Establishment Of A Model For Decapod Iridovirus Infection In Macrobrachium Rosenbergii And Screening Of Antiviral Drugs

Decapod iridescent virus 1（DIV1）is a DNA virus belonging to the genus Decapod iridovirus within the family Iridoviridae.DIV1 is the pathogen that causes the“white head disease”of M.rosenbergii,with a maximum mortality rate of over 80%,seriously restricting the development of the M.rosenbergii aquaculture industry.At present,the mechanism of action of DIV1 on M.rosenbergii is not clear,and there are no effective prevention and treatment drugs for this disease.This study aims to analyze the regulatory mechanism of M.rosenbergii on DIV1 by constructing a virus infection model;screen drugs with certain therapeutic effects on DIV1,elucidate their mechanism of action against DIV1,and provide basic data and theoretical support for the prevention and treatment of decapod iridovirus.The contents of this study are divided into four sections as follows:1.Construction of a DIV1 infection model for M.rosenbergiiThis study first determined the median lethal concentration(LD₅₀)of DIV1 on M.rosenbergii through injection sensitization experiment.Furthermore,by exploring the effects of different factors such as injection dose,water temperature,and body size on the infection of M.rosenbergii with DIV1,we constructed a DIV1 infection model for M.rosenbergii.The results showed that the optimal in vivo infection model conditions for DIV1 on M.rosenbergii included water temperature of 25°C,body size of 20 g,and injection dose of 0.1 m L 10 times LD₅₀.The clinical symptoms of M.rosenbergii infected with DIV1 mainly included redness of the body,yellowing of gill filaments,and hepatopancreatic enlargement.The histopathological changes involved in obvious contraction and deformation of the hepatic tubular stellate structure,and vacuolar degeneration of liver cells;The connection of gill filaments was loose,muscle fiber disordered,and there were vacuoles and cell swelling in the columnar epithelium of the upper layer of gill filaments.Compared with other tissues,DIV1 proliferated fast in the gill tissue of M.rosenbergii,and virus replication increased exponential growth after 24-72 hours infection.In addition,after infection with DIV1,immune related genes such as acid phosphatase（ACP）,alkaline phosphatase（AKP）,catalase（CAT）,copper-zinc superoxide dismutase（Cu/Zn SOD）,C-type lectin（CTL）and phenol oxidase progenitor（Pro PO）were up-regulated to varying degrees in the hepatopancreas of M.rosenbergii.2.Analysis of response mechanism of M.rosenbergii to DIV1 by transcriptome sequencingTo further explore the response mechanism of M.rosenbergii to DIV1,this study conducted transcriptome sequencing analysis on hepatopancreas tissues of M.rosenbergii infected group and control group based on the constructed DIV1 infection model.The results showed that the Q20ratio of raw reads in the six sequencing libraries was more than 97.67%,and the Q30 ratio was more than 93.56%,indicating that the transcriptome sequencing quality was good;The results of correlation analysis and cluster analysis indicated that there was good reproducibility between the samples of the infection group and the control group;A total of 1530 differentially expressed genes（DEGs）were identified through differential expression analysis,including 708 up-regulated expression genes and 822 down-regulated expression genes;Using GO and KEGG annotations,it was found that most DEGs were enriched in Warburg effect,signaling molecules and interaction related pathways,revealing that these pathways can participate in regulating the replication of DIV1 in M.rosenbergii;In addition,the reliability of transcriptome data was verified by q RT-PCR analysis of CTL,CAT,Cu/Zn SOD and Pro PO genes.3.Screening of antiviral drugs for M.rosenbergiiUsing the constructed virus infection model,this study investigated the anti-DIV1 function of 19 conventional drugs such as aloe-emodin.Firstly,the safety of the drug was determined through acute toxicity assay,and 19 drugs were co-incubated with DIV1 within the determined drug safety range;Then,0.1 m L of drug and DIV1 virus premix was injected into the base of the abdominal segment of M.rosenbergii,the cumulative mortality rate was calculated,and the protection rate of 19 drugs against M.rosenbergii was detected;Use q RT-PCR method,we determined the viral load of M.rosenbergii after drug administration,and screen effective drugs against DIV1.The results showed that the 19 drugs tested,including aloe emodin,did not cause death of M.rosenbergii within the range of 50 mg/kg.The results of antiviral efficacy showed that aloe-emodin,doxycycline hydrochloride and vinblastine hydrochloride had anti-DIV1 effects,among which aloe emodin had the best effect.At a concentration of 50 mg/kg,the protection rate of aloe-emodin against M.rosenbergii was 32.5%,and the inhibition rate of aloe-emodin against viruses was 77.06%.4.Study on the anti-DIV1 effect of aloe-emodinWith the optimal effective concentration of 25 mg/kg as the dosage,we explored the inhibition role of aloe-emodin on DIV1 virus at different administration times.The changes of histopathology of M.rosenbergii before and after administration were detected by H&E staining.We used q RT-PCR to investigate the regulatory effect of aloe-emodin on immune related genes and antioxidant related enzyme activity in M.rosenbergii,and the mechanism of aloe emodin against DIV1 was preliminarily elucidated.The results showed that after 24,48,and 72 hours of infection with DIV1,the virus inhibition rates of aloe-emodin on M.rosenbergii were 63.31%,83.19%,and 92.95%,respectively.After 72 hours of using aloe emodin,the pathological damage caused by virus infection was significantly improved.In addition,we found that aloe-emodin could significantly enhance the enzyme activities of AKP,ACP,SOD,and CAT（P<0.01）,thereby enhancing the antioxidant capacity of M.rosenbergii.Meanwhile,aloe-emodin could significantly up-regulate the expression levels of immune related genes AKP,ACP,CAT,and Pro PO,and inhibited the expression of C-type lectin（CTL）gene（P<0.01）.