Evaluation of immune response after LCDV-Sa infection in DNA-vaccinated gilthead seabream

The immune-related gene expression in vaccinated gilthead seabream after Lymphocystis Disease Virus 3 (LCDV-Sa) infection was analysed by using an OpenArray based on TaqMan qPCR. The DNA vaccine used in this study encodes the viral major capsid protein and confers protection against LCDV-Sa infection in juvenile gilthead seabream. Gilthead seabream juveniles were distributed into four experimental groups and intramuscularly injected with the vaccine (vaccinated group), the empty-plasmid (mock-vaccinated group), or PBS (control groups). Thirty days after vaccination, vaccinated and mock-vaccinated fish, as well as one of the control groups, were injected intraperitoneally with LCDV-Sa (106 TCID50/fish). Samples of head-kidney (HK) from 6 fish were individually collected 1 and 3 days post-infection (dpi). The relative expression levels of 49 genes related to the immune response and 4 reference genes were analysed using an OpenArray. Samples from the non-infected control group were used as calibrator. The number of genes differentially expressed (DEG) in HK at 1 dpi was higher in vaccinated fish compared with both mock-vaccinated and non-vaccinated animals. At 3 dpi, most DEG were upregulated, and the differences in their number among groups were minimized. The recombination-activating gene 1 (rag1), a mediator of development of B and T lymphocytes, was the only gene upregulated in HK samples at 1 dpi. This gene was also upregulated in non-vaccinated animals but at 3 dpi. In contrast, early mx induction was observed in non-vaccinated animals (upregulation of mx2 at 1 dpi) in comparison to vaccinated seabreams (upregulation of mx1 and mx2 at 3 dpi). The results that will be discussed could evidence the role of the DNA vaccine as regulator of the primary lymphoid tissues (HK) in gilthead seabream against LCDV-Sa infection, through downregulation of inflammation related-genes, early upregulation of rag1, and a later expression of interferon stimulated genes.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Immune response of vaccinated juvenile gilthead seabream (Sparus aurata) after LCDV-Sa infection

Lymphocystis disease is one of the main viral pathologies affecting cultured gilthead seabream in the Mediterranean area. In our group, a DNA-vaccine has been developed based on the major capsid protein (MCP) of the Lymphocystis Disease Virus 3 (LCDV-Sa). The aim of the present study is the evaluation of immune-related gene expression in vaccinated fish after viral infection to identify immunogenes involved in the vaccine-induced protection. To fulfil this objective an OpenArray® platform has been developed to study 49 genes related to the immune response. Reference and viral genes were also evaluated. Gilthead seabream specimens (5 g mean weight) were distributed into 3 experimental groups, inoculated with the vaccine at 0.1 µg/g fish dose, the empty plasmid at the same dose or PBS. Thirty days post-vaccination, fish were intramuscularly injected with the virus at 106 TCID50/fish dose. Samples of head-kidney, spleen, intestine and caudal fin from 6 fish were individually collected at 1, 2 and 3-days post-injection in all groups. The quantification of viral DNA in fins of fish challenged with LCDV-Sa were carried out by a qPCR assay targeting a viral structural gene (putative myristoylated membrane protein, MMP) alternative to the mcp gene contained in the vaccine. The results obtained showed an increase of genes deregulated within the haematopoietic organs between vaccinated and non-vaccinated fish. However, in the intestine and fin, the results showed the opposite trend. The global effect of fish vaccination was a diminished immune response compared to non-vaccinated fish, being 83 and 99 genes differentially expressed through the experiment, respectively. Moreover, viral replication decreased in groups of fish previously vaccinated. The modulation of the immune response provoked by the vaccination trial seems to control the progression of the disease.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Senegalese sole immune response against betanodavirus recombinants harboring modifications in the 3' terminal region of the RNA1

The nervous necrosis virus (NNV) is the etiological agent of the viral nervous necrosis (VNN), a disease affecting a high number of fish species worldwide. NNV genome is composed of two segments RNA1 and RNA2, encoding the RNA-dependent RNA polymerase and the capsid protein, respectively. NNV has been classified into four species: SJNNV, TPNNV, RGNNV and BFNNV. Furthermore, reassortants between RGNNV and SJNNV have been reported, such as wt160 isolated from Senegalese sole, which presents a RGNNV-RNA1 and a SJNNV-RNA2 type segments, and causes 100% mortality in this fish species. This isolate exhibited differences in the 3’ NCR of both genomic segments when compared to the reference strains of each genotype. In this study, the effect on virulence of the substitutions observed in the 3’NCR of the wt160-RNA1 has been evaluated, by the development of two recombinants harbouring mutations at position 3073 and 3093, which make the wt160-RNA1 similar to the reference RGNNV. Moreover, immune response of sole against the infection with these recombinants compared to the wild-type, has been evaluated using an OpenArray. The infection with the recombinants r3073 and r3093 decreased the mortality to 29.3% and 25.3%, respectively. Furthermore, the number of DEGs was higher at 3 days than at 2 days p.i., after the infection with the three viruses, being the number of DEG quite similar among viruses. Significant differences between DEG fold changes after infection with the mutants and the wt160 will be discussed. It should be highlighted that at 2 days p.i., the gene gig1 was not expressed after the infection with r3073 and r3093. However, at 3 days p.i. this gene was expressed at the highest level after the infection with the three viruses. Moreover, the infection with the wt160 induced the down-regulation of the genes gilt and magel2, which was not observed after the infections with r3073.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Actividad antagonista de aislados del Virus de la Septicemia Hemorrágica Viral (VHSV) frente al sistema del interferón tipo I de lenguado senegalés

Senegalese sole (Solea senegalensis) is susceptible to marine Viral Haemorrhagic Septicaemia Virus (VHSV) isolates, but it is not affected by freshwater VHSV isolates. In addition, the sole type I interferon (IFN I) response is lower after infection with a marine VHSV isolate than in response to a freshwater isolate. In order to disclose the reasons of such differential response, in this study, the antagonistic activity of both kinds of VHSV isolates against IFN I system was characterised using an in vitro experimental system consisting of RTG-2 cells stably transfected with the luciferase gene under the control of the Senegalese sole mx promoter, which is one of the most induced interferon-stimulated genes. Our results showed that both isolates exert a dose-dependent negative effect on the response triggered by type I interferon, acting in the signal cascade pathway induced by IFN I, since the transcription of the gene coding for this cytokine is not affected. However, much higher levels of the non-pathogenic freshwater isolate were necessary to detect such antagonistic activity. Therefore, the inefficient antagonistic activity of the freshwater VHSV isolate might be involved in the lack of virulence of this isolate to Senegalese sole. Resumen El lenguado senegalés (Solea senegalensis) es susceptible a aislados marinos del Virus de la Septicemia Hemorrágica Viral (VHSV), pero no a aislados de agua dulce, frente a los cuales, además, hay una respuesta del sistema del interferón tipo I (IFN I) más intensa. Para averiguar las razones de esta respuesta diferencial, el objetivo que se plantea para el presente estudio es caracterizar y comparar la actividad antagonista de ambos aislados de VHSV frente al sistema del IFN I. Con este fin se utilizó un sistema experimental in vitro consistente en células RTG-2 transfectadas de forma estable con el gen de la luciferasa bajo el control del promotor del gen mx, que es uno de los genes inducidos por IFN I que más se estimulan en respuesta a infecciones víricas. Los resultados obtenidos muestran que ambos aislados de VHSV producen una interferencia negativa sobre la respuesta desencadenada por el IFN I. Dicha interferencia ocurre tras la síntesis de IFN I, y es dependiente de la dosis vírica. Sin embargo, para detectarla es necesario infectar las células con una dosis mucho más alta del aislado de agua dulce que del marino. Por lo tanto, la ineficiente actividad antagonista del aislado de agua dulce podría estar implicada en la no virulencia de este tipo de aislados en lenguado.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Estudio in vitro de la actividad antagonista de aislados del virus de la septicemia hemorrágica viral (VHSV) de distinto origen sobre el sistema del IFN tipo I

El lenguado senegalés (Solea senegalensis) es susceptible a aislados marinos del virus de la septicemia hemorrágica viral (VHSV); sin embargo, los VHSV patógenos para especies de agua dulce no son virulentos para especies marinas. Esta diferencia puede deberse al mecanismo antagonista que presenta cada tipo de aislado frente al sistema del interferón tipo I (IFN I). El objetivo de este estudio es caracterizar y comparar la actividad antagonista de dos aislados de VHSV de distinto origen. Para ello, se desarrolló un sistema experimental in vitro, consistente en células RTG-2 (trucha arcoíris), transfectadas con el gen de luciferasa bajo el control del promotor mx de lenguado, un efector antiviral estimulado por IFN I. Ambos aislados mostraron actividad antagonista dependiente de la multiplicidad de infección (MOI) y de la virulencia del virus en lenguado. Así, el aislado marino antagoniza a una MOI menor que el de agua dulce. Además, el antagonismo del marino ocurre exclusivamente a nivel del promotor mx de lenguado, no a nivel de transcripción de ifn I, ni de la cascada de activación de los genes estimulados por interferón. Por el contrario, el aislado de agua dulce también antagoniza la transcripción de genes mx endógenos (de trucha), pero no la transcripción de ifn I. El gen viral nv, implicado en la actividad antagonista, mostró un patrón de transcripción idéntico al del n (nucleoproteína) para el aislado de agua dulce, mientras que el nv marino inicia la transcripción más tarde que n. En cambio, en la línea celular de origen marino SAF-1, no se detecta este retraso en la transcripción de nv del aislado marino. El comportamiento diferencial de VHSV en función del origen del aislado y de la línea celular es un interesante aspecto a considerar en el estudio de la interacción VHSV-hospedador.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Immunogene expression analysis in betanodavirus infected-Senegalese sole using an OpenArray® platform.

The transcriptomic response of Senegalese sole (Solea senegalensis) triggered by two betanodaviruses with different virulence to that fish species has been assessed using an OpenArray® platform based on TaqMan™ quantitative PCR. The transcription of 112 genes per sample has been evaluated at two sampling times in two organs (head kidney and eye/brain-pooled samples). Those genes were involved in several roles or pathways, such as viral recognition, regulation of type I (IFN-1)-dependent immune responses, JAK-STAT cascade, interferon stimulated genes, protein ubiquitination, virus responsive genes, complement system, inflammatory response, other immune system effectors, regulation of T-cell proliferation, and proteolysis and apoptosis. The highly virulent isolate, wSs160.3, a wild type reassortant containing a RGNNV-type RNA1 and a SJNNV-type RNA2 segments, induced the expression of a higher number of genes in both tested organs than the moderately virulent strain, a recombinant harbouring mutations in the protruding domain of the capsid protein. The number of differentially expressed genes was higher 2 days after the infection with the wild type isolate than at 3 days post-inoculation. The wild type isolate also elicited an exacerbated interferon 1 response, which, instead of protecting sole against the infection, increases the disease severity by the induction of apoptosis and inflammation-derived immunopathology, although inflammation seems to be modulated by the complement system. Furthermore, results derived from this study suggest a potential important role for some genes with high expression after infection with the highly virulent virus, such as rtp3, sacs and isg15. On the other hand, the infection with the mutant does not induce immune response, probably due to an altered recognition by the host, which is supported by a different viral recognition pathway, involving myd88 and tbkbp1.Funding for open access charge: Universidad de Málaga / CBUA. This work has been supported by the projects AGL2014-54532-C2 and RTI2018-094687-B-C22 form the Spanish Government (Ministerio de Economía y Competitividad), co-funded by the FEDER

Immune response of gilthead seabream infected with wild and mutant nervous necrosis virus reassortants.

Viral nervous necrosis (VNN) is a disease that affects farmed fish worldwide. Its etiologic agent is the nervous necrosis virus currently classified into 4 species: SJNNV, RGNNV, TPNNV and BFNNV. In Southern Europe, reassortants between RGNNV and SJNNV have been isolated from VNN outbreaks affecting Senegalese sole and gilthead seabream. The aim of the present study is to identify differentially expressed genes (DEGs) involved in gilthead seabream response against different NNV reassortants. Gilthead seabream juveniles were challenged by intramuscular injection using the wild-type (wt) strain Ss160.03, a highly virulent reassortant isolated from sole, and an attenuated mutant of this strain. Head-kidney and brain samples were collected at 24, 48 and 72 h post-challenge (hpc). Viral multiplication was analysed by qPCR, and the evaluation of the immune response against the infection was carried out using an OpenArray® with 56 gene targets. At the end of the experiment, viral multiplication in seabream was higher for the wt reassortant. The number of DEGs detected through the experiment was also higher in the wt-infected animals, being the kinetic of expression different between the organs analysed (early deregulation of genes in head-kidney compared to belated expression in brain). The main differences in gene expression found between fish infected with both reassortants occurred at 72 hpc, being all DEGs up-regulated in wt-infected fish, compared to the downregulation observed within the mutant-infected animals. In addition, the expression of toll-like receptor 9 (tlr9), interleukin 1 beta (il1β), and interferon (ifn) was detected exclusively in brain samples at 72 hpc, and the endothelial leukocyte adhesion molecule (elam) in head-kidney samples at the same time point. The different profiles of gene regulation detected at 72 hpc in gilthead seabream infected with both viruses could be related to the lower multiplication of the mutant strain.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Proyecto RTI2018-094687-B-C22, MCIUI y FEDER

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