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

The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus

“…The transcriptional levels of different immune genes were evaluated in fish immunized with the different plasmids by semi-quantitative reverse transcription (RT)-PCR assays, which have been used for statistically differentiating gene expression analysis (Yasuike et al, 2007;Chico et al, 2009). Total RNAs were isolated from the liver and spleen tissues taken from three fish in each treatment group at 1, 3, 7, 14 and 21 days after vaccination with TRIzol reagent, then treated with RNase-free DNase I.…”

“…The intensities of the amplification bands were quantified using GeneTools Version 4.01 software (Syngene, UK). Analysis of the expression for each gene was calculated as relative to the b-actin gene expression (expression relative to b-actin) estimated in the same sample using the formula: intensity of the target gene band/intensity of its corresponding b-actin gene band (Chico et al, 2009). The PCR products from all genes studied were sequenced to verify that only specific gene was amplified with each reaction.…”

Protection and antibody response induced by intramuscular DNA vaccine encoding for viral haemorrhagic septicaemia virus (VHSV) G glycoprotein in turbot (Scophthalmus maximus)

“…The plates were further incubated at 20 C for 2 days. As an additional control, the cells were also transfected with the previously characterised pMCV1.4-G 07.71 [29] encoding the cDNA sequence of the gpG of VHSV strain 07.71 (genotype I) isolated from rainbow trout Oncorhynchus mykiss [30].…”

“…Cell RNA extraction and cDNA synthesis were performed as previously described [29]. RT-qPCR was carried out using the gpG 860 gene primers and probe designed to detect specifically the gpG from the isolate VHSV 860 (forward 5 0 -GGTCTTCCTTCTATTGG-TACTTTGCT-3 0 ; reverse 5 0 -GAAATCCCGTAGTTTGGA ATAGG-3 0 ; Probe 5 0 -CTGGAGCCTCTGGCCGTCATTAGC A 3) by using the RTqPCR conditions previously described [32,33].…”

“…There are only 2 amino acid differences between the VHSV strains (underlined N283K and D298E, single letter aminoacid in 07.71 position amino acid in 860 strain, respectively). Briefly, sera (6 fish per group) from turbot injected with PBS, pMCV1.4 or pCMV1.4-gpG 860 were analysed and ELISA assays were carried out as previously described [29] but using the monoclonal antibody to turbot IgM, UR3, kindly donated by Dr. J.M Leiro and Dr. J. Lamas, University of Santiago de Compostela [35]. As a positive control 6 pools of sera (each containing serum from 5 different fish) obtained from turbots surviving to VHSV 860 infection were used.…”