Red seabream iridovirus (RSIV) infection causes significant mortality in cultured marine fishes. A formalin-inactivated vaccine for RSIV infection has been developed commercially, but and it is not efficient for protecting fish belonging to the genus Oplegnathus, including rock bream O. fasciatus. I...
Red seabream iridovirus (RSIV) infection causes significant mortality in cultured marine fishes. A formalin-inactivated vaccine for RSIV infection has been developed commercially, but and it is not efficient for protecting fish belonging to the genus Oplegnathus, including rock bream O. fasciatus. It was reported that “Poly(I:C) immunization with a live virus” and “fish immunization with live virus by controlling fish rearing temperature” confer protection of fishes from infections with homologous viruses. In the present thesis, both of the fish immunization methods with live virus were applied to RSIV infection in rock bream. Moreover, kinetics of RSIV in vivo and in vitro were investigated for practical application of the fish immunization with live RSIV at rock bream farms in Korea.
In the Chapter I, background of RSIV infections in marine fishes and status of developing vaccines for RSIV infection were briefly reviewed. In the next Chapter II, the optimization of qPCR targeting phosphatase gene of RSIV genome was performed for detection of RSIV in splenic tissues of rock bream. The regression line for quantitative detection of the RSIV genome was y = - 0.266x + 10.948 (amplification efficacy: 84.5%), which was available between Ct 8 and 33. The quantitative detection limit of the assay was 103.5 genomes/mg. In the Chapter III, the concept of "Poly(I:C) immunization with live virus" was applied to RSIV infection in rock bream. Unfortunately, rock bream administered Poly(I:C) were not protected from RSIV infection, although interferon (IFN) was well induced in the rock bream with Poly(I:C) administration. Moreover, no significant difference was observed in RSIV kinetics between the fish that did or did not receive Poly(I:C). It was concluded that RSIV was probably insensitive to the transient innate immune response induced by Poly(I:C) administration.
In the Chapter IV, the concept of “fish immunization with live virus by controlling fish rearing temperature” was applied to RSIV infection of rock bream. Mortalities in rock bream that were inoculated with RSIV and reared at 21 – 30°C were ≥ 90%, whereas no mortality was observed in fish that received an RSIV inoculation and were reared at ≤ 18°C. RSIV kinetics revealed that RSIV multiplied rapidly in fish reared at 24.3°C, and achieved the critical level for rock bream (approximately 109.0 genomes/mg) within 28 days. In contrast, the RSIV genome was detected on day 10 in fish that received an RSIV inoculation at 15.5°C, and peaked on day 28 at 105.91 ± 0.54 genomes/mg, decreased gradually, and then maintained under the detection level beginning on day 84 after RSIV inoculation. Furthermore, the fish surviving the RSIV infection at low rearing temperature were strongly protected from challenge with homologous RSIV; the threshold level of RSIV for rock bream to mount a protective immune response was ≤ 105.4 genomes/mg. Cohabitation experiments revealed that the spread of RSIV from rock bream vaccinated with a live RSIV could be low if it is limited to fish in the late stage (≥ 84 days of elapse) after vaccination. Thus, it was concluded that when rock bream are reared at ≤ 18°C and inoculated with RSIV, the survivors can mount a protective immune response against RSIV, suggesting a positive effect of a live RSIV vaccine for rock bream.
“Persistent infection of RSIV in immunized fish” remains as a problem for a practical application of the live RSIV vaccine. In the Chapter V, a new grunt fin (GF) cell line persistently infected with RSIV (PI-GFRSIV) was established by subculturing GF cells that survived RSIV inoculation. PI-GFRSIV cells were morphologically indistinguishable from naïve GF cells. They stably produced RSIV at approximately 104.9 ± 0.5 genome/μl after 24 passages over one and half years. The optimum temperature to produce RSIV in PI-GFRSIV cells was about 25°C, although these cells could produce RSIV at 15°C to 30°C. RSIV in PI-GFRSIV cells was expelled after several times of subcultures at 15°C or 30°C. The PI-GFRSIV cells expelling RSIV (Exp.GF cells) exhibited the same level of RSIV productivity as those of naïve GF cells.
Finally in the Chapter VI, I concluded the present researches on kinetics of RSIV in vivo and in vitro at different temperatures, and also I discussed on advantages and disadvantages for practical application of the “immunization of rock bream with live RSIV at a low temperature” at rock bream farms in Korea.
Red seabream iridovirus (RSIV) infection causes significant mortality in cultured marine fishes. A formalin-inactivated vaccine for RSIV infection has been developed commercially, but and it is not efficient for protecting fish belonging to the genus Oplegnathus, including rock bream O. fasciatus. It was reported that “Poly(I:C) immunization with a live virus” and “fish immunization with live virus by controlling fish rearing temperature” confer protection of fishes from infections with homologous viruses. In the present thesis, both of the fish immunization methods with live virus were applied to RSIV infection in rock bream. Moreover, kinetics of RSIV in vivo and in vitro were investigated for practical application of the fish immunization with live RSIV at rock bream farms in Korea.
In the Chapter I, background of RSIV infections in marine fishes and status of developing vaccines for RSIV infection were briefly reviewed. In the next Chapter II, the optimization of qPCR targeting phosphatase gene of RSIV genome was performed for detection of RSIV in splenic tissues of rock bream. The regression line for quantitative detection of the RSIV genome was y = - 0.266x + 10.948 (amplification efficacy: 84.5%), which was available between Ct 8 and 33. The quantitative detection limit of the assay was 103.5 genomes/mg. In the Chapter III, the concept of "Poly(I:C) immunization with live virus" was applied to RSIV infection in rock bream. Unfortunately, rock bream administered Poly(I:C) were not protected from RSIV infection, although interferon (IFN) was well induced in the rock bream with Poly(I:C) administration. Moreover, no significant difference was observed in RSIV kinetics between the fish that did or did not receive Poly(I:C). It was concluded that RSIV was probably insensitive to the transient innate immune response induced by Poly(I:C) administration.
In the Chapter IV, the concept of “fish immunization with live virus by controlling fish rearing temperature” was applied to RSIV infection of rock bream. Mortalities in rock bream that were inoculated with RSIV and reared at 21 – 30°C were ≥ 90%, whereas no mortality was observed in fish that received an RSIV inoculation and were reared at ≤ 18°C. RSIV kinetics revealed that RSIV multiplied rapidly in fish reared at 24.3°C, and achieved the critical level for rock bream (approximately 109.0 genomes/mg) within 28 days. In contrast, the RSIV genome was detected on day 10 in fish that received an RSIV inoculation at 15.5°C, and peaked on day 28 at 105.91 ± 0.54 genomes/mg, decreased gradually, and then maintained under the detection level beginning on day 84 after RSIV inoculation. Furthermore, the fish surviving the RSIV infection at low rearing temperature were strongly protected from challenge with homologous RSIV; the threshold level of RSIV for rock bream to mount a protective immune response was ≤ 105.4 genomes/mg. Cohabitation experiments revealed that the spread of RSIV from rock bream vaccinated with a live RSIV could be low if it is limited to fish in the late stage (≥ 84 days of elapse) after vaccination. Thus, it was concluded that when rock bream are reared at ≤ 18°C and inoculated with RSIV, the survivors can mount a protective immune response against RSIV, suggesting a positive effect of a live RSIV vaccine for rock bream.
“Persistent infection of RSIV in immunized fish” remains as a problem for a practical application of the live RSIV vaccine. In the Chapter V, a new grunt fin (GF) cell line persistently infected with RSIV (PI-GFRSIV) was established by subculturing GF cells that survived RSIV inoculation. PI-GFRSIV cells were morphologically indistinguishable from naïve GF cells. They stably produced RSIV at approximately 104.9 ± 0.5 genome/μl after 24 passages over one and half years. The optimum temperature to produce RSIV in PI-GFRSIV cells was about 25°C, although these cells could produce RSIV at 15°C to 30°C. RSIV in PI-GFRSIV cells was expelled after several times of subcultures at 15°C or 30°C. The PI-GFRSIV cells expelling RSIV (Exp.GF cells) exhibited the same level of RSIV productivity as those of naïve GF cells.
Finally in the Chapter VI, I concluded the present researches on kinetics of RSIV in vivo and in vitro at different temperatures, and also I discussed on advantages and disadvantages for practical application of the “immunization of rock bream with live RSIV at a low temperature” at rock bream farms in Korea.
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