Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR

A quantitative real-time RT-PCR (Q-RT-PCR) was developed to detect and determine the amount of viral hemorrhagic septicemia virus (VHSV) in organs of experimentally infected rainbow trout. Primers and TaqMan probes targeting the glycoprotein (G) and the nucleoprotein (N) genes of the virus were designed. The efficiency, linear range and detection limit of the Q-RT-PCR were assessed on cell cultured virus samples. VHSV N gene amplification was more efficient and more sensitive than the VHSV G amplicon. On cell culture grown virus, samples could be accurately assayed over a range of seven logs of infectious particles per reaction. To demonstrate the utility of Q-RT-PCR in vivo, bath infection trials were carried out and samples from fish spleen, kidney, liver and blood were harvested and tested for VHSV. Q-RT-PCR was a more reliable method than either conventional RT-PCR or the cell culture assay for virus diagnosis. Results of VHSV RNA detection in fish shortly after infection as well as on asymptomatic fish several weeks after experimental challenge are presented here. This is the first report showing the utility of Q-RT-PCR for VHSV detection and quantitation both in vitro and in vivo. The suitability of this method to test the efficacy of antiviral treatments is also discussed

Expression and antiviral activity of a β-defensin-like peptide identified in the rainbow trout (Oncorhynchus mykiss) EST sequences

The in silico identification of a β-defensin-like peptide sequence (omBD-1) in the rainbow trout (Oncorhynchuss mykiss) database of salmonid EST is reported here. We have studied the transcript expression of this β-defensin-like sequence in different organs and expressed the recombinant peptide in a fish cell line. Finally, we have demonstrated the in vitro antiviral activity of the recombinant trout β-defensin-like peptide against viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most devastating viruses for worldwide aquaculture. Thus, the resistance to VHSV infection of EPC cells transfected with pMCV 1.4-omBD-1 has been shown. Since EPC cells transfected with omBD-1 produced acid and heat stable antiviral activity and up regulation of Mx, a type I IFN-mediated mechanism of antiviral action is suggested. To our knowledge, this is the first report showing biological activity of a β-defensin-like peptide from any fish

The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences

A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5′ upstream sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp β-actin gene (pAE6-GVHSV) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-GVHSV). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-GVHSV. However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation

In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells

We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (GVHSV)-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of GVHSV protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9–CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding GVHSV gene are due to GVHSV rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants