Antimicrobial peptides as model molecules for the development of novel antiviral agents in aquaculture.

Antimicrobial peptides (AMPs) are one of the components of the non-specific immune system that operate first lines of protection in many animal species including fish. They exert broad-spectrum antimicrobial activity, apart from many other potential roles in innate immunity, and represent a promising class of antiviral agents. Recent advances in understanding the mechanisms of their antiviral action(s) indicate that they have a dual role in antiviral defence, acting not only directly on the virion but also on the host cell. Despite the acute problems of viral diseases and restrictions in using chemicals in aquaculture, few but successful attempts to assess the antiviral activities of fish AMPs have been reported. This review focuses on the antiviral activities and mechanisms of action of some AMPs, and their potential relevance in the aquaculture industry, one of the most important sources of fishery products in the near future. It is a matter of notable concern to understand whether the AMPs can be used as model molecules for designing antiviral drugs that might help to solve the problems with viruses in the fish farming industry worldwide. In addition, because fish rely more heavily on their innate immune defences than mammals, they might constitute a potential rich source of antiviral compounds for fighting against mammalian viral infections.

In vivo modulation of the rainbow trout (Oncorhynchus mykiss) immune response by the human alpha defensin 1, HNP1.

Recent studies have demonstrated that the synthetic human defensin-alpha1, also designated as human neutrophil peptide 1 (HNP1), not only has in vitro antiviral activity against viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, but can also modulate some immune activities of rainbow trout (Oncorhynchus mykiss) head kidney leucocytes. However, none of these HNP1 properties have been analysed in vivo so far. Thus, in the current work, we have studied the in vivo immunomodulatory capacity of HNP1 on the rainbow trout immune system as a first approach to evaluate the possible use of this family of antimicrobial peptides (AMPs) to increase fish resistance by enhancing non-specific defence mechanisms. The intramuscular injection of synthetic HNP1 induced the transcript expression of genes encoding both pro-inflammatory cytokines (IL-1beta, TNF-alpha1 and specially IL-8) and CC chemokines (CK5B, CK6 and CK7A) as well as of the genes related to type I interferon (IFN) production (Mx1, Mx2, Mx3 and IFN regulatory factor 3, IRF-3) in different trout tissues (muscle, head kidney and blood). Furthermore, the chemotactic capacity of HNP1 towards trout leucocytes has been clearly revealed. All together, these results demonstrate that in vivo HNP1 is active across species and can modulate fish immune responses. Therefore, in a moment when most pathogens have developed resistance to commonly used antibiotics, natural antimicrobial peptides with inter-specific activity, such as HNP1, might prove to be useful model molecules for the development of novel therapeutic agents that exhibit both microbicidal and immunoenhancing capabilities.

Identification of selective inhibitors of VHSV from biased combinatorial libraries of N,N'-disubstituted 2,5-piperazinediones.

A combinatorial strategy has been used to design and identify inhibitors of viral haemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus of economic importance. Two libraries of N,N'-disubstituted 2,5-piperazinediones (DKP), DKP-I and DKP-II were screened for inhibition of VHSV infectivity. Among the 98 DKP-derivatives (R1-DKP-R2) screened, a novel class of VHSV in vitro inhibitors was identified. Evidences are presented showing that the selected DKP-derivatives cause dose-dependent inhibition of VHSV infectivity in the absence of cellular toxicity. Preliminary characterization of its inhibition mechanism ruled out direct inactivation of the virus (virucidal effect) or interference with early viral replication steps. Furthermore, analysis of infection foci sizes, virus titers, viral protein accumulation and presence of cell free virus derived from VHSV-infected cell cultures in the presence of DKP-derivates suggested that virus assembly/release was impaired leading to a reduced virus spread in cell culture. New DKP-derivatives with a significant higher specific activity need to be developed to start testing its possible practical use but the selected DKP-derivatives described here may contribute to their further development as well as being tools to improve our knowledge on the fish rhabdovirus infection cycle.

Stable expression of bioactive recombinant pleurocidin in a fish cell line.

Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limanda limanda and cloned in a vector under the control of a non-viral promoter (the carp beta-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.