Thus, matrix protein and RNA-directed RNA polymerase L were not significantly increased in vaccinated fish at any of the tested time points, whereas the other three genes were only detected after 72 hours post-infection and in a lower level in comparison with the other two groups of non-vaccinated fish. This reduction in the number of viral transcripts in the host cells and, as consequence, the high survival rates obtained after vaccination, might be directly related to the presence of specific neutralizing antibodies against VHSV one month after immunization as was previously reported. However, we cannot rule out that some non-specific immune responses could also be contributing to the reduction of viral replication because it is known that the specific protection provided by VHSV and IHNV G gene DNA vaccines in fish is preceded by a protective nonspecific antiviral response, possibly related to interferon induced mechanisms. In fact, interestingly, turbot previously receiving only the empty plasmid and then challenged with VHSV one month later showed a significantly reduced expression of all VHSV genes when compared to the PBS-injected VHSV challenged group. This reduction in the transcription of viral genes could be related to the persistence of a non-specific immune response probably due to the induction of several immune factors by unmethylated CpG motifs present in the plasmid backbone. DNA vaccines are constructed from plasmids of bacterial DNA that are able to induce the maturation, differentiation and proliferation of the immune cells and, therefore, to increase the production of the several cytokines. Ritter et al. revealed that immunization using the empty plasmids pcDNA3 and pORF was able to Asperosaponin-VI reduce the number of colony-forming units of the bacteria Paracoccidioides brasiliensis in mice. Other publication showed some but modest protective effect against Mycobacterium tuberculosis in mice after ZL006 vaccination with the empty plasmid pGX10. With regard to fish viruses, a protective response against the Infectious Pancreatic Necrosis Virus in Atlantic salmon was also observed one week after CpG oligodeoxynucleotides stimulation, revealing the induction of a nonspecific immune response against virus.