RESUMEN
A rhabdovirus associated with a lethal hemorrhagic disease in cultured turbot Scophthalmus maximus Linnaeus was isolated. The virus induced typical cytopathogenic effects (CPE) in 9 of 15 fish cell lines examined and was then propagated and isolated from infected carp leucocyte cells (CLC). Electron microscopy observations revealed that the negatively stained virions had a typical bullet-shaped morphology with one rounded end and one flat base end. The bullet-shaped morphology was more obvious and clear in ultrathin sections of infected cells. Experimental infections also indicated that the S. maximus rhabdovirus (SMRV) was not only a viral pathogen for cultured turbot, but also had the ability to infect other fish species, such as freshwater grass carp. A partial nucleotide sequence of the SMRV polymerase gene was determined by RT-PCR using 2 pairs of degenerate primers designed according to the conserved sequences of rhabdovirus polymerase genes. Homology analysis, amino acid sequence alignment, and phylogenetic relationship analysis of the partial SMRV polymerase sequence indicated that SMRV was genetically distinct from other rhabdoviruses. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified SMRV revealed 5 major structural proteins, and their molecular masses were estimated to be about 250, 58, 47, 42, and 28 kDa. Significant serological reactivity differences were also observed between SMRV and its nearest neighbor, spring viremia of carp virus (SVCV). The data suggest that SMRV is likely a novel fish rhabdovirus, although it is closely related to rhabdoviruses in the genus Vesiculovirus.
Asunto(s)
Enfermedades de los Peces/virología , Peces Planos/virología , Infecciones por Rhabdoviridae/veterinaria , Rhabdoviridae/patogenicidad , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/metabolismo , Carpas/virología , Línea Celular , Efecto Citopatogénico Viral , Genes Virales/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Filogenia , Rhabdoviridae/clasificación , Rhabdoviridae/genética , Rhabdoviridae/ultraestructura , Infecciones por Rhabdoviridae/virología , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Virotherapy with oncolytic viruses is a highly promising approach for cancer therapy. To improve further the therapeutic effect of oncolytic viruses, therapeutic genes have been incorporated into these types of vectors. In this study, we have inserted hTRAIL (approved gene symbol TNFSF10) into the ZD55 vector, which was based on deletion of the adenoviral E1B 55-kDa gene and could replicate in and lyse p53-deficient tumors. Our data shows that infection of colorectal carcinoma cells with ZD55-hTRAIL resulted in tumor cell death that was much greater than that induced by ZD55 vector or replication-defective adenovirus expressing hTRAIL. In contrast to these, ZD55-hTRAIL did not induce any cytopathic effect in normal cells. Treatment of established tumor with ZD55-hTRAIL resulted in dramatic inhibition of tumor growth in an animal model of colorectal carcinoma. However, when the established tumors were treated by coadministration of ZD55-hTRAIL and Ad-k5, we observed complete eradication of the established tumors in all animals treated with the combined therapy. This strong anti-tumor activity was due to the fact that two genes may act with compensative (or synergic) effect through different mechanisms to kill tumors. Therefore, targeting dual gene-virotherapy may be one of the best strategies for cancer therapy if two suitable genes are chosen.
Asunto(s)
Adenoviridae/genética , Carcinoma/terapia , Neoplasias Colorrectales/terapia , Terapia Genética/métodos , Glicoproteínas de Membrana/genética , Fragmentos de Péptidos/genética , Plasminógeno/genética , Factor de Necrosis Tumoral alfa/genética , Proteínas E1B de Adenovirus/genética , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis , Carcinoma/genética , Neoplasias Colorrectales/genética , Vectores Genéticos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Ligando Inductor de Apoptosis Relacionado con TNF , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Replicación Viral/genéticaRESUMEN
The causative agent of lymphocystis disease that frequently occurs in cultured flounder Paralichthys olivaceus in China is lymphocystis virus (LV). In this study, 13 fish cell lines were tested for their susceptibility to LV. Of these, 2 cell lines derived from the freshwater grass carp Ctenopharyngodon idellus proved susceptible to the LV, and 1 cell line, GCO (grass carp ovary), was therefore used to replicate and propagate the virus. An obvious cytopathic effect (CPE) was first observed in cell monolayers at 1 d post-inoculation, and at 3 d this had extended to about 75% of the cell monolayer. However, no further CPE extension was observed after 4 d. Cytopathic characteristics induced by the LV were detected by Giemsa staining and fluorescence microscopic observation with Hoechst 33258 staining. The propagated virus particles were also observed by electron microscopy. Ultrastructure analysis revealed several distinct cellular changes, such as chromatin compaction and margination, vesicle formation, cell-surface convolution, nuclear fragmentation and the occurrence of characteristic 'blebs' and cell fusion. This study provides a detailed report of LV infection and propagation in a freshwater fish cell line, and presents direct electron microscopy evidence for propagation of the virus in infected cells. A possible process by which the CPEs are controlled is suggested.
