In this sense, only two studies have described DNA vaccines for IPNV [17] and [18]. Atlantic salmon intramuscularly injected with Antidiabetic Compound Library supplier two plasmids (one with the long segment A ORF and the other with VP2 gene) showed a 84% of survival after IPNV challenge whist only 29% of the salmons vaccinated with the plasmid coding for VP2 gene alone survived [18], indicating the importance of other viral proteins apart from VP2 in the immunogenicity. This is also demonstrated by the finding that although most of the neutralizing antibodies are directed to VP2, there is also some immune reaction against VP3 and VP4 [19] and [20]. More recently,
a new DNA vaccine including the VP2 gene of IPNV has shown to up-regulate the expression of interferon (IFN) and IFN-related genes as well as the generation of specific antibodies in vaccinated brown trout [17]. However, further experiments are
still needed to develop an optimal DNA vaccine for IPNV and to elucidate the mechanisms used to induce the fish immune response. Considering this background, we have generated ZD1839 cell line a DNA vaccine consisting of a plasmid encoding the IPNV polyprotein (pIPNV-PP) based on the long ORF of the segment A. We have evaluated the plasmid transcription in vitro and translation in cell-free transfection systems and in transfected fish cells. Through in vivo studies, rainbow trout specimens were intramuscularly injected with the plasmid and the effect on the innate (gene expression) and adaptive (neutralizing antibodies) immune system and the decrease of viral load upon a posterior challenge studied. Results are discussed trying to elucidate the protective mechanisms conferred by this vaccine Tolmetin and the differences compared to other DNA vaccines and IPNV vaccines tested. Rainbow trout (O. mykiss) of approximately 6–8 cm (4–12 g) obtained from Centro de Acuicultura El Molino (Madrid, Spain) were maintained at the Centro de Investigación
en Sanidad Animal (CISA-INIA) laboratory at 14 °C and fed daily with a commercial diet (Skretting). Prior to the vaccination experiments, fish were acclimatised to laboratory conditions for 2 weeks. The Sp serotype of IPNV obtained from the American Type Culture Collection (ATCC VR 1318) was propagated in the RTG-2 (ATCC CCL-55) rainbow trout cell line. Cells were cultured at 20 °C in RPMI (Gibco) supplemented with penicillin (100 IU ml−1), streptomycin (100 μg ml−1) and 10% foetal calf serum (FCS, Gibco). Virus was inoculated on confluent RTG-2 in RPMI with antibiotics and 2% FCS at 14 °C. When cytophatic effect was extensive, the supernatant was harvested and centrifuged to eliminate cell debris. These supernatants were used for the experiments and titrated in 96-well plates according to Reed and Muench [21].