Studie On The Effect Of Snakehead Vesiculovirus Was Attenuated By Cell Passage

Snakehead vesiculovirus（SHVV）is a fish rhabdvirus that infects snakeheads causing symptoms such as enlarged liver and blackened spleen,resulting in a high mortality rate,and there are currently no effective control measures for this virus disease.With the implementation of the concept of green and healthy farming,vaccination has become a popular research method to prevent and control viral diseases.Live attenuated vaccine has become an important direction in the preparation of fishery vaccine due to its long immunisation period and their ability to proliferate in vivo,and attenuation by cell passage to obtain weakness strains is the most classical method to develop it.In our laboratory,SHVV has been passaged on Channel catfish ovary cells（CCO）for 100 generations and we found that the pathogenicity of SHVV-P100 to snakeheads was less reduced compared to that of SHVV.In this study,we continued to pass the virus on CCO cells up to P160generation and added the inducer 5-fluorouracil（5-FU）to induce the virus mutation.The main results obtained were as follows:1.Passage of SHVV on CCO cellsWe serially pass SHVV-P100 on CCO cells up to the P160 generation and with or without the addition of 5-FU during the passaging process.The P1,P100,P130,P130-5FU,P160 and P160-5FU strains were inoculated with CCO cells at multiplicity of infection（MOI）=0.01,and the supernatants were collected at 24 h and 48 h to detect the virus titers.The results showed that the titer of the virus gradually increased with the increase in the number of passages,which indicated that the proliferation of the passaged strains on CCO cells gradually increased.2.Whole genome sequencing and mutation site analysis of SHVV strainsWhole-genome sequencing of the P130,P130-5FU,P160 and P160-5FU strain and comparison with the whole-genome sequence of P100 strain showed that,compared with P100,P130 strain observed 1 new nucleotide mutation and P160 strain observed 4 new nucleotide mutations,but neither observed any new amino acid mutation,while P130-5FU strain observed 26 new nucleotide mutations and 1nucleotide deletion.The P130-5FU strain observed 26 nucleotide mutations and 1nucleotide deletion,resulting in 11 amino acid mutations,and the P160-5FU strain observed 71 nucleotide mutations,resulting in 20 amino acid mutations.This indicates that the 5-FU-induced mutagenesis increased the mutation rate of nucleotides and amino acids in the transmission strain and accelerated the mutation of the strain.3.Pathogenicity analysis of SHVV heirloom strains in snakeheadsThe SHVV strains P1,P160 and P160-5FU were all injected intraperitoneally into snakehead at two doses(10⁵TCID₅₀and 10³TCID₅₀)and observed for 28 days.The results showed that the survival percentage of P1 was 26.7%and 66.7%after the snakehead was injected with two doses,the survival percentage of P160 was 66.7%and 96.7%,while the survival percentage of P160-5FU was 83.3%and 93.3%.This indicates that the virulence of the P160 and P160-5FU was reduced,with the P160-5FU being the least virulent.4.Immunoprotection of P160-5FU against snakeheadIn order to test the immunoprotective effect of the P160-5FU on snakehead,two doses of 10⁵TCID₅₀(10⁵TCID₅₀immunised group)and 10³TCID₅₀(10³TCID₅₀immunised group)were administered intraperitoneally to the snakehead and the antibody titres were measured by blood collection at 28 days.The results showed that the antibody titer in the 10⁵TCID₅₀immunised group reached 1:112,while no antibodies were detected in the sera of the 10³TCID₅₀immunised group.Furthermore,we infected the snakehead with 10⁵TCID₅₀dose of SHVV after 28 days of immunisation and set up a control group（not immune to infecting SHVV only）.The results showed that the viral loads in the liver and spleen of the snakehead in the 10⁵TCID₅₀immunised group were significantly lower than those in the 10³TCID₅₀immunised group and control groups（p<0.01）.The survival rate of control group was 27.3%,the 10⁵TCID₅₀immunised group was 63.2%and the relative immune protection rate was 50%,while the survival rate of the 10³TCID₅₀immunised group was 31.8%and the relative immune protection rate was 6.25%.In conclusion,P160-5FU is a candidate for the preparation of a live attenuated vaccine against SHVV.