Validation of two pmx-luciferase reporter systems to study virus - host interaction.

Viral diseases represent an important threat in aquaculture. Therefore, the development of strategies and tools to understand fish susceptibility, virus-host interaction, or to identify viral virulence markers is a priority. Fish possess an effective antiviral response mediated by type I interferon, IFN I, a cytokine that induces the expression of a set of genes, called IFN-stimulated genes (ISGs), which generate an antiviral state in infected and surrounding cells. Among ISGs, mx genes are considered markers of the IFN I response, since Mx proteins show direct antiviral activity and mx genes display strong and quick induction, which varies according to viral virulence. In addition, mx transcription is blocked by several viruses. Therefore, the level and time-course of mx induction reflect virus-host interaction. This idea prompted the development of in vitro experimental systems consisting of RTG-2 cells stably expressing luciferase under the control of mx promoters to study virus-host interplay. Specifically, Senegalese sole and sea bream mx promoters have been evaluated in this study. Both systems were inoculated with different doses of viral isolates relevant in aquaculture: Infectious Pancreatic Necrosis Virus, IPNV, Viral Haemorrhagic Septicaemia Virus, VHSV, and Nervous Necrosis Virus, NNV. Each isolate triggered a characteristic profile in each experimental system. The sensitivity of both experimental systems in detecting two NNV isolates in infected tissues was tested, and the NNV antagonistic activity was also characterized. The minimal viral dose required to detect pmx induction and/or blocking is a possible virulence marker for these isolates. Thus, both experimental systems have been validated and can be used to get more insight on virus-host interaction and contribute to fight viral infections in aquaculture. Acknowledgments This study has been supported by the Project UMA20-FEDERJA-020 (Regional Government).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Genomic characterization and transcription analysis of European sea bass (Dicentrarchus labrax) rtp3 genes.

Fish RTP3, belonging to the receptor-transporting protein family, display several functions, including a putative antiviral role as virus-responsive gene. In this work, we have identified and characterized two different European sea bass rtp3 genes. In addition, an in vivo transcription analysis in response to LPS, poly I:C and betanodavirus infection (RGNNV genotype) has been performed. The sequence analysis showed that European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively), located within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been found in the intron sequence of both genes, whereas rtp3 X1 also contains this motif in both untranslated regions. The transcription analyses revealed significant level of rtp3 X2 mRNA in brain and head kidney after LPS and poly I:C inoculation; however, the induction elicited by RGNNV infection was much higher, suggesting an essential role for this protein in controlling NNV infectionsFunding for open access charge: Universidad de Málaga / CBU

European sea bass RTP3 genes: genomic characterization and transcription analyses.

Introduction: Fish RTP3, belonging to receptor-transporting protein (RTP) family, has been described as an interferon-α (IFN-α)-responsive gene. However, little information is available about fish rtp3 gene structure and the role of RTP3 proteins during viral infections. NNV (Nodaviridae family, Betanodavirus genus) is the causative agent of the viral nervous necrosis, the main viral disease affecting European sea bass (Dicentrarchus labrax) culture. Betanodaviruses have been classified into four species, although RGNNV is the only one causing high mortalities in sea bass. Aim: The aim of the study has been to analyse the genomic structure of European sea bass rtp3, and its transcription profile after injection with LPS, poly I:C, or RGNNV infection. Methodology: A partial sequence of seabass rtp3 gene was used as alignment sequence within European sea bass genome database. The located sequences were used as templates to design primers for full-length rtp3 sequencing. In addition, rtp3 X1 and X2 transcription was analysed in brain and head kidney by relative qPCR. Results: European sea bass displays two rtp3 genes, X1 and X2, composed of two exons and a single intron (1007-bp and 888-bp long, respectively) within the ORF sequence. The full-length cDNA is 1969 bp for rtp3 X1, and 1491 bp for rtp3 X2. Several ATTTA motifs have been detected in the intron sequence of both genes, whereas rtp3 X1 also showed this motif in both untranslated regions. Regarding transcription analysis, the results revealed a significant level of rtp3 X2 transcription in brain and head kidney after LPS and poly I:C inoculation; however, the induction caused by RGNNV infection is much higher, suggesting an essential role of this protein in controlling NNV infections. Page | 38 Conclusion: The present study contributes to further characterize the European sea bass response against RGNNV, being the first step in elucidating the role of sea bass rtp3 in the course of infections.Project PID2020-115954RB100/AEI/10.13039/501100011033 (Spanish Government) and I+D+I Project UMA20-FEDERJA103 by the Operative Program FEDER Andalucía 2014-2020. In vivo challenges were conducted in the Center for Ecology and Microbiology of Controlled Aquatic Systems (CEMSAC). Thanks to Carmar Cultivos Marinos S.L. (Grupo CUMAREX) for providing the fish. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Setting out zebrafish (Danio rerio) as a model to study nervous necrosis virus-host interaction.

Viral nervous necrosis is responsible for important economic losses in aquaculture facilities. The causative agent is the nervous necrosis virus (NNV). Four NNV species have been described, although only RGNNV and SJNNV have been detected in the Mediterranean area. RGNNV- SJNNV reassortants have also been isolated from several species. In order to design strategies to improve fish resistance to NNV, in vivo studies in commercial and model species are required to study the mechanisms underlying fish susceptibility to viral isolates. The aim of this work was to set up zebrafish as model of NNV infection. To fulfil this aim, zebrafish susceptibility to three NNV isolates was determined, and viral replication and innate immune response were characterized. Three days post-fertilisation zebrafish larvae were infected by intracerebral injection with 107 TCID50/mL of SJ93Nag (SJNNV), Dl956 (RGNNV from seabass), and RG/SJ (from seabream). Larvae were daily monitored for 4 days to record clinical signs and mortality. At 1 and 4 days post-infection (dpi), 3 pools of 6 larvae were sampled for viral genome quantification. Innate immune response was also assessed. Transcriptional analyses were completed by in vivo 3D imaging approaches on a zebrafish transgenic line expressing GFP in neutrophils (Tg (mpx:GFP) to monitor neutrophils recruitment in brain. RGNNV was the most virulent isolate compared to SJNNV and RG/SJ. These observations were consistent with viral genome replication, as the highest number of viral genome copies was in RGNNV-infected larvae. The induction of immune-related genes and the recruitment of neutrophils in brain, was also higher in RGNNV-infected larvae. Therefore, further experiments can be designed in this successfully model to better understand the mechanisms underlying NNV virulence in its hosts. Acknowledgments: Projects PID2020-115954RB-100/AEI/10.13039/501100011033 (Spanish Government) and EU H2020 VBN_22_73 (VetBioNet project).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Understanding the role of European sea bass RTP3 protein in the course of betanodavirus infections.

Nervous necrosis virus (NNV, Betanodavirus genus) is the causative agent of the viral nervous necrosis, the main viral disease affecting European sea bass. Betanodavirus genome is composed of two single-stranded, positive-sense segments: RNA1 and RNA2, and this virus has been classified into four species, being RGNNV and SJNNV the viral species most frequently detected in the Mediterranean area. Numerous studies have pointed out the importance of the host immune response to defeat betanodavirus infections, highlighting the relevant transcription of a new virus responsive gene, rtp3. This gene encodes the RTP3 protein, which belongs to the receptor-transporting protein (RTP) family. Previously, our research group has described two rtp3 genes within the genome of European sea bass (rtp3X1, rtp3X2). The aim of the present study is to analyse the role of sea bass RTP3 proteins against betanodavirus infections, performing an in vivo transcription analysis and evaluating the RTP3 anti-NNV activity using an in vitro approach. The transcription profile of rtp3X1 and X2 was analysed by qPCR in sea bass injected with LPS, poly I:C, or RGNNV. The results revealed significant rtp3X1 and X2 transcription after LPS and poly I:C inoculation, although the values obtained for rtp3X2 were higher than those recorded for rtp3X1. The highest transcription of both rtp3 genes was induced by RGNNV infection, recording upregulation of rtp3X2 extremely high in brain. The anti-NNV activity was evaluated against RGNNV and SJNNV infections, inoculating E-11 cell lines permanently expressing the sea bass RTP3 proteins and quantifying viral replication in those cells, using E-11 cells as control. In RTP3 X1-expressing cells, the number of RGNNV RNA1 and RNA2 copies were significantly higher compared with values detected in E-11 cells, whereas in RTP3 X2-expressing cells a significant reduction of both viral segments was recordedUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NNV virulence-dependent immunogene response in European sea bass

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinant of RGNNV virulence to European sea bass.This study has been supported by the project AGL2017-84644-R (MINECO/AEI/FEDER, UE) (Spanish Government)

Shewanella putrefaciens Pdp11 extracts protect against betanodavirus infection.

Viral nervous necrosis is caused by the nervous necrosis virus (Betanodavirus genus), a naked virus with two positive-sense RNA segments. Betanodaviruses are classified into four species, being RGNNV predominant in the Mediterranean area. RGNNV causes high mortality in several fish species, including European seabass. There are two vaccines to protect seabass against RGNNV infection. In this regard, the development of strategies to protect different fish species against different viruses, such as the use of probiotics, is a key issue for the aquaculture industry. Shewanella putrefaciens Pdp11, SpPdp11, is a fish probiotic with proven positive effects on seabream and Senegalese sole, protecting those species against bacterial pathogens; however, its antiviral activity is unknown. This study evaluates the anti-RGNNV activity of sonicated-SpPdp11 extracts in vitro and in vivo. The in vitro evaluation was performed on E11 cells following 3 assays: (i) neutralization, (ii) 6-h pre-adsorption, (iii) post-adsorption, determining the inhibition of RGNNV-induced CPEs and quantifying viral replication. The immunostimulatory activity of SpPdp11 extracts was also examined, analysing the transcription of mx, hsp70, tnfα, e3 and tlr3 in E11 cells. For the in vivo evaluation, two European seabass groups were established: (i) control group, (ii) experimental group, fed with commercial pellet supplemented with SpPdp11 extracts. Animals were fed for 30 days and subsequently challenged by intramuscular injection. SpPdp11 extracts compromised RGNNV replication in E11 cells (67.3% and 55% CPE inhibition in pre-adsorption and post-adsorption assays, respectively), and modulated the transcription of all the immune-related genes examined. The highest induction was obtained for mx gene. Regarding the in vivo results, 82% of fish fed with the SpPdp11-supplemented diet survived to RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%.P18-RT-1067 (Proyecto de Excelencia, Junta de Andalucia), PID2020-113637RB-C22 (MINECO/AEI/FEDER, UE). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Inhibition of nervous necrosis virus replication by Shewanella putrefaciens Pdp11 extract

One of the most relevant infectious diseases affecting farmed fish is the viral encephalopathy and retinopathy, caused by the nervous necrosis virus (NNV). The two commercial vaccines available against this virus have been designed to protect European seabass against the infection with a particular NNV species, RGNNV. Therefore, the development of strategies to protect different fish species against different viruses is a key issue for the aquaculture industry. In this regard, there are numerous studies focused on the use of probiotics, although their antiviral activity has been poorly investigated. Shewanella putrefaciens Pdp11, SpPdp11, is a probiotic with proven positive effects on gilthead seabream and Senegalese sole, protecting those species against several bac- terial pathogens; however, its antiviral activity remains to be investigated. The current study is a step forward in the use of probiotics against viral infections, evaluating the anti-RGNNV activity of sonicated-SpPdp11 extracts both, in vitro and in vivo. According to our results, SpPdp11 extracts compromised RGNNV multiplication in E11 cells, affecting viral assembly and/or exit rather than genome replication. Furthermore, this antiviral activity may be produced by the capacity of the SpPdp11-extract to induce an immune response in treated cells. This antiRGNNV activity has been confirmed in vivo, since 82% of fish fed with the SpPdp11-supplemented diet survived an experimental-RGNNV infection, whereas the survival rate of fish fed with the control diet was 64%. These results suggest that SpPdp11-supplemented feeding can be a promising prophylactic tool against RGNNV and encourage further research on other fish species and viral pathogens.This study has been supported by the projects P18-RT-1067 (Proyecto de Excelencia, Junta de Andalucia, Regional Government) and PID2020-113637RB-C22 (MINECO/AEI/FEDER, UE) (Spanish Government). Daniel Álvarez-Torres has been granted by the project PTA2020-018984-I. Funding for open access charge: Universidad de Málaga/CBUA

Juvenile gilthead sea bream immune response against RGNNV or RGNNV/SJNNV infections

Viral nervous necrosis is one of the main threats for the aquaculture. It is caused by the nervous necrosis virus (NNV), with a genome composed of two positive-sense, ssRNA segments: RNA1, encoding the viral polymerase; and RNA2, encoding the capsid protein. NNV have been clustered into four species:RG, SJ, BF and TP. In addition, reassortment between RG and SJ has also been detected. NNV can affect a wide range of fish species; however, not all viral species are equally virulent to all fish species; this issue is relevant concerning European sea bass and gilthead sea bream, which are frequently co-cultured. Sea bass is susceptible to RG, whereas RG/SJ cause low mortality in this species. On the contrary, RG/SJ cause high mortality in sea bream and this fish is reluctant to RG infections. The outcome of viral infections depends on the specific virus-host interaction. Understanding the mechanisms responsible for this differential interaction is crucial to control viral diseases in aquaculture. This study focuses on the analysis of the immune gene transcription in brain of 3g sea bream experimentally infected with NNV isolated from sea bass (Dl, RG) or sea bream (Sa, RG/SJ). We have analysed genes suggested to be relevant in controlling RG infection in sea bass: inflammatory, apoptotic, stress and IFNI. Mortalities were not recorded in any group for 30 days, and the quantification of viral genome evidenced that only Sa replicated in sea bream brain. Principal component analysis clustered samples according to the viral isolate from 1 day post-infection onwards, and evidenced differences in the immune response against both viruses. Sea bream response against Dl is characterized by a higher rtp3 transcription early after the infection, a longer-lasting transcription of the antinflammatory gene il10 and a stronger induction of casp1 and hsp70. These genes should be targets for future studies in order to elucidate their role in hampering the replication of RG in sea breamAgencia Estatal de Investigación. Ref. PID2020-115954RB-I00 Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Shewanella putrefaciens Pdp11 extracts promote European sea bass resistance against RGNNV infection.

The viral nervous necrosis (VNN) is a neurotropic disease affecting different fish species, resulting in high mortality rates. The causal agent is the nervous necrosis virus (NNV), belonging to the Betanodavirus genus, Nodaviridae family. Betanodaviruses are classified into four species; however, commercial vaccines are only available for RGNNV, one of the most predominant viral species isolated in the Mediterranean area. Since preventive measures against NNV infections are scarce, the development of different strategies to avoid the disease is highly relevant. Functional diets play an important role, and, in this context, the addition of probiotics to fish feed has been reported to provide several benefits to fish health. However, there are safety concerns regarding feed supplementation with live bacterial cells. In this regard, the use of non-viable probiotics it is of great interest. Shewanella putrefaciens Pdp11, SpPdp11, is a probiotic isolated from healthy gilthead seabream, which has been described to promote antibacterial activity; however, its antiviral activity remains poorly understood. Recently, our research team evaluated the anti-RGNNV effect of SpPdp11 sonicated extracts in vitro. In the present study, the effect of feed supplementation with sonicated extracts of SpPdp11 has been evaluated in European sea bass, as well as its effect against an RGNNV challenge. Animals were fed for 30 days with the supplemented diet and the results were compared with a control diet. Changes in the intestinal microbiota and in the transcription of immune genes were assessed at 7 and 30 days within the feeding period. Animals fed with the supplemented diet show higher levels of prokaryotic diversity; however, these differences were not statistically significant. Regarding transcriptional analyses, only 8 of 56 genes were differentially transcribed in fish fed with the supplemented diet, all of them downregulated.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech