Identification And Pathogenicity Of Turbot Hemorrhagic Disease Pathogen

Turbot(Scophthalmus maximus)is an important mariculture economic fish in the northern coastal areas of China.With the rapid development of turbot farming,disease problems are becoming increasingly prominent,seriously threatening the healthy and sustainable development of the industry.A new disease,commonly known as"turbot hemorrhagic disease",with bleeding and ulcerated fins as the main clinical symptoms,broke out among farmed turbot in China in late 2019.The disease rapidly spread to all farming areas in China and has become the most severe disease-threatening turbot culture.In this study,we obtained a highly abundant circovirus sequence from the affected turbot by viral group sequencing.We identified the virus as a new fish circovirus named turbot circovirus(TCV)by viral group full-length sequencing and analysis.The details of the study are as follows:1.Epidemiologic survey of the hemorrhagic disease of turbot.From 2019 to date,our team conducted 69 stream surveys in several farms in the main turbot production areas of Shandong,Liaoning,and Jiangsu,and collected a total of 390 diseased fish with turbot hemorrhagic disease.The epidemiological survey results showed that the disease was highly prevalent from August to December,and the water temperature was between 12℃-22℃.In the early stage of the disease,turbot feeding was normal and vigorous and did not show any abnormalities;1-2 days before the onset of the disease,a large amount of foam appeared on the surface of the breeding pool,followed by fin bleeding,almost all turbot in the whole breeding pool died in about one week,and the cumulative mortality rate of dozens of breeding pools in the whole fish farm exceeded 90%in about one month.The onset of disease covered almost the entire breeding cycle,with a slow onset process at low temperatures and a particularly intense onset at high temperatures.No parasitic infection was found in the gill filaments and body surface mucus of the diseased fish through microscopic observation and common bacterial pathogens such as Edwardsiella piscida and Aeromonas salmonicida could be detected only in a small number of diseased fish,but the proportion of the total diseased fish was extremely low.The specific fragments of turbot iridovirus(TRBIV)and viral hemorrhagic septicemia virus(VHSV)were also not detected by PCR.Treatment with antibiotics such as Oxytetracycline,Enrofloxacin,and Florfenicol has not had any effect and may aggravate the occurrence of death.2.Superficial characteristics and histopathology.The main anatomical features of turbot hemorrhagic disease fish are(1)severe bleeding and lipstick in sick fish;(2)anemia of sick fish,the dark gray appearance of gills;(3)enteritis,ascites;(4)degenerative swim bladder with effusion;(5)when the disease is more serious,turbot kidneys show erosive changes.The results of histopathological studies showed that there were significantly more mast cells in the kidneys and spleen of sick fish than in healthy fish,a large number of lymphocytes were infiltrated,and the liver,heart,kidneys,and spleen of sick fish had different degrees of damage or necrosis.By transmission electron microscopy(TEM),spherical virions of the same shape and size can be found in multiple tissues of sick fish,with a diameter of about 20to 30 nm,of which the kidney and spleen tissues have the most virions.3.Taxonomic status and molecular biology identification of turbot circovirus.A highly abundant circovirus sequence was identified in the kidney samples of the diseased turbot by multiple viroid sequencing analyses.The whole gene sequence of the virus was obtained by PCR amplification,which had a total length of 1774 bp,and three major structural proteins were discovered and predicted,encoding replication-related protein(Rep),capsid protein(Cap),and ORF3 with unknown function.The predicted Cap protein sequence is about 30%similar to the reported circovirus protein,and the Rep protein is about 50%similar to the reported circovirus protein.Phylogenetic analysis showed that the virus belonged to the Circoviridae family,the genus Circovirus.In this study,based on the predicted cap gene of TCV,the synthesis of digoxin-labeled DNA probes into a length of 471 bp was obtained,and the results of in situ hybridization showed that strong positive signals of TCV in the spleen and kidney tissue sections of the diseased turbot,and no positive signal of TCV was detected in healthy turbot tissue sections.4.Establishment of Real-time PCR detection technology methods.In this study,a pair of specific primers were designed and synthesized based on the predicted TCV cap gene with a primer amplification fragment length of 150 bp.By modifying and optimizing the concentration of primers,annealing temperature,number of reaction cycles,etc.,the Real-time PCR detection method of TCV was successfully established.A plasmid(TCV–Cap)diluted with a 10-fold gradient was used as a positive standard,and the TCV SYBR Green standard curve was plotted.Its amplification efficiency is 99.2%,R~2=0.999,and the standard curve equation obtained according to the results is Y=-3.341X+37.594.The lower detection limit of this detection method is 101 copies/μL.Specific fragments of TCV were detected in the kidney and spleen tissues of diseased fish,but not in tissues of healthy turbot.The absolute quantitative detection method was used to detect 390 turbot and three kinds of frozen diet,Ammodytes personatus,Engraulis japonicus,and Enedrias fangi,the results show that all sick turbot can detect the TCV,viral loads up to 10~8-10~9copies/g;Both Ammodytes personatus and Enedrias fangi carry TCV,and it is speculated that chilled food may also be one of the transmission routes of TCV.The establishment of TCV detection technology can provide technical support for the study of the epidemic situation and transmission route of the virus,and provide support for the rapid diagnosis and prevention of“turbot hemorrhagic disease”.5.Pathogenicity of TCV to turbot.Tissue samples of fish naturally infected with"turbot hemorrhagic disease"was collected and tested for confirmation.Diseased tissues were ground and filtered through a 0.22μm filter membrane,and healthy turbot was injected for artificial infection experiments.The results showed that the characterization of artificially infected turbot was consistent with the symptoms of naturally onset turbot,infected fish had a mortality rate of up to 100%within 5 weeks and TCV could be detected in diseased fish.We detected the distribution of TCV in both naturally infected and artificially infected fish and found that specific fragments of TCV could be detected in the liver,spleen,kidney,muscle,heart,and brain tissues of infected fish.The virus copy numbers in the kidney and spleen were the highest.The number of copies of the virus in the kidney tissue of the artificially infected Turbot was 10~7-10~8copies/g and 10~6-10~7copies/g,respectively.Virus copy number in kidney tissue of artificially infected turbot was detected after the challenge,and it was found that the number of TCV increased continuously after infection.Epithelioma papulosum cyprini(EPC)cells,Chinook salmon embryo cells(CHSE-214),Spotted halibut kidney(SHK)cells,and Turbot kidney(TK)cells were infected with a typically pathogenic turbot tissue homogenate.Cell isolation experiments showed that no cytopathic effect(CPE)was observed in all of the above cell lines.Real-time PCR also did not detect specific fragments of TCV in cell isolation experiments,suggesting that TCV may not be sensitive to existing cell lines.The expression of immune-related genes in the head kidney of Turbot was analyzed in artificially infected turbot for 5,10,and 15 days respectively.The results showed that the transcription of MHCI,MHCII,Ig M,IL-1β,and IL-8R was decreased in the head kidney of infected turbot compared with that of healthy turbot.