Cyprinid herpesvirus 2 attenuated strain and application thereof

[0018] Example 1:

[0019] Isolation and identification of carp herpes virus type II CyHV-2DX2019

[0020] (1) Detection of carp herpes virus type II

[0021] The carp herpes virus type II CyHV-2DX2019 attenuated strain of the present invention was taken from silver carp in a crucian carp-silver carp polyculture pond in Jingzhou, Hubei. The silver carp kidney tissue was placed in a sterile petri dish and cut with sterile ophthalmic scissors Crush, add 10 times the volume (V / W) of phosphate buffer (PBS), transfer to a glass homogenizer, and grind into a tissue homogenate under ice bath conditions. The DNA of the tissue homogenate was extracted and amplified with primers CyHV2Hel-F: 5'-GGACTTGCGAAGAGTTTGATTTCTAC-3' and CyHV2Hel-R: 5'-CCATAGTCACCATCGTCTCATC-3'. The result of running gel of the amplified product showed that the test sample had a specific band at 360 bp, which was the same size as the band size of the carp herpes virus type II positive result. The amplified product was

[0029] Example 2:

[0030] Pathogenicity experiment of carp herpes virus type II CyHV-2DX2019 strain on crucian carp

[0031] Take 180 crucian carp, average weight (150.0±4.0) g, average body length (25.0±2.5) cm. Before the experiment, the crucian carp needs to be tested for viral nucleic acid to confirm that the carp herpes virus type II is negative before it can be used in the experiment. According to the test strains, the crucian carp was randomly divided into 6 groups, each with 30 tails, and inoculated with fresh cultures of the 6th to 10th generation test strains (CyHV-2DX2019 strain) by intraperitoneal injection, 0.2ml / tail, virus concentration ≥5×10 5 copies / ml, the control group was inoculated with PBS by the same injection method and observed continuously for 14 days.

[0032] The results showed that the behavior, appetite and other physiological indicators of the crucian carp vaccinated with different generations of virus were normal, and did not show any clinical symp

[0033] Example 3:

[0034] Preparation of Live Vaccine for Hematopoietic Organ Necrosis of Crucian Carp

[0035] The inoculated cells of the 10th and subsequent passages blindly transmitted in Example 1 were observed continuously for 14 days, and then the cell culture was harvested. After freezing at -80°C, it was thawed at room temperature. The freeze-thaw cycle was repeated 3 times, and then centrifuged at 4000r / min for 30min at 4°C. Take the supernatant to extract the viral DNA, and use digital PCR to detect the viral content. Make the virus concentration ≥5×10 5 The supernatant of copies / ml was diluted 100 times with PBS to prepare a live vaccine for hematopoietic necrosis of the crucian carp. The vaccine contains the carp herpes virus type II DX2019 strain at a concentration of ≥5×10 3 copies / ml.