Recomendaciones

The development of DNA vaccines has provided encouraging results for rhabdoviruses VHSV, IHNV and SVCV (Anderson et al., 1996a; b; Lorenzen et al., 1998; Lorenzen and La Patra, 2005; Sommerset et al., 2005a; Emmenegger and Kurath, 2008; Tonheim et al., 2008), which may enable a DIVA strategy if immunogenic epitopes of the G protein, for which some have been previously mapped for VHSV (Fernandez-Alonso et al., 1998), are absent from the expressed protein in the vaccine. Specific antibody responses to VHSV have been reported to be detectable > 6 months (Lorenzen and La Patra, 1999; Fregeneda-Grandes et al., 2008), which would be necessary for DIVA strategies to be implemented for this rhabdovirus. One of the very few reported studies of developing marker vaccines for fish

into pathogenic bacteria (Aeromonas salmonicida) as a vector. This induced differential antibody responses from immunised rainbow trout to the vaccine strain and pathogenic virus by western blot (Enzmann et al., 1998). In the same study a genetic DIVA vaccine was also constructed by utilising a variable region of the G-gene to develop a differentiating RT-PCR for an attenuated vaccine. Therefore detection of vaccine strain virus can be differentiated from wild-type virus (Enzmann et al., 1998). Another example was obtained by successful expression of exogenous foreign marker genes in IHNV, i.e. GFP, following a deletion of the non-structural NV protein gene by reverse genetics (Biaccesi et al., 2000). This differential gene expression could be utilised for genetic DIVA approaches.

Another study applied a different approach to positive marker vaccination for another aquatic RNA virus of the birnaviridae family, infectious pancreatic necrosis virus (IPNV).

Subviral particles (SVPs) are formed by structural virus proteins self-aggregating to form particles that do not mimic the native virus capsid (Dhar et al., 2010). These have been synthesised from infectious pancreatic necrosis virus (IPNV) VP2 protein (Allnutt et al., 2007). The subsequent recombinant VP2 (rVP2) particles were also able to carry foreign protein insertions, which reduced IPNV shedding in immunised rainbow trout and elicited specific antibodies to the foreign antigen, c-myc (human oncogene) and to VP2 (Dhar et al., 2010). If antibodies are also detectable to alternative IPNV proteins only in infected fish, e.g.

VP3, then such a vaccine could be utilised for DIVA vaccination.

Previously, an aquatic DNA virus, a member of the alloherpesviridae, channel catfish virus (CCV), has also been investigated for its ability to support the insertion of foreign genes, thus provide an effective vaccine vector (Zhang and Hanson, 1996). The foreign protein was found to induce a specific antibody response in vaccinated catfish, but following infection it would not be possible to indicate fish as uninfected using such a vaccine. A number of vaccines have been developed for KHV, but at present only live attenuated

vaccines have been commercialised (Ronen et al., 2003; KV3, KoVax; Cavoy® Novartis).

These vaccines do not, however, enable antibodies to be differentiated between infected and vaccinated, although genetic DIVA is available for the KoVax vaccine as a PCR was developed specific for an altered nucleotide sequence in the vaccine strain. This can be differentiated from the wild-type virus by PCR (KoVax).

A recently developed oral subunit vaccine for ISAV is based on the haemaggluttinin esterase (HE) protein (Dhar and Allnutt, 2011; Centrovet, Chile). This could potentially enable a DIVA approach by screening for antibodies against the nucleoprotein (NP) that is lacking in the vaccine as only infected fish would respond to this antigen. Indeed the NP protein has been reported as a highly immunogenic antigen (Falk pers. comm. cited in Wolf et al., 2013) and recombinant proteins developed for the HE protein (Krossøy et al., 2001;

Müller et al., 2008) would enable an indication of vaccine efficacy if coated on ELISA plates.

By using such marker vaccines in conjunction with their companion diagnostic test, it may be possible to implement DIVA strategies using serology whereby all fish within the infected site are destroyed, but all fish in the control and surveillance zones are ‘emergency vaccinated’ with the marker vaccine. Those populations of fish that are subsequently found to be positive for antibodies to the marker would be destroyed, while those negative may be spared (Fig. 1.2).

Figure 1.2 Schematic map of hypothetical ISA outbreak in Scotland with control zone and surveillance zones during a DIVA vaccination eradication programme.

Implementing a vaccination eradication programme during an outbreak of ISA, all fish at the infected site (A) are culled whilst those fish in farms within a 5-10 km radius of the infected site (Control zone) (B) are emergency vaccinated. Any fish positive for antibodies to the marker are immediately slaughtered, whilst negative fish are spared. Movement of stocks is still restricted within the control zone. All fish are also vaccinated within the surveillance zone (C) and movements are permitted as antibodies to the vaccine can be differentiated from those to infection. After McGill (2005)

Dhar et al. (2010) pertinently stated that “methods to reduce viral diseases in aquaculture will improve both the quality of life of the animal and make the industry more sustainable”. This could be achieved more effectively by DIVA vaccination. However, approaches to marker vaccine development against fish viruses are limited, and the feasibility of DIVA vaccination for fish has not been assessed. Differences in humoral immunity between higher and lower vertebrates must be taken into consideration as well as the DIVA

approach to particular viral pathogens to shed more light on the feasibility of this vaccination strategy for aquaculture.