Nodavirus Colonizes and Replicates in the Testis of Gilthead Seabream and European Sea Bass Modulating Its Immune and Reproductive Functions

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.

Immune-Endocrine Interactions in the Fish Gonad during Infection: An Open Door to Vertical Transmission

“…This behavior, together with the high number of species that it is able to infect, makes NNV a good model to study immune-endocrine interactions in fish [126]. Nodavirus has been detected in the male and female gonad of several species in different reproductive stages, including European sea bass, gilthead seabream, Pacific cod (Gadus microcephalus), shi drum (Umbrina cirrosa), and striped jack (Pseudocaranx dentex) [112][113][114][115]. The cellular localization of the virus in the male gonad has recently been reported for the first time [113].…”

“…Nodavirus has been detected in the male and female gonad of several species in different reproductive stages, including European sea bass, gilthead seabream, Pacific cod (Gadus microcephalus), shi drum (Umbrina cirrosa), and striped jack (Pseudocaranx dentex) [112][113][114][115]. The cellular localization of the virus in the male gonad has recently been reported for the first time [113]. Thus, the gene coding for the capsid protein of NNV was located in Sertoli cells of European sea bass and gilthead seabream, and also in tunica albuginea and germ cells of gilthead seabream testis.…”

“…Thus, the gene coding for the capsid protein of NNV was located in Sertoli cells of European sea bass and gilthead seabream, and also in tunica albuginea and germ cells of gilthead seabream testis. Those data suggested that NNV can be vertically transmitted to the offspring through the germ cells or gonadal fluids in both species [113]. Although infective particles of NNV were recovered from the testes of both species, only in the testes of gilthead seabream were viral proteins (the capsid and B2 protein) detected by immunohistochemistry techniques [113].…”

“…Those data suggested that NNV can be vertically transmitted to the offspring through the germ cells or gonadal fluids in both species [113]. Although infective particles of NNV were recovered from the testes of both species, only in the testes of gilthead seabream were viral proteins (the capsid and B2 protein) detected by immunohistochemistry techniques [113]. Several studies about the transmission mechanisms of NNV point to both horizontal and vertical transmission modes [92,94,[127][128][129][130][131].…”

“…Gilthead seabream is an asymptomatic carrier of the traditional strains of NNV and does not suffer signs of infection or mortalities when it is infected with NNV (RGNNV genotype) [113,132]. However, European sea bass is an especially susceptible species to this virus and is known to suffer mortalities of up to 100%, mainly in larvae and juveniles stages [127], which exhibit the typical signs of infection [113,132,133].…”