Summary auto-generated
This study reports the first successful purification of a herpes-like virus from the Japanese oyster Crassostrea gigas, a bivalve mollusc. Herpes-like viruses have been associated with high mortality rates in cultured oyster species since the 1970s, but diagnosis has relied on laborious histological and electron microscopy methods. The researchers developed a purification procedure using sucrose density gradient centrifugation to isolate virus particles from infected larvae. Transmission electron microscopy revealed both empty capsids (70-80 nm diameter) and enveloped virions (110-130 nm diameter). High molecular weight viral DNA was successfully extracted and characterized. Restriction enzyme digestion with EcoRI and BglII produced over 24 fragments each, and genome size was estimated at approximately 180 kilobase pairs by summing the largest restriction fragments, consistent with typical herpesvirus genome sizes. Partial cloning of the viral genome was achieved, and selected cloned fragments demonstrated specificity for virus DNA through dot blot hybridization, showing no hybridization to healthy oyster DNA. These purified viral particles and cloned genome fragments provide tools for developing improved diagnostic methods, such as PCR-based assays and immunological detection, to better understand this economically important oyster pathogen.
Key findings
- First successful purification of a virus pathogen from a bivalve mollusc using a sucrose gradient method that yielded capsids and enveloped viral particles
- Viral genome estimated at approximately 180 kilobase pairs, within the typical range for Herpesviridae, determined by restriction enzyme digestion and fragment analysis
- Partial genome cloning and characterization achieved with specific cloned fragments showing hybridization to infected oyster DNA but not to healthy oyster DNA
- Purified viral particles and cloned genome fragments enable development of rapid diagnostic techniques such as PCR assays to detect herpes-like virus infections in oyster stocks
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Abstract
First observed in 1972 in Crassostrea virginica, herpes-like viruses of bivalves were more recently found to be associated with high mortality rates in other cultured oyster species, such as Crassostrea gigas and Ostrea edulis. The diagnosis of herpes-like virus infections is performed currently by laborious histological and transmission electron microscope examinations. Preparation of specific reagents for use in more amenable diagnostic techniques prompted purification of virus particles and investigation of the viral genome. This paper is the first description of the purification of a virus pathogen from a bivalve mollusc. A procedure was developed which facilitated purification of large amounts of virus particles on the 40--50% interface of sucrose gradients. Transmission electron microscopy showed that a purified virus suspension contained capsids and enveloped virus particles. High molecular mass viral DNA was extracted, and the genome size was estimated by the summation of the sizes of restriction endonuclease fragments to be approximately 180 kbp. Partial cloning of the virus genome was achieved and the specificity of certain cloned fragments was established by dot blot hybridization.