Herpes-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes.
An in situ hybridization protocol for detecting the herpes-like virus which infects French Pacific oysters, Crassostrea gigas, was developed. Two DNA probes were synthesized by incorporation of digoxigenin 11-dUTP during PCR. Two oyster herpes-like virus specific primer pairs, A5/A6 and C1/C6, were used. Both DIG-labelled probes were able to detect 50 pg of herpes-like virus PCR amplified DNA in Southern blot hybridizations. The probes hybridized with viral DNA in paraffin sections of infected C. gigas spat. No non-specific binding was observed. The ability of the defined in situ hybridization technique to diagnose herpes-like virus infections in oysters was compared with light and transmission electron microscopy techniques in infected and non-infected spat. In situ hybridization assays were also conducted on paraffin sections to determine virus distribution within the host and to study the pathogenesis infection. In situ hybridization confirmed that the expression pattern of the herpes-like virus was restricted to connective tissues as described previously by light and transmission electron microscopy. However, this technique also allowed the detection of viral DNA in the oyster nervous system. Some labelled cells were observed in the visceral ganglion of infected oyster spat.
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Laboratoire de Genetique et Pathologie, IFREMER, 17390, La Tremblade, France.
Renault
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D000818:Animals; D015139:Blotting, Southern; D015342:DNA Probes / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D004076:Digoxigenin / Q000031:analogs & derivatives* / Q000737:chemistry*; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity / Q000648:ultrastructure; D017403:In Situ Hybridization / Q000379:methods*; D010125:Ostreidae / Q000648:ultrastructure / Q000821:virology*; D010140:Pacific Ocean; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; C070202:digoxigenin-11-deoxyuridine triphosphate; D004076:Digoxigenin