| In recent years,reptiles,amphibians,and fish diseases caused by ranavirus have been prevalent in the Americas,Europe,Asia and Australia,and because of its wide range of host species,high morbidity rates and high mortality rates,ranaviral disease is considered to be the most direct cause of several decline even extinction in the number of amphibians.Of the five genera in the family of Iridiviridae,the genus Ranavirus is the most widely distributed,and its representing species is Frog Virus 3(FV3),which can infect kinds of economic and pharmaceutical animals in fish,reptiles and amphibians,so it has caused serious damage to these animals’ wild populations and aquaculture industries.In view of the serious damage caused by ranavirus infection,the World Organisation for Animal Health(OIE)has included ranairus as a list of OIE-Listed diseases,infections and infestations in force.In addition,due to the rapid onset and short course of ranaviral infection,and the 100%mortality rate after oneset,no better treatment has been developed now.China is one of the countries with the most amphibious resources in the world,so in the global context where the number of amphibians is rapidly falling,the protection of amphibians in China needs to be more widely popularized and developed.In this study,using the method of epidemiological survey combined with pathology,cytology,molecular biology,high-throughput sequencing technology,the ranavirus isolated from Rana dybowskii in the Heilongjiang province of China was studied,including the virus isolated and the host transcriptome studies,and finally the screening work of medicines that are resistance to ranavirus was carried out;this study not only helped fully understandding the distribution of ranavirus in the wild population of R.dybowskii in Heilongjiang area,but also laying the foundation for the study of diagnosis,prevention and treatment for ranavirus,which is of great significance for the protection of local ecological health of cold-blooded vertebrate populations.1.The the viral isolation in the wild population of R.dybowskii in Heilongjiang province,ChinaUsing the rapid detection method of PCR and LAMP established in this study,the distribution of ranavirus in the wild population of R.dybowskii was re-investigation.The survey area was selected from the area of Heilongjiang which ranavirus showed up before,including a total of 3 sampling site-Hebei,Heihe and Dongfanghong forest farm,and the detection samples were a total of 511 liver tissue samples,5 ranavirus positive samples were detected in this investigation.According to the method of isolation and identification of ranavirus recommended by OIE,the ranavirus positive samples from the survey were identified and isolated.Based on the sequencing results of these positive samples,the multiple sequence alignment and system phylogenetic analysis were performed using the MCP gene of ranavirus,it was found that the positive samples had a 99%identity with the FV3 sequences and were closer to FV3 in the taxonomic status,so it was confirmed to belong to the genus Ranavirus;using EPC cell line,the virus was isolated and studied,and a titer of 107.231 TCID50 was obtained by cell culture and virus titer determination;besides under the electron microscope,the ranaviras particles and their replication in cells were observed:its virus assembly area was seen in the cytoplasm,and the assembled virus particles were hexagonal and in crystalline arrangement,and some mature virus particles were released from the cells by sprouting2.Transcriptome study of R.dybowskii exposed to ranavirusUsing high-throughput sequencing techniques,all transcripts information of R.dybowskii were obtained for the first time,with a total of 153,979 gene sequences.According to the results of various gene database comparisons,gene annotation analysis were performed on these genes;using the comparative transcriptomics,the analysis of differentially expressed genes and signal pathways that they involved in R.dybowskii exposed to ranavirus was performed and found 58 differentially expression genes like Gene MHC-I,TGFBR2,PARP and so on;41 involved signal pathways were also obtained,including some important immunological correlations In laboratory independent infection experiments,qPCR was used to verify the results of the transcriptome of R.dybowskii.Six differentially expressed genes were selected and their mRNA expression changes were studied.Through the verification study,the results are in accordance with the transcriptome conclusions,confirming that the transcriptome results of this study are credible;besides,the expression of mRNA expression changes of three immune-related genes at different time points and in different tissues exposed to ranavirus were studied and analyzed.This study provided research ideas and basis for studying the mechanism of ranavirus infection and host immune response.3.Study on the anti-ranavirus effect of two commonly used antiviral Chinese medicine preparationsUsing the established models of ranavirus infection in vitro and in vivo,the antiviral effects of two antiviral Chinese medicine preparations(Astragalus and radix isatidis injection)were evaluated.In an in vitro model of ranavirus-infected EPC cells,CCK8 and apoptotic flow cytometry were used to detect the antiviral effects of the two drugs at different concentrations and in different ways(prevention and treatment).After treatment with radix isatidis(30mg/ml),the survival of EPC cells increased and the apoptosis rate decreased significantly.In experimental animal infection experiments,qPCR was used to detect the changes of frog virus load in R.dybowskii and fold-changes of mRNA expression of three related immune genes,it was also found that Radix Isatidis reduced the infection load of ranavirus in the liver tissues and increased the mRNA expression of these immune genes in the treatment and prevention of ranavirus infection.According to the results of in vitro and in vivo experiments,radix isatidis has believed to have a certain antiviral effect on ranavirus. |
