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J Quer

Publications and source records attributed to J Quer.

At least 19 recordsLinked to original sources
PubMed1999

Red queen dynamics, competition and critical points in a model of RNA virus quasispecies.

RNA viruses offer a unique opportunity for the study of evolution at the molecular level. Recent experiments involving clonal populations of RNA viruses have shown that competition among virus strains of approximately equal relative fitness can result in the eventual competitive exclusion of one of the species. As competition proceeds in time, both the winners and the losers exhibited absolute gains in fitness, consistent with the "Red Queen" hypothesis of evolution. Further experiments involving closely related evolving quasispecies revealed a highly predictable nonlinear behavior suggesting a deterministic component in the underlying quasispecies dynamics. This is apparently in contradiction with the standard view of RNA virus evolution as a highly unpredictable process. In this paper we present a simple model which allows previous hypothesis to be tested and provides an interpretation for the observed experimental results.

Cloning, Molecular↗
PubMed1999

Hepatitis C viral quasispecies.

Analysing significant numbers of cDNA clones of the hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. This way of organizing the genetic information is referred to as quasispecies. Throughout HCV infection, the number and composition of the variants in the viral population keeps changing owing to environmental influences, resulting in a virus that is constantly redefining itself both genetically and phenotypically. Therefore, the virus has often been investigated in population terms. Many clinical studies have tried to unravel, through the parameters that characterize the HCV quasispecies, prognostic markers of the disease and its response to treatment. Other investigations have focused on discovering how the virus and host interact during chronic infection. The consensus sequence, the rate of fixation of mutations and the complexity of the viral population are useful parameters for describing the viral population behaviour and its interaction with the host. In addition to sequencing, several other methods, based on electrophoretic mobility, have been used to study these parameters, such as temperature gradient-gel electrophoresis, single-strand conformation polymorphism and gel-shift analysis. The viral region examined, the source of clinical specimen, as well as the methodology employed, will be decisive in interpreting the information obtained.

Genetic Variation↗
PubMed1999

Evaluation of RNA and E2 antibodies in prospectively followed recipients of hepatitis G virus-infected blood.

BACKGROUND: Hepatitis G virus (HGV) has recently been cloned and tests for HGV RNA and envelope antibodies (anti-E2) have been developed. HGV infection is widespread among blood donors worldwide, but the clinical and serologic outcome of transfusion-associated HGV infection has not been fully characterized. STUDY DESIGN AND METHODS: Consecutive blood donors (n = 2210) were investigated for HGV markers (RNA and anti-E2). The recipients of HGV RNA-positive blood were followed for 1 year after transfusion. RESULTS: Forty-two blood donors (1.9%) were positive for HGV RNA. Eight recipients of HGV RNA-positive blood were retrospectively identified within 2 weeks of transfusion and prospectively followed. In four patients, the presence of anti-E2 before transfusion or an early antibody response protected them from reinfection or prevented HGV persistence, while, in the remaining four patients, transient or persistent viremia was detected shortly after exposure. None of the infected recipients had any evidence of liver disease. CONCLUSION: These results do not support the screening of donors to prevent transfusion-associated HGV infection.

Blood Donors↗
PubMed1999

Genetic alterations in the S gene of hepatitis B virus in patients with acute hepatitis B, chronic hepatitis B and hepatitis B liver cirrhosis before and after liver transplantation.

BACKGROUND: Several studies have shown that hepatitis B immunoglobulin (HBIG) imposes a selection pressure on the hepatitis B virus (HBV) S gene, and that the emergence of mutations in this region would make reinfection after orthotopic liver transplantation (OLT) possible. AIMS: This study was undertaken to analyze the presence of HBV S-gene mutations in the different stages of HBV infection and the relationship between HBIG therapy and the emergence of mutations in liver transplant recipients. METHODS: The frequency and location of mutations in the coding region of the HBV S gene were studied by PCR and direct sequencing in 30 patients (7 with acute self-limited hepatitis B, 16 with chronic hepatitis B and 7 recipients of (OLT) for HBV-related end stage liver disease who became reinfected). RESULTS: The average number of amino acid changes was higher in patients with a more advanced stage of disease, 0.57 mutations/100 positions in acute hepatitis B and 1.57 in chronic hepatitis B (1.28 in HBeAg-positive and 1.8 in anti-HBe-positive patients). The average number of substitutions in the transplanted patients was 2.7 before OLT and 3 after OLT. No amino acid substitutions were detected in the "a" determinant of HBsAg in acute hepatitis B, however, 8 substitutions were observed in 6 chronic patients. In 3 OLT patients, 4 substitutions were observed in samples before and after OLT. One of these patients, who had protective levels of anti-HBs, showed 3 additional new amino acid substitutions after OLT, suggesting escape mutant selection by the effect of HBIG therapy. No changes were observed between the consensus sequences obtained several years before and after transplantation, indicating consensus sequence stability. CONCLUSION: These results show that there is an accumulation of HBV S-gene mutations in HBV-related end-stage liver disease. Prophylaxis with HBIG mainly obtained from acute self-limited hepatitis patients who have a highly homogeneous viral population, may be one factor underlying the reinfection after liver transplantation.

Acute Disease↗
PubMed1999

High-throughput real-time reverse transcription-PCR quantitation of hepatitis C virus RNA.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5' noncoding region (5' NCR) of HCV. The probe contains a fluorescent reporter at the 5' end and a fluorescent quencher at the 3' end. The use of such a probe combined with the 5'-3' nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5' NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10(3) to 10(7)). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

Adult↗
PubMed1999

Exponential fitness gains of RNA virus populations are limited by bottleneck effects.

Fitness is a parameter that quantitatively measures adaptation of a virus to a given environment. We have previously reported exponential fitness gains of large populations of vesicular stomatitis virus replicating in a constant environment (I. S. Novella et al., Proc. Natl. Acad. Sci. USA 92:5841-5844, 1995). In this paper, we report that during long-term passage of such large viral populations, fitness values reached a high-fitness plateau during which stochastic fitness variations were observed. This effect appears likely to be due to bottleneck effects on very high fitness populations.

Adaptation, Biological↗
PubMed1998

Hepatitis delta genotypes in chronic delta infection in the northeast of Spain (Catalonia).

BACKGROUND/AIMS: Based on genetic analysis of variants obtained around the world, three genotypes of the hepatitis delta virus have been defined. Hepatitis delta virus variants have been associated with different disease patterns and geographic distributions. To determine the prevalence of hepatitis delta virus genotypes in the northeast of Spain (Catalonia) and the correlation with transmission routes and clinical disease, we studied the nucleotide divergence of the consensus sequence of HDV RNA obtained from 33 patients with chronic delta hepatitis (24 were intravenous drug users and nine had no risk factors), and four patients with acute self-limited delta infection. METHODS: Serum HDV RNA was amplified by the polymerase chain reaction technique and a fragment of 350 nucleotides (nt 910 to 1259) was directly sequenced. RESULTS: Genetic analysis of the nucleotide consensus sequence obtained showed a high degree of conservation among sequences (93% of mean). Comparison of these sequences with those derived from different geographic areas and pertaining to genotypes I, II and III, showed a mean sequence identity of 92% with genotype I, 73% with genotype II and 61% with genotype III. At the amino acid level (aa 115 to 214), the mean identity was 87% with genotype I, 63% with genotype II and 56% with genotype III. Conserved regions included the RNA editing domain, the carboxyl terminal 19 amino acids of the hepatitis delta antigen and the polyadenylation signal of the viral mRNA. CONCLUSIONS: Hepatitis delta virus isolates in the northeast of Spain are exclusively genotype I, independently of the transmission route and the type of infection. No hepatitis delta virus subgenotypes were found, suggesting that the origin of hepatitis delta virus infection in our geographical area is homogeneous.

Adolescent↗
PubMed1998

Identification and molecular characterization of the new alpha-1-antitrypsin deficient allele PI Y barcelona (Asp256-->Val and Pro391-->His). Mutations in brief no. 174. Online.

To characterize the molecular basis of the "new" alpha1-antitrypsin (alpha1AT) deficient variant, PI Y barcelona, DNA sequence analysis of the coding exons of the alpha1AT gene was carried out using an amplification DNA technique and direct sequencing. The PI Y barcelona allele differs from the normal M1(Val213) allele sequence by two point substitutions: a transversion of GAT TO GTT in exon III in the codon for residue 256, resulting in the amino acid change of Asp256 to Val256, and a transversion of CCC to CAC in exon V in the codon for residue 391, resulting in the amino acid substitution of Pro391 to His391. On isoelectric focusing analysis these substitutions result in a cathodal migration of the "new" variant close to the PI Z. The index case, diagnosed with severe obstructive pulmonary disease, initially phenotyped a PI ZZ, was homozygous for PI Y barcelona. The patient's serum alpha1AT level was 16 mg/dL (normal values 115-220 mg/dL). Inheritance of the PI Y barcelona was confirmed by family study. Amino acid substitution in postion 391 occurs in the C-terminal peptide region, which shows a high degree of homology with the family of serpins. Pro391 is considered to have special relevance in the secretion of alpha1AT.

Alleles↗
PubMed1996

Reproducible nonlinear population dynamics and critical points during replicative competitions of RNA virus quasispecies.

RNA virus evolution is generally considered to be highly unpredictable, but tests of determinism in the evolution of competing populations during viral infections have not been performed. Here we study the fate of two closely related evolving quasispecies of vesicular stomatitis virus, by determining the relative concentration of a wild-type clone and a surrogate marked virus subclone (MARM-C) upon extensive competitive replication in a constant cell culture environment. A highly predictable nonlinear behaviour of the two competing populations was found. In addition, the presence of critical points, which are defined as points from which viral competitions may follow different trajectories, has been documented. Critical points were reached after nearly constant periods of time. The dynamics of relative fitness values for both competing populations were calculated during the replication passages. Concomitant with expected fitness gain of both competing viral populations (which follow the Red Queen hypothesis) a tendency for the MARM-C to gain less fitness than the wild-type was observed. Although fitness variations were noisy, this tendency was seen in all evolutionary replicas. Thus, despite the stochastic process of mutation that leads to a continuous generation of mutant genomes during RNA virus replication, a nonlinear, nearly deterministic evolutionary behaviour has been observed. It is proposed that such a behaviour is mediated by a low-pass filter (averaging of mutational noise signals) due to competitive selection among variants.

Analysis of Variance↗
PubMed1996

Transmission of hepatitis C virus by a cardiac surgeon.

BACKGROUND: In the course of a study conducted in 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients. METHODS: Of 222 of the surgeon's patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype. RESULTS: The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusion-associated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P < 0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients. CONCLUSIONS: Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery.

Adult↗
PubMed1996

Basic concepts in RNA virus evolution.

A hallmark of RNA genomes is the error-prone nature of their replication and retrotranscription. The major biochemical basis of the limited replication fidelity is the absence of proofreading/repair and postreplicative error correction mechanisms that normally operate during replication of cellular DNA. In spite of this unique feature of RNA replicons, the dynamics of viral populations seems to follow the same basic principles that classical population genetics has established for higher organisms. Here we review recent evidence of the profound effects that genetic bottlenecks have in enhancing the deleterious effects of Muller's ratchet during RNA virus evolution. The validity of the Red Queen hypothesis and of the competitive exclusion principle for RNA viruses are viewed as the expected result of the highly variable and adaptable nature of viral quasispecies. Viral fitness, or ability to replicate infectious progeny, can vary a million-fold within short time intervals. Paradoxically, functional and structural studies suggest extreme limitations to virus variation. Adaptability of RNA viruses appears to be based on the occupation of very narrow portions of sequence space at any given time.

Animals↗
PubMed1995

Efficacy of screening donors for antibodies to the hepatitis C virus to prevent transfusion-associated hepatitis: final report of a prospective trial.

Routine screening of blood donors for anti-hepatitis C virus (HCV) has been implemented in most developed countries. However, the independent efficacy of such screening has not been established in a controlled, prospective study. We tracked 478 patients transfused with anti-HCV-negative blood by first-generation enzyme-linked immunoassay (EIA) between July 1989 and May 1990 and compared the incidence of transfusion-associated hepatitis and HCV infections with that found among 280 patients transfused with blood unscreened for anti-HCV during the immediately preceding year. Of the 280 patients who had received transfusions before donors were screened for anti-HCV, 27 (9.6%) developed posttransfusion hepatitis and 1 additional patient seroconverted to anti-HCV without evidence of hepatitis, for a risk of posttransfusion HCV infection of 10.7% (28 of 262 recipients seronegative for anti-HCV before transfusion). Of the 478 patients transfused after July 1989 with blood screened for anti-HCV, only 9 (1.9%) developed posttransfusion hepatitis for a risk reduction of 80%. Seven of the 9 residual cases of hepatitis were caused by HCV (7 of 456 recipients seronegative before transfusion or 1.5%) for a risk reduction of transfusion-associated HCV infection of 86%. In retrospect, an anti-HCV positive donor was detected by second-generation immunoassay in 4 (57%) of the 7 HCV cases from the study cohort and in 19 of the 23 (83%) cases from whom all donor samples were available for testing in the historical cohort. No additional infectious donors were detected by third-generation immunoassay or serum HCV-RNA by polymerase chain reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Hepatitis C virus infection in renal transplant recipients: epidemiology, clinical impact, serological confirmation and viral replication.

The presence and significance of hepatitis C virus infection was evaluated in 241 renal transplant recipients from our hospital. Hepatitis C virus antibodies were tested by second-generation enzyme immunoassay, followed by second- and third-generation immunoblot assays (RIBA-2 and RIBA-3); hepatitis C virus RNA was measured by nested polymerase chain reaction. Hepatitis C virus antibodies, which were detected in 46.5% of patients, were mainly present before transplantation and independently associated with the total amount of transfused blood, time in hemodialysis and duration of posttransplant follow up. Liver dysfunction (alanine aminotransferase elevation) was observed in 50% of antibody-positive recipients, and 92.5% of patients with chronic liver disease without hepatitis B infection were infected with hepatitis C virus. Most antibody-positive patients (78.4%) tested positive by RIBA-2, but 21.6% were indeterminate; RIBA-3 was positive in 90% of these indeterminates. Hepatitis C virus RNA detection was positive in 96% of antibody-positive cases tested, in 20% of patients who were already anti-HCV negative before transplantation and also demonstrated persistence of HCV infection in all cases who, being antibody positive prior to transplantation, lost these antibodies during follow up (9% of transplanted patients). In conclusion, hepatitis C virus infection is extremely prevalent in renal transplant recipients from Spain and is the main cause of chronic liver disease in these patients. Confirmation by supplemental assays of anti-HCV antibodies is not necessary, but hepatitis C virus RNA testing is indispensable to detect those cases who lose or do not develop hepatitis C virus antibodies.

Adult↗
PubMed1995

Extreme fitness differences in mammalian and insect hosts after continuous replication of vesicular stomatitis virus in sandfly cells.

Continuous, persistent replication of a wild-type strain of vesicular stomatitis virus in cultured sandfly cells for 10 months profoundly decreased virus replicative fitness in mammalian cells and greatly increased fitness in sandfly cells. After persistent infection of sandfly cells, fitness was over 2,000,000-fold greater than that in mammalian cells, indicating extreme selective differences in the environmental conditions provided by insect and mammalian cells. The sandfly-adapted virus also showed extremely low fitness in mouse brain cells (comparable to that in mammalian cell cultures). It also showed an attenuated phenotype, requiring a nearly millionfold higher intracranial dose than that of its parent clone to kill mice. A single passage of this adapted virus in BHK-21 cells at 37 degrees C restored fitness to near neutrality and also restored mouse neurovirulence. These results clearly illustrate the enormous capacity of RNA viruses to adapt to changing selective environments.

Animals↗
PubMed1994

Etiology of acute sporadic hepatitis in Spain: the role of hepatitis C and E viruses.

The aim of this study was to determine the prevalence of hepatitis viruses and analyze the epidemiological features in acute hepatitis in Spain. Consecutive serum samples from 341 patients with acute hepatitis were tested for IgM anti-hepatitis A virus, IgM anti-hepatitis B core antigen and HBsAg. The antibody to hepatitis C virus was determined using a second-generation enzyme immunoassay. In selected cases, serum was tested for antibodies for hepatitis E virus by enzyme immunoassay and hepatitis C virus-RNA by polymerase chain reaction. One hundred and fourteen (33.2%) cases of acute hepatitis were due to hepatitis A virus, 68 (20%) to hepatitis B virus, 21 (6%) to hepatitis D virus and 74 (21.8%) to hepatitis C virus infection. Of the 74 hepatitis C virus infections, 48 (65%) were anti-HCV positive at the time of diagnosis, while 26 (35%) seroconverted to anti-HCV later during follow-up. No case of hepatitis E virus infection was detected. The remaining 64 cases were negative to all acute serological markers and were classified as non-A, non-B, non-C, non-E hepatitis. Hepatitis C virus was implicated in 65/119 (54.6%) of the cases among intravenous drug users but in only in 9/199 (4.5%) of the sporadic cases (p < 0.01). Progression to chronic hepatitis was observed more frequently in anti-hepatitis C virus-positive than in antibody-negative-cases (42/70; 60% vs. 3/52 or 5.5%) (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Incidence and outcome of hepatitis C virus infection after liver transplantation.

The objective of this study was to determine the incidence and outcome of hepatitis C virus (HCV) infection after liver transplantation (OLT). Fifty-two transplanted patients were studied. Serum samples were examined for antibodies to HCV (anti-HCV) and HCV-RNA by PCR, before and after OLT. Patients were distributed into two groups: group 1 consisted of 24 patients (pretransplant anti-HCV positive) and group 2 consisted of 28 patients (pretransplant anti-HCV negative). One year after OLT, HCV-infected patients were evaluated by liver biopsy. HCV-RNA was detected in 28 of the 52 (53.9%) patients after OLT. Twenty-two patients in group 1 (96%) were reinfected. In group 2, acquired HCV infection was detected in six (21.4%) patients. At 6 and 12 months, one and five of six patients had seroconverted, respectively. Liver biopsy in 23 HCV-infected patients showed chronic hepatitis in 18 (78%) cases (2, chronic persistent hepatitis; 3, chronic lobular hepatitis and 13, chronic active hepatitis). Fourteen of the 23 (60.8%) patients were asymptomatic. Most symptomatic patients had chronic hepatitis with cholestasis. Overall, 18 of 20 cases of chronic hepatitis diagnosed in OLT recipients were HCV related. Mortality beyond 6 months after OLT was slightly higher in the HCV-infected group (P = 0.055). In conclusion, HCV reinfection is almost universal. Acquired HCV infection post-OLT is frequent. HCV-infected patients frequently develop chronic hepatitis. Most chronic hepatitis after transplantation are HCV related.

Adult↗
PubMed1994

Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.

Amino Acid Sequence↗