Studies On The Tissue Tropisms And Pathogenicities Of Mollusc Herpesviruses

In this paper,two strain of known mollusc herpesviruses,Ostreid herpesvirus 1(Os HV-1)and Haliotid herpesvirus 1(Ha HV-1)were used a pathogens to infected Scapharca broughtonii,Haliotis diversicolor supertexta and Haliotis discus hannai by artificial infection,respectively.Molecular biological detection,histopathological analysis,transmission electron microscopy and in situ hybridization technology were used to analyze the histopathological changes caused by Os HV-1 and Ha HV-1 infection,the infected site of parasitic tissue,tissue tropism,the type of infected cells and pathological characteristics,and the pathogenicity of two viruses to mollusc were studied and discussed.Ⅰ.Tissue tropism and infection process of Os HV-1 to S.broughtoniiReal-time quantitative PCR was used to detect the viral load in different tissues of S.broughtonii infected with Os HV-1.The viral load of each tissue increased rapidly after 24 h of infection,peaked at 48 h,and gradually decreased at 72 h.Histopathological and in situ hybridization results showed that Os HV-1 positive signal was observed in hepatopancreas,mantle,gill,foot and adductor muscle at 48 h and 72 h,the type of infected tissues and pathogenesis time was consistent with that of in situ hybridization,tissue tropism of virus was mainly connective tissue,and the types of infected cells are fibroblasts,myocytes and infiltrating blood cells.In the course of virus infection,hepatopancreas,gills and mantle were most seriously infiltrated by blood cells,and showed strong positive signals,followed by adductor muscle and foot.Ⅱ.Susceptibility of two abalone species,Haliotis diversicolor supertexta and Haliotis discus hannai,to Haliotid herpesvirus 1 infectionTwo cultivated abalone species,H.diversicolor supertexta and H.discus hannai were challenged with Ha HV-1-CN2003 collected in 2003 in China using three different methods(injection,immersion infection,cohabitation infection).Results showed that H.diversicolor supertexta was highly susceptible to Ha HV-1,the cumulative mortality rate after infection for4 days reaches 100%.On the contrary,H.discus hannai was not susceptible to the viral infection.Different methods of infection had no effect on host infectivity.Different infection methods have no effect on host susceptibility.Histopathology combined with transmission electron microscopy showed that the tropism of Ha HV-1 includes both neural tissue and haemocytes.III.Tissue tropism and infection process of Ha HV-1 to H.diversicolor supertextaH.diversicolor supertexta is highly susceptible to Ha HV-1.The tropism and infection process of Ha HV-1 on different tissues of H.diversicolor supertexta were investigated by real-time PCR,histopathology and in situ hybridization technology.The number of virus copies increased rapidly after infection for 24 h,the viral load reached the peak at 48 h after infection,and decreased after 72 h of infection.The infected tissues of H.diversicolor supertexta were mainly nerve,mantle,hepatopancreas and gill.Virus positive signal were observed in nerve tissue and mantle at 48 h after infection.But positive signal were detected in hepatopancreas and gill at the late infection(72 h).It can be seen that the first stage of Ha HV-1 infection was nerve and mantle,finally hepatopancreas and gill.The results showed that tropism tissues and cells of Ha HV-1 includes nervous centralis and peripheral nervous,neurogliocyte and haemocytes.Ⅳ.Infection of abalone hemolymph in vitroTo establish a method for suspension infection of abalone hemolymph with Ha HV-1 in vitro,Ha HV-1 tropism for abalone hemolymph was confirmed.In vitro infection results showed that the hemolymph of H.diversicolor supertexta were susceptible to Ha HV-1,and the viral load could reach 10~7 copies/ng of total DNA at 60 h after infection,while the hemolymph cells of H.discus hannai were not susceptible to Ha HV-1,the results were consistent with the challenge infection abalone in vitro.After the primary culture of abalone hemolymph cells is infected with the virus,cell aggregation,fragmentation,irregularity,etc.Giemsa staining can roughly divide hemolymph cells into three categories:granulosa cells,clear cells,lymphoid-like cells,the exact type of cells that are susceptible to viral probiosis and infection remains to be determined.V.Establishment and application of indirect in situ hybridization PCR for detection of shellfish herpesvirusTo detect the localization of shellfish herpesvirus in tissues of shellfish host,a sensitive method was firstly established based on indirect in situ polymerase chain reaction(ISPCR)technology.Then the method was employed to investigate Os HV-1 distribution pattern in different types of organs of S.broughtonii.To establish the detection method,suitable amplification cycles for indirect ISPCR were selected and the concentrations of proteinase K for digesting different types of tissues were also optimized.The results showed the optimum number of amplification cycles were 20,the optimum concentration of proteinase K were 20μg/m L.We used the optimized indirect ISPCR method to detect and analyze the distribution pattern of Os HV-1 in five different organs of ark shells(mantle,gill,hepatopancreas,foot and adductor muscle).The results showed that the virus positive signals were mainly distributed in infiltrated hemocytes and fibrobalsts in connective tissues of the mantle,infiltrated hemocytes in hepatopancreas and gills,necrotic muscle cells in foot and adductor muscle.These results showed that the established indirect ISPCR method for detecting had the advantages of high sensitivity and specificity in fixing the viral position.The method could be used for studying the distribution of shellfish herpesvirus in different tissues and targeted cell types by investigating positive signals of viral nuclear acid in tissue sections.The method could be used in for confirmatory diagnosis of shellfish herpesvirus infection,the tissue tropism,invasion routes and pathogenic mechanism of shellfish herpesvirus.