Ferruccio Bonino

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

A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in formalin-fixed, paraffin-embedded liver sections by a nonisotopic in situ hybridization (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 were 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 sections, 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.

Hepatitis delta virus heterogeneity : a study by immunofluorescence

Hepatitis delta virus (HDV)-RNA shows a microheterogeneity at nucleotide 1015 resulting in the production of two co-terminal forms of the HDV-associated antigen (HDAg), referred to as p24 and p27. In vitro experiments have shown that p24 is localized within the nucleolus, while p27 expression is probably confined to the nucleoplasm. The sequential appearance of these two proteins may play a role in the HDV-induced cell damage. We have examined 12 formalin-fixed human liver biopsies taken at different phases of the natural course of HDV infection. Specimens were assayed for HDAg by direct immunofluorescence with a human IgG reactive towards both p24 and p27. Positive cells were then examined for cytopathological features after hematoxylin/eosin staining. All biopsies contained intranuclear HDAg. However, three different patterns of fluorescence were seen: (1) nucleolar with weak nucleoplasmic; (2) homogeneous nucleoplasmic with negative nucleolus; (3) intense fluorescence diffuse to the whole nucleus. Pattern 1 was found in 15-50% of positive nuclei of all samples, irrespective of the phase of infection, while the remaining positive nuclei mostly showed another pattern (2) of fluorescence. Neither pattern was associated with cytoplasmic eosinophilia. Degenerated hepatocytes, when infected (5%), showed pattern 3 of HDAg fluorescence but were mostly negative for HDV. Therefore, the intranuclear distribution of HDAg, as assayed in vivo, does not seem to correlate with peculiar phases of HDV infection. Different phases of the viral life style, instead, are asynchronously represented within each liver sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis Delta Virus in Acute and Chronic Liver Disease

Hepatitis delta virus (HDV) is a defective hepatotropic RNA agent that replicates only in hosts who are simultaneously infected with hepatitis B virus (HBV).1,2 The virion is a spherical 36-nm particle, composed of a lipoprotein envelope. The surface antigen of HBV (HBsAg) contains delta antigen (HDAg) and an RNA molecule of 1.7 kilobases (kb) (HDV-RNA).1,2 The detection of HDAg requires treatment with detergents, but nucleocapsidic structures are not seen electron microscopically within the HBsAg envelope of the particle.3 HDAg is composed of two major proteins, 24,000–27,000 M r and 27,000–29,000 M r molecular weight.3,4 HDV-RNA is considered the genome of the defective agent. It was reverse transcribed to make complementary DNA (cDNA) that was cloned using appropriate vectors.5–11 DNA clone fragments of different length, spanning the entire HDV genome, were obtained and sequenced.5–11 No significant homology was observed between HDV-RNA and HBV-DNA or between HDV-RNA and host DNA. HDV-RNA contains 1679–1683 nucleotides and five open reading frames (ORF) of more than 100 amino acids in both genomic and antigenomic strands.2’5–11 The largest ORF, present on the antigenomic strand (+) and coding for 214 amino acids, was expressed in bacteria; the resultant fusion protein reacted with human antibodies to HDAg (anti-HD) by Western blot analysis.5

Incidence and significance of different types of connective tissue antibodies in adult and pediatric gastroenterological disorders.

The incidence of connective tissue antibodies was assessed in 3,000 adults and in 388 pediatric patients with gastroenterological disorders. Following a previous classification, connective tissue antibodies were distinguished in 5 different types, R1 and R2 reacting with reticulin components, KC with Kupffer cells, AC and Rs with intra- and extracellular mesenchymal antigens. R1 predominated in children and was observed only in patients with malabsorption; the great majority of them had celiac disease and an abnormal small bowel mucosa. Ac was rarely seen in children and occurred in several unrelated gastrointestinal diseases of the adult; its incidence, however, was significantly higher in disorders with idiopathic and secondary malabsorption. R2, KC and Rs were seen only occasionally in unrelated gastroenterological disorders without malabsorption. R1, therefore, seems to be strongly suggestive of a malabsorption syndrome with abnormal small bowel mucosa and AC is a nonspecific reaction occuring predominantly in primary and secondary malabsorption; the other connective tissue antibodies have no clinical significance in gastroenterological disorders.

Hepatitis C Virus Heteroduplex Tracking Assay

The heteroduplex tracking assay (HTA) is a tool that can be used for determining genotype, quasispecies analysis, molecular evolution, and epidemiological studies (1-7). By hybridizing a labeled, single-stranded DNA probe to colinear, reverse transcriptase (RT) PCR products from a sample of interest, the probe will either form a homoduplex with identical molecules or a heteroduplex with nonidentical sequences. The hybridization products are separated on MDE or polyacrylamide gels and visualized. Delwart et al., the developers of the HTA technique (1,2,7), have previously shown that the migration of heteroduplexes relative to the homoduplex on gels are approximately proportional to the percent nucleotide divergence between two species, and therefore, the genetic distance between two species can be determined. Genetic rearrangements, deletions, and/or insertions can alter the migration of heteroduplexes in a manner that disturbs the direct relationship between relative migration and genetic distance. Typically, heteroduplexes of 0.176-1.8 kb containing >1.4-3% to ~30% nucleotide substitutions, which lack genetic alterations, can be identified as unique species on MDE gels (1,4,6). The number and distribution of unique bands indicates the genetic complexity of viral species in each sample.

Hepatitis B Virus Precore Mutants

Genetic heterogeneity is a common feature of viruses and can influence the outcome and course of infections. Hepatitis B virus (HBV) genetic heterogeneity can be an important determinant of the variability of both infection and replication efficiencies of the virus and of the host’s antiviral immunoreactions. Important mutations were found in the genes of HBV structural proteins, HBsAg and HBc/eAg. Mutants, unable to secrete the soluble form of HBV nucleocapsid protein, HBeAg, prevail over wild-type HBVs in patients with severe acute and chronic hepatitis B and in chronic HBsAg carriers at the time of HBeAg/anti-HBe seroconversion. These mutants are positively selected by the antiviral immuno-reaction. Behaving as escape mutants, they appear to be involved in HBV pathogenenicity and associated with a poor response to interferon. However, the absence of HBeAg-defective mutants in some cases of fulminant hepatitis B, as well as their detection in some asymptomatic carriers of HBsAg, should not be surprising. The severity of hepatitis B is influenced by many other factors: number of virus-infected cells, competence and genetic heterogeneity of the immune system, vigor and extent of nonspecific inflammatory response, and number of hepatocytes endangered by coincident diseases or infected with other hepatotropic viruses.

Modeling the Dynamics of HCV Infected Cells to Tailor Antiviral Therapy in Clinical Practice: Can This Approach Fit for Neoplastic Cells?

The always present need to predict therapy outcomes and to understand complex phenomena in medicine can be addressed by the use of mathematical models that simulate which relevant events may be expected at the single patient level. In chronic hepatitis C patients undergoing effective antiviral treatment, we found that a model specifically developed to compute the infected cells surviving at the end of the treatment, was able to predict the final outcome of the therapy better than the single measures of the viral load decline after 4 and 12 weeks. This approach, that showed in a prospective trial to improve the cost efficacy of standard peg-interferon/ribavirin treatment, could become very useful after the first month of treatment to select patients in whom adding a more potent antiviral drug is actually required, as standard therapy outcome is unlikely favorable or its predicted duration is too long. Similarly, in patients with hepatocellular carcinoma where the efficacy of the novel biological treatments combined to standard ablation approaches is difficult to assess, modeling the dynamics of the neoplastic cells using experimental data from imaging techniques integrated with measures of liver cell function and proliferation, should become available to help clinical management of these patients.