THE FIRST OUTBREAK OF VIRAL ENCEPHALOPATHY AND RETINOPATHY IN FARMED SEA BASS (Dicentrarchus labrax) IN SLOVENIA

Viral encephalopathy and retinopathy (VER) is considered a serious disease of several marine fish species, caused by RNA virus belonging to the family Nodaviridae, genus Betanodavirus. The disease is spread almost worldwide and causes significant losses among diseased fish. It is characterised by vacuolation of the central nervous system and the retina. In July 2018, behavioural abnormalities i.e. altered swimming, swirling and vertical floating as well as lethargy and anorexia were observed in farmed sea bass (Dicentrarchus labrax) in the Gulf of Piran (Slovenia), associated with significant mortality. The disease initially occurred in juvenile sea bass, but later market-sized fish also became affected. Diseased fish displayed ocular opacity and multifocal skin ulceration on the head. Emaciation in some fish was also evident. Histopathology revealed characteristic vacuolation in the brain and retina. Performing a RT-PCR and RT-qPCR techniques, we have identified and confirmed the presence of betanodavirus nucleic acid in ocular and brain tissues. In addition, concentrations of the causative agent of VER in spleen and kidney did result in significantly higher viral yield than expected. Phylogenetic analysis showed that Slovenian isolate belongs to RGNNV species of betanodaviruses. Based on the clinical signs, gross and typical microscopic lesions and results of molecular analyses, we can conclude that farmed sea bass from the Gulf of Piran were affected with VER. To the best of our knowledge, this is the first report of VER in Slovenia.Key words: viral encephalopathy and retinopathy; betanodavirus; sea bass; histopathology; RT-qPCR PRVI IZBRUH VIRUSNE ENCEFALOPATIJE IN RETINOPATIJE PRI GOJENIH BRANCINIH (Dicentrarchus labrax) V SLOVENIJI Izvleček: Virusna encefalopatija in retinopatija (VER) je nevarna bolezen številnih vrst morskih rib, ki jo povzroča nevrotropni RNA virus iz družine Nodaviridae, rod Betanodavirus. Bolezen je razširjena skoraj po vsem svetu in povzroča visok pogin okuženih rib. Zanjo so značilne vakuole v centralnem živčnem sistemu in retini. Konec julija 2018 so v ribogojnici v Piranskem zalivu pri brancinih opazili nepravilno plavanje, vrtenje in postavljanje v vertikalno smer ter letargijo in neješčnost, brancini so množično poginjali. Bolezen se je najprej pojavila pri mladicah, nato tudi pri konzumnih kategorijah brancinov. Obolele ribe so imele sivo-motna očesna zrkla ter multifokalne kožne razjede na glavi, posamezne so bile shujšane. S histopatološko preiskavo smo ugotovili značilne vakuole v možganih in retini. Z molekularnima metodama RT-PCR in RT-qPCR smo potrdili prisotnost nukleinske kisline betanodavirusa v očesnem zrklu in možganih. Koncentracije virusa, ki so bile signifikantno višje od pričakovanih, smo ugotovili tudi v vranici in ledvicah. Na podlagi kliničnih znakov, makroskopskih in tipičnih histopatoloških sprememb ter rezultatov molekularnih preiskav lahko zaključimo, da so gojeni brancini v ribogojnici v Piranskem zalivu zboleli za VER. Opisani izbruh je prvi potrjeni primer te bolezni v Sloveniji.Ključne besede: virusna encefalopatija in retinopatija; betanodavirus; brancin; histopatologija; RT-qPCR

Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.

Immune Response of Senegalese Sole against Betanodavirus Mutants with Modified Virulence

Nervous necrosis virus (NNV), genus Betanodavirus, the etiological agent of the viral encephalopathy and retinopathy (VER), presents a genome with two positive-sense single-stranded RNA segments. Striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV), together with reassortants RGNNV/SJNNV, are the betanodaviruses predominantly isolated in Southern Europe. An RGNNV/SJNNV reassortant isolated from Senegalese sole (wt160) causes high mortalities in this fish species. This virus presents differences in the sequence of the 3’ non-coding region (NCR) of both segments compared to RGNNV and SJNNV reference strains. Previously, it has been reported that the reversion of two of these differences (nucleotides 1408 and 1412) in the RNA2 3’NCR to the SJNNV-type (recombinant r1408-1412) resulted in a decrease in sole mortality. In the present study, we have applied an OpenArray® to analyse the involvement of sole immune response in the virulence of several recombinants: the r1408-1412 and two recombinants, developed in the present study, harbouring mutations at positions 3073 and 3093 of RNA1 3’NCR to revert them to RGNNV-type. According to the correlation values and to the number of expressed genes, the infection with the RNA2-mutant provoked the most different immune response compared to the immune response triggered after the infection with the rest of the viruses, and the exclusive and high upregulation of genes related to the complement system. The infection with the RNA1-mutants also provoked a decrease in mortality and their replication was delayed at least 24 h compared to the wt160 replication, which could provoke the lag observed in the immune response. Furthermore, the infection with the RNA1-mutants provoked the exclusive expression of pkr and the downregulation of il17rc.

Betanodavirus and VER Disease: A 30-year Research Review

The outbreaks of viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), represent one of the main infectious threats for marine aquaculture worldwide. Since the first description of the disease at the end of the 1980s, a considerable amount of research has gone into understanding the mechanisms involved in fish infection, developing reliable diagnostic methods, and control measures, and several comprehensive reviews have been published to date. This review focuses on host–virus interaction and epidemiological aspects, comprising viral distribution and transmission as well as the continuously increasing host range (177 susceptible marine species and epizootic outbreaks reported in 62 of them), with special emphasis on genotypes and the effect of global warming on NNV infection, but also including the latest findings in the NNV life cycle and virulence as well as diagnostic methods and VER disease control.