Abstract
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 cases.
Highlights
Viruses and viral diseases have become one of the unsolved problems in modern aquaculture resulting in major economic loses
In the testis and brain of gilthead seabream and European sea bass specimens experimentally infected in vivo with nervous necrosis virus (NNV) were determined the transcription levels of the mRNA coding for the two main proteins of the virus, the RdRP and the coat protein (CP) proteins, using a specific probe for each, by TaqMan real-time PCR
Once we knew that the NNV colonized and/or replicated in the testis and brain of gilthead seabream and European sea bass, we studied the pattern of expression of pro-inflammatory cytokines tnfa, il6 and il1b and T and B lymphocyte markers genes in both tissues and species upon in vivo infection and in the testis upon in vitro infection (Fig 4) and found that, in gilthead seabream (Fig 4A–4E), all the cytokine genes were unchanged in the testis (Fig 4A–4E), whilst in the brain, the tnfa and il1b gene expression was down-regulated after 7 days of infection (Fig 4A and 4C) and the il6 gene expression was increased after 15 days of infection (Fig 4B)
Summary
Viruses and viral diseases have become one of the unsolved problems in modern aquaculture (since there are no effective preventive measures available to control them) resulting in major economic loses. NNV is a small, naked icosahedral virus, composed of 2 positive single stranded RNA fragments, RNA1 and RNA2, which are capped but not polyadenylated [3]. The capsid is composed of multiple units of a single protein, the coat protein (CP) [4] coded by the RNA2 [3, 5] and involved in host specificity. It has been recently described that each units of the capsid protein (CP) shows three major domains: the N-terminal arm, the shell domain (S-domain) and the protrusion domain (Pdomain) formed by three-fold trimeric protrusions with hypervariable surface regions that contribute to host binding and specificity [6]. The RNA3 of betanodavirus has been considered to have a single open reading frame encoding for protein B2 [7]. B2 is only detected when the virus is actively replicating, but not in persistent infections [10]
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