Preliminary Study On The Cause And Mechanism Of White Spot Syndrome Virus(WSSV) Pathogenicity To Chinese Mitten Crab(Eriocheir Sinensis)

White spot syndrome virus?WSSV?is highly infectious which belongs to the Whispovirus family.It is one of the most serious aquatic pathogens of crustaceans,such as shrimp and crab.In 2015,it was reported that the Chinese mitten crabs?Eriocheir sinensis?in ponds have been infected with WSSV,causing highly mortality.Although partial pathogenic mechanism of WSSV has been explored,there is still no effective method of prevention and treatment.Studies have shown that autophagy plays a key role in the interaction between virus and host.This study focused on the pathological changes of hepatopancreas,pancreas,and intestine after infection with WSSV.Western-blot and immunofluorescence techniques were used to detect autophagy induced by WSSV.The WSSV-induced blood cell autophagosome formation and possible activation mechanisms were studied.We have obtained the following research results:WSSV and Aeromonas veronii AVZ01 were isolated from natural diseased Procambarus clarkii and Litopenaeus vannamei.The same symptoms appeared after re-injecting the extracted WSSV virus fluid into healthy Procambarus clarkii and the target band was expanded.After the artificial infection test with strain AVZ01 from the diseased shrimp,the symptoms of shrimp were similar to those of natural disease,the LD₅₀ was counted to 8.7×10⁵ CFU/mL.According to the morphological characteristics,biochemical characteristics and 16S rRNA gene sequence analysis,the pathogen was identified as A.veronii..No abnormal symptoms were observed after injection of the WSSV virus into the zebrafishs.The Chinese mitten crabs infected with WSSV had no obvious symptoms in 1-2 days.After 3-7 days,they became unresponsiveness,low food intake,and showed the weakness of appendages and no evasion and aggressive behavior.It was difficult to recover quickly after manual inversion for the Chinese mitten crabs.It was found that the back shell was easy to separate,the hemolymph was not easy to coagulate,the color of the cocoon was dark,and the hepatopancreas was light yellow.After WSSV infection 72 h,the results of histopathology showed that in the hepatopancreas,the basement membrane completely detached from the hepato-pancreatic cells,the voids increased,the vacuoles began to rupture,and some of the hepatocytes disintegrated in the lumen,and the hepatic tubules only showed a blurred outline.The appearance of the hepatopancreas was an off-white paste;Occurrence of keratinocytes and epithelial cells in the temporal lobe,and folds in the stratum corneum,granuloma in the iliac crest,swelling in the iliac crest,and some areas were normal in branchial lamella thickness 2-3 times,part of the nuclei overflowed from the folds,the tendon tissue swelled,the columnar epithelial cells fell off,and the nuclei became swollen;Part of the intestinal villi was degenerated and shedding,forming a gap with the intestinal epithelium.As the infection time increases,the intestinal villi were completely deformed,the intestinal villi were dissolved,the nucleus falled off and passed through the intestinal epithelium,the lumen of the intestine was blurred and impurities appear.Autophagy plays an important role in the immune system.It can both sense the virus and have an antiviral effect.In this study,the cytosolic microtubule-associated protein 1A/1B-light chain 3?LC3?-I?LC3-I?esterification to LC3-II and the formation of autophagosome were observed after 24 h treatment of autophagic activator Rapamycin in blood cells of E.sinensis.LPS treatment for 24 h resulted in LC3-II conversion in blood lymphocytes.Significantly higher than the PBS treatment group.Injection of WSSV in vivo can also induce the formation of blood cell LC3-II and autophagosome,and WSSV-induced autophagy is a process that increases first?0-12 h?,then decreases?12-24 h?,and then rises?24-48 h?and decreases?48-72 h?.Rapamy-activated autophagy is the process of rising first?0-24 h?and then decreasing?24-72 h?.RT-PCR analysis showed that the expression levels of two genes involved in autophagy?EsGabarap and EsAtg7?were significantly increased in WSSV-infected blood cells from Chinese mitten crab in 12-24 h.In summary,this paper studied the pathological changes in WSSV-infected Chinese mitten crab and the WSSV-induced autophagy and autophagy-associated genes in the haemolymphocyte,in order to further understand the pathological damage changes and mechanism caused by WSSV infection.The interaction between autophagy and WSSV provided important basic knowledge and a new reference for the effective prevention and treatment of WSSV in the future.