| Cyprinid herpesvirus type Ⅱ（CyHV-2）,a genus of Cyprinivirus in the family Herpesviridae,is an important pathogen of hematopoietic organ necrosis in goldfish and crucian carp,which is spreading worldwide and causing significant economic losses in goldfish and crucian carp aquaculture.The possibility of virus level transmission in the water environment can’t be ignored,however,the virus level in the aquaculture water environment is often too low to be accurately determined,so the establishment of an efficient enrichment method for concentrating virus in large volume water samples can help monitor the virus level in the aquaculture water environment,and the timely use of green and effective disinfectants to interrupt the horizontal transmission of virus is also important for the biosecurity of aquaculture industry.This project is aimed at developing a method for the detection of C In this study,an iron flocculation method was developed for the enrichment and concentration of CyHV-2 and combined with real-time fluorescence quantitative PCR to further investigate the kinetics of horizontal transmission of CyHV-2 in goldfish infected in the aquaculture water environment and also to provide preliminary answers on the effectiveness of different disinfectants in blocking the horizontal transmission of CyHV-2.The results of the study were as follows:1.Study on the enrichment of CyHV-2 in concentrated aqueous environment by iron flocculation.CyHV-2 with different concentration gradients was concentrated and recovered by iron flocculation,the virus copy number before and after recovery was detected by real-time real-time PCR,the sensitivity and recovery gradient range of iron flocculation method were determined,and the GiCF cell line was used to culture the concentrated recovered CyHV-2.The results showed that when the concentration of CyHV-2 was 103.0～107.0 copies/m L,the iron flocculation method could efficiently recover CyHV-2 in the water,and when the virus copy numbers were added 103.0,104.0,104.8,105.5,106.0,107.0 copies/m L（3 parallel per gradient）,the average copy numbers after concentration and recovery were 103.16,104.17,104.76,105.51,respectively.105.93、106.58 copies/m L.The fitting curve of the added virus and the recovered viral copy number was established by the above concentration gradient,and R2=0.994showed a good linear relationship.All concentration gradients recovered by iron flocculation can produce cytopathic effects in GiCF cells.Observation of concentrated recovery under transmission electron microscopy showed that intact CyHV-2 virus particles were observed.2.CyHV-2 horizontal propagation kinetics.We immersed healthy goldfish in a serial dilution of CyHV-2 virus added with107～103 copies/L,and found that the virus concentration in the aquatic environment as low as 104 copies/L can also effectively cause infection of healthy goldfish,and the highest mortality rate occurred 8～10 days after immersion.The iron flocculation method established in Chapter I was used to monitor the virus level in the aquaculture water environment.A significantly high level of 1×104.5 copies/L is reached on day 4of the immersion experiment and then drops to a lower level,and a maximum level of1×105 copies/L is reached on day 8 of the immersion experiment and then drops again to a lower level of 103.2 copies/L until day 10 post-infection.Our experimental results demonstrated that the level of virus shed by CyHV-2 infected goldfish was sufficient to cause horizontal transmission of CyHV-2 in the goldfish culture water environment.Real-time PCR determined the genome copy number of kidney and spleen virus infected with dead fish for different days after immersion,and in the two parallel experiments,the viral load of the surviving fish was quite different,and the virus shedding ability of individual fish was significantly different.3.Study on the effect of different disinfectants in blocking CyHV-2Commercial disinfectants complex sodium chlorite,hydrogen peroxide,povidone-iodine,sodium perperiodate have been widely used in aquaculture to control microbial infections by splashing directly in water.The results of in vitro inhibition of CyHV-2 by compound sodium chlorite powder showed that the inactivation rate of 62.5μg/L（close to the safe concentration of 60μg/L for goldfish）was only 87.8431%for virus titers of 3.89 Log10 TCID50/m L.In the detection experiment of disinfectant on cell viability,the recommended concentrations of hydrogen peroxide,povidone-iodine and sodium periodate had no significant effect on GiCF cells,and the viability detection results of several disinfectants were higher than 94%after incubating GiCF cells at the recommended concentrations of aquatic products of several disinfectants,while in the in vitro inhibition of CyHV-2experiments on hydrogen peroxide,povidone-iodine and sodium periodate,except for100μg/L and 200μg/L of povidone-iodine,the inactivation rate of CyHV-2 was less than 90%Among them,hydrogen peroxide has the worst disinfection and inactivation effect,all of which are less than 50%.Therefore,povidone-iodine and sodium periodate were selected as alternative disinfectants for aquaculture water,and the results of horizontal transmission interruption experiments in aquaculture water showed that all immersion infection groups were consistent with the positive control group,reaching a high mortality rate of 100%,indicating that horizontal transmission during virus outbreaks is very harmful,and the effect of disinfectants as therapeutic drugs to block horizontal transmission and reduce the mortality of aquatic animals is not ideal.Overall,our data provided quantitative data to demonstrate the shedding strength of CyHV-2 in water,and the four aquatic disinfectants of sodium chlorite,hydrogen peroxide,povidone-iodine,and sodium periodate have been shown to be ineffective in blocking the horizontal transmission of CyHV-2 in goldfish in aquaculture water.The virus concentration protocol and viral suppression tests established here also pave the way for evaluating the effectiveness of more aquatic disinfectants in interrupting transmission at CyHV-2 levels. |
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