Hubert E. Blum

Detection of Viral Nucleic Acids by in Situ Hybridization

Publisher Summary Methods to adapt hybridization techniques for preparations of cells and chromosomes were devised and introduced in 1969 and used successfully to localize highly reiterated or amplified genes. Improvements in both hybridization techniques and labeling of specific probes have extended the compass of in situ hybridization to detection of single genomes of viruses in cells and single genes in chromosomes. These developments herald a new era in the analysis of viral infections and the role of viruses in chronic diseases and present some exceptional opportunities to examine the expression of genes in individual cells in other disciplines, such as developmental biology. This chapter provides descriptions of methods, presents examples of the use of in situ hybridization in virology, and discusses some of the important issues of which solution will be greatly influenced by in situ hybridization. In in situ hybridization, a nucleic acid probe, labeled radioactively or with a reporter molecule, is annealed to suitably prepared cells or chromosomes, and the formation of hybrids is assessed by autoradiography, by fluorescence, or by histochemical means. The great increase in sensitivity achieved in current techniques is the result of the increased signal generated by contemporary probes and the increased efficiency of hybridization. This chapter reflects these improvements, first, in the preparation of probes and, second, in the treatment of cells and conditions for hybridization.

Molecular pathogenesis of hepatitis B virus infection: simultaneous detection of viral DNA and antigens in paraffin-embedded liver sections.

Hepatitis B virus (HBV) DNA and viral antigens were simultaneously identified by immunohistochemical staining of formalin-fixed, paraffin-embedded liver sections followed by in situ hybridisation. In the developed radioautographs, silver grains indicate the location of viral DNA in the cell and the immunohistochemical stain marks sites of accumulation of viral antigen. In liver from a patient with chronic active hepatitis serologically positive for hepatitis B surface antigen and e antigen (HBsAg/HBeAg) viral nucleotide sequences, representing actively replicating DNA species, were demonstrated predominantly in the cytoplasm. Viral core antigen (HBcAg) was expressed in the liver cell nuclei. HBcAg was not detectable in most hepatocytes with high levels of viral replication. Conversely, most liver cells in which HBcAg was found did not contain replicating HBV. HBcAg and replicating viral DNA species were not detectable in hepatocytes undergoing pathological changes, such as ground glass cells. Because no pathological changes could be identified either in hepatocytes with high levels of HBV replication or expression of nuclear HBcAg, the liver cell damage in this patient with chronic hepatitis B was presumably induced by other mechanisms. The simultaneous observation of viral DNA, antigens, and pathological changes at the single cell level and their correlation with clinical findings should contribute to the understanding of the molecular mechanisms underlying HBV-induced liver cell injury.

Asymmetric replication of hepatitis B virus DNA in human liver: Demonstration of cytoplasmic minus-strand DNA by blot analyses and in situ hybridization

In situ and blot hybridization techniques have been used with strand- and region-specific probes to characterize the forms of hepatitis B virus (HBV) DNA in the liver of a patient with chronic active hepatitis B. The hepatocytes contain a heterogeneous population of rapidly migrating DNA species in the 0.5-1.4 kb position that are localized predominantly in the cytoplasm and are of minus-strand polarity. The findings indicate that the replication is asymmetric, with separate pathways for plus- and minus-strand synthesis of HBV DNA; that viral DNA synthesis is initiated at a site near the nick in the minus strand of virion DNA; and that actively replicating forms of HBV DNA can be identified at the cellular level by in situ hybridization.

Synthesis in cell culture of the gapped linear duplex DNA of the slow virus visna

Visna virus is a nontransforming retrovirus that causes slow infections in animals and a rapidly progressive-lytic infection in cell culture. The results of an analysis of the synthesis of viral DNA in cell culture are reported. Region- and strand-specific probes cloned in M13 have been used to define the dynamics of DNA synthesis and the major nucleic acid species formed. It is shown that (i) within the first hours of infection, a full-length copy of the viral RNA genome is synthesized by reverse transcription, (ii) early in infection a major species of DNA is formed that extends from a site near the center of the molecule to the 3 end, (iii) somewhat later a second major species of plus-strand DNA is generated that extends from the 5 end to the middle of the genome. As a consequence, most viral DNA molecules consist of a full-length minus strand, and two plus strands separated by a gap or nick in the center of the molecule (J. D. Harris, J. V. Scott, B. Traynor, M. Brahic, L. Stowring, P. Ventura, A. T. Haase, and R. Peluso (1981). Virology 113, 573-583). The implications of this viral DNA structure for one unusual aspect of the lentivirus life cycle, the production of viral RNA, and virions from extrachromosomal DNA are discussed (J. D. Harris, H. Blum, J. Scott, B. Traynor, P. Ventura, and A. T. Haase (1984). Proc. Natl. Acad. Sci. USA 81, 7212-7215).

Combined macroscopic and microscopic detection of viral genes in tissues

A hybridization technique has been devised for detecting and quantitating viral genes in tissues that combines macroscopic and microscopic analyses in the same section. The method is based on dual labeling virus-specific probes with 125I and 35S to generate signals that can be detected both with X-ray films and nuclear track emulsions. The regions of increased hybridization evident in the X-ray film serve as a guide to the portion of the section that warrants microscopic examination. Detection of viral RNA in tissues with visna virus and viral DNA with hepatitis B virus are illustrated, and potential applications of this technique in virology and other disciplines are discussed.

Interaction of aflatoxin and hepatitis B virus in the pathogenesis of hepatocellular carcinoma

Aflatoxin-B1 (AFB) and chronic hepatitis B virus (HBV) infection epidemiologically correlate with the geographic distribution of hepatocellular carcinoma (HCC). Integration of HBV DNA into the cellular genome of HCCs and the in vivo formation of adducts between AFB and nucleic acids lead us to suggest that hepatocytes with integrated HBV DNA preferentially accumulate AFB; the AFB-adducts formed may then initiate cell transformation by modifying the expression of critical host genes. The altered molecular biology of liver cells in HCC is evidenced by the fact that HBV does not replicate in HCC tissues or cell lines. The effect of AFB on the expression of cellular genes such as endogenous retrovirus(es) and possibly cellular oncogene(s) can be analyzed in HCC cell lines with and without integrated HBV DNA. In addition, human HCC tissues can be probed for HBV sequences and AFB-DNA adducts at the single-cell level. The presence of HBV and AFB can be correlated with the expression of putative transforming genes, providing a new insight into the interaction between liver cells, HBV and AFB in the pathogenesis of HCC.