Plasmid pcDNA3.1-s11 constructed based on the S11 segment of grass carp reovirus as DNA vaccine provides immune protection

Although some commercial vaccines against grass carp reovirus (GCRV) are available, given the many varieties of GCRV and limited types of vaccines, the disease caused by GCRV remains a major problem, which leads to economic losses in grass carp aquaculture. A reovirus strain (GCRV-HN14) was recently isolated from local diseased fish in our laboratory. The S11 segment of GCRV-HN14 was speculated to encode the virus capsid protein VP35. In our study, the S11 segment was cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1-s11, which was then transfected into CIK cells, and the VP35 protein was successfully expressed. Grass carp was immunized with pcDNA3.1-s11, and the in vivo distribution and expression of the pcDNA3.1-s11 plasmids were analyzed by PCR and Western blot. Recombinant plasmids were detected in the blood, liver, spleen, kidney, and muscle. However, protein expression could only be detected in the muscle. The immune protection of the pcDNA3.1-s11 plasmid in grass carp was evaluated using a series of experiments. Results showed that the population of white blood cells significantly increased at 1, 7, and 14 days post-immunization (dpi) and reached a peak with (9.58 ± 0.72) × 107/ml at 7 dpi (P < 0.01 or P < 0.05). The percentage of neutrophils reached a peak with (24.13 ± 2.38)% at 7 dpi (P < 0.01), whereas the lymphocytes peaked with (93.30 ± 4.71)% at 14 dpi (P < 0.05). Serum antibody levels were significantly enhanced in immunized fish at 14, 21, and 28 dpi (P < 0.01). The mRNA expression levels of type I interferon, immunoglobulin M, Toll-like receptor 22, and major histocompatibility complex class I were significantly up-regulated in the head kidney and spleen of immunized fish (P < 0.05). Grass carp immunized with pcDNA3.1-s11 exhibited a higher survival percentage (70.4%-73.3%) than the controls (5%-13%). Overall, as a DNA vaccine, the pcDNA3.1-s11 plasmid could induce immune protection against GCRV.