Evaluation Of An Attenuated Strain Of Carp Herpesvirus 2 As A Candidate Strain For Live Vaccine

Herpesviral hematopoietic necrosis disease causes by cyprinid herpesvirus 2(Cy HV-2)infection is a high mortality disease that leads to great economic damage to gibel carp,Carassius auratus gibelio aquaculture.In this study,an attenuated strain of Cy HV-2 GRP7 was achieved by subculture on Ryu F‐2 cells derived from the fin of Ryukin‐variety goldfish and Gi CF cells derived from fin of gibel carp.As the attenuated vaccine candidate,there are no clinical symptoms of gibel carp that immersion or intraperitoneal injection with G-RP7 strain.After immersion and intraperitoneal injection of G-PR7 to immunize gibel carp,the protection rate after being challenged with virulent strain was92% and 100%,respectively.The dynamic of virus DNA in each tissue of G-RP7 vaccination fish increased within 1,3,and 5 days post-immunization,and subsequently decreased and stabilized within 7 and 14 days.In addition,the increase of anti-virus antibody titer was detected both immersion and injection immunization fish 21 days after vaccination by ELISA.These results demonstrated that G-RP7 can be a promising live attenuated vaccine candidate against the disease.In the test for virulence reversion,the candidate was propagated through gibel carp six times by intraperitoneal injection with kidney and spleen homogenate of the inoculated fish.During in vivo passages in gibel carp,no abnormality and mortality of the inoculated fish were observed,and the virus DNA copies maintain a low level from the first passage to the sixth passage.In vatro transmission of gibel carp,the safety of healthy fish is highly,In the parallel propagation experiment,it was verified that G-RP7 may have a risk of transmission in water.In order to understand the immune mechanism of the vaccine candidate strain in the gibel carp after inoculation,the gibel carp was inoculated with G-RP7 strain by injection and immersion.The kidneys of the gibel carp were taken on the 3rd,7th and 14 th days and sent to the transcriptome for sequencing.Through transcriptome data screening,a large number of key genes in immune related pathways were obtained,and the significance of specific and non specific immune genes in allogynogenetic crucian carp was analyzed.Three immune genes,STAT2,IL-8,and C3,were selected for quantitative validation.The results showed that C3 participated in specific immunity in the injection group,but did not stimulate specific immunity in the soaking group.After infected G-RP7,IL-8 reacted to virion immediately in vivo,playing an inflammatory role,and kept rising at 7 days,playing an anti-inflammatory role.At 14 days,there was still a significant difference,indicating that IL-8 promoted specific immune response stimulation.Both the soaking group and the injection group showed significant differences starting from 3 days,and there was no difference between 7 and 14 days,indicating that STAT2 plays a non-specific immunity role.