Molecular Diagnosis of Epstein-Barr Virus-Related Diseases

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Presentation transcript:

Molecular Diagnosis of Epstein-Barr Virus-Related Diseases Margaret L. Gulley The Journal of Molecular Diagnostics Volume 3, Issue 1, Pages 1-10 (February 2001) DOI: 10.1016/S1525-1578(10)60642-3 Copyright © 2001 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 A: H&E stain of invasive gastric adenocarcinoma surrounded by normal surface epithelium. B: EBER in situhybridization reveals EBER transcripts in the nucleus of the carcinoma cells, but not in the overlying normal surface epithelium, nor in the surrounding benign stromal cells. C: EBER is localized to dysplastic gastric epithelium but not to adjacent normal-appearing glands, implying that EBV infection is an early event in gastric carcinogenesis. D: EBER is localized to the nucleus of a single small lymphoid cell, representing the rare infected lymphocyte that might be found in any previously infected individual. Original magnifications, ×50 (A and B), ×80 (C), and ×150 (D). The Journal of Molecular Diagnostics 2001 3, 1-10DOI: (10.1016/S1525-1578(10)60642-3) Copyright © 2001 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 The EBV clonality assay evaluates clonality with respect to the structure of the EBV genome. The assay is based on the presence of variable numbers of tandem repeat sequences (shown as open boxes) at the ends of the linear viral genome. On infection of a cell, these ends join to form an episome by fusing up to 20 terminal repeat sequences. When an infected cell undergoes malignant transformation, the same fused terminal repeat structure is inherited by all progeny of the malignant clone. The clonality assay is accomplished by Southern blot analysis of DNA extracted from patient tissue and digested with BamHI restriction endonuclease (shown by arrows) to cut the EBV genome at sites flanking the terminal repeats. This results in restriction fragments that are recognized by a DNA probe (black bar). Examination of the band pattern on Southern blots reveals that infectious virions produce a ladder array of small bands. In contrast, a monoclonal tumor exhibits a single band of high molecular weight, and an oligoclonal tumor has several such bands. The Journal of Molecular Diagnostics 2001 3, 1-10DOI: (10.1016/S1525-1578(10)60642-3) Copyright © 2001 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 The EBV viral load assay is accomplished by coamplification of EBV DNA and a control sequence that is spiked into the sample before DNA extraction. In the experiment shown here, assay linearity was tested on serial twofold dilutions of EBV DNA. PCR products at the endpoint of amplification were evaluated by agarose gel electrophoresis. In lanes 1–8, the EBV product is seen as a 210-bp band at template levels as low as 5 copies. The control product, visible at 260 bp, ensures that no inhibitors are present, and it also serves as a gauge by which to extrapolate the amount of EBV template in each sample. A molecular weight (MW) marker is shown on the left, and lanes 13 and 14 represent control reactions to which no template was added. The Journal of Molecular Diagnostics 2001 3, 1-10DOI: (10.1016/S1525-1578(10)60642-3) Copyright © 2001 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Primary EBV infection in healthy hosts is accompanied by an orchestrated serological response. IgM antibody against viral capsid antigen (VCA) rises first. Antibodies against EBNA appear at least 1 month after primary infection and are measured, along with IgG anti-VCA, as markers of prior infection and as indicators of EBV reactivation. Titers against early antigen (EA) rise on primary infection and again in pathological states of EBV reactivation. The Journal of Molecular Diagnostics 2001 3, 1-10DOI: (10.1016/S1525-1578(10)60642-3) Copyright © 2001 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions