Establishment and application of indirect in situ polymerase chain reaction for detection of Ostreid herpesvirus-1 (OsHV-1)

To detect the localization of Ostreid herpesvirus-1 (OsHV-1) in tissues of shellfish host, a sensitive method was first established based on indirect in situ polymerase chain reaction (ISPCR) technology. Then the method was employed to investigate OsHV-1 distribution pattern in different types of organs of Scapharca broughtonii. To establish the detection method, suitable PCR primers and amplification cycles for indirect ISPCR were selected, which could generate stable and specific amplification of viral DNA in fixed tissues on sections. The concentrations of proteinase K for digesting different types of tissues were also optimized. Finally, in situ hybridization was conducted with digoxigenin-labeled nucleic acid probes prepared using the same primers, and the distribution of OsHV-1 in the sample tissues was shown by immunoenzyme labeling technology. The results showed the optimum number of amplification cycles was 20, and the optimum concentration of proteinase K was 20 μg/mL. We used the optimized indirect ISPCR method to detect and analyze the distribution pattern of OsHV-1 in five different tissues 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 OsHV-1 had the advantages of high sensitivity and specificity in fixing the viral position. The method could be used for studying the distribution of OsHV-1 in different tissues and targeted cell types by investigating positive signals of viral nuclear acid in tissue sections. The method could be used for confirmatory diagnosis of OsHV-1 infection, the tissue tropism, invasion routes and pathogenic mechanism of OsHV-1.