Variability of the PreS1/PreS2/S regions of hepatitis B virus in Hungary.

Infection with the hepatitis B virus can occur perinatally, parenterally, or sexually, and it can cause acute or chronic liver diseases. Phylogenetic analysis of the virus has led to its classification into eight genotypes (A-H), which show a characteristic worldwide distribution. The aim of this study was to reveal the HBV genotypes present in Hungary and to investigate a nosocomial and an intrafamilial outbreak. The collected samples were tested by nested PCR, and a 650-nucleotide-long segment of the preS1/preS2/S region was sequenced. As no previous genotype data were available from Hungary, sera of 24 HBsAg-positive patients were collected from different regions of the country. They also served as control samples for the molecular epidemiologic study. Nineteen of them carried genotype D of hepatitis B virus, and five of them carried genotype A. Twenty-nine patients from a haemato-oncology unit were affected in a nosocomial outbreak. The patients had haematological and/or oncological diseases, most of them were immunosuppressed. In twenty-eight cases, based on phylogenetic analysis of the viruses, there was presumably a common source of infection, and an epidemiological investigation showed that the infections seemed to be hospital-acquired. In the intrafamilial outbreak, two asymptomatic carrier children infected their foster mother. The three sequences were totally identical.

A soluble factor(s) released by MRC-5 cells early and late after human cytomegalovirus infection induces maturation of monocyte-derived dendritic cells.

A human cytomegalovirus (HCMV) strain passaged 10 times on MRC-5 human fibroblast cells failed to express immediate early (IE) antigens in immature dendritic cells (iDCs) after infection. However, both the early and the late HCMV conditioning medium, harvested from MRC-5 cells at 24 h or 7-9 days after infection, respectively, induced a higher ratio of DCs expressing maturation markers (CD40, CD83, CD86 and HLA-DR) on the surface of the cells. HCMV conditioning medium, ultracentrifuged to remove virus particles, exhibited a similarly enhanced expression of DC maturation markers. DCs treated with HCMV conditioning medium harvested late after infection increased the percentages of autologous CD4+ and CD8+ cells of seropositive donors to produce IFN-gamma and stimulated HCMV-specific lymphoproliferative responses. The early HCMC conditioning medium was also able to induce the functional maturation of DCs, as demonstrated by supplementing this medium with a Chlamydia pneumoniae antigen.

Genotypic distribution of human herpesvirus-8 strains circulating in HIV-positive patients with and without Kaposi's sarcoma in Hungary.

Open reading frame (ORF) 26 of human herpesvirus-8 (HHV-8) from peripheral blood samples of 15 Hungarian HIV-positive patients with or without Kaposi's sarcoma (KS) were amplified and sequenced. Four variants of HHV-8 were identified according to ORF 26 genotyping. Most of the samples were shown to be subtype A3, however, subtypes A, B3/C2/C2', and C3 (ORF 26 region) were also identified. The ORF 26 subtypes A and C3 of HHV-8 were only recovered from patients with KS while A3 was dominant in KS negative cases. The amplification of the hypervariable ORF K1 gene was successful only from 2 of the same 15 patients. Sequence analysis of the amplified ORF K1/VR1 regions identified subtype A3 from 2 patients with AIDS-associated KS. A novel ORF K1/VR1 variant belonging to subgroup A' was detected in a different sample in one of them. Amplification of the ORF K15, another representative locus for HHV-8 genotyping, was not successful from any of the peripheral blood samples. Unsuccessful amplification of the terminal K1 and K15 ORFs from peripheral blood samples suggests that KS biopsy specimens are needed for complete genotyping of HHV-8 strains from Hungary.

Epigenetics of latent Epstein-Barr virus genomes: high resolution methylation analysis of the bidirectional promoter region of latent membrane protein 1 and 2B genes.

We analysed the methylation patterns of CpG dinucleotides in a bidirectional promoter region (LRS, LMP 1 regulatory sequences) of latent Epstein-Barr virus (EBV) genomes using automated fluorescent genomic sequencing after bisulfite-induced modification of DNA. Transcripts for two latent membrane proteins, LMP 1 (a transforming protein) and LMP 2B, are initiated in this region in opposite directions. We found that B cell lines and a clone expressing LMP 1 carried EBV genomes with unmethylated or hypomethylated LRS, while highly methylated CpG dinucleotides were present at each position or at discrete sites and within hypermethylated regions in LMP 1 negative cells. Comparison of high resolution methylation maps suggests that CpG methylation-mediated direct interference with binding of nuclear factors LBF 2, 3, 7, AML1/LBF1, LBF5 and LBF6 or methylation of CpGs within an E-box sequence (where activators as well as repressors can bind) is not the major mechanism in silencing of the LMP 1 promoter. Although a role for CpG methylation within binding sites of Sp1 and 3, ATF/CRE and a sis-inducible factor (SIF) cannot be excluded, hypermethylation of LRS or regions within LRS in LMP 1 negative cells suggests a role for an indirect mechanism, via methylcytosine binding proteins, in silencing of the LMP 1 promoter.

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes.

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.

Genetic subtypes of HIV type 1 in Hungary.

We examined the diversity of HIV-1 subtypes in 11 adults from Hungary, using the heteroduplex mobility assay (HMA) and DNA sequencing. HMA results showed that HIV-1 gp120 sequences from 10 patients were of subtype B, whereas 1 patient, infected in Africa, carried a subtype C strain. DNA sequencing confirmed the HMA results and revealed a high intrasubtype diversity in the C2V3 region of env in different clade B isolates, which suggests multiple introduction of subtype B to Hungary. This study shows that subtype B is the predominant HIV-1 clade in Hungary.

Analysis of the genetic variability of the 1st (CCC/ACC, P52T) and the 10th exons (bp 1012-1704) of the TSH receptor gene in Graves' disease.

We determined the genetic variability of the 1st (CCC/ACC, P52T polymorphic variant) and 10th exons (bp 1012-1704) of the TSH receptor (TSHR) gene in Graves' disease. A total of 101 Graves' patients and 163 control subjects were screened. The A253 mutant allele was carried by nine patients with Graves' disease (8.91%) and 13 control subjects (7.98%) in heterozygous genotype. No significant difference in the frequency of the mutant allele was found between Graves' patients and control subjects. These results provide evidence that the A253 polymorphism has no genetic relevance in Graves' disease. Moreover, the DNA nucleotide sequence of 693 bp of the 10th exon (bp 1012-1704) of the TSHR gene was determined in 15 Graves' patients. Six patients were homozygous for the wild-type allele and nine were heterozygous for the mutant allele at the 253rd nucleotide of the first exon. No polymorphism was found in the DNA sequences obtained from leukocytes of Graves' patients, similarly to the sequences obtained from the nine control subjects. None of the nine patients carrying the A253 polymorphism in the 1st exon of the TSHR had polymorphism in the examined part of the 10th exon, including two additional patients whose thyroid tissue was directly analysed. In all likelihood, the polymorphisms of the examined regions of either the 1st or the 10th exon of the THSR gene do not contribute to the genetic susceptibility to Graves' disease.

Nucleotide sequences and mutations of the 5'-nontranslated region (5'NTR) of natural isolates of an epidemic echovirus 11' (prime).

An echovirus 11' (prime) virus caused an epidemic in Hungary in 1989. The leading clinical form of the diseases was myocarditis. Hemorrhagic hepatitis syndroms were also caused, however, with lethal outcome in 13 newborn babies. Altogether 386 children suffered from registered clinical disease. No accumulation of serous meningitis cases and intrauterine death were observed during the epidemic, and the monovalent oral poliovirus vaccination campaign has prevented the further circulation of the virus. The 5'-nontranslated region (5'-NTR) of 12 natural isolates were sequenced (nucleotides: 260-577). The 5'-NTR was found to be different from that of the prototype Gregory strain (X80059) of EV11 (less than 90% identity), but related to the swine vesicular disease virus (D16364) SVDV and EV9 (X92886) as indicated by the best fitting dendogram. The examination of the variable nucleotides in the internal ribosomal entry site (IRES) revealed, that the nucleotide sequence of a region of the epidemic 5'-NTR was identical to that of coxsackievirus B2. Five of the epidemic isolates were found to carry mutations. Seven EV11' IRES elements possessed identical sequences indicating, that the virus has evolved before its arrival to Hungary. The comparative examination of the suboptimal secondary structures revealed, that no one of the mutations affected the secondary structure of stem-loop structures IV and V in the IRES elements. Although it has been shown previously, that the echovirus group is genetically coherent and related to coxsackie B viruses the sequence differences in the epidemic isolates resulted in profound modification of the central stem (residues 477-529) of stem-loop structure No.V known to be affecting neurovirulence of polioviruses. Two alternate cloverleaf (stem-loop) structures were also recognised (nucleotides 376 to 460 and 540 to 565) which seem to mask both regions of the IRES element complementary to the 3'-end of the 18 S rRNA (460 to 466 and 561 to 570), thus probably diminishing initiation of translation. The possible biological importance of the alternative cloverleaf structures is supported by the fact that neither the 17 variable nucleotides nor the two mutations of epidemic isolates within the regions seem to modify the predicted alternative secondary structures in EV11, SVDV and CBV1-4.

Periodic reappearance of bovine herpesvirus type 4 DNA in the sera of naturally and experimentally infected rabbits and calves.

A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.

Human herpesvirus 8 in hematologic diseases.

Human herpesvirus type 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV) is a new member of the g-herpesvirus family. It is an unusual herpesvirus in that it carries a large number of genes that encode oncoproteins or cell signaling proteins. In addition to being the causative agent of both HIV-associated and non-HIV-associated Kaposi's sarcoma this DNA tumor virus has been implicated in the pathogenesis of several diseases. These include multiple myeloma (MM), Waldenstöm's macroglobulinemia (WM), multicentric Castleman's disease (MCD), body cavity-based lymphoma (BCBL), and various other conditions such as sarcoidosis and pemphigus. While the causative role of the viral infection is fairly certain in the development of BCBL and multicentric Castleman's disease, HHV-8 may act through a different mechanism to induce plasma cell malignancies. It has been suggested though the finding is still controversial - that infection of bone marrow stromal dendritic cells by HHV-8 might be a key factor in the etiology and pathogenesis of monoclonal gammopathies. The aim of this review is to provide a short introduction into the tumorigenic potential of HHV-8 as well as to detail the available data and possible mechanisms on the involvement of this virus in different hematologic diseases.

Characterisation of an echovirus type 11' (prime) epidemic strain causing haemorrhagic syndrome in newborn babies in Hungary.

Echovirus 11' (prime) isolates from an epidemic of haemorrhagic syndrome in departments of obstetrics in Hungary have been characterised. The leading component of the clinical disease was carditis and its lethal outcome occurred in 13 newborn babies. Maternal immunity was found to be absent even in women of 41 years of age. The application of monovalent oral poliovirus type 1 vaccine prevented the progress of the epidemic within two weeks. Nevertheless, a serological survey among primovacinees of 3-15 months of age revealed that 20% of the babies seroconverted without clinical symptoms during the epidemic. Serological evidence showed that the echovirus 11' infection was unable to interfere with the efficacy of oral poliovirus vaccine (OPV), since seroconversion rates of primovaccinees did not differ significantly from those in the group seroconverted also to echovirus 11' during the vaccination campaign. A 440 nucleotide (nt) fragment of the 5'-non-translated region of 12 epidemic echovirus 11' isolates and 26 echovirus prototype strains was amplified by a nested reverse transcription-polymerase chain reaction (RT-PCR) and analysed using three different restriction endonucleases. The 5'-regions of the echovirus 11' isolates were found to be identical to each other but different from that of the prototype echovirus 11 (Gregory) strain. The results indicate that echovirus 11' isolates underwent genetic changes in the 5'-end and P1 region of the genome before the onset of the epidemic.

Molecular biological characterization of adenovirus DNA.

The agarose gel electrophoresis of DNA, the ethidium bromide fluorescence detection of DNA fragments and restriction endonucleases were discovered at the end of the '60s. The methodological progress enabled institutions equipped with less sophisticated technology to achieve also unique experimental and scientific results in the field of viral DNA research. The team working on virus DNA within the adenovirus research group has constructed several new restriction endonuclease maps of the genomes of human and animal adenoviruses; contributed to the methodology of the determination of specific endonuclease sites, and genome polarity; discovered new restriction endonucleases, adenovirus subtypes, new empty capsid, and genome subpopulations; participated in cooperations leading to novel, although hypothetical approaches in AIDS therapy, taxonomic definition of viruses, and evolutionary origins of adenovirus replication and encapsidation strategy.

[Clinical immunological features and interferon therapy in chronic hepatitis C]. / Klinikai immunológiai megfigyelések és interferon terápia krónikus C hepatitisben.

Clinical and immunological findings of 74 patients with chronic hepatitis C have been reported and experiences with interferon-alpha treatment of 31 patients are summarized. In addition, the first results of anti-HCV screening of blood donors are also briefly described. Transfusion in the history was noted in 69% of patients and the time, elapsed from the transfusion to the diagnosis was a mean of 7.15 +/- 8.1 years. Concerning the severity of the liver disease, chronic persistent hepatitis was established in 40%, active hepatitis in 45% and cirrhosis in 15% of the patients, respectively. Cholestasis was recorded in 32% of the cases. A significant elevation of serum immunoglobulin levels was noted in 83%, an antibody to liver specific protein (anti-LSP) has occurred in 80%, cryoglobulinaemia in 44% and circulating immune complexes in 33% of the patients. Natural killer cell activity of peripheral blood mononuclear cells significantly decreased. HLA B8 and DR3 antigens were found with elevated frequency (36.6% and 42.1%). Recombinant interferon-alpha at a weekly dose of 3MU thrice, for six months, has normalized serum alanine aminotransferase in 45% of patients and a sustained remission was found in 26%. The treatment resulted in the clearance of HCV-RNS from the serum in 40% of patients and that well correlated with the complete remission. In the good responders, a decrease in CD4+ cell count and a moderate decrease in CD8+ cell count as well as a transient rise in B cell count were seen during the treatment. Mitogen-induced lymphoproliferative response and natural killer cell activity increased. Predictors of response were as follows: female sex, shorter time elapsed from transfusion, absence of HLA, A1, B8, DR3 and serum anti-HBc negativity. Anti-HCV has been found in 0.33--0.38% of blood donors screened, and it is suggested, that a liver disease accompanied with elevated serum alanine aminotransferase, may be present in about 25-30% of anti-HCV positive symptom-free persons.

Detection of transfusion-associated hepatitis caused by non-A, non-B, non-C flavivirus.

Sera of patients suffering from acute hepatitis, and different forms of chronic hepatitis were found to be reactive to reagents prepared from the yellow fever virus (YF) vaccine strain. Serum samples of 1974 patients were tested, and 133 of them were positive. Hepatitis C virus specific antibodies were absent from the majority of them. The frequency of antibodies to other flaviviruses (tick-borne encephalitis, West Nile) and hepatitis B virus markers was similar to that measured among the population in Hungary positive for any of the surrogate markers of hepatitis infections. Results of both immunofluorescence tests, and Western blots suggest that there is a non-A, non-B, non-C hepatitis virus circulating among the Hungarian population, which possesses antigenic cross-reactivity with the yellow fever virus, but the identity to any of the known flaviviruses could not be verified yet. No history of yellow fever vaccination could be revealed in any of the patients included into this study. The anamnestic data on previous transfusions or surgical operations can be verified only in the case of the half of YFV-positive patients, nevertheless, the sexual transmission seems to be very infrequent. Attempts are continued in order to detect the viral RNA using polymerase chain reaction, and clone cDNA sequences for sequence analysis.

[Human cytomegalovirus, its significance in immune deficiency states, laboratory diagnosis, therapeutic possibilities]. / Humán cytomegalovirus, jelentösége immunhiányos állapotokban, laboratóriumi diagnózis, terápiás lehetöségek.

The authors report on the virological findings of 59 transplant recipients. The following procedures were used for the detection of human cytomegalovirus (HCMV) infection: detection of antiviral antibodies by ELISA, the detection of virus-coded antigens in the patients' leucocytes (HCMV antigenemia test), "accelerated" virus isolation using immunofluorescence (IF). Serial examinations revealed the HCMV infection in 12 patients following organ transplantation. The antigenemia test proved to be positive in all cases. Two third of the cases suffered from viremia. The virus specific serology possess diagnostic value only in every second acute illness. Since the antigenemia test used to be successful in the earliest phase of acute illnesses, the chance of effective chemotherapy can be increased significantly. The virus serological examinations are of essential importance during the pretransplantation screening of donors and recipients. The "accelerated" procedure of virus isolation experiments indicates the presence of infective HCMV within 1 to 4 days. Transplant recipients obtain new life perspectives, nevertheless, the modern diagnostic procedures may only support the prevention of life-threatening virus infections under the conditions of immunosuppression. The excellent mutual cooperation of the clinicians and diagnostic virologists seems to be at least as important condition of successful transplantation medicine as the high technology in surgery and clinical diagnostics.

[Transfusion-associated non-A, non-B, non-C hepatitis caused by flaviviruses]. / A non-A, non-B, non-C flavivírus által okozott inokulációs hepatitis.

Hepatitis C virus was shown to be a member of the flavivirus family. Tick-borne encephalitis virus and West Nile virus, members of the same family occur in Hungary, too. Serum samples from patients suffering from transfusion associated hepatitis were tested with yellow fever virus antigens for specific IgG, and IgM using immunofluorescence test. Eight hundred serum samples were tested. Yellow fever virus related IgG antibodies were found in 232 sera. In the case of 72 patients specific IgM antibodies could also be detected. The majority of the IgM positive patients underwent surgical operation and/or blood transfusion 1 to 2 months before the onset of the disease. Fifty-four sera positive for yellow fever virus-related antibodies were tested with HCV reagents, but only 13 were found to be positive, or cross-reacting. The 20 patients with yellow fever related antibodies were controlled with tick-borne encephalitis antigens, too. Nevertheless, no measurable cross-reaction could be detected. No measurable cross-reaction could be detected with the West Nile virus. The hepatitis B markers also were tested in 44 sera positive for yellow fever antibodies. There was only one, which contained HBsAg, and 10 of them proved to be positive for anti-HBcAg. The results indicate, that a non-A, non-B, non-C flavivirus is also present in the Hungarian population, which can be detected on the basis of the antigenic cross-reactivity with the attenuated yellow fever virus. This virus seems to be responsible for every 11th transfusion associated hepatitis examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple enlargements in the right inverted terminal repeat of the DNA of canine adenovirus type 2.

The Manhattan strain of canine adenovirus type 2 (CAV 2) was examined. Restriction endonuclease analysis and blot hybridization experiments revealed the heterogeneity of the viral DNA. At least 9 unequally expanded species of the viral genome have been recognized. This diversity is caused by different enlargements in the right inverted terminal repeat (ITR) of the virus. The differences between the individual enlargements were shown to be the different multiples of 150 base pairs. Relatedness of CAV 2 DNA to the DNA of bovine adenovirus type 2 (BAV 2) and human adenovirus type 2 (HAV 2) has also been observed during DNA hybridization experiments.

Characterization of an intermediate adenovirus strain.

An intermediate strain of human adenovirus of subgenus D was investigated by type specific serological reactions and restriction endonuclease analysis. The latter method showed the strain identical to the prototype strain of human adenovirus type 9 as well as did serum neutralization tests. In contrast with the previous methods haemagglutination inhibition tests showed the strain related to both the prototypes strains of human adenovirus 9 and 13.

Molecular genetics of PKU in eastern Europe: a nonsense mutation associated with haplotype 4 of the phenylalanine hydroxylase gene.

Phenylketonuria (PKU) is a genetic disorder secondary to a deficiency of hepatic phenylalanine hydroxylase (PAH). Several mutations in the PAH gene have recently been reported, and linkage disequilibrium was observed between RFLP haplotypes and specific mutations. A new molecular lesion has been identified in exon 7 of the PAH gene in a Hungarian PKU patient by direct sequencing of PCR-amplified DNA. The C-to-T transition causes the substitution of Arg243 to a termination codon, and the mutant allele is associated with haplotype 4 of the PAH gene. The mutation is present in two of nine mutant haplotype 4 alleles among Eastern Europeans and is not present among Western Europeans and Asians. The rarity of this mutant allele and its restricted geographic distribution suggest that the mutational event occurred recently on a normal haplotype 4 background in Eastern Europe.