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L Cova

Publications and source records attributed to L Cova.

At least 19 recordsLinked to original sources
PubMed1997

In vivo selection of duck hepatitis B virus pre-S variants which escape from neutralization.

To better understand the role of specific residues within the duck hepatitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutralization. A highly neutralizing monoclonal antibody (Mab 900) which recognizes an epitope 83IPQPQWTP90 localized previously on the DHBV pre-S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only two in vivo neutralization rounds with Mab 900, five different pre-S mutant genomes were identified, which harbored point mutations affecting only proline residues located at position 90 within this epitope (83IPQPQWTP90) and/or at a distance at position 5. We have shown that a single (P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity nor in vivo infectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P90H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapid in vivo selection and identification of DHBV pre-S variants and provide evidence that residues within and at a distance from the neutralization epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity.

Animals↗
PubMed1997

Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes.

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.

Amino Acid Sequence↗
PubMed1997

Sequence heterogeneity of heron hepatitis B virus genomes determined by full-length DNA amplification and direct sequencing reveals novel and unique features.

So far, only a single heron hepatitis B virus genome (HHBV-4) has been cloned and sequenced. Therefore, neither the significance of its sequence divergence from other avian hepadnaviruses nor the sequence variability of HHBV genomes in general are known. Here we have analysed the sequence heterogeneity of HHBV genome populations in several sera from naturally infected herons. A highly sensitive PCR method for full-length HHBV genome amplification was established which allowed direct sequencing of entire HHBV populations without prior cloning. Sequences of HHBV genomes from four sera were thus obtained which differed from those of HHBV-4 by up to 7%. Some of the divergent nucleotides and the corresponding amino acids of the predicted viral proteins were conserved in all four new HHBV isolates and varied only in HHBV-4. This indicates that the HHBV-4 genome is not in all aspects representative of this class of viruses. Interestingly, a highly conserved ORF upstream of the C-gene present in a position analogous to that of the mammalian hepadnavirus X-gene became apparent in all HHBV genomes. In contrast to the duck hepadnaviruses, the small (sAg-S) instead of the largest (sAg-L) envelope protein of all HHBVs has a myristylation site. These data confirm the significant sequence divergence of HHBV from other avian hepadnaviruses. Moreover, they show that HHBV has low sequence variability and indicate two new and unique features not evident in other avihepadnaviruses: an additional, highly conserved gene and potential myristylation of the sAg-S instead of the sAg-L envelope protein.

Amino Acid Sequence↗
PubMed1996

Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.

It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties.

Action Potentials↗
PubMed1996

Early stages of rabbit haemorrhagic disease virus infection monitored by polymerase chain reaction.

In order to define more accurately the initial events that take place during rabbit haemorrhagic disease virus (RHDV) infection, different organs of experimentally infected rabbits were analysed for the presence of the virus and correlated with histopathological observations. A total of 24 rabbits were intranasally inoculated with a viral suspension, and tissue samples were taken from the liver, spleen, kidney, lung, thymus, lymph node and tonsil at different intervals post-inoculation (2, 4, 6, 12, 18, 24, 30, 36, 48, 50, 51, 70 and 72 h). Histopathological observations revealed the presence of the first significant lesions at 30 h post-inoculation (p.i.) in the liver. Using an ELISA and a haemagglutination test (HAT), the virus was detected in the liver at 36 h p.i. The reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the RHDV RNA was present as early as 18 h p.i. in the liver and spleen, whereas thymus, kidney, tonsil and lymph node were found to be positive after more than 36 h p.i. The lungs presented a variable positivity between 0 and 36 h p.i., but remained positive after this time.

Animals↗
PubMed1995

Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS.

Stem cells isolated from the CNS of both embryonic and adult mice undergo extensive proliferation in the presence of epidermal growth factor (EGF). Removal of EGF determines the differentiation of these cells into neurons and glia. We have recently demonstrated that basic fibroblast growth factor (bFGF) regulates the proliferation of EGF-generated progenitors of the embryonic mouse striatum. We report here that bFGF induces proliferation of some EGF-generated precursors of the adult mouse striatum which, in turn, differentiate in vitro into cells possessing neuron-like morphology and neuronal antigenic properties. These results demonstrate that EGF and bFGF can act sequentially to regulate the de novo generation of neurons from the adult mouse CNS in vitro and suggest the existence of a lineage relationship between EGF- and bFGF-responsive progenitor cells of the adult murine brain.

Animals↗
PubMed1995

Detection of rabbit haemorrhagic disease virus isolates and sequence comparison of the N-terminus of the capsid protein gene by the polymerase chain reaction.

At present there is no sensitive method for the detection of rabbit haemorrhagic disease virus (RHDV), a calicivirus causing high mortality in rabbit populations. For this purpose a reverse transcriptase polymerase chain reaction (RT-PCR) was established in the N-terminal portion of the RHDV capsid region. The RT-PCR was 10(4)-fold more sensitive than ELISA testing for the detection of the virus and was able to detect as few as 12 copies of template cDNA. By using the RT-PCR test and sequencing, 96.6 to 98.7 per cent homology was demonstrated in the N-terminal portion of the capsid protein of three isolates from geographically and temporally separate outbreaks of viral haemorrhagic disease, indicating that this portion of the RHDV capsid protein is highly conserved.

Amino Acid Sequence↗
PubMed1995

Spectrum of liver disease and duck hepatitis B virus infection in a large series of Chinese ducks with hepatocellular carcinoma.

The striking difference in the geographical distribution of liver cancer in ducks raised the question of whether duck hepatitis B virus (DHBV), like mammalian hepadnaviruses, could be an oncogenic agent. Hepatocellular carcinomas (HCCs) have been found only in domestic ducks in Qidong, China, where hepatitis B virus infection and aflatoxin B1 (AFB1) are both risk factors and where a high frequency of human HCCs has been reported. To date, the study of liver pathology occurring in Chinese ducks has been hampered by the small number of samples available. We describe here a series of 59 Chinese brown duck livers that were collected in Qidong more than 20 years ago and formalin fixed. Thirty-six HCCs, which ranged from well-differentiated trabecular to highly anaplastic type, were identified in relatively young ducks (average age, 3.3 years). Several unique features not previously reported, such as tumor giant cells, tumor necrosis, tumor thrombi in blood vessels, and inactive cirrhosis, were observed. Bile ductule proliferation, known to be a prominent feature of AFB1 exposure in ducks, was present in 86% of livers. Using polymerase chain reaction (PCR) and two primer pairs, located within conserved portions of the DHBV S and C genes, we demonstrated the presence of DHBV DNA in 23 of 34 HCCs analyzed (68%). The spectrum of liver pathology that we report in brown ducks from Qidong was never observed in Pekin ducks of the same age chronically infected with DHBV and followed under controlled conditions outside China, suggesting that causative factors other than virus infection may be involved in duck liver carcinogenesis observed in this area.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Identification of major antigenic domains of duck hepatitis B virus pre-S protein by peptide scanning.

Neutralization epitopes of duck hepatitis B virus (DHBV) have been previously mapped within the N-terminal portion of the pre-S protein using monoclonal antibodies. However, the immune response of ducks to this region is not well characterized at the amino acid level. To this end, we have immunized adult Pekin ducks with either DHBV positive serum or bacterially expressed DHBpre-S polypeptide representing the N-terminal portion of the DHBV pre-S region. We have demonstrated that adult ducks inoculated with either antigen developed antibodies to the DHBV pre-S region starting 5 to 10 days postinjection. The sera of all ducks, irrespective of the immunogen used, exhibited a significant protective activity against DHBV, as assessed in vivo. To identify which pre-S domains bind antibodies from these duck sera, we have used the Pepscan methodology with overlapping octapeptides spanning the DHBV pre-S sequence from amino acids 1 to 145. Using this approach, five major antigenic domains, 7KSMDVRRI14, 22NQLAGRMIP30, 58TLQNQGAW65, 71RRVGLSNPT79, and 127GDDPLLGNQ135 were identified within the DHBV pre-S region.

Amino Acid Sequence↗
PubMed1994

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↗
PubMed1994

Duck hepatitis B virus infection, aflatoxin B1 and liver cancer in domestic Chinese ducks.

The oncogenicity of Duck hepatitis B virus (DHBV) is unclear since hepatocellular carcinomas (HCCs) have been reported only in domestic ducks in Qidong, an area of China where hepatitis B virus (HBV) and aflatoxin B1 (AFB1) are risk factors for liver cancer in man. In order to better define the association between DHBV infection, AFB1 and HCC we analysed a series of 16 duck liver samples collected from local farms in Qidong. HCC was found in eight and cirrhosis in one of these samples. Furthermore bile duct proliferation, characteristic of AFB1 exposure in ducks and other animal species, was found in these ducks. Integration of DHBV DNA into cellular DNA was observed in only one out of four DHBV positive HCCs, indicating that viral integration is not prerequisite for tumour development. In four remaining HCCs the polymerase chain reaction (PCR) failed to show any DHBV DNA suggesting that liver tumours do occur in polymerase chain reaction (PCR) failed to show any DHBV DNA suggesting that liver tumours do occur in these ducks in the absence of DHBV infection. In addition, AFB1-DNA adducts were detected by hplc-immunoassay in one such DHBV-negative tumour. In summary we demonstrate that risk factors other than DHBV, including AFB1 exposure, may be important in duck liver carcinogenesis in Qidong.

Aflatoxin B1↗
PubMed1994

Absence of p53 mutation at codon 249 in duck hepatocellular carcinomas from the high incidence area of Qidong (China).

Dietary aflatoxin and hepatitis B virus infection may play a role in generating the p53 tumor suppressor gene codon 249 hotspot mutation found in human hepatocellular carcinomas (HCCs) from Qidong (China) and southern Africa. No data are available on the HCC site-specific mutation of the p53 gene in hepadnavirus-infected animals exposed to AFB1. We have searched for the presence of p53 gene codon 249 mutations in both duck hepatitis B virus (DHBV) positive and negative HCCs of domestic ducks from Qidong, where the human p53 hotspot is so prevalent, as well as in duck HCCs experimentally induced by AFB1. Direct sequencing of DNA amplification products encompassing p53 codon 249 did not reveal any mutations in 11 HCCs from Qidong ducks, regardless of the status of DHBV infection. In addition no mutation was detected in four HCCs from AFB1-treated ducks. This contrasts with the human data; however, in humans, the mutation and the preferential binding of AFB1 to codon 249 occurs at the third nucleotide G, while in duck, the codon 249 lacks this G residue. The DNA sequence of adjacent codons is also different in the two species even though the amino acid sequence is identical. This may explain the low frequency of mutation we have observed. In addition, species differences in metabolism and DNA repair could influence the occurrence of codon 249 mutations.

Aflatoxin B1↗
PubMed1993

Fine mapping of neutralization epitopes on duck hepatitis B virus (DHBV) pre-S protein using monoclonal antibodies and overlapping peptides.

To define the residues involved in duck hepatitis B virus (DHBV) neutralization at the amino acid level, we have used a procedure combining monoclonal antibodies (MAbs) and overlapping octapeptides (Pepscan). Two neutralizing MAbs (SD20 and 900), specific for the pre-S protein were shown to reduce DHBV infectivity in vivo by 75 and 90%, respectively, while complete protection of ducklings was achieved with a polyclonal antiserum raised against the bacterially expressed first 131 amino acids of the DHBV pre-S region (DHBpre-S). Using fusion polypeptides, the binding sites of these MAbs were localized between aa 77 and 100 on pre-S protein. We have used octapeptides spanning the pre-S sequence from aa 64 to 115 for fine mapping of these epitopes. Within the sequence scanned, the polyclonal anti-DHBpre-S antiserum recognized a region exclusively limited to the residues E82-K95, suggesting immunodominance of this region in the sequence aa 64-115. The epitope recognized by Mab 900 was mapped within the same region, whereas the epitope recognized by Mab SD20 was localized downstream from this region. To define the amino acids essential for binding to the highly neutralizing Mab 900, we have used single amino acid replacement and demonstrated that two residues Q87 and W88 were important for antibody recognition.

Amino Acid Sequence↗
PubMed1993

Duck hepatitis B virus (DHBV) as a model for understanding hepadnavirus neutralization.

The role of the immune response to the human hepatitis B virus (HBV) envelope proteins in neutralization of viral infectivity has been well documented. The similarity between HBV, prototype member of the hepadnavirus family, and the closely related duck hepatitis B virus (DHBV) has allowed, use of the latter as a convenient model for the study of molecular mechanisms of HBV replication and neutralization. In this brief review, we will examine the HBV and DHBV envelope proteins and their role as targets for virus neutralization.

Amino Acid Sequence↗
PubMed1993

Duck hepatitis B virus infection, aflatoxin B1 and liver cancer in ducks.

The association between chronic infection by hepadnaviruses isolated from human (HBV), woodchuck (WHV), ground squirrel (GSHV) and development of hepatocellular carcinoma (HCC) in their respective hosts is well established (reviewed in [11, 15, 17]). By contrast, the association of duck hepatitis B virus (DHBV) infection with HCC is less documented. Pekin ducks congenitally infected with DHBV and followed for several years throughout the world do not develop liver tumors: HCC has been found only in domestic ducks from a single area of China, Qidong. Several factors such as DHBV carrier rate, breed and age of ducks, subtype of DHBV and environmental carcinogens are suspected to contribute to this striking difference between the geographical repartition of liver cancer in DHBV-carrier ducks. In this brief review we will consider successively the role of these different factors in duck liver oncogenesis.

Aflatoxin B1↗
PubMed1993

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↗
PubMed1993

Molecular dosimetry of aflatoxin exposure: contribution to understanding the multifactorial etiopathogenesis of primary hepatocellular carcinoma with particular reference to hepatitis B virus.

Aflatoxin exposure and hepatitis B virus infection have been implicated as major risk factors for primary hepatocellular carcinoma (PHC) in high-incidence regions of the world. Investigations using the assay of aflatoxin bound to peripheral blood albumin have shown that exposure can occur throughout the life span of the individual, including during the perinatal period, in high-incidence areas such as The Gambia, Senegal, Kenya, and The People's Republic of China. The possibility of measuring aflatoxin exposure at the individual level permits an investigation of the putative mechanisms of interaction of this carcinogen with HBV in the etiopathogenesis of PHC. Animal models, e.g., Pekin duck and HBV-transgenic mice, have also been used to study these questions, and the available data are reviewed.

Aflatoxin B1↗