 22, 24, 48, 52, 72 and 76 h after infection the cultures were submitted to a 1-h starvation period, followed by a 2-h pulse with 10 microCi/ml of [3H]-uridine. The homogenates of virus-infected cultures treated or not with IPS were submitted to phenol/chloroform extraction followed by polyacrylamide gel electrophoresis. The amount of radioactivity in viral RNA eluted from the gel strips was determined. Inhibition of viral RNA synthesis was highest at the IPS concentration of 1,000 microg/ml at 72 h after infection, corresponding to 78 percent inhibition. Although the results obtained in vitro suggest that IPS may be useful for the treatment of rotavirus infection, an in vivo demonstration of its efficacy is needed.
Asunto(s)
Técnicas In Vitro , Inosina Pranobex/farmacología , Rotavirus/efectos de los fármacos , Replicación Viral , Rotavirus/crecimiento & desarrolloRESUMEN
Utilizando a cepa de rotavírus SA-11, cultivada em células MA-104 e purificada em gradiente de CsCl, prepararam-se soros antirotavírus em cobaias para uso no diagnóstico de gastroenterites por rotavírus, através da reaçäo de imunofluorescência indireta. Num total de 268 casos, obteve-se uma porcentagem de positividade de 14.93 por cento (40 amostras). A comparaçäo desta prova com o ensaio imunoenzimático (EIERA) e a eletroforese em gel de poliacrilamida(EGPA), revelou boa concordância entre os três ensaios, com valores de kappa entre 0.72 e 0.76.
Asunto(s)
Rotavirus/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Rotavirus/metabolismo , Electroforesis en Gel de PoliacrilamidaRESUMEN
Resumo: O teste de coaglutinaçäo foi utilizado para a detecçäo de rotavírus em fezes de origem humana e de suínos. Suspensäo de Staphylococcus aureos, produtor de proteina A, foi sensibilizada com uma diluiçäo seriada de antissoro anti-rotavirus do grupo A mostrando que quando foi utilizada a diluiçäo a 1:20, o teste foi capaz de detectar tanto antígeno SA11 como também o extrato fecal, ambos diluídos. Um total de 89 amostras de fezes absorvidas com S. aureus foram testadas por coaglutinaçäo e por um ensaio imunoenzimático. A análise estatística dos resultados obtidos mostrou uma concordância de 0,91 entre os dois testes o que levou-nos a conluir que a coaglutinaçäo é um método simples, rápido, sensível e pouco dispendioso para a detecçäo de rotavírus diretamente do material fecal. Além da utilizaçäo do soro diluído para a sensibilizaçäo de s> aureus ficou demonstrado também que, esta mistura, quando estocada a 4 graus C pode ser utilizada por até 6 meses após o seu preparo, sem implicar em resultados falso-positivos (au)
Asunto(s)
Animales , Staphylococcus aureus/efectos de los fármacos , Rotavirus/efectos de los fármacos , HecesRESUMEN
Las enfermedades diarreicas están difundidas y causan todos los años la muerte de más de 4,5 millones de menores de 5 años, la importancia que presentan los rotavirus como agentes virales ha originado que se desarrollen estrategias inmunoprofilácticas, siendo de ellas la utilizadas en el desarrollo de una vacuna anti-rotavirus que consisten en el uso de una cepa animal relacionada antigenicamente y que sea capaz de crecer de manera eficiente en cultivos celulares. En este estudio fueron seleccionados 54 niños con edades entre 4 y 10 meses y recibieron 2 dosis de vacunas y el placebo. Fueron tomadas muestras de heces diariamente durante el período de observación. Los resultados preliminares de este estudio demostraron que la vacuna no produce reacciones secundarias en los diferentes grupos etarios estudiados, en especial en el grupo menor de 4 meses, en el grupo de niños de 2 a 3 meses se determinó la excreción viral en un 75