Potentiality of a live vaccine with nervous necrosis virus (NNV) for sevenband grouper Epinephelus septemfasciatus at a low rearing temperature.

Nervous necrosis virus (NNV) is the causative agent of viral nervous necrosis (VNN), one of the most serious diseases in over 30 species of cultured marine fishes worldwide. Although several kinds of NNV vaccines have been developed, none of these vaccines have been yet marketed. Here, we demonstrate the potentiality of a live NNV vaccine for sevenband grouper Epinephelus septemfasciatus at a low rearing temperature (17°C). Moreover, we investigated the kinetics of NNV infectivity titer in fish reared at low and optimum temperatures (17°C and 26°C) for VNN onset to determine why sevenband grouper reared at 17°C survive NNV infection. In pathogenicity tests of NNV, fish mortality was reduced by decreasing the fish rearing temperature, and no mortality was observed in fish reared at 17°C regardless of the infection method. During fish acclimation to the optimum temperature of VNN onset (26°C), increased mortalities were observed in the survivors from the 1st NNV-infection. Little or no mortality was observed in the 2nd NNV-infection. Thus, it was demonstrated that the survivors from the 1st NNV-infection mounted a specific protective immune response against NNV. Especially, in the fish infected with NNV by immersion at 17°C, only two out of 30 fish died until the end of the 2nd infection (total survival rate: 93.3%), suggesting a positive potentiality for a live NNV vaccine. In the analysis of NNV kinetics in the fish reared at 26°C, NNV rapidly multiplied up to ≥ 10(9)TCID(50)g(-1) before fish began to die, and the critical level of NNV was around 10(10)TCID(50)g(-1). Probability of NNV multiplication reduced by decreasing the inoculated NNV dose, but NNV multiplication rate was independent of the NNV dose. The threshold of NNV for fish mounting a protective immune response was around >10(4)TCID(50)g(-1). Against this, in the fish reared at 17°C, NNV slowly multiplied in comparison with that in fish at 26°C. NNV titer in the peak was at 10(7.1 ± 1.4)TCID(50)g(-1), which was far behind the critical level of NNV but still greatly above the threshold level (10(4)TCID(50)g(-1)). Thus, it was demonstrated that the multiplication rate of NNV in vivo was strongly correlated to NNV virulence and fish mortality, and down-regulation of NNV multiplication in fish reared at 17°C enabled control of VNN onset for development of a live NNV vaccine.

Live vaccine of viral hemorrhagic septicemia virus (VHSV) for Japanese flounder at fish rearing temperature of 21°C instead of Poly(I:C) administration.

The process of "Poly(I:C) immunization" involves immunization of fish with a pathogenic live virus, followed by administration of Poly(I:C), which induces a transient, non-specific antiviral state. As a result, fish in an antiviral state survive the initial immunization with live virus. Moreover, these fish are able to mount a specific protective immune response against the injected pathogenic virus. In the present study, we investigated the optimum temperature for Poly(I:C) immunization of Japanese flounder Paralichthys olivaceus with live viral hemorrhagic septicemia virus (VHSV). It was revealed that the optimum temperature was around at 17°C for Poly(I:C) immunization in Japanese flounder. Furthermore, the protection efficacy of Poly(I:C) immunization was significantly decreased by elevation of fish rearing temperature, and no efficacy was observed at a fish rearing temperature of 25°C. Interestingly, no mortality by VHSV infection was observed in fish reared at 21°C and 25°C even when those fish were not administered Poly(I:C). All of the survivors from the first VHSV-challenge at 21°C were strongly protected from re-challenge with VHSV. However, almost all of the survivors (≥82.6%) from the first challenge at 25°C were lost by the second challenge with VHSV. It was thus concluded that by rearing fish at 21°C and challenging with live VHSV, it is possible to induce strong specific immunity in Japanese flounder without Poly(I:C) administration.

Protection of Japanese flounder Paralichthys olivaceus from viral hemorrhagic septicemia (VHS) by Poly(I:C) immunization.

In immunization of fish with polyinosinic-polycytidylic acid (poly[I:C], a synthetic double-stranded RNA, injection of Poly(I:C) followed by challenge with a live virus induces a transient, non-specific antiviral state by interferon activity. When exposed to a virus while in this antiviral state, the fish acquire a specific and protective immunity against the corresponding viral disease and survive. In the present study, the effiacy of Poly(I:C) immunization was investigated in japanese flounder Paralichthys olivaceus using viral hemorrhagic septicemia virus (VHSV) as a model; the minimum dose of Poly(I:C) required for inducing protection and the duration of the antiviral state were determined, and a potentially curative effect of Poly(I:C) administration was assessed. The antiviral state was induced by administration of Poly(I:C) doses ranging from 12.5 to 200 microg fish(-1). Minimum dose to induce the antiviral state (relative percentage survival, RPS: 90%) was 12.5 microg fish(-1). No curative effect of Poly(I:C) was observed in fish pre-infected with VHSV. Fish injected with 200 microg Poly(I:C) fish(-1) were highly protected (RPS: 100%) from an artificial challenge with VHSV, and specific antibodies against VHSV were detected. The corresponding high level of antiviral state against VHSV was attained 1 d post Poly(I:C) injection, lasted for 6 d and susequently decreased. Moreover, the surviving fish were highly protected from re-challenge with VHSV (RPS: 100%). Thus, it was considered that an immunity against viral hemorrhagic septicemia was induced in the Japanese flounder by injecting live VHSV following Poly(I:C) administration.

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus.

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.