European sea bass brain DLB-1 cell line is susceptible to nodavirus: A transcriptomic study

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the fish central nervous system. It is considered one of the most serious viral diseases in marine aquaculture, the European sea bass (Dicentrarchus labrax) being amongst the most susceptible. We have evaluated the European sea bass brain derived cell line (DLB-1) susceptibility to NNV genotypes and evaluated its transcriptomic profile. DLB-1 cells supported NNV gene transcription and replication since strains belonging to the four NNV genotypes produce cytopathic effects. Afterwards, DLB-1 cells were infected with an RGNNV strain, the one which showed the highest replication, for 12 and 72 h and an RNA-seq analysis was performed to identify potential genes involved in the host-NNV interactions. Differential expression analysis showed the up-regulation of many genes related to immunity, heat-shock proteins or apoptosis but not to proteasome or autophagy processes. These data suggest that the immune response, mainly the interferon (IFN) pathway, is not powerful enough to abrogate the infection, and cells finally suffer stress and die by apoptosis liberating infective particles. GO enrichment also revealed, for the first time, the down-regulation of terms related to brain/neuron biology indicating molecular mechanisms causing the pathogenic effect of NNV. This study opens the way to understand key elements in sea bass brain and NNV interactions.Versión del edito

Generación de una línea celular de cerebro de lubina (DBL-1). Aplicación en estudios de toxicología y virología

Las líneas celulares son herramientas fundamentales para diversos estudios de Biología. En peces existen pocas líneas celulares y muy pocas de peces marinos. Por ello, hemos generado y caracterizado una línea celular (DLB-1) a partir de cerebro de lubina (Dicentrarchus labrax) y evaluamos su posible aplicación en Toxicología y Virología de peces. La incubación con distintos metales pesados (Cd, Hg, As o Pb) produce mortalidad en las células DLB-1 de manera dosis-dependiente, siendo muy tóxicos el Cd, Hg y As y muy poco el plomo. Además, se indujo la expresión del gen de la metalotioneína. Por otro lado, hemos evaluado la susceptibilidad de las células DLB-1 a nodavirus, el principal virus que afecta a lubinas. Los datos muestran que las células DLB-1 son susceptibles a los 4 genotipos de nodavirus. Esta línea celular puede ser muy útil para diversos estudios de la Biología de peces, concretamente de la lubina

Nodavirus colonizes and replicates in the testis of gilthead seabream and European sea bass modulating its immune and reproductive functions

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the casesMINECO and FEDER (AGL2010-20801-C02-01; AGL2010-20801-C02-02; AGL2013-43588-P); Fundación Séneca (04538/GERM/06)Versión del editor4,411