| Green turtle（Chelonia mydas）is a flagship and umbrella species,which is regarded as a health indicator of marine ecosystems for a long time.As a result of long-term overuse,green turtle becomes an endangered species,which is listed as the first class national key protected wild animals.It is of great significance to study the pathogen susceptible to green turtles and the detection of novel viruses is the weakest link in the protection of green turtles.At present,a large number of release activities have not carried out effective risk assessment on the pathogen carrying status of green turtles,which poses a serious threat to the health of the green turtle population.The rescue process before release helps all kinds of pathogens to reproduce.Due to the limited space and other factors,the space for rescue of green turtles is often very cramped.In this cramped space,high-density captive breeding makes many pathogens break through the immune defense of green turtles.From 2018 to 2022,unexplained population deaths of green turtles occurred in captive sites in Wenchang and Qionghai of Hainan Province,and the surviving individuals also gradually died of diseases.In this study,NGS（Next Generation Sequencing）sequencing method was used to detect green turtles in Hainan,and the detection results were compared with a normal stranded green turtle.The complete genome sequence of a new type green turtle hepatitis C virus was obtained,which was tentatively named ChHCV,the virus is most likely closely linked to the deaths of green sea turtles.The sequence of ChHCV was verified by Sanger sequencing,then we compared and analyzed the ChHCV with the known viruses,established the detection method of ChHCV,after that,we carried out epidemiological study on the captive,rescue and wild green turtle in Hainan area,and preliminarily found out the epidemiology the virus.This study will serve as a demonstration for detecting unknown susceptible viruses in marine turtles.Main study results and innovations:1.Complete genome sequence of ChHCV abtained based on NGS methodA number of captive dead green turtles in Wenchang and Qionghai of Hainan province were collected for necropsy,and brain,heart,liver,spleen,lung,kidney and tissue fluid were selected as samples for NGS sequencing.The same operation was also performed on another green turtle which was stranded and dead on the beach.The results of NGS sequencing showed that only the ubiquitous virus,such as ChHV5,were found in the dead green turtle which was stranded on the beach,novel viruses of flaviviridae were also detected in the dead green turtles which under captive conditions.The complete genome sequence of a novel green turtle hepatitis C virus was obtained by using bioinformatics technology after the NGS sequencing data were processed by the independently developed analytical process for identifying unknown viruses.The discovery of hepatitis C virus in green turtles is the first report in the world and the first discovery of flaviviridae virus in marine turtles.2.Complete genome sequence analysis and validation of ChHCVThe NGS sequencing method was used to obtain a novel type of hepatitis C virus,with a complete genome sequence of 9430 bp.Phylogenetic tree analysis shows that ChHCV in the green turtle is a novel species of flaviviridae virus.Then,the complete genome sequence was verified by Sanger sequencing,and a pair of primers were designed every 1000 bp.The degenerated bases in the presequence results were detected,and the NGS sequencing results were completely verified.The full length of one ChHCV genome was 9430 bp with GC content of 45%.According to Sanger sequencing results,20 bp of NGS sequencing results were modified and determined,the consistency between Sanger sequencing results and NGS sequencing results was 99.79%.3.Establishment of ChHCV detection methodA realtime RT-PCR method for detection of ChHCV was established.In view of the conserved sequence of ChHCV complete genome,several pairs of primers and probes were explored and one pair of primer and probe was selected to establish the detection method,which can accurately detect ChHCV.The method can effectively distinguish ChHV5（Chelonid herpesvirus 5,ChHV5）,RSIV（Red sea bream iridovirus,RSIV）,Vibrio Parahemolyticus(Vibrio Parahemolyticus,Vp AHPND)with high specificity and except ChHCV,the results did not show typical“S”curve in realtime RT-PCR reaction.The method also showed high sensitivity and the minimum detection limit is 2.6 copies/μL.The coefficient of variation was 0.200%～0.664%.This method can be used to detect ChHCV in captive,stranding and wild green turtles.4.Epidemiological study of ChHCVAccording to the virus detection method established in this study,ChHCV detections were performed on green turtles from various captive sites,included captive,stranded and wild individuals in Hainan Province.Among them,77 were collected in captive sites,and25 were positive.26 rescue individuals were collected,and 8 of them were positive.No ChHCV was detected in one stranded individual and two wild individuals.A total of 106green turtles were collected in this study,and 33 were positive,with a total detection rate of31.1%.ChHCV infection has not been found in pure wild green turtles,and captive condition may be the main factor for the occurrence and transmission of the novel virus,so it is urgent to regulate the release of marine turtles. |
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