Virus 'quasispecies': making a mountain out of a molehill?
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Animals↗
Biomedical subjects
D B Smith
Publications and source records attributed to D B Smith.
Discrimination of hepatitis G virus/GBV-C geographical variants by analysis of the 5' non-coding region.
We have investigated the ability of different subgenomic fragments to reproduce the phylogenetic relationships observed between six complete genome sequences of GBV-C/hepatitis G virus (HGV). While similar relationships were observed following analysis of part of the 5' non-coding region (5'NCR), for the coding region they were not accurately reproduced for some large fragments or for the majority of fragments of 300 or 600 nucleotides. Analysis of 5'NCR sequences from a large number of isolates, including newly obtained sequences from Pakistan, Zaïre and Scotland, produced separate groupings of Asian, African and European/North American variants. These groupings are associated with specific polymorphisms in the 5'NCR, many of which were covariant and consistent with a proposed secondary structure for this region. The relatively low level of amino acid sequence variation observed between these geographically and phylogenetically defined groups of variants suggests that they are unlikely to display significant biological differences.
Base Sequence↗
Investigation of the pattern of diversity of hepatitis C virus in relation to times of transmission.
The rate of sequence change of HCV in vivo was used to date the spread of HCV genotype 1b in European, USA and Japanese populations. Silent substitution rates of 0.0011 and 0.0017 substitutions per site per year were observed in the NS5 and E1 regions by sequence comparisons from a cohort of individuals infected from a common source of infection 17 years previously. Mean silent substitution frequencies of 0.169 and 0.224 in NS5 and E1, respectively, were observed amongst type 1b variants infecting epidemiologically unrelated individuals from several countries. This predicted a time of divergence from a common ancestor of 64-69 years. The absence of country-specific groupings by phylogenetic analysis of these sequences suggested that the spread of this genotype occurred on a worldwide basis at a similar time. Dates for the spread of other genotypes varied from around 80 years (type 2a) to 54 years (type 3a), suggesting that different variants spread into communities at different times this century. By extrapolating the silent substitution rate, the time of divergence of type 1a from 1b can be estimated at 200-300 years, but even this is likely to be an underestimate of the true time due to inequalities on the transition and transversion ratios, and the greater frequency of G <--> A transitions, compared with C <--> U, which are not considered in the current analysis. Thus, the diversity of the globally distributed genotypes such as type 1b and 3a suggests a relatively recent origin for HCV in Western countries and Japan, and contrast with the much greater diversity of specific genotypes in Central Africa (type 4) and South East Asia (type 6). These data may assist in understanding the global epidemiology of HCV and the mechanisms by which it has spread.
Genetic Variation↗
Review: molecular epidemiology of hepatitis C virus.
Molecular techniques have been used to investigate the epidemiology of hepatitis C virus (HCV) at several different levels. At a global level, the time of divergence of the diverse HCV genotypes isolated from different geographical regions has been estimated from the rate of divergence observed among a cohort of individuals infected from a common source. Estimates of more than 300 years for virus subtypes and more than 500-2000 years for virus types are consistent with their current geographical distributions. Analysis of virus sequences has also provided evidence for a common source of infection in several large-scale outbreaks of HCV infection, although where there is evidence that the implicated source contains more than one variant it may be difficult to distinguish individuals infected by different sources. Finally, sequence analysis has been used to investigate the vertical or horizontal transmission of HCV between pairs of individuals. The hypervariable region of the E2 gene is the most informative region to study if samples are available soon after the transmission event, but evidence for more distant events can still be obtained from analysis of genes such as NS5b and E1. Interpretation of some studies is complicated by the conservation of the gene region studied, or by the failure to make comparisons with sequences from epidemiologically unrelated viruses.
Disease Outbreaks↗
Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.
Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.
Base Sequence↗
Mechanomyographic responses to maximal eccentric isokinetic muscle actions.
The purpose of the present investigation was to examine the mechanomyographic (MMG) responses to maximal eccentric isokinetic muscle actions. Eight adult male volunteers [age 22 +/- 2 (SD) yr] performed maximal eccentric muscle actions of the leg extensors at 60, 90, 120, and 180 degrees /s on a Cybex 6000 isokinetic dynamometer. MMG was detected by a piezoelectric crystal contact sensor placed over the vastus lateralis muscle. Test-retest intraclass correlations ranged from R = 0.88 to 0.97 for peak torque and from R = 0.97 to 0.98 for root mean square MMG amplitude values. There was no significant (P > 0.05) velocity-related change in eccentric peak torque; however, there was a significant (P < 0.05) increase in MMG between 60 [119 +/- 44 (SE) mV] and 180 degrees/s (302 +/- 128 mV). These findings indicated a velocity-related dissociation between MMG and peak torque for maximal eccentric isokinetic muscle actions.
Adult↗
Endoscopic ultrasound-guided real-time fine-needle aspiration biopsy of the pancreas in cancer patients with pancreatic lesions.
BACKGROUND: Endoscopic ultrasound (EUS) is an important new tool in the staging of pancreatic malignancies. Using new curved linear-array instruments, real-time fine-needle aspiration biopsy (RTFNA) of pancreatic lesions can be performed. METHODS: Forty-five patients with pancreatic lesions (22 males and 23 females) underwent staging with the Olympus EUM-20 (Olympus America Corp, Melville, NY) followed by EUS-RTFNA with the Pentax FG-32PUA (Pentax-Precision Instrument Corp, Orangeburg, NY) and the 22-gauge GIP needle (GIP Medizin Technik, Grassau, Germany). RESULTS: EUS tumor stages were as follows: TO, n = 1; T1, n = 8; T2, n = 9; and T3 n = 27. Aspiration attempts were unsuccessful in four patients (two technical failures and two inadequate specimens). The remaining 41 lesions (mean size, 3.3 cm) were aspirated under EUS guidance (median passes, three) and the cytologic diagnoses were 25 definite adenocarcinoma, five suspicious for adenocarcinoma (three subsequently confirmed and two clinical course consistent with adenocarcinoma), and 11 negative for malignancy. Of 11 negatives, two were found to have adenocarcinoma, seven were confirmed benign at surgery (four cystadenomas and three inflammatory), one had a benign pseudocyst, and one had abundant inflammatory cells on RTFNA and follow-up time greater than 12 months with computed tomographic (CT) scans consistent with resolving inflammation. There were no false-positive RTFNAs. There were no procedure-related complications. Among those with diagnostic EUS-RTFNA (91%), the sensitivity for malignancy (confirmed plus suspicious) was 94% and negative predictive value 82%. CONCLUSION: EUS-guided RTFNA is a safe and accurate method for performing pancreatic biopsy. It should be considered in patients with suspected pancreatic malignancies in whom a tissue diagnosis is required or when other modalities have failed. EUS-RTFNA allows for local staging and tissue diagnosis in one procedure.
Biopsy, Needle↗
High-resolution magnetic resonance imaging to characterize the geometry of fatigued porcine bioprosthetic heart valves.
BACKGROUND AND AIMS OF THE STUDY: Porcine bioprosthetic heart valves (PBHV) continue to suffer from limited long-term durability. Failure of PBHV occurs mainly in the cusps and is characterized by mechanical damage, usually in conjunction with calcification. Mechanisms underlying calcification have received considerable attention, yet mechanical damage phenomena remain poorly understood. The structural response of PBHV cusps to in-vivo cyclic loading involves three primary factors: (i) mechanical properties; (ii) fiber architecture; and (iii) 3D geometry. Previous finite element studies have shown cuspal stress distribution to be highly sensitive to subtle changes in geometry, yet to date, cusp geometry has been largely ignored in studies of PBHV durability. METHODS: A non-destructive method was developed to quantify PBHV 3D geometry using high-resolution magnetic resonance (MR) imaging. Images were obtained in three orthogonal planes from virgin and accelerated tested (50 x 10(6) and 200 x 10(6) cycles) PBHVs to fully capture 3D cuspal geometry. Surface curvatures were computed using a local biquadric surface patch approach. RESULTS: Results indicated a tendency for cusps to permanently deform with accelerated testing, manifesting primarily as sagging of the cusp. This sagging induced areas of high curvature from the central belly region upwards to the nodulus of Aranti, corresponding to known locations of tissue failure. CONCLUSIONS: It is likely that the observed changes in cuspal geometry induce deleterious alterations in the stress distribution, independent of those related to mechanical properties and fiber structure, and contribute to valve failure. Our results suggest that PBHV designers should attempt to compensate for the deleterious geometric changes that occur post-implantation.
Animals↗
Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid.
Structure-activity relationships in the region of the phthalide ring of the inosine monophosphate dehydrogenase inhibitor mycophenolic acid have been explored. Replacement of the lactone ring with other cyclic moieties resulted in loss of potency, especially for larger groups. Replacement of the ring by acyclic substituents also indicated a strong sensitivity to steric bulk. A phenolic hydroxyl group, with an adjacent hydrogen bond acceptor, was found to be essential for high potency. The aromatic methyl group was essential for activity; the methoxyl group could be replaced by ethyl to give a compound with 2-4 times the potency of mycophenolic acid in vitro and in vivo.
Animals↗
In vivo expression of full-length human dystrophin from adenoviral vectors deleted of all viral genes.
Adenoviral vectors have been shown to effect efficient somatic gene transfer in skeletal muscle and thus offer potential for the development of therapy for Duchenne muscular dystrophy (DMD). Efficient transfer of recombinant genes has been demonstrated in skeletal muscle using recombinant adenoviruses deleted of E1. Application of this vector system to the treatment of DMD is limited by the vector immunogenicity, as well as by size constraints for insertion of recombinant genes, precluding the incorporation of a full-length dystrophin minigene construct. We describe in this study the use of helper adenovirus to generate a recombinant vector deleted of all viral open reading frames and containing a full-length dystrophin minigene. We show that this deleted vector (delta vector) is capable of efficiently transducing dystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo. Our modification of adenoviral vector technology may be useful for the development of gene therapies for DMD and other diseases.
Adenoviridae↗
Mesenteric cystic lymphangioma: radiologic appearance mimicking cystic teratoma.
Lymphangiomas are benign multiloculated cystic tumors. We present radiologic and pathologic findings of a mesenteric lymphangioma containing calcification and mimicking a mesenteric teratoma. This may broaden the differential diagnosis of a cystic mesenteric mass containing calcification.
Adolescent↗
A weekly alternating chemotherapy regimen with low toxicity for the treatment of aggressive lymphoma.
A total of 50 consecutive adult patients with newly diagnosed aggressive non-Hodgkin's lymphoma were treated with a weekly alternating combination chemotherapy schedule, BEMOP/CA, including bleomycin, etoposide, methotrexate, vincristine, cyclophosphamide and Adriamycin. Two-thirds of the patients were over 60 years old or had stage 4 disease. Clinical remission was achieved in 56% of cases. The 3-year survival is 53% (95% confidence interval, 39-66%). The presence of B symptoms and a serum albumin value of <33 g/l at presentation were poor prognostic indicators for survival in a multivariate proportional-hazards model. Overall, the response rate and survival for this group of patients with intermediate- and high-grade lymphomas is similar to results previously reported. The BEMOP/CA treatment was brief (16 weeks) and associated with a low fatal toxicity (one early death and one late fatality from Pneumocystis pneumonia), and the drug costs are equivalent to those for eight cycles of CHOP.
Adolescent↗
Heterogeneity of hepatitis C virus.
A great deal of information on the molecular heterogeneity of hepatitis C virus (HCV) has been achieved since its discovery in 1989. However, little is known about the clinical significance of these variations. Based on the degree of sequence variation, HCV has been classified into six major groups or types, differing by 31-34% at the nucleotide level over the entire virus genome. Each type is divided into several subtypes that differ by 20-23% in nucleotide sequence. Viruses within the same subtype are up to 10% divergent and, within infected individuals, vary by up to 1.5%. Genotype distributions are not homogeneous around the world and may reflect both historical and recent parenteral routes of transmission. The clinical implication of these genomic variations are not yet fully elucidated: genotype 1b has been associated with end-stage liver disease, including liver cirrhosis and hepatocellular carcinoma, but this finding might rather reflect its earlier introduction to the populations studied. Consistent evidence exists that types 2 and 3 have a higher response rate to interferon treatment than type 1, although the interplay between genotype and viral load in determining the response is still unclear. Immunohistochemical studies indicate a stronger activation of the endogenous interferon system in the liver of patients infected with type 1 compared to those infected with types 2 and 3, explaining, at least in part, its low responsiveness to exogenous interferon treatment. Biological, sequence-dependent variations of genotypes have been poorly investigated to date, but differential efficiency of translation activity of the 5' non-coding region has been reported. The availability of "in vitro" systems for evaluating pathogenetic aspects and neutralization mechanisms will improve the present knowledge on this world-wide infectious disease and on the clinical usefulness of distinguishing between genotypes.
Genotype↗
Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.
Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.
Animals↗
Differential antibody recognition of FC27-like Plasmodium falciparum merozoite surface protein MSP2 antigens which lack 12 amino acid repeats.
Three alleles of the FC27-type allelic family of the MSP2 gene of the malaria parasite Plasmodium falciparum have been sequenced from parasites from the field (The Gambia and Tanzania). These alleles lack the 12 amino acid repeat units which are usual in this family of MSP2 alleles. We have investigated the recognition by sera from an endemic area (The Gambia) of three recombinant MSP2 proteins that have 5, 1 and no copies of this repeat region. Antibody recognition of these recombinant proteins varied according to the number of repeats present. High titre antibody levels were seen with most sera using the recombinant protein with 5 x 12-mer repeats, whereas only low responses were measured using proteins containing 1 or no 12-mer repeats. Several sera entirely failed to recognise the protein which lacked 12-mer repeats. The data suggest that variation in the number of tandem repeat sequences could allow the parasite to avoid high avidity antibody binding and this may allow escape from immune recognition.
Alleles↗
Effect of workbout duration on the physical working capacity at fatigue threshold (PWCFT) test.
The purpose of this study was twofold: (1) to determine the effect of increasing the duration of the workbout at each power output during the physical working capacity at fatigue threshold (PWCFT) test from 2 to 3 or 4 min, and (2) to examine the time to exhaustion during continuous workbouts at the PWCFT. Twelve adult males (means +/- SD = 22.4 +/- 3.0 years) volunteered to perform three PWCFT tests using workbout durations of 2, 3, and 4 min. Following the determination of the PWCFT values, nine of the subjects performed continuous workbouts at PWCFT2 and PWCFT4 for as long as possible. The mean PWCFT value using 4-min workbouts (PWCFT4 = 168.8 +/- 45.1 W) was significantly less (p < 0.05, 19.1%) than that using 2-min workbouts (PWCFT2 = 208.9 +/- 59.0 W). However, only two subjects were able to complete 60 min at PWCFT4 and none of the subjects were able to complete 60 min at PWCFT2. Therefore, although increasing the duration of the workbout at each power output resulted in a lower PWCFT4, these findings do not support a recommendation for a change in the PWCFT test protocol.
Adult↗
Levels of hepatitis C virus in blood donors infected with different viral genotypes. International HCV Collaborative Study Group.
The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P<.001) but did not correlate with donor age (P=.06) or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment.
Blood Donors↗
Evolutionary analysis of variants of hepatitis C virus found in South-East Asia: comparison with classifications based upon sequence similarity.
Variants of hepatitis C virus (HCV) have been classified by nucleotide sequence comparisons in different regions of the genome. Many investigators have defined the ranges of sequence similarity values or evolutionary distances corresponding to divisions of HCV into types, subtypes and isolates. Using these criteria, novel variants of HCV from Vietnam, Thailand and Indonesia have been classified as types 7, 8, 9, 10 and 11, many of which can be further subdivided into between two to four subtypes. In this study, this distance-based method of virus classification was compared with phylogenetic analysis and statistical measures to establish the confidence of the groupings. Using bootstrap resampling of phylogenetic trees in several subgenomic regions (core, E1, NS5) and with complete genomic sequences, we found that one set of novel HCV variants ('types 7, 8, 9 and 11') consistently grouped together into a single clade that also contained type 6a, while 'type 10a' grouped with type 3. In contrast, no robust higher-order groupings were observed between any of the other five previously described HCV genotypes (types 1-5). In each subgenomic region, the distribution of pairwise distances between members of the type 6 clade were consistently bi-modal and therefore provided no justification for classification of these variants into the three proposed categories (type, subtype, isolate). Based on these results, we propose that a more useful classification would regard all these variants as subtypes of type 6 or type 3, even though the level of sequence diversity within the clade was greater than observed for other genotypes. Classification by phylogenetic relatedness rules out simple sequence similarity measurements as a method for assigning HCV genotypes, but provides a more appropriate description of the evolutionary and epidemiological history of a virus.
Asia, Southeastern↗