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

SIMPLE SUMMARY: Viral Encephalopathy and Retinopathy (VER) is caused by an RNA virus named Betanodavirus representing a major problem in Mediterranean marine aquaculture resulting in great economic losses. Control of VER currently relies on effective biosecurity measures and vaccines. Natural non-toxic biopolymers such as chitosan are produced commercially from crab and shrimp shell waste offering useful biomolecules for a wide range of applications for their antimicrobial and antioxidant activity, immunostimulant effect and bioactive coating. This study evaluates the in-vitro antiviral activity of chitosan extracted from the exo-skeleton of Parapenaeus longirostris, focusing on its ability to modulate the innate immune response in E-11 cells challenged with nodavirus as well as its impact on nodavirus RNA2 gene expression. Using qPCR, results reviewed here demonstrated a modulation of the innate immune gene expression and a reduction in virus load suggesting that chitosan was found to be a suppressor of nodaviral infection in fish cell systems. ABSTRACT: 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 10(6.74) TCID(50) 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.