Isolation And Identification Of Snakehead Vesiculovirus Strain 2019 And Its Attenuation Via Cell Culture Passage

Snakehead vesiculovirus（SHVV）is a kind of fish rhabdovirus,which causes serious economic losses to the snakehead fish culture in China.At present,there is still a lack of prevention and control measures against SHVV.Due to the replication in animal body,long immune duration,and easy operation,live attenuated vaccines has become an important way for vaccine research.Cellular passage is a traditional method for preparing live attenuated vaccine.In this study,a rhabdovirus was isolated from the diseased snakehead fish in 2019.It was named SHVV-2019.Whole genome sequencing of SHVV-2019 was performed,and SHVV-2019 was serially passaged on channel catfish ovary（CCO）cells for 100 generations.The genome sequence and pathogenicity to snakehead fish of the passaged strains were compared with those of SHVV-2019.The details are as follows:In September 2019,morbidity and death occurred in cultured snakehead fish in Shunde district,Foshan city,Guangdong province.Its appearance was normal,but the spleen was black and the intestine was hemorrhage.Obvious cytopathic effect appeared24 hours after the tissue homogenate of the diseased snakehead fish was incubated with CCO cells.Reverse transcription-polymerase chain reaction（RT-PCR）amplification and sequence analysis revealed that this virus strain shared more than 99%sequence similarity to that of SHVV-2014 strain,so the virus was named as SHVV-2019 strain.We sequenced the whole genome of the SHVV-2019 strain and compared it with the SHVV-2014 strain.We found that compared with the SHVV-2014 strain,there are 320different bases in the SHVV-2019 strain,62 of which cause amino acid differences.In order to prepare a live attenuated vaccine of snakehead vesiculovirus,we passed the SHVV-2019 strain on CCO cells for 100 consecutive generations,and each generation was collected 24 hours after the challenge and passed to the next generation.CCO cells were infected with the P1,P30,P50,P70,and P100 strains at a multiplicity of infection（MOI）of 0.01.The cytopathic effect was observed for 24 hours and the cell supernatant was collected for TCID₅₀determination.The results showed that as the number of passages increased,the degree of cytopathic effect and virus titer increased significantly,indicating that the passaged viruses were gradually adapted to CCO cells.We sequenced the whole genome of the P50 and P100 strains and compared them with the genome sequence of the SHVV-2019 strain.The results showed that five base mutations and one base deletion were observed in P50 strain compared with SHVV-2019strain,of which the five base mutations led to four amino acid mutations(M_N141D,G_K247R,G_R254Q,G_K485N),the P100 strain retained the mutations of the P50 strain,and at the same time,six more base mutations were apperaed,which led to six amino acid mutations（MM154L,GA59V,GK190Q,GQ253R,GE427D,LR1117K）.We infected snakehead fish with the P1,P50 and P100 strains,with 50 fish in each group.The morbidity and mortality of the snakehead fish were observed every day.It was found that the fish in P1 group died from the fifth day after infection,and all fish died at the eighth day;the fish in P50 group died from the seventh day after infection,and all fish died at the ninth day;the fish in P100 group died from the seventh day after infection,and all fish died at the tenth day.At the seventh day after infection,three dead snakehead fish were randomly selected from each group for dissection.The liver,spleen,kidney,heart and brain tissues were taken,and the G gene of SHVV was amplified by RT-PCR.The results showed that among the 15 tissue samples in each group,G gene could be detected from13 tissues in the P1 group,9 tissues in the P50 group,and only 8 tissues in the P100group.In conclusion,the pathogenicity of SHVV-2019-P100 strain to snakehead fish is weaker than that of SHVV-2019-P1 strain.