Preliminary Investigation On Cross-species Infection Of Covert Mortality Nodavirus And Pathogenic Mechanism On Apostichopus Japonicus

Apostichopus japonicus,as the representative economic animal species of echinodermata,possesses a special evolutionary position and its aquaculture production value occupies a relatively high proportion in the aquaculture industry.Recently,the increasingly serious disease problems have severely restricted the green and high-quality development of the aquaculture industry of A.japonicus.It is of great theoretical value and practical significance to carry out indepth research on sea cucumber diseases and its pathogens.Covert mortality nodavirus(CMNV),a viral aquatic pathogen that causes viral covert mortality disease(VCMD)in shrimps,is highly pathogenic to cultured shrimps.Notably,CMNV possesses the strong ability of cross-species transmission and can infect a variety of crustaceans and teleostean.In a systematic investigation of CMNV natural hosts and vectors,the author’s laboratory found the suspected transmission of CMNV in A.japonicus,and based on this,systematic studies was carried out using virus purification,protein profiling identification,artificial infection experiment,molecular biology,histopathology and transcriptome sequencing.In this thesis,the author verified the natural infection of CMNV in A.japonicus,analyzed the pathogenicity of CMNV to cultured A.japonicus,studied the expression changes of vital functional genes in A.japonicus infected with CMNV,discussed the process of CMNV infection and its pathogenic molecular mechanism,and investigated the prevalence of CMNV in the main culture areas of A.japonicus in Shandong province,to clarify the potential threat of CMNV to the farmed A.japonicus industry,reveal the pathogenic mechanism of CMNV cross-species infection of A.japonicus,and provide technical support and theoretical basis for the prevention and control of farmed A.japonicus viral diseases.The main achievements of the research are as follows:Firstly,CMNV natural infection in disease A.japonicus was confirmed by RT-n PCR,in situ hybridization(ISH)detection,protein profile analysis,transmission electron microscopy(TEM)and histopathological analysis.The CMNV detection and analysis of vital tissues and organs of A.japonicus revealed that the intestine,respiratory trees,and gonad were the target tissues of CMNV natural infection in sea cucumber,and the infection could lead to the massive karyopyknosis and vacuolation of the coelomic epidermis of intestine and respiratory trees,the atrophy and necrosis of spermatozoa,spermatocytes and vitellogenic oocytes.The above results indicated that CMNV might be one of the potential pathogens of cultured A.japonicus and reminded that the risk of vertical transmission of CMNV in A.japonicus.Secondly,to understand the distribution and prevalence of CMNV in the main culture areas of A.japonicus in Shandong,ISH detection and hihiopathological analysis were performed on A.japonicus collected from different areas of Shandong from 2018 to 2021 in this study.The results showed that from 2018 to 2021,CMNV infection and prevalence were all found in the important A.japonicus breeding areas(Qingdao,Weifang,Yantai and Weihai)of Shandong,and all the CMNV positive individuals showed serious tissue lesions.These results suggested that we should concern the existence and prevalence of CMNV and other viral pathogens in A.japonicus diseases and pay more attention to the prevalence of CMNV in other sea cucumber culture areas(Liaoning,Hebei and Fujian)in China.Third,the experiment of CMNV artificial infection on healthy A.japonicus was carried out,and the symptoms,incidence rate,area of lesion,mortality and pathological changes caused by CMNV infection were analyzed by using technological means such as RT-n PCR,Taq Man RTq PCR,ISH and histopathological analysis.The results showed that the A.japonicus artificially infected with CMNV showed the symptoms of ulcerated skin on the back,increased mucus on the body surface,deteriorate contraction of the ginseng body,and weakened or lost adsorption capacity of the abdominal tube feet.Additionally,the pathological changes of the target tissues and organs in the challenge group were consistent with those in the naturally infected CMNV A.japonicus;The incidence and area of lesion caused by CMNV infection in A.japonicus were positively correlated with the CMNV load in sea cucumbers and CMNV possessed strong pathogenicity to A.japonicus;And we also found that "12-36 h after infection" might be the critical period for CMNV to exert pathogenic effects on A.japonicus.The above results provided theoretical basis for the diagnosis and analysis of the pathogens of A.japonicus diseases in summer,meanwhile,were helpful for the study the infection process and pathogenic mechanism of diseases.Fourth,transcriptomic analysis of intestine and respiratory trees of A.japonicus in the control and challenge group of artificial infection experiment was conducted by highthroughput sequencing,and we identified the differential expression gene between the control and the infected group,then performed the GO functional annotation and KEGG metabolic pathway analysis of the differential expression gene.Then we found that the functional genes involved in coding matrix metalloproteinases,collagenases,cathepsins and serine proteases in A.japonicus artificially infected with CMNV were all significantly up-regulated and these results were verified by q RT-PCR detection,meanwhile,we speculated that the up-regulated expression of these genes intensifies the damage to collagen protein and collagenous fiber,eventually lead to the symptom such as ulcerated skin,increased mucus on the body surface,deteriorate contraction of the ginseng body,and weakened adsorption capacity;The expression of genes encoding proteins such as apoptosis and nerve signaling also increased significantly and this change was also subsequently verified by the q RT-PCR test results,and we speculated that the significant increase in the expression of apoptosis and neural signal transduction related protein genes was the internal reason of the skin fester,karyopyknosis and vacuolation;In addition,the overall analysis of the transcriptome data found that the differential genes in the intestine were far more than those in the respiratory trees,which suggested that the intestinal epithelial cells were more sensitive to CMNV infection and the intestine was more suitable as target tissue for mechanism research such as pathogenic mechanisms.The above results revealed the changes in host genes and metabolic pathways caused by CMNV infection,clarified the key differential expression genes of the host during the viral infection process,and provided a theoretical basis for studying the corresponding complex relationship between CMNV infection and the host.Fifth,according to the above results,in the repeated experiments of CMNV artificial infection of A.japonicus,three time points of “12 h,24 h and 36 h” were selected for the sample collection and analysis of tube feet,intestines and respiratory trees of A.japonicus.Based on the results of Taq Man RT-q PCR,in situ hybridization and histopathology,the replication and proliferation of CMNV in A.japonicus showed a trend of first decline and then rise(24 h <12 h<36 h);The severity of tissue lesions was positively correlated with the proliferation of CMNV,and showed a tendency to weaken and then aggravate after acute infection(24 h <12 h ≤ 36 h);We speculated that CMNV began to proliferate in A.japonicus at 0-12 h and was accompanied by stress response caused by virus infection,then during 12-24 h,the non-specific immune effect in A.japonicus led to a slight decrease in the expression of CMNV in A.japonicus,and the symptoms were also alleviated;The expression of CMNV increased significantly during 24-36 h and was much higher than that at 12 h,which might be presumably due to the stronger pathogenicity caused by the continuous proliferation of CMNV;Furthermore,the symptoms of the diseased A.japonicus was compared with the proliferation of CMNV using transcriptome sequencing and found that the up-regulation of genes encoding proteins involved in innate immunity at 12-24 h was much greater than that at 0-12 h and 24-36 h,which provided more evidence to confirm the above speculation about the course of infection,and these results were also supported by q RT-PCR assay data;Moreover,the analysis of differential functional gene during the critical period of CMNV infection in A.japonicus further verified that these genes encoding matrix metalloproteinases,collagenases,cathepsins and other proteins play an important role in the pathogenesis of A.japonicus.The above results laid a foundation for further study of the molecular mechanism of CMNV on the pathogenic of A.japonicus.In conclusion,this paper confirmed the existence of natural CMNV infection in cultured A.japonicus,investigated the prevalence and distribution of CMNV in the main A.japonicus breeding areas in Shandong Province,and clarified the pathogenicity of CMNV to A.japonicus and the main target tissues of CMNV infected ponds.We also found the expression changes of important host genes in the process of CMNV infection in A.japonicus,and analyzed the key differential expression genes in sea cucumbers during CMNV infection,and clarified the process of CMNV infection in A.japonicus and the characteristics of transcriptomic changes in the main target tissues during CMNV infection.The above research results provided massive data support for clarifying the viral pathogens of A.japonicus disease,and also provided theoretical basis for revealing the pathogenic mechanism of CMNV on A.japonicus.