RESUMEN
Southern rice black-streaked dwarf virus (SRBSDV) is a rice pathogen that had an outbreak in southern China in 2010 and caused significant crop losses. Therefore, screening for effective antiviral drugs against SRBSDV is very important. This study used rice suspension cells infected with SRBSDV by polyethylene glycol-mediated uptake for screening antiviral drugs. SRBSDV P7-1, which is coded by the S7-1 gene, has an intrinsic ability to self-interact to form tubules that play an important role in viral infection. Therefore, relative expression level of the SRBSDV S7-1 gene in infected rice suspension cells was assayed by real-time quantitative polymerase chain reaction to evaluate the antiviral activities of various drugs. Dufulin displayed the highest inhibitory activity against SRBSDV S7-1 expression. In addition, changes in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) activities were determined in inoculated and noninoculated cells. The results showed that both POD and PPO activities increased upon dufulin treatment. Furthermore, the validity of this approach was confirmed in an in vivo experiment in which dufulin was found to effectively inhibit SRBSDV.
Asunto(s)
Antivirales/farmacología , Oryza/virología , Enfermedades de las Plantas/virología , Virus de Plantas/efectos de los fármacos , Reoviridae/efectos de los fármacos , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Benzotiazoles/farmacología , Técnicas de Cultivo de Célula , Evaluación Preclínica de Medicamentos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Virus de Plantas/genética , Virus de Plantas/metabolismo , Reoviridae/genética , Reoviridae/metabolismo , Proteínas Virales/metabolismoRESUMEN
Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.
Asunto(s)
Mariposas Nocturnas/virología , ARN Polimerasa Dependiente del ARN/genética , Reoviridae/genética , Proteínas Virales/genética , Animales , Clonación Molecular , Análisis por Conglomerados , Coenzimas/farmacología , ADN Complementario/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Peso Molecular , Filogenia , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Temperatura , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Although the concept of using viruses as antineoplastic agents dates back nearly a century, recent advances in the fields of molecular biology, genetics, and virology have enabled investigators to engineer viruses with greater potency and tumor specificity. Further enhancements involve arming these viruses with therapeutic transgenes, and combining the traditional modalities of chemotherapy and radiation therapy with oncolytic viral therapy in hopes of reducing the chance of developing resistant tumor cell clones. Another means of augmenting the antineoplastic effect of these viruses involves modulating the immune response to minimize antiviral immunity, while at the same time maximizing antitumor immunity. A better understanding of mechanisms that viruses use to overcome cellular defenses to achieve robust replication within the cell will lead to development of oncolytic viruses with better tumor specificity and reduced toxicity. Initial clinical studies have shown that oncolytic viral therapy for metastatic disease is safe and well tolerated. In addition, using similar genetic modification strategies, these viruses have demonstrated antineoplastic effects in humans similar to those seen in preclinical animal models.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Neoplasias/terapia , Virus Oncogénicos/genética , Adenoviridae/genética , Animales , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Ensayos Clínicos Fase III como Asunto , Terapia Combinada , Evaluación Preclínica de Medicamentos , Terapia Genética/tendencias , Humanos , Neoplasias/genética , Virus de la Enfermedad de Newcastle/genética , Reoviridae/genética , Simplexvirus/genética , Virus Vaccinia/genética , Proteínas Virales/genética , Replicación Viral/genéticaRESUMEN
Only one strand of each double-stranded (ds) RNA segment of the Kemerovo virus genome was 5'end-labelled using gamma-32P-ATP and T 4 polynucleotide kinase after preceding dephosphorylation of 5'ends by calf intestinal alkaline phosphatase. This suggests a 5'-terminal modification of the one of complementary strands in the ds RNA segments.