Showing papers by "Peter Simmonds published in 2004"

Genetic diversity and evolution of hepatitis C virus--15 years on.

Abstract: In the 15 years since the discovery of hepatitis C virus (HCV), much has been learned about its role as a major causative agent of human liver disease and its ability to persist in the face of host-cell defences and the immune system. This review describes what is known about the diversity of HCV, the current classification of HCV genotypes within the family Flaviviridae and how this genetic diversity contributes to its pathogenesis. On one hand, diversification of HCV has been constrained by its intimate adaptation to its host. Despite the >30 % nucleotide sequence divergence between genotypes, HCV variants nevertheless remain remarkably similar in their transmission dynamics, persistence and disease development. Nowhere is this more evident than in the evolutionary conservation of numerous evasion methods to counteract the cell's innate antiviral defence pathways; this series of highly complex virus-host interactions may represent key components in establishing its 'ecological niche' in the human liver. On the other hand, the mutability and large population size of HCV enables it to respond very rapidly to new selection pressures, manifested by immune-driven changes in T- and B-cell epitopes that are encountered on transmission between individuals with different antigen-recognition repertoires. If human immunodeficiency virus type 1 is a precedent, future therapies that target virus protease or polymerase enzymes may also select very rapidly for antiviral-resistant mutants. These contrasting aspects of conservatism and adaptability provide a fascinating paradigm in which to explore the complex selection pressures that underlie the evolution of HCV and other persistent viruses.

Detection of genome-scale ordered RNA structure (GORS) in genomes of positive-stranded RNA viruses: Implications for virus evolution and host persistence.

Abstract: Discrete RNA secondary and higher-order structures, typically local in extent, play a fundamental role in RNA virus replication. Using new bioinformatics analysis methods, we have identified genome-scale ordered RNA structure (GORS) in many genera and families of positive-strand animal and plant RNA viruses. There was remarkably variability between genera that possess this characteristic; for example, hepaciviruses in the family Flaviviridae show evidence for extensive internal base-pairing throughout their coding sequences that was absent in both the related pestivirus and flavivirus genera. Similar genus-associated variability was observed in the Picornaviridae, the Caliciviridae, and many plant virus families. The similarity in replication strategies between genera in each of these families rules out a role for GORS in a fundamentally conserved aspect of this aspect of the virus life cycle. However, in the Picornaviridae, Flaviviridae, and Caliciviridae, the existence of GORS correlated strongly with the ability of each genus to persist in their natural hosts. This raises the intriguing possibility of a role for GORS in the modulation of innate intracellular defense mechanisms (and secondarily, the acquired immune system) triggered by double-stranded RNA, analogous in function to the expression of structured RNA transcripts by large DNA viruses. Irrespective of function, the observed evolutionary conservation of GORS in many viruses imposes a considerable constraint on genome plasticity and the consequent narrowing of sequence space in which neutral drift can occur. These findings potentially reconcile the rapid evolution of RNA viruses over short periods with the documented examples of extreme conservatism evident from their intimate coevolution with their hosts.

Biological Analysis of Human Immunodeficiency Virus Type 1 R5 Envelopes Amplified from Brain and Lymph Node Tissues of AIDS Patients with Neuropathology Reveals Two Distinct Tropism Phenotypes and Identifies Envelopes in the Brain That Confer an Enhanced Tropism and Fusigenicity for Macrophages

Abstract: Complete envelope genes were amplified from autopsy brain tissue of five individuals who had died of AIDS and had neurological complications. Lymph node samples were included for two of the patients. Nineteen different envelope clones from the five patients had distinct V1V2 sequences. Thirteen of the envelopes were functional and conferred fusigenicity and infectivity for CD4 + CCR5 + cells. Infectivity and cell-cell fusion assays showed that most envelopes used both CCR5 and CCR3. One brain-derived envelope used a broad range of coreceptors, while three other brain envelopes from one individual were restricted to CCR5. However, there was no correlation between tissue of origin and coreceptor use. Envelopes showed two very distinct phenotypes depending on their capacity to infect macrophages and to exploit low levels of CD4 and/or CCR5 for infection. Envelopes that were highly fusigenic and tropic for macrophages were identified in brain tissue from four of the five patients. The enhanced macrophage tropism correlated with reduced sensitivity to inhibition by Q4120, a CD4-specific antibody, but not with sensitivity to the CCR5 inhibitor, TAK779. The highly macrophage-tropic envelopes were able to infect cells expressing low levels of CD4 and/or CCR5. Comparison with several well-characterized macrophage-tropic envelopes showed that the four identified patient envelopes were at the top limit of macrophage tropism. In contrast, all four lymph node-derived envelopes exhibited a non-macrophage-tropic phenotype and required high levels of CD4 for infection. Our data support the presence of envelopes that are highly fusigenic and tropic for macrophages in the brains of patients with neurological complications. These envelopes are able to infect cells that express low levels of CD4 and/or CCR5 and may have adapted for replication in brain macrophages and microglia, which are known to express limited amounts of CD4.

Spread of Hepatitis C Virus among European Injection Drug Users Infected with HIV: A Phylogenetic Analysis

Abstract: To describe the spread of hepatitis C virus (HCV) among HCV/human immunodeficiency virus (HIV)-coinfected injection drug users (IDUs), the molecular epidemiology of HCV was studied among 108 IDUs from 7 European countries. Phylogenetic analysis based on the NS5B region showed great sequence variation of HCV within each country and no clear phylogenetic clustering by geographic region. The most prevalent subtypes were 1a and 3a, but the percentage of genotype 4 was also relatively high, ranging from 7% in northern Europe to 24% in southern Europe. Genotype 4 consisted mainly of subtype 4d and has entered the majority of the IDU populations studied. The significantly lower evolutionary distances within subtype 4d suggest that this subtype may have entered the European IDU population relatively recently. In conclusion, HCV exchange between European IDU populations has occurred on a large scale, and, overall, country-specific clustering for HCV was less than that shown for HIV.

Rapid growth of hydrofluorocarbon 134a and hydrochlorofluorocarbons 141b, 142b, and 22 from Advanced Global Atmospheric Gases Experiment (AGAGE) observations at Cape Grim, Tasmania, and Mace Head, Ireland

Abstract: [1] An update of in situ Advanced Global Atmospheric Gases Experiment (AGAGE) hydrofluorocarbon (HFC)/hydrochlorofluorocarbon (HCFC) measurements made at Mace Head, Ireland, and Cape Grim, Tasmania, from 1998 to 2002 are reported. HCFC-142b, HCFC-141b, HCFC-22 and HFC-134a show continued rapid growth in the atmosphere at mean rates of 1.1, 1.6, 6.0, and 3.4 ppt/year, respectively. Emissions inferred from measurements are compared to recent estimates from consumption data. Minor updates to the industry estimates of emissions are reported together with a discussion of how to best determine OH concentrations from these trace gas measurements. In addition, AGAGE measurements and derived emissions are compared to those deduced from NOAA-Climate Monitoring and Diagnostics Laboratory flask measurements (which are mostly made at different locations). European emission estimates obtained from Mace Head pollution events using the Nuclear Accident Model (NAME) dispersion model and the best fit algorithm (known as simulated annealing) are presented as 3-year rolling average emissions over Europe for the period 1999–2001. Finally, the measurements of HCFC-141b, HCFC-142b, and HCFC-22 discussed in this paper have been combined with the Atmospheric Lifetime Experiment (ALE)/Global Atmospheric Gases Experiment (GAGE)/AGAGE measurements of CFC-11, CFC-12, CFC-113, CCl4, and CH3CCl3 to produce the evolution of tropospheric chlorine loading.

Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods.

Abstract: There is accumulating evidence from bioinformatic studies that hepatitis C virus (HCV) possesses extensive RNA secondary structure in the core and NS5B-encoding regions of the genome. Recent functional studies have defined one such stem–loop structure in the NS5B region as an essential cis-acting replication element (CRE). A program was developed (structur_dist) that analyses multiple rna-folding patterns predicted by mfold to determine the evolutionary conservation of predicted stem–loop structures and, by a new method, to analyse frequencies of covariant sites in predicted RNA folding between HCV genotypes. These novel bioinformatic methods have been combined with enzymic mapping of RNA transcripts from the core and NS5B regions to precisely delineate the RNA structures that are present in these genomic regions. Together, these methods predict the existence of multiple, often juxtaposed stem–loops that are found in all HCV genotypes throughout both regions, as well as several strikingly conserved single-stranded regions, one of which coincides with a region of the genome to which ribosomal access is required for translation initiation. Despite the existence of marked sequence conservation between genotypes in the HCV CRE and single-stranded regions, there was no evidence for comparable suppression of variability at either synonymous or non-synonymous sites in the other predicted stem–loop structures. The configuration and genetic variability of many of these other NS5B and core structures is perhaps more consistent with their involvement in genome-scale ordered RNA structure, a structural configuration of the genomes of many positive-stranded RNA viruses that is associated with host persistence.

Halogenated greenhouse gases at the Swiss High Alpine site of Jungfraujoch (3580 m asl): Continuous measurements and their use for regional European source allocation

Abstract: [1] At the high Alpine site of Jungfraujoch (3580 m asl), 23 halogenated greenhouse gases are measured quasi-continuously by gas chromatography-mass spectrometry (GCMS). Measurement data from the years 2000–2002 are analyzed for trends and pollution events. Concentrations of the halogenated trace gases, which are already controlled in industrialized countries by the Montreal Protocol (e.g., CFCs) were at least stable or declining. Positive trends in the background concentrations were observed for substances which are used as CFC-substitutes (hydrofluorocarbons, hydrochlorofluorocarbons). Background concentrations of the hydrofluorocarbons at the Jungfraujoch increased from January 2000 until December 2002 as follows: HFC 134a (CF3CH2F) from 15 to 27 ppt, HFC 125 (CF3CHF2) from 1.4 to 2.8 ppt, and HFC 152a (CHF2CH3) from 2.3 to 3.2 ppt. For HFC 152a, a distinct increase of its concentration magnitude during pollution events was observed from 2000 to 2002, indicating rising European emissions for this compound. Background concentrations of all measured compounds were in good agreement with similar measurements at Mace Head, Ireland. On the other hand, peak concentrations were significantly higher at the Jungfraujoch. This finding is due to the proximity to potent European sources, foremost in southern Europe. The average ratio of halocarbons versus carbon monoxide (CO) concentrations above their baseline values was used to estimate source strengths for the part of Europe which most influences the Jungfraujoch during pollution events. HFCs emission estimates from Jungfraujoch tend to be higher than figures at Mace Head (Ireland) from the end of the 1990s, which either reflects the increased use of these compounds or the closer location of Jungfraujoch to major southern European sources. Transport of polluted European boundary layer air masses to the high Alpine site was observed especially during frontal passages, foehn events, and thermal lifting of air masses in summer. The measurement data during the periods when the Jungfraujoch was under the influence of the polluted boundary layer were used in combination with concurrent air mass trajectories to allocate above baseline halocarbon concentrations to specific European source regions.

A comparative method for finding and folding RNA secondary structures within protein-coding regions

Abstract: Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins, and evidence is accumulating that this phenomenon may also be found in Eukaryotes. We here present the first comparative method, called RNA-DECODER, which explicitly takes the known protein-coding context of an RNA-sequence alignment into account in order to predict evolutionarily conserved secondary-structure elements, which may span both coding and non-coding regions. RNA-DECODER employs a stochastic context-free grammar together with a set of carefully devised phylogenetic substitution-models, which can disentangle and evaluate the different kinds of overlapping evolutionary constraints which arise. We show that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD.

Allelic variation of HERV-K(HML-2) endogenous retroviral elements in human populations

Abstract: Human endogenous retroviruses (HERVs) are the remnants of ancient germ cell infection by exogenous retroviruses and occupy up to 8% of the human genome. It has been suggested that HERV sequences have contributed to primate evolution by regulating the expression of cellular genes and mediating chromosome rearrangements. After integration approximately 28 million years ago, members of the HERV-K (HML-2) family have continued to amplify and recombine. To investigate the utility of HML-2 polymorphisms as markers for the study of more recent human evolution, we compiled a list of the structure and integration sites of sequences that are unique to humans and screened each insertion for polymorphism within the human genome databases. Of the total of 74 HML-2 sequences, 18 corresponded to complete or near-complete proviruses, 49 were solitary long terminal repeats (LTRs), 6 were incomplete LTRs, and 1 was a SVA retrotransposon. A number of different allelic configurations were identified including the alternation of a provirus and solitary LTR. We developed polymerase chain reaction-based assays for seven HML-2 loci and screened 109 human DNA samples from Africa, Europe, Asia, and Southeast Asia. Our results indicate that the diversity of HML-2 elements is higher in African than non-African populations, with population differentiation values ranging from 0.6 to 9.8%. These findings denote a recent expansion from Africa. We compare the phylogenetic relationships of HML-2 sequences that are unique to humans and consider whether these elements have played a role in the remodeling of the hominid genome.

AGAGE Observations of Methyl Bromide and Methyl Chloride at Mace Head, Ireland, and Cape Grim, Tasmania, 1998–2001

Abstract: In situ AGAGE GC-MS measurements of methyl bromide (CH3Br) and methyl chloride (CH3Cl) at Mace Head, Ireland and Cape Grim, Tasmania (1998–2001) reveal a complex pattern of sources. At Mace Head both gases have well-defined seasonal cycles with similar average annual decreases of 3.0% yr−1 (CH3Br) and 2.6% yr−1 (CH3Cl), and mean northern hemisphere baseline mole fractions of 10.37 ± 0.05 ppt and 535.7 ± 2.2 ppt, respectively. We have used a Lagrangian dispersion model and local meteorological data to segregate the Mace Head observations into different source regions, and interpret the results in terms of the known sources and sinks of these two key halocarbons. At Cape Grim CH3Br and CH3Cl also show annual decreases in their baseline mixing ratios of 2.5% yr−1 and 1.5% yr−1, respectively. Mean baseline mole fractions were 7.94 ± 0.03 ppt (CH3Br) and 541.3 ± 1.1 ppt (CH3Cl). Although CH3Cl has astrong seasonal cycle there is no well-defined seasonal cycle in the Cape Grim CH3Br record. The fact that both gases are steadily decreasing in the atmosphere at both locations implies that a change has occurred which is affecting a common, major source of both gases (possibly biomass burning) and/or their major sink process (destruction by hydroxyl radical).

Does drug abuse influence the microglial response in AIDS and HIV encephalitis

Abstract: Objectives: To investigate the pathological evidence for a possible interaction between drugs of abuse and HIV infection in terms of microglial responses in early and late HIV/AIDS, and to discuss the possible long-term consequences of microglial activation in chronic HIV infection. Design: This brain pathology study compared age and sex-matched control patients with HIV-negative intravenous drug users, and with HIV-positive drug users both in the presymptomatic stage and with AIDS. A further group of non-drug-using AIDS patients was included. All the AIDS patients had HIV encephalitis (HIVE) but no other significant HIV-associated brain pathology. Methods: Microglia/macrophages were identified in the grey and white matter of the frontal and temporal lobes and the thalamus, using antibodies to CD68 and MHCII. Objective quantitation was used to compare subjects in the different groups. Results: AIDS patients showed a significant increase in activated microglia/macrophages in both the grey and white matter of all areas compared with non-AIDS patients. Drug users with HIVE tended to have more activated microglia than non-drug-using comparison groups, but this difference was not found in all brain areas studied. Conclusion: Drug misuse appears to enhance the microglial activation resulting from HIV infection in some individuals. Other factors such as the severity of HIVE, or systemic immune factors, are also likely to affect the degree of microglial activation. The implications for drug-using patients who survive long term with HIV/AIDS are discussed, particularly in relation to premature neurodegeneration.

High Levels of Human Immunodeficiency Virus Infection of CD8 Lymphocytes Expressing CD4 In Vivo

Abstract: Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.