DNA vaccination by immersion and ultrasound to trout viral haemorrhagic septicaemia virus.

This work reports preliminary data on the application of a novel method, ultrasound, for the DNA vaccination of rainbow trout. First, the best formulations were selected that increased the transfer by immersion of a plasmid coding for the green fluorescent protein (GFP) gene into trout fry. Quantification of GFP expression by fluorescence in the fin cells was used to study time course, DNA concentration dependence and comparison of different formulations. The best GFP expression results were obtained with short pulses of ultrasound, DOTAP liposomes and recombinant bacteria or bactofection. Other liposomes or microencapsulation formulations resulted in a GFP fluorescence similar to background values. Second, DNA immersion-vaccination of immunocompetent fingerling trout with the selected formulations was performed by using a plasmid coding for the glycoprotein G gene of the viral haemorrhagic septicaemia virus (VHSV). The immunization of fingerling trout was estimated by measuring humoral antibody, lymphoproliferation and VHSV challenge responses. Short pulses of low intensity ultrasound were the only method by which both humoral antibody responses and survival after VHSV challenge were obtained. Immersion DNA-vaccination using short pulses of ultrasound could eventually lead to a practical way to vaccinate small fish.

In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine fever virus (ASFV) by extracts from marine microalgae.

We have screened for in vitro inhibition of viral replication with extracts from the following marine microalgae: Porphyridium cruentum, Phaeodactylum tricornutum, Tetraselmis suecica, Chlorella autotrophica, Dunaliella tertiolecta, Dunaliella bardawil, Isochrysis galbana, Isochrysis galbana var Tiso, Ellipsoidon sp. and Tetraselmis tetrathele. We have used as viral models two enveloped viruses of significant economic importance, the viral hemorrhagic septicemia virus (VHSV) of salmonid fish and the African swine fever virus (ASFV). The aqueous extracts from P. cruentum, C. autotrophica and Ellipsoidon sp., produced a significant inhibition of the in vitro replication of both viruses in a dose-dependent manner. That this inhibition could be due to sulfated polysaccharides was suggested because the same pattern of viral inhibition was obtained by using exocellular extracts from microalgae enriched in these compounds and/or dextran sulfate of high molecular weight. However, the inhibition of viral replication did not correlate with the percentage of sulfatation of the exocellular polysaccharides. Extracts from marine microalgae may have prophylactic utility against fish and mammalian viral diseases.

Structure, binding and neutralization of VHSV with synthetic peptides.

The phosphatidylserine binding region p2 of VHSV was characterized and was shown to be involved with fusion. Synthetic peptides corresponding to this region interact with phospholipids by penetrating into the membrane and changing to a beta sheet configuration. Computer modeling of this region shows the possible ways by which the interaction with the membranes can succeed. Inhibitory peptides are presently being sought by studying possible interactions within heptad repeats located in other regions of the G protein of VHSV. The heptad repeat region that includes the phosphatidylserine binding domain p2 has been cloned and preliminary experiments show that under certain conditions, peptides from this region can inhibit VHSV infectivity.