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Cold-adapted strain GCRV-GD108ca of grass carp reovirus type II and application thereof

A technology of reovirus, gcrv-gd108ca, applied in antiviral agents, virus antigen components, virus/bacteriophage, etc., can solve the problems of unsatisfactory control effect and inability to effectively prevent the attack of type II epidemic strains, and achieve good protection effects, effects of significant economic value and social significance

Pending Publication Date: 2021-04-16
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no specific drug for the treatment of grass carp haemorrhagic disease, and immunization is the most effective means of prevention and treatment of the disease. At present, the type I grass carp reovirus vaccine has been applied in the market, but because of its genetic level and type II virus There are large differences, so that the existing type I vaccine cannot effectively prevent the attack of type II epidemic strains, and the control effect is not ideal

Method used

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  • Cold-adapted strain GCRV-GD108ca of grass carp reovirus type II and application thereof
  • Cold-adapted strain GCRV-GD108ca of grass carp reovirus type II and application thereof
  • Cold-adapted strain GCRV-GD108ca of grass carp reovirus type II and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Cultivation and identification of cold-adapted virus strains

[0034] (1) The cold-adapted passage of virus strain GD108 weakens

[0035] The virus culture adopts the subculture inoculation method, that is, after the PSF cells grow to a monolayer, dilute the GD108 virus solution with serum-free M199 medium, inoculate the cell bottle with 1ml / bottle, and incubate at 28°C for 1 hour after the inoculation, discard the incubated virus solution, Add M199 medium cell maintenance solution containing 2% serum, culture in a constant temperature biochemical incubator at 20°C, and freeze and thaw twice at -20°C for 7 days, as the next generation of subcultured virus; continue to subculture for 30 generations according to the above method Finally, the virus can be stably expressed at 20°C, and the virus obtained by low-temperature culture is named GCRV-GD108ca strain, and the virus content is measured every time it is subcultured.

[0036] At 20°C, the GD108 strain wa...

Embodiment 2

[0087] Example 2 Identification of cold-adapted phenotypes

[0088] Take 6 bottles of PSF cells covered with a single layer, inoculate three bottles of cold-adapted P30 generation GCRV-GD108ca strain and GD108 strain, and the inoculation dose is MOI0.1. After inoculation, take one bottle of GCRV-GD108ca strain and GCRV GD108 strain The inoculated cells were cultured in biochemical incubators at 20°C, 28°C, and 35°C for 7 days, and the virus content of GCRV-GD108ca strain and GCRV GD108 cultured at 20°C, 28°C, and 35°C were measured by QPCR method, and each sample was repeated three times. The experimental data is the mean ± SD of three toxicity experiments. Cold adaptation is cold adaptation (ca), which means that the virus titer ratio of the virus strains cultured at 28°C and 20°C is less than 100 times, indicating that the strain can grow effectively at 20°C; temperature sensitivity is temperature sensitive (ts), It means that the titer ratio of the virus strains cultured a...

Embodiment 3

[0091] Embodiment 3 Immunity potency experiment and protective effect

[0092] Take the 30th-generation virus strain GCRV-GD108ca strain that was cold-adapted to culture at low temperature and dilute it 100 times with 0.65% normal saline, and the virus content after dilution is ≥1×10 4 copy / ml, divide 10±2cm long grass carp into 3 groups, 50 fish in each group, and keep them in 28°C water temperature for a week. The remaining two groups were respectively used as positive and negative control groups to inject normal saline, the injection dose was 0.2ml / tail, fasted for 24 hours before and after injection, observed daily after immunization, and recorded the death and morbidity of experimental fish. After 21 days, the immunization group and The positive control group was challenged. The challenge virus strain was GCRV GD108 strain, and the challenge dose was 50 LD 50 / 0.2ml, continue to observe day by day for 15 days after the challenge, record the morbidity and death conditions...

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Abstract

The invention discloses an II-type grass carp reovirus cold-adapted strain GCRV-GD108ca and application thereof, and belongs to the technical field of prevention and treatment of viral hemorrhagic diseases of fishes. The cold-adapted attenuated vaccine strain GCRV-GD108ca disclosed by the invention has a good protection effect on viral hemorrhagic disease of grass carp, and can effectively resist attack of homologous or heterologous type II grass carp reovirus. A safety experiment shows that the GCRV-GD108ca strain is safe to a target animal, is injected into grass carps in an over-dose manner, and does not cause morbidity and death phenomena of experimental fishes. Continuous passage is carried out on cells and fish bodies and susceptible animal toxicity attacking experiments are carried out, wherein the results show that the attenuated strain is stable in toxicity and free of toxicity enhancement; besides, symbiosis infection experiments show that the cold-adapted strain is safe to different grass carp polyculture varieties such as silver carp and crucian carp, does not generate horizontal propagation phenomenon and has no side effect. The invention provides a technical scheme for preventing and treating viral hemorrhagic disease of grass carp, and has important economic value and social significance.

Description

technical field [0001] The invention relates to the technical field of prevention and treatment of fish viral hemorrhagic disease, in particular to type II grass carp reovirus cold-adapted strain GCRV-GD108ca and application thereof. Background technique [0002] Grass carp hemorrhage virus (GCHV, referred to as reovirus ofgrass carp, GCRV by the International Committee on Taxonomy of Viruses) is the first fish virus isolated in China, belonging to the family Reoviridae and the genus Reovirus of aquatic animals. 70-80nm in diameter, 20-hedron spherical particles, containing 11 fragments of double-stranded RNA. Different strains exist in different regions. So far, 10 isolates have been reported. [0003] The virus mainly causes haemorrhagic disease in the fingerling stage of grass carp, the main species of freshwater aquaculture in China, with a mortality rate of over 90%, causing huge losses to the aquaculture industry. The pathogen is a Reovirus belonging to the family R...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/765A61K39/15A61K9/19A61K47/42A61K47/26A61K47/46A61P31/14G01N33/569
Inventor 赵长臣江小燕陈总会巩华刘春花王庆黄志斌
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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