Synthetic Peptides Suppress Nervous Necrosis Virus Absorption and Improve Survival Rates in European Sea Bass

Abstract

With few preventive strategies available against nodavirus (NNV) in aquaculture, therapeutic applications remain underexplored. This study aimed to peptide-based treatments disrupting critical stages of its viral life cycle. Thus, we designed and synthesized seven low-molecular-weight peptides (P1-P7) based on predicted binding regions of the capsid protein from the red-spotted grouper nervous necrosis virus (RGNNV) genotype to mimic viral capsid regions. Although in silico predictions suggested limited direct antiviral activity, in vitro assays using the E-11 cell line and in vivo trials in RGNNV-infected European sea bass (Dicentrarchus labrax) juveniles yielded promising results. The peptides, particularly when co-administered individually or as P3 + P4 and P5 + P6 combinations with the virus, disrupted RGNNV attachment in vitro. Moreover, they exhibited cross-reactivity against the striped jack nervous necrosis virus (SJNNV) genotype and both RGNNV/SJNNV and SJNNV/RGNNV reassortants. Treatment of RGNNV-infected sea bass significantly increased the relative percent survival, ranging from 81.3% for P4 to 62.5% for P3 and P3 + P4, while reducing viral load within 48 h post-treatment without altering systemic antiviral immune responses, tested through the transcriptional levels of mx gene in the head-kidney. Notably, peptide P4 partially inhibited viral replication in vitro at the same time-point when cells were pre-treated for 24 h, likely through modulation of host immune responses. These findings highlight the potential of targeted peptide-based therapies as a promising antiviral therapeutic strategy against NNV infections.

Keywords: Adsorption; Betanodavirus; European sea bass; Replication; Synthetic peptides; Viral cycle.

Fig. 1

Tridimensional structure of the RGNNV capsid protein under the homotrimeric model highlighting potential binding regions used for the P1 to P7 peptide synthesis. The model was constructed through the SwissModel server and analysed with Chimera as previously described (Cárdenas et al. 2020)

Fig. 2

Designed peptides, single or combined, reduce the NNV absorption in vitro. NNV parental or reassortant genotypes were incubated with 500 μg/mL of the synthetic peptides for 15 min. Then, the viruses were titrated in the E − 11 cell line. Control samples consisted on virus incubated with the same amount of phosphate buffer saline. (A) Viral titer (TCID₅₀/mL) and (B) % of virus inhibition with respect to the controls are represented. Data are representative of two independent assays. RGNNV, red-spotted grouper nervous necrosis virus parental strain; SJNNV, striped jack nervous necrosis virus parental strain; RGNNV/SJNNV and SJNNV/RGNNV reassortant strains (RNA1/RNA2 from parental strains)

Fig. 3

The pre-treatment with P4 synthetic peptide partially disrupts the replication of RGNNV in vitro. (A) Intracellular replication kinetics; (B) Viral production in cell supernatants; and (C, D) Genome synthesis in cell lysates by means of transcriptional levels of rdrp (C) and cp (D) genes. Monolayers of E-11 were incubated with 500 µg/mL of P4 or untreated for 24 h and then infected with red-spotted orange grouper nervous necrosis virus (RGNNV) (n = 3). Supernatants and cell lysates were collected at 8, 12, 24, 48 h post-infection (hpi). Additionally, supernatants were sampled at 168 hpi for extracellular replication kinetic analysis. Infectious titers were determined by the end-point titration method (Reed and Müench 1938) and expressed as mean Log₁₀ TCID₅₀/mL ± standard error of the mean (SEM). Relative expression was presented as the mean ± SEM (n = 3). Asterisks indicate significant differences among groups according to the Student-t test (p < 0.05)

Fig. 4

Therapeutic administration of synthetic peptides improves survival and symptomatology in red spotted orange nervous necrosis virus (RGNNV)-infected European sea bass juveniles. Fish were infected by bath with RGNNV (3.16 × 10⁴ TCID₅₀/mL) for 2 h and immediately intramuscularly injected with PBS (RGNNV group) or P2, P3, P4, P7, P3 + P4 or P5 + P6 synthetic peptides (1 μg peptide/g of fish). A mock-infected group was treated twice with only PBS. (A) Kaplan–Meier survival curves; (B) Heat-map representing the cumulated number of fish showing clinical signs of VER disease attending to their severity: (1) changes of the color of the skin, slower rhythm of swimming and/or slower reaction to external stimuli as feeding; (2) alterations in the swimming balance and/or erratic swimming spasms; (3) continuous erratic swimming; and (4) complete incapacity to keep balance, swim and/or move without external stimuli. VER, viral encephalopathy and retinopathy

Fig. 5

Therapeutic administration of synthetic peptides decreased viral loads in the brain without additional mx gene regulation in the sea bass juveniles head-kidney infected with red-spotted grouper nervous necrosis virus (RGNNV). Fish were infected by bath with RGNNV (3.16 × 10⁴ TCID₅₀/mL) for 2 h and immediately intramuscularly injected with PBS (RGNNV group) or P2, P3, P4, P7, P3 + P4 or P5 + P6 synthetic peptides (1 μg peptide/g of fish). A mock-infected group was treated twice with only PBS. (A–C) Transcription levels of RGNNV cp (A) and rdrp (B) genes in the brain and (C) mx gene in the head-kidney. Data represent the relative gene expression mean ± SEM (n = 6). Different letters indicate significant differences among groups according to the ANOVA (p < 0.05)