Assessment of a natural grass carp reovirus genotype II avirulent strain GD1108 shows great potential as an avirulent live vaccine.

Vaccine immunization is currently the only effective way to prevent and control the grass carp haemorrhagic disease, and the primary pathogen in these infections is grass carp reovirus genotype II (GCRV-II) for which there is no commercial vaccine. In this study, we evaluated the safety of the GCRV-II avirulent strain GD1108 which isolated in the early stage of the laboratory through continuously passed in grass carp. The immunogenicity and protective effects were evaluated after immunization by injection and immersion. The avirulent strain GD1108 could infect and replicate in the fish which did not revert to virulence after continuous passage. No adverse side effects were observed and the vaccine strain did not spread horizontally among fish. Two routes of immunization induced high serum antibody titers of OD450nm value were 0.79 and 0.76 and neutralization titers of 320 and 320 for the injection and immersion routes of inoculation, respectively. The expression of immune-related genes increased after immunization and the levels were statistically significant. Challenge of immunized fish with a virulent GCRV-II strain resulted in protection rates of 93.88% and 76.00% for the injection and immersion routes, respectively. The avirulent strain GD1108 revealed good safety and immunogenicity via two different inoculation routes. Although the injection route provided the best immune effect, two pathways provided protection against infection with virulent GCRV-II strains in various degrees. These results indicated that the avirulent strain GD1108 can be used for the development and application as live vaccine.

Comparison of the blood parameters and histopathology between grass carp infected with a virulent and avirulent isolates of genotype II grass carp reovirus.

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) is the most important disease for grass carp aquaculture. Its typical clinical symptom is haemorrhaging, although the mechanism was remained unclear. In this study, we investigated the differences in blood parameters and histopathological features between grass carp infected with a virulent and avirulent isolates of genotype II GCRV. Infection with the virulent isolate resulted in increases in 8 routine blood and 2 serum biochemical parameters (P < 0.05); while 9 routine blood and 5 biochemical parameters were significantly decreased (P < 0.05) compared with fish infected with the avirulent isolate. The majority of these alterations were related to hemorrhage, inflammatory reactions and organic damage. The histopathologic changes were primarily vasodilation and hyperaemia in multiple organs, lymphocyte and macrophage infiltration as well as severe vacuolar degeneration in spleen, kidney and liver. The histopathology changes in fish infected with the avirulent isolate were minimal. These results indicated that the pathogenicity of GCRV was primarily reflected in destruction of the blood circulatory system and parenchymatous organs. This study lays the foundation for further research on the pathogenesis of bleeding caused by GCRV infection and the use of blood parameters and histopathology as tools for disease diagnosis.

Development of a simple and rapid reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay for sensitive detection of tilapia lake virus.

Recently, substantial mortality of farmed and wild tilapia caused by tilapia lake virus (TiLV) infection has been observed worldwide. However, sensitive and reliable diagnostic method is limited. A reverse transcription-loopmediated isothermal amplification (RT-LAMP) assay has been applied for the detection of TiLV nucleotide sequence. Six primers targeting two locations on the target gene based on a highly conserved sequence in the segment 1 (S1) region of the TiLV genome have been designed. The optimized RT-LAMP reaction was maintained at the isothermal condition of 63°C for 45 min. And the amplifications could be verified by turbidity or a colour change with the addition of SYBR Green I. Subsequently, RT-LAMP products could be observed by a ladder pattern following gel electrophoresis. The species-specific assay showed that the method was sensitive enough to detect as low as 1.6 copies of viral particle, and the assay was highly specific because no cross-reactivity was observed with other pathogens, and had a diagnostic sensitivity and specificity of 100% when TiLV-positive samples and non-target virus were tested. In summary, all the results demonstrate that this RT-LAMP is a rapid, effective and sensitive method for TiLV detection in tilapia aquaculture.

Cell Culture-Derived Tilapia Lake Virus-Inactivated Vaccine Containing Montanide Adjuvant Provides High Protection against Viral Challenge for Tilapia.

Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and ß-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that ß-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 108, 107, and 106 50% tissue culture infectious dose (TCID50)/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The ß-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.