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PubMed2007

Progression of astrocytomas and meningiomas: an evaluation in vitro.

OBJECTIVES/BACKGROUND: In biological terms, progression means that malignancy increases as genetic mutations accumulate leading to increased proliferation and invasion capacity. By verifying the proliferation capacity, human telomerase reverse transcriptase (hTERT) expression and in vitro invasion, in a group of highly malignant glioblastomas, benign meningiomas and astrocytomas, at the initial stage of progression, we have analysed putative progression in vitro for proliferation and telomerase expression. MATERIALS AND METHODS: The relative proliferation status (visualized with Ki-67 antibodies) and presence of hTERT protein was analysed in 27 intracranial tumours (6 astrocytomas, 8 glioblastomas and 13 meningiomas) by immunohistochemistry on paraffin-embedded biopsy tissue, as well as on primary tumour-derived cell cultures. A confrontation model was used to analyse invasiveness in vitro. RESULTS: The mean proliferation indices were 22.3 (SD = 18.1) for glioblastomas and 2.1 (SD = 1.9) for low-grade (LG) astrocytomas. The group of benign meningiomas had a labelling index of 2.2 (SD = 2.7). Mean percentages of staining for hTERT varied between 36.5 (SD = 28.4) for glioblastomas and 10.2 (SD = 8.6) for LG astrocytomas. The group of benign meningiomas had a labelling index of 12.4 (SD = 19.2) for hTERT. A significant difference was seen for Ki-67 (P < 0.05) and hTERT (P < 0.001) in vivo versus in vitro. No difference was seen between the group of invasive and non-invasive tumour-derived cell cultures for the histopathological markers Ki-67 and hTERT (P > 0.05) in vitro. CONCLUSIONS: The elevated expression of hTERT and Ki-67 in vitro provides a potential prognostic tool for early detection of the progression of brain tumours. As tumour cells require telomerase for continued proliferation, the expression of hTERT may mark immortality, leading to indefinite life span. On the other hand, hTERT expression and cell proliferation are not linked directly to invasion in vitro.

Adolescent↗
PubMed2006

Reproductive performance in grey seals: age-related improvement and senescence in a capital breeder.

1. Three hypotheses have been advanced to account for age-related improvement in performance: the selection hypothesis predicts improved due to the loss of lower quality phenotypes, the constraint hypothesis predicts individuals improve function, and the restraint hypothesis predicts younger individuals forego or reduce effort because of mortality risks. A decline in age-related performance (i.e. senescence) is predicted by mutation accumulation, antagonistic pleiotropy and disposable soma (wear and tear) hypotheses. 2. Using five measures of performance - birth rate, maternal and pup birth mass, pup weaning mass, weaning success and lactation length - we tested these hypotheses concerning age-related change in reproduction in 279 female grey seals (Halichoerus grypus), ages 4-42 years, over a 23-year period between 1983 and 2005 on Sable Island, Nova Scotia. These females produced 2071 pups. 3. Although body mass of primiparous females increased with age (4-7 years) birth mass of their pups did not, but pup weaning mass did. Second- and third-parity females of the same age as primiparous females gave birth to and weaned heavier pups. However, parity and age were dropped from models when maternal body mass was included. 4. The proportion of females giving birth varied significantly with maternal age, increasing in young females and then declining late in life. Weaning success rate also increased rapidly to about 8 years and subsequently declined in females > 32 years. 5. Generalized additive models indicated nonlinear changes in 3 day body mass (i.e. approximately birth mass) and weaning mass of pups as a function of maternal age, after accounting statistically for the effects of maternal body mass. Mixed-effects, repeated-measures models fitted to longitudinal data further supported the conclusion that pup birth mass and weaning mass vary nonlinearly with maternal age and indicated nonlinear changes in lactation duration. 6. We found some support for the constraint hypothesis, but our findings were not consistent with the selection hypothesis or the restraint hypothesis as the basis for improvement in reproductive performance. 7. Senescence was evident in multiple female and offspring traits, indicating the degeneration in function of several physiological systems as predicted by the disposable soma hypothesis.

Aging↗
PubMedDate not supplied

Invention and mystery in hypotrich DNA.

Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly, are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.

Animals↗
PubMed1977

Lethal and mutagenic effects of 5-iodouracil on bacteriophage T4td8rII.

Evidence was obtained which indicates that the lethal effect of 5-iodouracil (IUra) on bacteriophage T4 is not due to a mutagenic process. T4td8rII (thymine requiring, rapid lysis) double mutants were constructed. Reversion of T4td8rII to r(+) was measured. First, reversion by growth in the presence of the structural analogues chlorouracil (ClUra) and bromouracil (BrUra) did not correlate with their relative lethal effects (for mutagenesis: IUra </= ClUra </= BrUra; for lethality: ClUra < BrUra < IUra). Second, reversion frequencies of T4td8rII in infected cells increased linearly with time of growth in the presence of IUra, whereas the frequency of lethality was constant with time. Third, reversion frequencies increased markedly at low levels of IUra substitution, whereas lethal effects were apparent only with extensive IUra substitution. Fourth, the reversion frequency of the nonviable fraction of IUra-substituted T4td8rII (as examined by multiplicity reactivation) did not differ significantly from that of the viable IUra-substituted T4td8 fraction. If mutagenesis caused lethality, then the nonviable T4td8rII fraction should accumulate mutations and have a higher reversion frequency.

Coliphages↗
PubMed1999

Variation in the composition and pore function of major outer membrane pore protein P2 of Haemophilus influenzae from cystic fibrosis patients.

We investigated the relationship between susceptibility to beta-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulated Haemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinct H. influenzae strains from two patients extensively treated with beta-lactam antibiotics. The variants differed in their susceptibilities to at least two beta-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to beta-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences of ompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.

Amino Acid Sequence↗
PubMed2003

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution.

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k(cat), the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

Benzothiazoles↗
PubMed1994

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

Animals↗
PubMed2002

Genetic variability and stability of Anaplasma phagocytophila msp2 (p44).

Anaplasma (Ehrlichia) phagocytophila's major immunodominant surface protein antigen, Msp2 (P44, 44-kDa antigen), is encoded by a family of paralogous genes characterized by conserved sequences flanking a hypervariable region. The antigenic profiles of most strains of A. phagocytophila are different, and the differences are principally related to Msp2 expression. To date, multiple unique msp2 gene paralogs have been found in A. phagocytophila isolates, but the overall number in the genome of a single strain is not yet known. Changes in msp2 expression may be related to antigenic variability; thus, we examined the minimal complement of msp2 genes or pseudogenes in two strains of A. phagocytophila and the number of transcriptionally active msp2 gene paralogs during low-passage, steady-state, in vitro propagation. Of 15 BDS strain clones, 1 had a hypervariable region identical to the region in a clone obtained from a BDS strain genomic library previously prepared from organisms after only two horse passages. When 124 Webster strain clones were examined, 18 unique hypervariable regions were identified. Of 64 Webster strain cDNA clones, 56 (87.5%) were derived from a single gene, and transcripts from six additional msp2 genes were also identified. The sequences of several hypervariable regions that were > or = 97% similar to regions present in other strains were identified by performing a BLAST analysis of sequences deposited in the GenBank database. These findings suggest that antigenic variability results from transcription of one or a few of the multiple paralogs and not from genetic instability that results in random accumulated mutations, although the possibility that gene recombination plays a role cannot be eliminated. The predominant Msp2 pattern in vitro is determined by transcription from a single gene.

Anaplasma↗
PubMed1990

Heat mutagenesis in bacteriophage T4: another walk down the transversion pathway.

Extracellular nonreplicating bacteriophage T4 particles accumulate mutations as functions of temperature, time, pH, and ionic environment via two mechanisms: 5-hydroxymethylcytidine deamination produces G.C----A.T transitions while a guanosine modification produces transversions. Neither frameshift mutations nor mutations at A.T base pairs are appreciably induced. We now show that heat induces G.C----T.A transversions which we suggest may arise via a G*.A mispair, in which G* is a modified guanosine that has experienced a glycosylic bond migration. The rate of this reaction at 37 degrees C is sufficient to present a genetic hazard, particularly to large genomes; thus, the lesion is probably efficiently repaired in cellular genomes.

Base Composition↗
PubMed1991

The RSF1 gene regulates septum formation in Saccharomyces cerevisiae.

Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum. We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene. However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum. This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START. The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis. We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.

Cell Cycle↗
PubMed1992

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.

Amino Acid Sequence↗
PubMed1997

Development of a rapid assay for detecting gyrA mutations in Escherichia coli and determination of incidence of gyrA mutations in clinical strains isolated from patients with complicated urinary tract infections.

The MICs of ofloxacin for 743 strains of Escherichia coli isolated from 1988 to 1994 were determined by testing. The strains were from patients with urinary tract infections complicated by functional or anatomical disorders of the urinary tract. Those determined to be ofloxacin resistant (MIC, > or =12.5 microg/ml) comprised 3 of 395 strains (1.3%) from the 1988 to 1990 group, 2 of 166 strains (1.2%) from the 1991 to 1992 group, and 7 of 182 strains (3.8%) from the 1993 to 1994 group. The incidence of resistant strains increased significantly during this period. The percentage of isolates with moderately decreased susceptibilities to ofloxacin (MIC, 0.39 to 3.13 microg/ml) also rose during the same period. To determine the incidence of gyrA mutations in urinary-tract-derived strains of E. coli, we developed a simple and rapid assay based on PCR amplification of the region of the gyrA gene containing the mutation sites followed by digestion of the PCR product with a restriction enzyme. Using this assay, we examined all 182 strains isolated in 1993 and 1994 for the presence of mutations at Ser-83 and Asp-87 in the gyrA gene. Of these strains, 33 (18.1%) had mutations in the gyrA gene. The incidences of mutations at Ser-83, at Asp-87, and at both codons were 10.4 (19 strains), 4.4 (8 strains), and 3.3% (6 strains), respectively. To determine the correlation of the mutations in the gyrA gene with susceptibilities to quinolones (nalidixic acid, ofloxacin, norfloxacin, and ciprofloxacin), we further examined 116 strains for which the MICs of ofloxacin were > or =0.2 microg/ml that were chosen from the isolates in the 1988 to 1992 group. The MICs of nalidixic acid for the strains without mutations at either Ser-83 or Asp-87 were < or =25 microg/ml, whereas those for the strains with single mutations or double mutations were from 50 to >800 microg/ml. For the fluoroquinolones, significant differences in the distributions of the MICs were observed among the strains without mutations, with single mutations, and with double mutations. The accumulation of mutations in the gyrA gene was associated with an increase in fluoroquinolone resistance. Ofloxacin MICs for the majority of the strains with single and double mutations were 0.39 to 3.13 and 6.25 to 100 microg/ml, respectively. This study demonstrates a chronological increase in the percentage of not only highly fluoroquinolone-resistant strains, corresponding to those with double mutations in the gyrA gene, but also strains with moderately decreased susceptibilities to fluoroquinolones, corresponding to those with single mutations. This increase in the incidence of strains with a single mutation in the gyrA gene portends a further increase in the incidence of strains with clinically significant resistance to fluoroquinolones.

Anti-Infective Agents↗
PubMed2004

SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability.

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.

Animals↗
PubMed2006

The hypervariable immunodominant NP418-426 epitope from the influenza A virus nucleoprotein is recognized by cytotoxic T lymphocytes with high functional avidity.

Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A- and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP(418-426) epitope was significantly higher than that of CTL recognizing other epitopes (P < 0.01). It was speculated that the high functional avidity of NP(418-426)-specific CTL was responsible for the diversification of this influenza A virus CTL epitope.

Amino Acid Sequence↗
PubMed2006

Evolutionary mechanisms of persistence and diversification of a calicivirus within endemically infected natural host populations.

In order to understand the evolutionary mechanisms of persistence and diversification within the Caliciviridae, we have been exploiting endemic infection of feline calicivirus within five geographically distinct household groups of cats. By sequencing immunodominant and variable regions of the capsid gene, we identified the relative contribution of the different evolutionary processes employed by the virus to ensure its long-term survival in the host population. Such strategies included progressive evolution of a given variant of a strain through mutation accumulation within an individual, sequential reinfection with either a variant of the same strain or with a different strain, and mixed infection. Recombination between different strains in this study has been reported in detail elsewhere (K. P. Coyne et al., J. Gen. Virol. 87:921-926, 2006). Here, we provide evidence to suggest that true long-term persistent infection in individuals is relatively rare, with the majority of apparent viral carriers undergoing a combination of progressive evolution and cyclical reinfection. Progressive evolution at the individual level and variant reinfection at both the individual and population levels were associated with positive selection. Two measures of evolution rate were determined; for a virus progressively evolving within an individual (1.32 x 10(-2) to 2.64 x 10(-2) substitutions per nucleotide per year, i.e., no transmission) and for a strain circulating within a population (3.84 x 10(-2) to 4.56 x 10(-2) substitutions per nucleotide per year, i.e., including transmission). Reiteration of both progressive evolution and variant reinfection appeared to lead to a gradual increase in the diversity of a given strain of virus, both in the individual and in the population, until eventually new strains emerged.

Animals↗
PubMed2002

Intrapatient diversity and its correlation with viral setpoint in human immunodeficiency virus type 1 CRF02_A/G-IbNG infection.

The human immunodeficiency virus type 1 (HIV-1) viral setpoint during the disease-free interval has been strongly associated with future risk of disease progression. An awareness of the correlation between viral setpoint and HIV-1 genetic evolution over time is important in the understanding of viral dynamics and infection. We examined genetic diversity in HIV-1 CRF02_A/G-IbNG-infected seroincident women in Dakar, Senegal; determined whether a viral setpoint kinetic pattern existed for CRF02_A/G-IbNG during the disease-free interval; and correlated viral load level and diversity. Samples were drawn during the disease-free interval from consenting CRF02_A/G-IbNG-infected, antiretroviral therapy-naïve female commercial sex workers in Dakar, Senegal. Based on sequential plasma RNA values, low and high viral setpoint groups were established. Intrapatient diversity and divergence over time was determined from earlier and later time point DNA samples from each person. Most individuals followed the viral setpoint paradigm. For each 1/-/log(10) copy/ml of plasma increase in viral load, intrapatient diversity increased by 1.4% (P = 0.028). A greater diversification rate was observed in the high viral setpoint group than in the low viral setpoint group (P = 0.01). Greater nucleotide (P = 0.015) and amino acid (P = 0.048) divergences and a greater nucleotide divergence rate (P = 0.03) were found in the high viral setpoint group. There was no difference between the groups in the ratio of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site. The greater intrapatient diversity, divergence, and diversification rates observed in the high viral setpoint group supports the notion that diversity is driven by cycles of viral replication resulting in accumulated mutations. Recognizing diversity potential based on viral load levels in individuals may inform the design of vaccines and therapies.

Base Sequence↗
PubMed2004

Functional compensation of a detrimental amino acid substitution in a cytotoxic-T-lymphocyte epitope of influenza a viruses by comutations.

Influenza A viruses accumulate amino acid substitutions in cytotoxic-T-lymphocyte (CTL) epitopes, allowing these viruses to escape from CTL immunity. The arginine-to-glycine substitution at position 384 of the viral nucleoprotein is associated with escape from CTLs. Introduction of the R384G substitution in the nucleoprotein gene segment of influenza virus A/Hong Kong/2/68 by site-directed mutagenesis was detrimental to viral fitness. Introduction of one of the comutations associated with R384G, E375G, partially restored viral fitness and nucleoprotein functionality. We hypothesized that influenza A viruses need to overcome functional constraints to accumulate mutations in CTL epitopes and escape from CTLs.

Amino Acid Substitution↗
PubMed2006

Dynamics of hepatitis B virus resistance to lamivudine.

Lamivudine was the first approved inhibitor of hepatitis B virus (HBV) reverse transcriptase (RT). Lamivudine resistance develops in 53% to 76% of patients after 3 years of treatment. We extensively characterized the dynamics of HBV quasispecies variant populations in four HBV-infected patients who developed lamivudine resistance. Virological breakthrough was preceded by 2 to 4 months by the emergence of quasispecies variants bearing amino acid substitutions at RT position 204, i.e., within the YMDD catalytic motif (rtM204V/I). Three patients had a gradual switch from a YMDD variant population at baseline to a 100% lamivudine-resistant variant population, whereas the remaining patient had a fluctuating pattern of resistance variant dynamics. Careful analysis of amino acid substitutions located outside domain C of HBV RT, including those known to partially restore replication capacities in vitro, showed that the in vivo replication of HBV variants is driven by multiple forces, including intrinsic replicative advantages conferred by mutations accumulating outside domain C and the changing environment in which these variants replicate. Our findings also suggest that individual treatment optimization will require sensitive methods capable of detecting the emergence of viral resistance before the relevant variants acquire optimal replicative capacities.

Adult↗