Localization of Epstein-Barr Virus-Encoded Small RNA-1 by in situ Reverse Transcription: Demonstration of cDNA Generation in Formalin-Fixed Paraffin-Embedded Tissue Sections.

Reverse transcription (RT) followed by polymerase chain reaction (RT-PCR) has been commonly used to detect viral and cellular transcripts in whole cell extracts. Application of this technique to tissue sections requires the in situ generation of cDNA. In this study, we selected an abundant transcript, Epstein-Barr virus (EBV)-encoded small RNA (EBER-1), as a model template to demonstrate cDNA generation in tissue sections. Using both digoxigenin-dUTP and primers which are complementary to EBER-1, we demonstrated specific EBER-1 cDNA generation both in vitro, and in tissue sections taken from formalin-fixed paraffin-embedded cell blocks of an EBV-infected cell line, B95-8. Furthermore, we utilized in situ RT in sections of EBV-associated nasopharyngeal carcinomas, and identified EBER-1 cDNA specifically in neoplastic cells, but not in the surrounding nonneoplastic stroma. EBER-1 cDNA was localized to the nucleus of these cells, with relative sparing of the nucleolus and the cytoplasm. No specific signal was evident if the reverse transcriptase was omitted, if 'sense' primers were used, or if RT was preceded by RNase digestion. The specificity of EBER-1 cDNA was further confirmed by in situ hybridization using the sense riboprobe, which has the same polarity as the EBER-1 transcript. Our results provide a successful example of using nonradioactive nucleotide analogue for cDNA generation in formalin-fixed, paraffin-embedded tissue sections. This approach would provide a visible assay to monitor RT in tissue sections, and allow further optimization of conditions for cDNA generation in tissue sections. Therefore, it potentially can be helpful for the future development of RT-PCR in tissue sections. Copyright 1995 S. Karger AG, Basel

Constriction of the umbilical cord by an amniotic band, with fetal compromise illustrated by reverse diastolic flow in the umbilical artery. A case report.

Constriction of the umbilical cord by an amniotic band is a rare entity, associated with a high rate of fetal mortality and congenital anomalies. A case occurred with early detection and successful intervention for fetal compromise caused by the amniotic band, which was associated with first-trimester amniocentesis. This is the first known report of ultrasound Doppler flow studies in a case of umbilical cord constriction by an amniotic band, showing reverse diastolic flow in the umbilical arteries.

Detection of a neuron-specific 9.0-kb transcript which shares homology with antisense transcripts of HIV-1 gag gene in patients with and without HIV-1 infection.

The pathogenesis of the neurologic abnormalities associated with the acquired immune deficiency syndrome (AIDS) is poorly understood. Although human immunodeficiency virus type 1 (HIV-1) transcripts have been detected in endothelial cells and macrophages of the central nervous system in patients with AIDS, infection of neuronal cells by HIV-1 has not been established. The purpose of this study was to localize HIV-1 transcripts in the central nervous system. 3H and digoxigenin-UTP-labeled riboprobes generated from a 942-bp fragment of DNA from the 5' end of the HIV-1 gag sequence were used for in situ hybridization. The antisense riboprobe hybridized to lymphoid cells in the sections of kidney and spleen obtained from patients with AIDS, as well as to the HIV-1-infected A3.01 cell line. The control sense probe did not hybridize to these same cells. In contrast, no detectable hybridization was observed to neuronal cells when the antisense probe was applied to sections of brain obtained from patients with and without AIDS. To our surprise, however, specific hybridization was observed to neuronal cells when the control sense probe was applied. This hybridization with the control sense probe was seen in both patients with and without HIV-1 infection. Northern blot analysis confirmed the in situ hybridization results; a unique 9.0-kb transcript was detected exclusively in brain tissue. These data suggest that there is a neuron-specific 9.0-kb transcript that shares extensive homology with antisense gag HIV-1 sequences and that this transcript is expressed in neuronal cells of both HIV-1-infected and noninfected individuals. The biological significance of this 9.0-kb transcript is unknown, but it may play an important role in the interactions of HIV-1 with neuronal cells.

Virus-associated RNAs (VA-I and VA-II). An efficient target for the detection of adenovirus infections by in situ hybridization.

The identification of adenovirus in tissue can be difficult. In situ hybridization for adenovirus nucleic acids may aid in the demonstration of adenovirus infections. To develop a probe against adenovirus, a 978 bp fragment of DNA containing the VA-I, VA-II, and a portion of the L-1 regions of the adenovirus type 2 genome was cloned into the SK+ vector. These regions were selected because they are generally conserved among adenoviruses and are abundantly transcribed during the lytic cycle. Sense and antisense tritium or Digoxigenin-labeled riboprobes were generated using in vitro transcription and applied to formalin-fixed paraffin-embedded sections of HeLa cells infected with adenovirus type 2. Extensive in situ hybridization of the antisense riboprobe to HeLa cells with cytopathic changes was found. The number of cells to which the probe hybridized decreased proportionately with dilution of infected with noninfected cells. The control sense riboprobe showed only scattered breakthrough hybridization and in these cells hybridization was mainly located in the nucleus. Northern blot analysis of RNA from infected HeLa cells confirmed the in situ hybridization results. No hybridization was detected when cultured cells infected with herpes simplex virus, Epstein-Barr virus, cytomegalovirus, or human immunodeficiency virus were examined. Specific hybridization was detected in tissues obtained at autopsy from four patients with culture proven adenovirus infection. These observations suggest that this probe is useful in the diagnosis of adenovirus in formalin-fixed paraffin-embedded material.