Prediction of RNA secondary structure in hepatitis C and related viruses

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognised. In this thesis several computational methods to detect RNA secondary structure in the coding regions of hepatitis C virus (HCV), hepatitis G virus (HGV)/GB virus C (GBV-C) and related viruses have been used. These include thermodynamic prediction of folding free energies (FFEs), evolutionary conservation of minimum energy structures between virus genotypes, suppression of synonymous variability and analysis of covariant and semi covariant substitutions in thermodynamically favoured structures. Each of the predictive methods provided evidence for conserved RNA secondary structure in the core and NS5B encoding regions of HCV and throughout the entire coding region of HGV/GBV-C.Positions in the HCV genome with predicted RNA structure localise precisely to regions of marked suppression of variability at synonymous sites, indicating that RNA structure constrains sequence change at what are generally regarded as phenotypically neutral sites. Combining these methods, the computational data obtained in this thesis demonstrates the existence of at least ten conserved stem loop structures within the NS5B coding region and three in that coding for the core protein both within the coding region of HCV. Analysis of the NS5B coding region and 3' untranslated region (3'UTR) of HGV/GBV-C indicates an even greater degree of RNA secondary structure. Remarkably, it appears from analysis of FFEs that extensive RNA secondary structure may exist along the entire length of both the HCV and HGV/GBV-C genomes, a finding with considerable implications for future functional studies.The existence of predicted RNA structures in the HCV genome was determined using controlled nuclease mapping of RNA transcripts from the core and NS5B regions under conditions which retained potential long-range RNA interactions. The pattern of cleavage sites of nucleases specific for single and double stranded RNA provided strong experimental support for structures previously predicted in this study. Electron microscopy was also used to directly visualise the RNA folding structure of HGV/GBV-C and provided some evidence for at least four structures within the NS5B coding region and long range RNA folding across the length of the virus genome.The degree of structural conservation between diverse HCV and HGV/GBV-C genotypes and related viruses suggests roles in virus replication, and/or RNA packaging for the discrete structures identified in this thesis. Whilst this role and that of the genome wide structure identified is currently not understood the structures predicted in this work are providing a starting point for such functional studies using the HCV replicon

The identifying extended Kalman filter: parametric system identification of a vehicle handling model

This article considers a novel method for estimating parameters in a vehicle-handling dynamic model using a recursive filter. The well-known extended Kalman filter - which is widely used for real-time state estimation of vehicle dynamics - is used here in an unorthodox fashion; a model is prescribed for the sensors alone, and the state vector is replaced by a set of unknown model parameters. With the aid of two simple tuning parameters, the system self-regulates its estimates of parameter and sensor errors, and hence smoothly identifies optimal parameter choices. The method makes one contentious assumption that vehicle lateral velocity (or body sideslip angle) is available as a measurement, along with the more conventionally available yaw velocity state. However, the article demonstrates that by using the new generation of combined GPS/inertial body motion measurement systems, a suitable lateral velocity signal is indeed measurable. The system identification is thus demonstrated in simulation, and also proved by successful parametrization of a model, using test vehicle data. The identifying extended Kalman filter has applications in model validation - for example, acting as a reference between vehicle behaviour and higher-order multi-body models - and it could also be operated in a real-time capacity to adapt parameters in model-based vehicle control applications

Постспленектомічний спленоз, як предиктор розвитку рецидиву хвороби Верльгофа

Спленектомія (СЕ) у хворих на хворобу Верльгофа (ХВ), виконується у випадку неефективності консервативного лікування цього тяжкого захворювання. Не зважаючи на високий ризик оперативного втручання, пов'язаний з притаманним для цього захворювання порушеннями в системі згортання крові, СЕ продовжує активно використовуватися , так як є найбільш ефективним методом лікування ХВ

A twist in the tail : SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication

The RNA structure and long-range interactions of the SL9266 cis-acting replication element located within the NS5B coding region of hepatitis C virus (HCV) were determined using selective 2′-hydroxyl acylation analysed by primer extension. Marked differences were found in the long-range interactions of SL9266 when the two widely used genotype 2a JFH-1 (HCVcc) and genotype 1b Con1b sub-genomic replicon systems were compared. In both genomes, there was evidence for interaction of the sub-terminal bulge loop of SL9266 and sequences around nucleotide 9110, though the replication phenotype of genomes bearing mutations that disrupted this interaction was fundamentally different. In contrast, a ‘kissing loop’ interaction between the terminal loop of SL9266 and sequences in the 3′-untranslated X-tail was only detectable in JFH-1-based genomes. In the latter, where both long-range interactions are present, they were independent, implying that SL9266 forms the core of an extended pseudoknot. The presence of the ‘kissing loop’ interaction inhibited the formation of SL9571 in the 3′-X-tail, an RNA structure implicated in genome replication. We propose that, SL9266 may contribute a switch function that modulates the mutually incompatible translation and replication events that must occur for replication of the positive-strand RNA genome of HCV

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

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