Chitosan Treatment of E-11 Cells Modulates Transcription of Nonspecific Immune Genes and Reduces Nodavirus Capsid Protein Gene Expression.

This study explores whether crustacean products inhibit viral infections in aquaculture. Chitosan (CHT) was extracted from waste products of Parapenaeus longirostris. Biochemical composition, viscosity measurement, molecular weight, structure and cytotoxicity tests were used to characterize the extracted chitosan. Cultures of E-11 cells derived from snakehead Ophicephalus striatus were inoculated with 106.74 TCID50 of an isolate of betanodavirus genotype RGNNV (redspotted grouper nervous necrosis virus) after being treated with solutions of 0.3% CHT for 1 h at room temperature. The antiviral effect of CHT was assessed by comparing the ability of RGNVV to replicate and produce cytopathic effects on CHT-treated cell cultures. The change in RNA expression levels of the nodavirus capsid protein gene and three mediator genes in infected cells with or without CHT treatment was evaluated by qPCR. Changes in gene expression compared to control groups were monitored at 6, 24, 48 and 71 h post treatment in all target gene transcripts. The CCR3 expression in CHT treated cells showed a significant increase (p < 0.05) until day 3. On the other hand, the expression of TNF-α decreased significantly (p < 0.05) in CHT treated cells throughout the experimental period. Likewise, the expression of the IL-10 gene showed a significant downregulation in CHT treated cells at all time points (p ≤ 0.05). As further evidence of an antiviral effect, CHT treatment of cells produced a reduction in virus load as measured by a reduced expression of the viral capsid gene and the increase in RQ values from 406 ± 1.9 at hour 1 to 695 ± 3.27 at 72 h post inoculation. Statistical analysis showed that the expression of the viral capsid gene was significantly lower in cells treated with chitosan (p ≤ 0.05). These results improve our knowledge about the antiviral activity of this bioactive molecule and highlight its potential use in fish feed industry.

Phylogeography of betanodavirus genotypes circulating in Tunisian aquaculture sites, 2012-2019.

The purpose of this study was to determine the phylogenetic relationships among the primary betanodavirus strains circulating in Tunisian coastal waters. A survey was conducted to investigate nodavirus infections at 15 European sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata farming sites located along the northern and eastern coasts of Tunisia. The primary objective of the study was to create epidemiological awareness of these infections by determining phylogenetic relationships between the main betanodavirus strains circulating during the period 2012-2019, using RNA1 and/or RNA2 genome segments. Approximately 40% (118 of 294) tissue pools tested were positive for betanodavirus. Positive pools were distributed across all of the sampling sites. While fish mortalities were always correlated with the presence of virus in sea bass, a severe outbreak was also identified in sea bream larvae in 2019. Phylogenetic analysis revealed that almost all Tunisian strains from both sea bass and sea bream irrespective of outbreaks clustered within the RGNNV genotype. It is noteworthy that samples collected during the 2019 outbreak from sea bream contained both RNA1 and RNA2 fragments belonging to the RGNNV and SJNNV genotype, respectively, an indication of viral genome reassortment. To our knowledge, this is the first report of reassortant betanodavirus in Tunisia.

Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates.

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Detection of Sapoviruses in two biological lines of Tunisian hospital wastewater treatment.

The efficiency of rotating biodisks and natural oxidizing lagoon procedures is investigated at a Tunisian semi-industrial pilot plant, El Menzeh I, where the wastewater is mainly provided by three different neighbouring hospital clinics. Throughout 2011, 102 wastewater samples were collected from the two mentioned wastewater treatment procedures. Results showed that the Sapovirus (SaV) frequency was approximately 29.4% using the real-time reverse transcription polymerase chain reaction (RT-PCR) technique, and about 16.6% using the conventional RT-PCR. Also, the SaV genogroups and genotypes were identified and genotyping revealed that all of the four Tunisian SaV strains obtained belonged to the two genogroups GIV.1 and GGI.3. In addition, two new genotypes, D and C, were detected. A moderate decrease in the SaV frequencies was observed at the exit of the two treatment processes and the SaV removal rate was around 90% in the natural oxidizing lagoons and 94% in the rotating biodisks procedure showing the temperate sensitivity of these viruses to the implemented biological wastewater. Therefore, an urgent disinfection process should be implemented downstream of the two biological treatment procedures for safe release of treated effluent in the different natural environments. Abbreviations: NoV: Noroviruses; SaV: Sapoviruses; EC: Electrical Conductivity; COD: Chemical Oxygen Demand; BOD5: Biological Oxygen Demand; SS: Suspended Solids; NH4-N: Ammonium Nitrogen; P-PO4: Ortho-Phosphate; AlCl3: aluminum chloride.

Viral encephalopathy and retinopathy of Dicentrarchus labrax and Sparus aurata farmed in Tunisia.

Viruses belonging to the Nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. In the present study, a total of 69 farmed Tunisian sea bass (Dicentrarchus labrax) and 24 sea bream (Sparus aurata) samples were tested monthly for the detection of betanodavirus. The virus was identified in both species using indirect immunofluorescence assays (IFAT) and RT-PCR. In addition sequence analysis of part of the coat protein gene indicated that both species were infected by highly related, but distinct, strains belonging to the RGNNV genotype. The sequence of the coat protein gene of several strains was identical but up to 9 different sequences were detected in a single farm. In addition, viral sequences obtained from fish that were held at lower temperature (<20 degrees C) were distinct from the rest of the sequences.