Pathogenicity Analysis Of Cyprinid Herpesvirus ? And Its Temperature Sensitive Effect Of Carassius Auratus Gibelio

Carassius auratus gibelio is one of the main economic fish species in China,and it accounts for a large proportion of the freshwater aquaculture fishery structure in China.In 2012,Crucian Carp Hematopoietic Necrosis by Cyprinid herpesvirus 2 was erupted in Yancheng City,Jiangsu Province.The disease spread rapidly and the mortality rate was high,which seriously affected the healthy development of the Chinese carp culture industry.Epidemiological surveys show that CyHV-2 infection has a certain relationship with water temperature,mostly in the spring and autumn,and the symptoms caused by the virus vary with temperature.However,the effect of temperature on CyHV-2 infection in carp and its mechanism are unclear.In this paper,a CyHV-2was isolated from the diseased Carassius auratus gibelio,and the effects of different temperatures on CyHV-2 infection of Carassius auratus gibelio were analyzed.The differentially-responsive gene TNF-?was expressed in E.coli.The main research contents are as follows:(1)A case of diseased gibel carp was collected from a farm in Dafeng District of Yancheng City.The diseased fish showed symptoms such as mandibular and fin base congestion,swelling and bleeding of the ankle.Tissue sections showed Pathological changes,severe degeneration and necrosis of spleen and kidney..Conventional PCR assay showed that the spleen DNA of the diseased fish was CyHV-2 positive.The BLAST alignment showed that the sequence similarity between the PCR Amplification product and the CyHV-2 published in GenBank was 100%,indicating that the virus was a CyHV-2 isolate.We named it CyHV-2 YC17strain.Through the phylogenetic tree,it was found that the YC17 strain had close genetic relationship with CyHV-2 BY-JS1,YZ-01,ZRX001 and other isolates.Artificial infection experiments showed that YC17 strain had strong pathogenicity to gibel carp,and its median lethal dose LD50 was about 4.63×10~6 copies/mg tissue.(2)In view of the different development of CyHV-2 infection in different water temperatures,the CyHV-2 YC17 strain was used to infect gibel carp at 10°C,15°C,20°C,25°C,30°C,and the cumulative mortality was observed.They are 0%,10%,95%,100%,and75%,respectively.Real-time PCR showed that the viral load in the spleen of each temperature group was 25°C,20°C,30°C,15°C,10°C in descending order.At 25°C,interferon-related genes such as Mx,IFN?1,PKR,and MDA5 showed a trend of increasing first and then decreasing,but no significant response was observed at 15°C;At 25°C,FAS,Caspase 8,Bcl-2and other apoptosis-related genes were observed.The peak reached 72 h after infection,but no significant change occurred at 15°C.At 25°C,inflammatory cytokines such as TNF-?and IL-1?were significantly up-regulated 12 h after infection.However,when the virus infection reached 72h,TNF-?was significantly up-regulated at 25°C,while TNF-?was not up-regulated at 15°C.At 15°C and 25°C,the activities of antioxidant enzymes such as SOD,ACP,CAT and MDA all decreased first and then increased.The above results indicate that CyHV-2 normally infects squid at 25°C,and CyHV-2 is latently infected in carp at 15°C.(3)To study the role of squid TNF-?in the process of CyHV-2 infection of gibel carp,we performed prokaryotic expression of TNF-?in E.coli expression system.The total DNA of the spleen of the gibel carp was extracted,and the full length of the TNF-?gene of the gibel carp.(4)was Amplified by PCR.The specific band was obtained at 696 bp,which was consistent with the expected size.This was ligated into the expression vector pET-28a to obtain a positive expression-producing bacterium.After induction by IPTG,a distinct protein band appeared at 25 kDa.The purified protein with higher purity was purified by Ni column affinity chromatography,and the protein mainly existed in the form of inclusion bodies.After renaturation by urea gradient,the results of Western blot showed that the refolded protein showed obvious reaction bands at 25kDa,indicating that the recombinant protein was successfully expressed.(5)In summary,this paper isolated a strain of CyHV-2 YC17 from the diseased gibel carp,which has strong pathogenicity to the gibel carp,which can cause mandibular and fin-based congestion in the carp.Is a typical symptom of hematopoietic necrosis.Artificial infection experiments at different temperatures showed that the cumulative mortality of gibel carp and the viral load in spleen at 25°C were significantly higher than those in other temperature groups.The non-specific immunity of gibel carp after CyHV-2 infection at 15°C and 25°C was further studied.The results showed that TNF-?and IL-1?in spleen showed significant up-regulation in the early stage of infection at 15°C.There was no apparent response subsequently,suggesting that latent infection occurred after CyHV-2 infection of gibel carp at 15°C,which may be responsible for the different course of disease at 15°C and 25°C.In addition,we successfully used the E.coli expression system to successfully carry out the prokaryotic expression of the TNF-?gene of the gibel carp,providing materials for the later study of the function of TNF-?in the process of CyHV-2 infection.The results of this study will help to reveal the mechanism of CyHV-2 infection and lay the foundation for the prevention and control of hematopoietic necrosis.(6)was Amplified by PCR.The specific band was obtained at 696bp,which was consistent with the expected size.This was ligated into the expression vector pET-28a to obtain a positive expression-producing bacterium.After induction by IPTG,a distinct protein band appeared at 25kDa.The purified protein with higher purity was purified by Ni column affinity chromatography,and the protein mainly existed in the form of inclusion bodies.After renaturation by urea gradient,the results of Western blot showed that the refolded protein showed obvious reaction bands at 25kDa,indicating that the recombinant protein was successfully expressed.In summary,this paper isolated a strain of CyHV-2 YC17 from the diseased gibel carp,which has strong pathogenicity to the gibel carp,which can cause mandibular and fin-based congestion in the carp.Is a typical symptom of hematopoietic necrosis.Artificial infection experiments at different temperatures showed that the cumulative mortality of gibel carp and the viral load in spleen at 25°C were significantly higher than those in other temperature groups.The non-specific immunity of gibel carp after CyHV-2 infection at 15°C and 25°C was further studied.The results showed that TNF-?and IL-1?in spleen showed significant up-regulation in the early stage of infection at 15°C.There was no apparent response subsequently,suggesting that latent infection occurred after CyHV-2 infection of gibel carp at 15°C,which may be responsible for the different course of disease at 15°C and 25°C.In addition,we successfully used the E.coli expression system to successfully carry out the prokaryotic expression of the TNF-?gene of the gibel carp,providing materials for the later study of the function of TNF-?in the process of CyHV-2 infection.The results of this study will help to reveal the mechanism of CyHV-2 infection and lay the foundation for the prevention and control of hematopoietic necrosis.