Detection of hepatitis delta virus RNA by a nonradioactive in situ hybridization procedure

Detection of Hepatitis Delta Virus RNA by a Nonradioactive In Situ Hybridization Procedure DONATELLA PACCHIONI, MD, FRANCESCO NEGRO, MD, ELISABETTA CHIABERGE, BS, MARIO RIZZETTO, MD, FERRUCCIO BONINO, MD, AND GIANNI BUSSOLATI, MD A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in form&n-fixed, paraffin-embledded liver sections by a nonisotopic in situ hyhridization (NISH) procedure. Twenty-three liver samples from chronically HDV-infected patients were studied. Eight liver specimens from humans and chimpanzees without markers of active HDV infection served as negative controls. In three samples, the NISH findings werse compared with characteristic nuclear features and with the distribution of the HDV encoded antigen, HDAg, as detected by direct immunofluorescence. All samples from HDV-infected patients were positive for HDV RNA by NISH. The viral genome was exclusively observed within the hepatocytic nuclei. No enzymatic reaction was detected after hybridization with the negative controls. “Sanded” nuclei, a cytopathologic change associated with HDV infection, were HDV RNA-positive, but only a small percentage of infected cells showed that feature. Hepatocytes containing the HDV RNA were sometimes binucleated or exhibited giant nuclei. When HDAg and HDV RNA were sequentially detected within the same sectionls, the localization of the viral antigen almost completely overlapped with the expression of the HDV transcripts, and vice versa. In conclusion, detection of intrahepatic HDV RNA by NISH is a rapid, sensitive, and specific technique that is easily applicable to routine histopathology and allows correlation of HDV with the morphology of hepatocyte nuclei. HUM PATHOL 23:557561. Copyright (C 1992 by W.B. Saunders Company

especially for the evaluation of patients treated with antivirals. Several assays for the direct assessment of the production of HDV virions in the liver are available, the most sensitive being the detection of HDV RNA by the polymerase chain reaction assay.“.’ Llrifortunately, the pc!l~merase chain reaction poses a significant risk of obtaming false positive results, which mav limit its routine application, and requires the extraction of nucleic acids from the liver tissue. Detection of viral expression by immunohistochernistry and in situ hybridization might represent a valid alternative in cases with low levels of viral replication and allows histopathology studies through preservation of the liver n~orphology. In this report we describe a novel, nonisotopic in situ hybridization (NISH) procedure for detection of intrahepatic HDV RNA, using a digoxigenin-tailed oligodeoxynucleoticte, MATERIALS

AND METHODS

Liver Samples Twenty-three specimens from patients chronically infected with HDV were used. All patients had high titers of antibody to HDAg (anti-HD) in the serum and HDAg in the liver as signs of HDV replication. The diagnoses were chronic persistent hepatitis in two cases, chronic active hepatitis (CAH) in 14 cases, and <:AH with cirrhosis in seven cases. Two specimens showing C4H with cirrhosis at histology were taken at autopsy. Negative controls included four liver biopsies from patients with chronic hepatitis B virus infection and CAH, one normal hurnan liver- taken at autopsy, and two open-air liver biopsies from chimpanzees with experimental acute hepatitis C. All subjects lacked serum anti-HD and therefore were presumed not to have been infected by HDV. An additional liver biopsy came from a patient who had had a self-lirniting acute delta hepatitis in the past and was still circulating low-titer anti-HD; histology showed minimal lesions. All samples were fixed in formalin and embedded in paraffin. Five-micron sections were applied to slides previously coated with 1%) poly-I,-Lysine (Sigma Chemical (:o, St Louis, MO).

Hepallitis delta virus (HDV) is a defective hepatotropic human pathogen that requires coinfection with a H~,budmmirus to support its life cycle.’ Hepatitis delta virus is comprised of an outer coat, expressing the surface antigen of the helper Hepadnnuil-us, and an internal small circular RNA (HDV RNA) associated with an HDV-encoded antigen (HDAg).’ Infection with HDV is often associated with severe and progressive liver disease (delta hepatitis) and rnay negatively affect the course of the Hepadnnvirus-induced damage, when present.’ Diagnosis of active HDV replication is therefore important,

From ttle Department of Biomedical Scientes and Human Onccrloh7 and the Instituw of. Internal Medicine, L’niversitv of Turin Srhool of .Medicine. Turin, Italy; and the Division of (;astrr,~.rrterolo~?, Molinette Hospital, Turin, Italy. Accepted for publicationJuly 8. 199 1. Supporled in px’ by (bntract No. 52OtiO12,‘1!~00 between the Division of t;;,lstroent~rology. Molinette Hospital, Turin, Italy, and thr Istituto Superior-e della Sanita’. Rome, Italy; and in part by grant\ loom the Ministn of University and Scientific Research. the Region 01 l’iemonte. and the “Assoc-iazione Iraliana per la Ricerca wl (Iancrc~“. Milan, lraly. Krv urortls: hepatitis delt,~ viwa. viral hepatiris. in bi(u hvtnklization, sanded nurlci. r\ddresa c ornspondrnt e and rcpl-int requests lo F~-arr~C~I Negro. MD. Division of (;astrocntel-olo~. Molinette Hospital. (:ors~r Bram;mIe X8. IO 125.Turin. Italy. (:opyright @I 1
Preparation

of the Probe

A 27-base oligodeoxynucleotide specific for genomic HDV RNA was synthesized with the phosphoramidite method and used throughout all the experiments. The oligomer has the sequence fi’-(=(;<:(:.A<;‘TcAATAAAC;CC;C;~‘T’I‘TCCACT-:I’ and corresponds to positions 557

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936 to 962 of HDV RNA according to the proposed numbering.“,” Twenty-five picomoles of the oligomer were labeled for 1 hour at 37°C using 25 U of 3’-terminal deoxynucleotidyl transferase (Promega Biotech, Madison, WI) in a tailing mixture containing 1 mmol/L digoxigenin11-deoxyuridine triphosphate (Boehringer Mannheim GmbH, Mannheim, Germany); 100 mmol/l, cacodylate bufIer, pH 6.8; 1 mmol/I, cobalt chloride; 0.1 mmol/I, dithiothreitol: and 0.1 mg/mL bovine serum albumin. The efficiency of the tailing reaction has been described elsewhere.’ The labeled probe was stored at -20°C until use. Nonisotopic

In Situ Hybridization

I,iver sections were deparaffinated with xylene and rehydrated through the series of ethanols to phosphatebuffered saline (PBS). Samples were then digested in 0.5 mg/mL proteinase K (DNase-and RNase-free, Merck, Darmstadt, Germany) in PBS for 15 minutes at 37°C and rinsed in PBS for 5 minutes. The following protocol essentially follows the guidelines recommended by the manufacturer (Boehringer Mannheim GmbH) for digoxigenin-tailed oligodeoxynucleotide probes. The prehybridization mixture contained 50% formamide, 4X SSC, 5% dextran sulphate, 1 X Denhardt’s solution, 500 pg/mL sheared salmon sperm DNA, and 250 pg/mL yeast tRNA, and was performed for 1 hour at room temperature (RT). Hybridization was run overnight at 37°C by adding the labeled probe to the prehybridization mixture at a final concentration of 3.3 nmol/L. After hybridization, slides were washed in 2X SSC for 1 hour at RT. then in 1X SSC for 1 hour at RT, in 0.5X SSC for 30 minutes at 37°C. and finally in 0.1 X SSC for 30 minutes at RT. After a further, brief wash in 100 mmol/L Tris-hydrochloric acid, pH 7.5, 150 mmol/L sodium chloride, sections were incubated twice in this same buffer containing first 2% normal sheep serum and 0.3% Triton X-100 (30 minutes at RT) and then 1% normal sheep serum, 0.3% Triton X-100, and a I:500 diluted sheep antidigoxigenin antibody conjugated with calf intestine alkaline phosphatase (Boehringer Mannheim GmbH; 2 hours at RT). Unbound antibody was washed off by immersing the slides twice for 15 minutes in the above buffer. Sections were finally washed in 100 mmol/L Tris-hydrochloric acid, pH 9.5, 100 mmol/L sodium chloride, 50 mmol/L magnesium chloride for 2 minutes. The color was developed using nitroblue tetrazolium salt as a substrate, following the manufacturer’s suggestions (Boehringer Mannheim GmbH). The development solution contained 0.024% tetramisole (Sigma Chemical Co). The reaction was stopped in 10 mmol/L. Tris-hydrochloric acid pH 8.0, 1 mmol/L ethylenediamine tetraacetic acid, and the sections counterstained in methyl green. Slides were then dehydrated and mounted. To compare the localization of genomic HDV RNA as detected by the above procedure with the presence of “sanded” nuclei, a cytopathologic feature recently found to be associated with HDV infection,x we first developed the alkaline phosphatase reaction according to Cordell et al.” As substrate, 50 mg of Fast Red TR 558

salt (Sigma Chemical Co; dissolved in 1 ml, of N,N’dimethylformamide), 18 mg of tetramisole-hydrochloric acid, and 25 mg of naphthol-AS-BI-phosphate (Serva, Heidelberg, Germany; dissolved in 1 ml, of N,N’-dimethylformamide) were added to 50 mL of 70 mmol/ I, 1,3-propanediol buffer, pH 9.5. This solution was then filtered before use and the color allowed to develop fol 10 minutes. Slides were then mounted in 50% glycerine. Fields containing HDV RNA-positive hepatocytes were photographed and the coordinates recorded. After unmounting, slides were briefly soaked in 70% ethanol and then in 95% ethanol to remove the red precipitate secondary to the alkaline phosphatase reaction. After rehydration, sections were stained in hematoxylin for 1 minute, dehydrated, and mounted. The same fields as above were then identified and rephotographed. to allow a comparison between the positivity for HDV RNA and the nuclei morphologic features. Sequential Hepatitis Delta Antigen/Hepatitis Delta Virus RNA Detection Antigens to hepatitis delta and HDV RNA were sequentially stained in three of the above samples. One of these was taken at autopsy of a patient with chronic HDV infection, known for at least 7 years. The other two samples were taken, 1 month apart, from a subject

FIGURE 1. Detection of HDV RNA by nonisotopic in situ hybridization. The alkaline phosphotase reaction (see text) was developed using the nitroblue tetrazolium salt as substrate. (Counterstain: methyl green: original magnification x40.)

IN SITU HYBRIDIZATION

FOR HDV RNA (Pacchioni et al)

FIGURE 2. (Left) lmmunohistochemical staining of HDV RNA within hepatocyte nuclei (arrows), using Fast Red a:, substrate for We alkaline phosphatase reaction. (Right) After removing the red precipitate, the section was stained with hemotoxylin and the two HDV RNA-positive nuclei showed a “sanded” appearance. (Magnification n 165.)

l>atocytes (Figs 1. 2A, and 3). The percentage of infected cells varied hetween 5% and 30%. without correlation with the histologic diagnosis. The NISH signal was exclusively confined to the nuclei. The onI>, cell type supporting HD\! RNA replication was the hepatocyte. No staining was observed in the liver samples from the patients lacking markers of current HD\r infection. When 3 section of a liver expressing intranuclear HDV RNA, as evidenced b! the Fast Red precipitate, ~83s first destained and then restained with hematoxylin, a small proportion (about 5%) 01’ HDV-infected nuclei showed a “sanded” appearance (Fig Z), it. a homogeneous acidophilic inclusion displacing the chromatin and the nucleolus at the periphery of the nucleus.’ Essentially all “sanded” nuclei were sho\c.n to contain HDV RNA in large amounts, based on the intensity of the histochemical reaction. Moreover, a significant proportion of HD\‘-infected hepatocytes appeared to be hinucleated or contained a giant nucleus (Fig I ). Sequential detection of HDAg and HDV RNA expression in three samples showed that the two viral markers mostly overlapped withill the same nuclei. However, while in all three cases the i~~~Ill~lIlofllIo~‘esc.ellt nuclei were also NISH-positive and vice \;ersa. in one of the samples (the autopsy specimen) about 5% of the

who was experiencing acute hepatitis after having undergone liver transplantation because of end-stage, HDV-related liver disease. After proteinase I( digestion of the deparaffinized sections, the specimens were incubated with an optimally diluted, fluorescein isothyocyanate-~ol!jugated human anti-HD immunoglobulin (; for 30 minutes at 37°C. After thorough washing in PBS, slides were mounted in 50%) glycerine and observed under the Iluorescence microscope. Random fields were photographed and coordinates recorded to localize HDAg-positive hepatocytes. Slides were then mimounted, rinsed in PBS for 5 minutes, and hybridized according to the procedure described above. Following the nitroblue tetrazoliunl reaction, sections were counterstaineti in methyl green. Pictures were then taken of the same fields photographed after the immunofluorescent detection of HDAg, to compare the distribution of the viral antigen and genome. At least 100 HDVinfected nuclei were examined per sample. RESULTS individuals All liver specimens from HDV-infected showed a variable number of HDV RNA-positive he559

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FIGURE 3. Detection of intrahepatic HDAg by direct immunofluorescence (left) followed on the same section by NISH for HDV RNA (right). Some hepatocytes contain the viral RNA without expressing the antigen (arrows). (Original magnification r,90.)

nl~clei

without

exhibited the presence of the viral genome detectable HDAg (Fig 3).

alone,

DISCUSSION The in situ hybridization technique represents a very sensitive and specific way to detect low levels of viral replication. Our report essentially confirms prethe expression and distrihuvious findings concerning tion of tissue HDV RNA.“‘,” Clnlike those in situ hybridization techniques based on autoradiography, out NISH allows direct comparison with nuclear pathologic patterns and therefore their interpretation. Nuclei appearance were confirmed to showing a “sanded” contain high amounts of replicating HDV.’ This peculiar finding was observed several years ago in livers from but has only recently been HBcAg-positive individuals,” associated with HDV infection, and tnore precisely with The affected nucleus ty$ically the HDAg expression.’ shows a ground-glass, acidophilic inclusion, with peripheral disposition of both the heterochromatin and the nucleolus. Conceivably. these are physically displaced by the huge amount of viral products being packed within the infected nucleus.“‘,“’ It is noteworthy that only a small proportion of HDV RNA-positive nuclei presented with this feature, namely those showing the highest degree of positivjty. based on the intensity of the Fast Ked staining. Posltlve nuclei frequently showed a giant size. Moreover, infected hepatocytes often were hinucleated. All these findings can be easily observed in conditions of accelerated cell turn-over and regeneration, eg. in the case of virus-induced necrosis. The results obtained with the negative control biopsies suggest that our NISH assay is specific. Essentially no background staining was observed. This is partly due to the fact that digoxigenin is completely absent from animal cells and therefore should be preferred to biotin when used for NISH. It has been shown that the choice of biotin as a reporter molecule may lead to false 560

positive results, especially on liver tissue’ ‘.I’, (;ind 0111‘ unpublished observations). These may be secondary to the binding between the biotin-targeted reagents (avidin, antibodies) and the endogenous biotin or, possibly, to the binding between the biotin incorporated within the probes and the surface antigen of HBV.“,” Nonisotopic in situ hybridization with digoxigenin-labeled probes also shows a better signal-to-noise ratio on different tissues, as compared with that obtained with biotinylated probes, ‘K” ancl has been shown to be as sensitive as “‘Slabeled probes.” The specificity of our assay also depends on the sequence of the oligomer. Thus, to clecrease the chance of (.I-oss-hybriclizatioll between the probe and known cellular sequences, the HDV RNA region to be hybridized (the signal recognition particle RNA) was chosen outside of a conserved domain pantially homologous to a small cellular RNA.” As suggested previously,‘“.” detection of HDV RNA by in situ hybridization seems to be more sensitive than the inlrnunohistochernical staining of HDAg. The asymmetrical distribution of HDV RNA anti HDAg, which involves a small proportion of cells, may reflect different stages of the viral replicative cycle. It has been shown recently that H DAg is expressed early in the viral life cycle and that this protein may act in trans to boost HDV RNA replication.‘!’ On the contrary, our data suggest that, under certain conditions. HDV replication may proceed in the absence of detectable HDAg. This finding should be investigated further. The sensitivity of our assay is also a function of the si;le of the probe. Small probes penetrate more easily within the section,“’ thus increasing the hybridization eficiency, and the use of labeled oligodeoxynucleotides does not decrease the signal-to-noise ratio if compared with larger, genomic probes.” As a result, oligomers are becoming the probe of choice for several tissue detection assays involving a11 in situ hybridization procedure.‘“-” To increase the sensitivity, we chose to detect genomic-sense HDV transcripts, due to their higher abundance relative to the antigenomic, replicative intermediate RNAs. “‘,‘x 1,oss in sensitivity due to genetic heterogeneity ancl consequent mismatches hetween probe and target sequence were minimized by choosing a region known for being invariant among all of the HDV RNA isolates sequenced to date.‘;’ In conclusion, detection of intrahepatic HDV RNA b,y,NISH appears to be a safe, simple, specific, and sensltlve assay. Its applicability to formalin-fixed, paraffinembedded sections makes it suitahle for routine use in clinical patholom, especially for the evaluation of patients undergoing antiviral therapy. Finally, the imrnunoenzymatic technique allows a correlation between the presence of HDV RNA replication and the morphology of liver cell nuclei. ,-\rk~~ou~lrd~~r~P,tl. The authors wish to thank I)r Maul-o Pay,otli fitr Providing helpful cr-iticisrn, Dr- Patrizia Gugtiot~a

for invaluable suggestions concerning the histochenGcal reactions. and Manuela Gallo and Nino Fermro for excellent technical help. The open-air liver biopsies from chimpanzees with experimental acute hepatitis C: were kindly provided hy Dr. R. H. Purcell, National Institutes of Health, Bethesda, MD.

IN SITU HYBRIDIZATION

FOR HDV RNA (Pacchioni et al)

561