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In situ hybridisation in herpetic lesions using a biotinylated DNA probe
1Department of Pathology, University Hospital, Lund, Sweden.
Insights
This study demonstrates a herpes simplex virus (HSV) DNA probe accurately detects HSV in various tissues. The probe differentiates HSV from varicella-zoster virus, even without characteristic nuclear changes.
Area of Science:
- Virology
- Pathology
- Molecular Biology
Background:
- Herpes simplex virus (HSV) infections can manifest with diverse clinical presentations.
- Accurate viral detection is crucial for diagnosis and differentiating between herpesviruses.
- Traditional methods for identifying viral inclusions may not always be present or definitive.
Purpose of the Study:
- To evaluate the efficacy of a biotinylated DNA probe for in situ hybridization in detecting HSV.
- To determine if the probe can identify HSV in the absence of characteristic cytopathic effects.
- To assess the probe's ability to differentiate HSV from varicella-zoster virus (VZV).
Main Methods:
- In situ hybridization using a biotinylated HSV DNA probe on formalin-fixed, paraffin-embedded tissue sections.
- High-temperature denaturation of tissue sections.
- Detection using an alkaline phosphatase/nitroblue tetrazolium system.
- Analysis of lung, brain, ganglion, and squamous epithelia tissues.
Main Results:
- The HSV probe successfully hybridized with viral material in cases of HSV encephalitis and viral pneumonitis, correlating with nuclear inclusions.
- Positive hybridization signals were observed in neurons, glia, and satellite cells in HSV encephalitis and trigeminal ganglionitis, even without visible nuclear changes.
- No staining occurred in trigeminal zoster or progressive multifocal leucoencephalopathy.
- HSV-infected epithelial cells in herpetic blisters showed strong hybridization, while VZV lesions were negative.
- Non-specific staining was noted in corpora amylacea and keratin-containing cells, requiring careful interpretation.
Conclusions:
- The HSV DNA probe enables specific detection of the virus in diverse tissues and cell types.
- The method is effective even when characteristic viral nuclear inclusions are absent.
- This technique allows for differentiation between HSV and VZV infections.
- Awareness of potential non-specific binding is essential for accurate interpretation of results.
Abstract:
In situ hybridisation was performed with a biotinylated DNA probe for herpes simplex virus (HSV) using high temperature denaturation on formalin fixed, paraffin wax sections of lung, brain, ganglion and keratinising and non-keratinising squamous epithelia. Eosinophilic viral nuclear inclusions or characteristically moulded multiple nuclei with altered chromatin, which were present in two cases of HSV encephalitis and one case of viral pneumonitis, all showed complete hybridisation visualised by an alkaline phosphatase/nitroblue tetrazolium detector system. HSV encephalitis and trigeminal ganglionitis, which were confirmed serologically or clinicopathologically but lacked nuclear changes, also gave positive dense nuclear signal in neurons, glias and satellite cells. No staining was present in the ganglion cells in trigeminal zoster, the glia in progressive multifocal leucoencephalopathy, or in a variety of cells in a lung coinfected with cytomegalovirus. In 10 herpetic blisters of squamous epithelia, infected cells hybridised strongly, while morphologically similar herpes zoster lesions remained negative. In neural tissues non-hybridisation staining was most obtrusive in corpora amylacea and seemed to reflect nonspecific probe adherence. In squamous epithelium, major non-hybridisation staining was caused by probe and antibody possibly adhering to intracellular keratin. The HSV probe permits specific detection of virus in the absence of characteristic nuclear changes and allows varicella zoster virus to be differentiated from HSV, provided that the aforementioned problems with non-hybridisation staining are borne in mind.