RESUMO
In the present study, we have developed a continuous cell line from the heart tissue of the Oreochromis niloticus and used for studying susceptibility to tilapia lake virus (TiLV). The cell line, designated as OnH, has been subcultured up to 82 passages. The optimal growth of OnH cells was observed at 28-32 °C in iL-15 medium supplemented with 20 % fetal bovine serum. Karyotype analysis revealed that the modal chromosome number of OnH cells was 44. Partial amplification and sequencing of 16S rRNA gene confirmed the origin of OnH cell line from O. niloticus. Immunophenotyping revealed that OnH cells were of epithelial origin. These cells were successfully transfected with pAcGFP1-N1 mammalian expression vector. OnH cells showed cytopathic effects following inoculation with TiLV. The virus titration study indicated that the cells were highly susceptible to TiLV with TCID50 value of 105.3/mL. The qRT-PCR studies revealed that the optimal temperature for TiLV replication in OnH cells was 28 °C. Further, transmission electron microscopy of TiLV-infected OnH cells showed a number of electron-dense virus particles measuring 60-90 nm diameter, which were enclosed in the vesicles in the cytoplasm. Therefore, the newly established OnH cell line provides a valuable tool for isolation of viruses from disease cases suspected to be of viral etiology in this candidate species' and also for transgenic and genetic manipulation studies.
Assuntos
Ciclídeos , Doenças dos Peixes , Vírus de RNA , Tilápia , Vírus , Animais , Linhagem Celular , RNA Ribossômico 16SRESUMO
El Virus de la Tilapia del Lago (TiLV), es un patógeno causante de mortalidades masivas tanto en poblaciones de tilapias cultivadas y silvestres alrededor del mundo. El desarrollo de una vacuna efectiva contra este patógeno emergente es imperativo para prevenir pérdidas económicas. En este trabajo se diseñó y evaluó un vector de expresión como una potencial vacuna de ADN contra este virus. Inicialmente, se realizó un análisis de enhebramiento para predecir las estructuras tridimensionales y las funciones de las proteínas del TiLV. Se encontraron homologías estructurales entre las proteínas correspondientes al segmento genómico 1 y al segmento genómico 4 del TiLV, con las proteínas de ARN polimerasa dependiente de ARN del virus de la influenza B (56%) y la proteína neuraminidasa que pertenece a la cápside del virus de la influenza A (12%), respectivamente. Se insertó el producto de PCR del gen neuraminidasa viral en el vector plasmídico de expresión pCMV. Finalmente, se inyectó el constructo plasmídico en juveniles de la tilapia del Nilo Oreochromis niloticus y se midió su expresión mediante RT-PCR en tiempo real a las 8h, 16h, 24h, 72h después de la segunda inyección inmunizante. Se logró detectar expresión génica en los cuatro tiempos evaluados, con mayor expresión a las 16 horas post inyección. Estos resultados constituyen el primer paso para el desarrollo de una vacuna efectiva para la protección de los stocks de tilapias alrededor del mundo.
Tilapia Lake Virus (TiLV) is a pathogen that causes massive mortalities in both cultured and wild tilapia populations around the world. The development of an effective vaccine against this emerging pathogen is imperative to prevent economic losses. In this work an expression vector was designed and evaluated as a potential DNA vaccine against this virus. Initially, a threading analysis was done to predict the threedimensional structures and functions of the TiLV proteins. Structural homologies were found between the TiLV proteins corresponding to the genomic segment 1 and the genomic segment 4, with the RNA-dependent RNA polymerase proteins of the influenza B virus (56%) and the neuraminidase protein belonging to the influenza A virus capsid (12%), respectively. The PCR product of the viral neuraminidase gene was inserted into the expression plasmid vector pCMV. Finally, the plasmid construct was injected into juveniles of the Nile tilapia Oreochromis niloticus and its expression was measured by real time RT-PCR at 8h, 16h, 24h, and 72h after the second immunizing injection. It was possible to detect gene expression in the four evaluated times and greater expression at 16 hours post injection. These results are the first step in the development of an effective vaccine for the protection of tilapia stocks around the world.