Search PubMed⌕ Search

Biomedical subjects

L Vitvitski

Publications and source records attributed to L Vitvitski.

At least 37 records · Page 2Linked to original sources

Detection of small and large genomes of hepatitis D virus in serum of patients with hepatitis D.

By combining the polymerase chain reaction (PCR) with restriction enzyme digestion technologies, we characterized the genomes for the small and large delta proteins of HDV in retrospective analysis of sera from 10 patients with varied clinical outcomes. Both small and large genomes of HDV were present in all 13 serum samples from the 6 acute and 4 chronic cases studied, while the specific HDV proteins (P24 and P27) could be detected by immunoblot analysis in only 4 of them. The relative amounts and ratios of the genomes for the large and the small proteins of HDV were different for each individual. The molecular ratio of large to small HDV genomes in serum correlated with viral replication. When the replication of HDV RNA increased, the ratio decreased and vice-versa. No specific correlation, however, was found between the ratio of both molecular forms and the clinical outcome.

Adolescent↗

Interferon therapy for hepatitis C.

Initial trials indicated that around 50% of patients respond to recombinant alpha interferon by normalizing alanine aminotransferase (ALT) at the end of therapy and that half of these relapsed within 6 months following cessation of treatment. Both dose and duration of treatment are critical in the response to therapy. Higher doses and longer duration have been suggested to be more effective than the current recommendations of 3 MUI thrice weekly for 6 months based on results of these initial studies which used ALT and histological scores to evaluate the efficacy of interferon therapy. Following studies using virological markers have shown that improvements in clinical features of disease are associated with decrease or loss of hepatitis C virus (HCV) from serum and liver. The heterogeneity of the response rates between clinical centers using identical protocol emphasizes that the selection of the patients treated was as important for the outcome that the therapy regimen itself with better responses in cases without cirrhosis and with low levels of HCV RNA. Furthermore, the genotype of HCV seems to be also critical for the response rate. Virological evaluations appears therefore crucial to assess not only HCV infection but also for the indication and monitoring of therapy.

Alanine Transaminase↗

[Limits of immunoserologic and molecular diagnosis of hepatitis C].

Hepatitis C is the most common cause of post-transfusion hepatitis, as well as of the viral chronic liver disease in the western world. However since it is even more often asymptomatic than HBV, this is not truly recognized. The detection of hepatitis C can only rely on serological and virological methods and require their extensive use in screening programs. Following the molecular identification characterisation of HCV, it became possible to detect virus specific antibodies. The first generation Elisas were limited in their scope and have been replaced by second and third generation tests with better sensitivity and specificity. These assays detect antibodies to several sets of HCV protein including the C22 core, the C33 and C100, which correspond to the non structural regions (NS3 and NS4 respectively). More recently, NS5 proteins have also been added and synthetic peptides have replaced some of the recombinant proteins used initially. In spite of improved sensitivity and specificity, last generation Elisas still require confirmation by supplemental assays which can be of different types (immunoblot or combined Elisas) and include sets of structural and non structural recombinant proteins or peptides. New tests are needed to improve sensitivity and proficiency of this mandatory confirmation procedure. It is unclear at this stage whether the dogma inherited from HIV to request two sets of reactive antibodies will be also warranted by experience in HCV infection. The biggest limitation of present HCV tests is the delayed appearance of anti-HCV following primary infection. Even more worrisome is the fact that 10% of chronic infection with liver disease still remain seronegative, despite circulating HCV RNA in serum and/or liver as well as expressing HCV antigen demonstrable in liver tissue by immunostaining. Such a proportion is even more common in settings with immune deficiencies including organ transplantation and HIV infection. DNA amplification methods, such as PCR or others, must be used in order to demonstrate HCV RNA in combination with reverse transcription steps. This new powerful technology must be however applied under stringent quality control procedures and cannot be yet considered for screening or routine diagnosis although it can detect viremia as early as a week after exposure and help to monitor interferon treatment. During acute hepatitis, the delay in the appearance of anti-HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness, and a third one distinct from the previous two. Two major HCV genotypes, F1 and F2, corresponding to HCV type I and II (USA prototype and Japanese) with prevalence of 45% and 55% respectively, were found in France. F1 infected patients were younger and more often male than F2 group. Nine of 28 patients in F1 genotype infected group had history of drug abuse but none i

Antibodies, Viral↗

Clinical relevance of the detection of hepatitis delta virus RNA in serum by RNA hybridization and polymerase chain reaction.

Hepatitis delta virus nucleic acid was detected by dot-blot hybridization using RNA probe and reverse transcription/polymerase chain reaction amplification in 223 serum samples from 66 patients with hepatitis D virus infection. Seven cases with chronic hepatitis D virus infection were treated with interferon: six for 3 months and one for 7.5 years. By using the primers located in the putative conserved regions, the technique of reverse transcription/polymerase chain reaction amplification was 10(3) to 10(4) times more sensitive than that of dot-blot hybridization. The main findings of this study are: (i) HDV RNA could be detected in the absence of any other serological hepatitis D virus marker in serum from acute hepatitis patients with IgM anti-HBc; (ii) high titer anti-HD antibodies (IgM and total anti-HD) persisted in patients during short-term interferon treatment, and in one patient during long-term interferon treatment, despite clearance of serum HDV RNA even after 3 years; (iii) total anti-HD alone was detected in the absence of IgM anti-HD and serum HDV RNA. These observations indicate that the detection of HDV RNA by molecular techniques in serum is a useful, sensitive and non-invasive technique for the early diagnosis and follow up of hepatitis D virus infection, as well as for the monitoring of antiviral therapy. In addition, total anti-HD antibody in the absence of HDV RNA may be the only residual marker of past infection. Finally, the choice of the technique for hepatitis D virus detection is important for the optimal assessment of the clinical stage and monitoring of antiviral therapy in hepatitis D virus-infected patients.

Alanine Transaminase↗

Hepatitis C virus genotypes in France: comparison of clinical features of patients infected with HCV type I and type II.

Two major hepatitis C virus genotypes, F1 and F2, corresponding to hepatitis C virus type I and type II respectively, were found in France. To investigate the correlation between infection with these genotypes (F1 and F2) and clinical features of patients, serum samples proven to be hepatitis C virus positive by polymerase chain reaction amplification on 5' non-coding region were further amplified in the NS3 region with nested polymerase chain reaction. The NS3-polymerase chain reaction products were Southern blotted and hybridized with specific probes to identify the genotype of hepatitis C virus. Of 70 samples 64 were NS3-polymerase chain reaction positive. Twenty-eight (40%) samples were hepatitis C virus type I (F1) and 34 (49%) were hepatitis C virus type II (F2), while one sample (HB) hybridized with both probes and another (HN) hybridized with neither. Some samples were sequenced, with results consistent with those of hybridization. The HB sample was related more to hepatitis C virus type II than to type I and the HN sample was divergent from both type I and type II genotypes. Clinical profiles of patients infected with hepatitis C virus type I and type II were compared. Type I infected patients were younger (p < 0.01) and more often male (p < 0.05) than those of the type II group. Nine of 28 patients in the type I infected group had a history of drug abuse, whereas none did in the type II group. Five of 22 (23%) type I infected patients and 19 of 32 (59%) type II infected patients had cirrhosis (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Heterogeneity of hepatitis C virus genotypes in France.

The genotypes of French hepatitis C virus (HCV) isolates were investigated by amplification of a domain from the non-structural region 3 (NS3) using nested PCR, followed by hybridization with two genotype-specific probes, F1 (HCV type I-specific) and F2 (HCV type II-specific). Among 119 HCV RNA-positive sera, 91% of samples were NS3 PCR positive. Most samples (83.2%) hybridized with one or the other probe only, whereas a few samples (4.2%) hybridized with both F1 and F2 probes (HB). A small percentage (3.4%) of samples appeared unable to hybridize with either probe (HN). For some of these samples (HB1, HB2, HN1, HN2, HN3, HN4), part of the NS3, core and envelope regions were sequenced and the corresponding deduced consensus sequences were compared with those of prototype isolates of the four HCV genotypes (types I to IV). A phylogenetic tree was constructed to illustrate the relationship between these isolates. The results obtained showed that (i) HN4 appears to be more closely related to type III than to type IV HCV genotypes, which suggests that in France there may exist additional although minor genotypes besides the two major types, F1 and F2. (ii) HB1, HB2, HN1, HN2 and probably HN3 belong to the type II HCV genotype. The association between sequence diversity and putative biological difference for isolates within the same genotype remains to be elucidated.

Africa, Northern↗

Serological responses to different genotypes of hepatitis C virus in France.

The relationship between hepatitis C virus (HCV) genotypes and antibody status was studied in 104 chronic non-A, non-B hepatitis patients and asymptomatic HCV-infected blood donors. On the basis of amplification of the nonstructural protein 3 (NS3) coding region by PCR and hybridization with specific probes, 55 and 42 patients were identified as being infected with type I and type II, respectively, according to the classification by H. Okamoto, K. Kurai, S. Okada, K. Yamamoto, H. Lizuka, T. Tanaka, S. Fukuda, F. Tsudaand, and S. Mishiro (Virology 188:331-341, 1992). All samples were tested for antibodies to 5.1.1, C-100, C-33, and C-22 proteins by a second-generation recombinant immunoblot assay. Among 97 patients with known HCV genotypes, 31 of 42 patients infected with type II and 24 of 55 infected with type I had antibodies against all four antigens (P < 0.01). In the type II-infected group, more patients had detectable antibodies to 5.11, C-33, and C-22 proteins than in the type I group (P < 0.05). No difference was found in the serological response to C-100 between the two groups.

Base Sequence↗

Discovery of a novel point mutation changing the HDAg expression of a hepatitis delta virus isolate from Central African Republic.

None of the mutations so far discovered in several hepatitis delta virus (HDV) isolates appears to determine important changes in HDV specific protein (HDAg) expression, except for a putative mutation at nucleotide 1012 converting an amber stop codon (TAG) to a codon for tryptophan (TGG). Here we present the characterization of an HDV obtained from the liver of a woodchuck inoculated with sera from fulminant HDV patients in Central African Republic (CAR). By restriction enzyme analysis and sequencing of HDAg-coding region cDNA clones, we found that this HDV isolate bears a novel mutation (T to A) at nucleotide 1013 which converts the amber stop codon (TAG) to a codon for lysine (AAG). Comparison of these nucleotide sequences with those available from American, Japanese, Taiwanese, French, Italian and Nauru isolates showed a variability of 1.7 to 21.5% and 1.9 to 28.7% at the nucleic acid and amino acid levels, respectively. The HDAg-encoding sequence of the CAR isolate is closely related to that of the Italian HDV isolate. The in vitro expression of this HDV isolate resulted in a unique HDAg species (28K) which was identical with that characterized in vivo.

Amino Acid Sequence↗

Hepatitis B virus genotype A rarely circulates as an HBe-minus mutant: possible contribution of a single nucleotide in the precore region.

The emergence of HBe-minus hepatitis B virus (HBV) mutants, usually through a UAG nonsense mutation at codon 28 of the precore region, helps the virus to survive the anti-HBe immune response of the host. Host and viral factors that predispose to the emergence of such mutants are not well characterized. The fact that the precore region forms a hairpin structure essential for the packaging of viral pregenomic RNA may explain the extremely high prevalence of the UAG mutation at codon 28. It converts a wobble U-G pair in the packaging signal between nucleotide 3 of codon 15 (CCU) and nucleotide 2 of codon 28 (UGG) into a U-A pair. Since genotype A of HBV has a CCC sequence at codon 15, the UAG mutation would, instead, disrupt a C-G pair present in the wild-type virus. This alteration was shown by transfection experiments to greatly compromise the packaging of pregenomic RNA. The implication of this finding was elucidated by molecular epidemiological studies. Genotype A was found to be the most prevalent genotype in the wild-type virus populations in France but was found in only 1 of the 46 isolates of HBe-minus mutants found there. These mutants were contributed chiefly by genotype D, the second most prevalent genotype in France, which is characterized by a CCU sequence at codon 15. The role of the single nucleotide at codon 15 was confirmed by the finding of the single genotype A isolate in which both wild-type and mutant viruses were present. Interestingly, nearly all of the mutants had a codon 15 sequence of CCU instead of the CCC present in the wild-type viruses. Our results suggest that genotype A of HBV rarely circulates as HBe-minus mutants, probably because of a requirement for a simultaneous sequence change at codon 15. These data, together with the virtual absence of genotype A in the Chinese samples examined, may provide some insights into the uneven prevalence of HBe-minus mutants in the world.

Base Composition↗

Evidence for a base-paired region of hepatitis B virus pregenome encapsidation signal which influences the patterns of precore mutations abolishing HBe protein expression.

In two natural HBe-minus hepatitis B virus mutants, expression of HBe protein was abrogated by a nonsense mutation at precore codon 28 and a frameshift mutation at codon 29, respectively. Both mutants contained an additional nucleotide substitution(s) which was found by transfection experiments to be required for efficient packaging of pregenomic RNA. The observed mutational profiles were consistent with the presence of a base-paired region of the pregenome encapsidation signal overlapping the HBe-coding sequence. Results obtained with artificial mutants with significant changes in the primary sequence suggested that base pairing is required but insufficient for efficient pregenome packaging. However, the predicted first four base pairs of the stem are dispensable.

Base Composition↗

Diagnostic markers of viral hepatitis B and C.

Hepatitis B virus (HBV) serology has become extremely refined. As well as the recognised hepatitis B surface (HBs), hepatitis B core (HBc), and hepatitis B e (HBe) antigen-antibody systems, new markers have been introduced including pre-S1, pre-S2 for the envelope and the functional X protein. New automates have been introduced allowing flexibility in the different tests according to precise needs. The monitoring of pre-S1 antigen provides a relevant correlate of viral replication. The quantitative determination of HBV-DNA, pre-S1 Ag, and IgM anti-HBc seem most useful for the decision to use, and the monitoring of, antiviral treatment. Second generation ELISAs detect antibodies to three sets of hepatitis C virus (HCV) protein including the c22 core, and c33, and c100, which correspond to the non-structural regions (NS3 and NS4, respectively). Second generation ELISAs require confirmation by supplement assays, but their biggest limitation is the delayed appearance of anti-HCV after primary infection. In addition 10% of chronic infections with liver disease still remain seronegative despite circulating HCV RNA in serum or liver, or both. Much progress still has to be made before HCV serology can reach the level of sophistication of HBV.

Biomarkers↗

Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal.

The emergence of hepatitis B virus variants unable to express HBe protein during late stage of viral infection may represent an important mechanism of viral persistence. The molecular mechanisms responsible for the elimination of HBe expression are nonsense or frameshift mutations or initiation codon mutations in part of its coding sequence, the precore region. So far only 2 of the 29 precore amino acid codons have been found mutated to stop codons in nature, although a total of 10 codons are convertible to stop codons by single nucleotide changes. Since the HBe-coding sequence is largely overlapped by the pregenome encapsidation signal (epsilon signal), a recently found cis-acting element required for the packaging of pregenomic RNA, the absence of other potential nonsense mutants could result from their impairment of the epsilon signal. Seven such potential stop codon mutants were constructed and tested for replication capacities by transfection into a hepatoma cell line. Five mutants were replication competent, but at levels lower than that of a prevalent natural stop codon mutant. The remaining two mutants were completely defective in DNA replication, which clearly explained why these two mutants are not found in nature. Northern blot analysis revealed wild-type levels of RNA transcription by these two mutants but complete lack of packaged pregenomic RNA. Additional studies lent further support to the importance of the epsilon signal in pregenome encapsidation and suggested relaxed sequence requirements for the computer-predicted hexanucleotide bulge region as compared to the hexanucleotide loop of the signal.

Base Sequence↗

[Importance of PCR in the diagnosis of hepatitis C].

The identification of hepatitis C virus, based on DNA amplification, gives a precise estimation of the prevalence of the most frequent agent of NANB hepatitis. The first ELISA allowing the detection of anti-HCV antibodies, had too many false positive results and required the development of more sensitive and specific assays to confirm its results. PCR, allowing the hepatitis C virus diagnosis by showing directly HCV RNA sequences, offers a complementary approach to immunoserological tests. In blood donors with anti-HCV antibodies and with indeterminate or negative confirmatory tests, the finding of HCV RNA sequences reveals serum infectivity. During acute hepatitis, the delay in the appearance of anti HCV hampers acute phase diagnosis. The early detection of HCV RNA in peripheral blood, confirms the diagnosis and opens up therapeutic possibilities. In chronic hepatitis, the diagnosis of seronegative forms may only be resolved by PCR. Moreover, the presence of HCV RNA in peripheral blood represents the only marker of on-going viral replication and coincides with the severity of liver damage. During treatment with interferon, the follow up of HCV RNA sequences makes it possible to monitor its efficacy. The search for HCV RNA sequences directly in liver tissue shows that HCV may replicate in the liver in the absence of viremia. The presence of HCV RNA in the liver and the serum of liver transplanted patients is essential for the etiological diagnosis and management of hepatitis and bone marrow failure occurring after transplantation. Epidemiological study using PCR is a major tool in documenting vertical transmission between mother and child. Finally, PCR is important for the analysis of the HCV genome. Thus, in France there are at least three main strains, one close to the US prototype, the other close to the Japanese strain, possibly responsible for a more severe illness and a third one distinct from the previous two. However, its limits and constraints imply that PCR must not be considered as a routine assay. This emphasizes the need for more simple and rapid diagnostic tests, allowing the detection of HCV antigens and, as in hepatitis B, the progressive unravelling of the life cycle of HCV.

Animals↗