Typescript. Primarily to identify a virus which was isolated from a single Gladiolus plant early in 1962 which showed some resemblance to Cucumber mosaic virus. Exemplifies the large differences which may occur in the biological properties of viruses, even though their physico-chemical properties may be similar.

My research focuses on structures of the genomes of single-stranded RNA viruses. The first project is concerned with the sequence and secondary structure of HIV-1 RNA. Based on the secondary structure that Watts et al. determined, I performed a series of analysis and the results suggested that the abundance of Adenosines at the wobble position of the codons leads to an unusual structure with numerous unpaired nucleotides. The findings indicated how the virus balances evolutionary pressures on the genomic RNA secondary structure against pressures on the sequence of the viral proteins. The second project is the modeling of satellite tobacco mosaic virus (STMV). STMV is a T=1 icosahedral virus with a single piece of RNA that has 1058 nucleotides. X-ray crystallography studies of this RNA have revealed a structure containing 30 helices. The linkers between the helices, the possible structures at the interior of the icosahedron, and the sequence of the RNA were all missing in the crystal structure. To explore how the genome is organized within the protein capsid, I built a 3D model based on the RNA secondary structure predicted by Susan Schroeder. Being the first all-atom model of any virus, this model is highly correlated with the crystal structure; and the comparison with the in vitro structure of the same RNA supports the hypothesis that capsid protein plays an important role in RNA folding during assembly. The third project includes the modeling of bacteriophage MS2 (MS2) and the examination of the compactness of RNA in different viruses. MS2 is a T=3 RNA virus, and the cryo-EM studies have revealed a double-shell conformation of the genome. My final model of MS2 recaptures the double-shell structure of the RNA presented in the cryo-EM density. In addition, the predicted secondary structure that I used for the construction of the model shares a strong similarity with the in vitro structure determined in 1980s. This similarity contrasts with the striking difference between in vivo and in vitro RNA structures observed in STMV. Inspired by this finding, I examined the compactness of the RNA of several different viruses. The results strongly suggest that the RNAs of viruses requiring packaging signals have evolved to be structurally compact, which facilitates post-replicational RNA packaging. In contrast, viruses that do not depend on packaging signals probably adopt co-replicational RNA packaging.

The presented work is the application of recent methodologies on modeling and simulation of single stranded RNA viruses. We first present the methods of modeling RNA molecules using the coarse-grained modeling package, YUP. Coarse-grained models simplify complex structures such as viruses and let us study general behavior of the complex biological systems that otherwise cannot be studied with all-atom details. Second, we modeled the first all-atom T=3, icosahedral, single stranded RNA virus, Pariacoto virus (PaV). The x-ray structure of PaV shows only 35% of the total RNA genome and 88% of the capsid. We modeled both missing portions of RNA and protein. The final model of the PaV demonstrated that the positively charged protein N- terminus was located deep inside the RNA. We propose that the positively charged N- terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into an icosahedral virus. Third, we simulated T=1 empty capsids using a coarse-grained model of three capsid proteins as a wedge-shaped triangular capsid unit. We varied the edge angle and the potentials of the capsid units to perform empty capsid assembly simulations. The final model and the potential are further improved for the whole virus assembly simulations. Finally, we performed stability and assembly simulations of the whole virus using coarse-grained models. We tested various strengths of RNA-protein tail and capsid protein-capsid protein attractions in our stability simulations and narrowed our search for optimal potentials for assembly. The assembly simulations were carried out with two different protocols: co-transcriptional and post-transcriptional. The co-transcriptional assembly protocol mimics the assembly occurring during the replication of the new RNA. Proteins bind the partly transcribed RNA in this protocol. The post-transcriptional assembly protocol assumes that the RNA is completely transcribed in the absence of proteins. Proteins later bind to the fully transcribed RNA. We found that both protocols can assemble viruses, when the RNA structure is compact enough to yield a successful virus particle. The post-transcriptional protocol depends more on the compactness of the RNA structure compared to the co-transcriptional assembly protocol. Viruses can exploit both assembly protocols based on the location of RNA replication and the compactness of the final structure of the RNA.

The characteristic trait of the Borna disease virus is that it is a complicated single negative stranded RNA virus that is capable of infecting a wide array of mammalian species including human beings. It has been implicated in a diverse variety of human neuropsychiatric diseases. The infection capability, mechanism of infection and range of protein action of this virus remain to be identified. The purpose of the present study was to determine (1) whether the previous Bornaviridae family classification is indeed accurate as the action of BDV indicates that it is related to other viruses and (2) to estimate the number of synonymous (nucleotide substitution) and non-synonymous (amino acid change) evolutionary mutation rates of proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix protein) exhibited by various Borna disease virus host species and the proteins (nucleoprotein, phosphoprotein, glycoprotein, matrix and X protein) of three Borna disease virus strains. The latter study would give an indication as to which proteins are subjected to positive selection. Phylogenetic methods were used to determine the accuracy of the Bornaviridae classification. Phylogenetic trees obtained through an alignment and analysis of the polymerase protein, which displays a uniquely conserved GDN motif, of various RNA negative single stranded viruses using neighbourhood and parsimony methods enabled comparison with other RNA virus families. A method adapted from Ina, (1995) for estimating the synonymous and non-synonymous evolutionary mutation rate was applied to various BDV proteins in order to provide more information on inter (host virus) and intra (virus) mutation rate. This information in turn was used to create an evolutionary model to clarify the positive and neutral evolutionary trend of the inter- and intra-virus proteins examined, which may help clarify and enhance the lack of current knowledge relating to species infection and the epidemiological nature of the virus. The results obtained by the polymerase alignment analysis indicates the presence of two newly discovered BDV motifs, v and vi, confirmed by three diverse alignment programmes. An analysis of the alignment of BDV proteins indicated that the BDV nucleoprotein nuclear localization signal aligns the BDV nucleoprotein between motifs IV and vi of the BDV polymerase. The results obtained by the phylogenetic analysis indicate that the Rabies virus and the Vesicular stomatitis virus are the most closely related animal viruses to BDV, whereas the Rice transitory yellowing (unclassified Rhabdovirus) and Sonchus yellow net plant virus are closet to BDV than other animal Rhabdoviridae raising intriguing questions on the evolutionary origins of the Borna disease virus. The phylogenetic analysis indicates that the Borna disease virus does not fall into a separate Bornaviridae family classification, and suggests that BDV may be more appropriately placed into a separate subfamily in the family Rhabdoviridae. The results of the evolutionary analysis indicate considerable diversity between BDV host virus (inter-species) and BDV virus (intra-species) protein sequences. In the host virus sequence comparison analysis all of the proteins examined displayed a high pattern of non random evolution, which is in contrast to the intra species comparison in which only three proteins; the BDV glycoprotein, nucleoprotein and X protein; displayed a non random pattern of evolution. The positive selection effect displayed by the inter-species (host) proteins may be attributed to antigenic variation displayed by the inter-species sequences and a super infection hypothesis, which indicates that positive selection on host variants could arise during the course of an infection as a result of specific immune responses. The positive and neutral selection trend of the proteins displayed by the intra-species (virus) sequences may be a result of a pattern of nucleotide substitution that is physio-chemically conservative. Conservation may be evident in volume, polarity, hydrophilicity, or molecular weight of amino acids of the proteins. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2002.

Paukščių gripas yra virusinė infekcija, kuri lengvai plintanti pavojinga užkrečiamoji paukščių liga, šia liga serga laukiniai ir naminiai paukščiai. Patys pavojingiausi ir patogeniškiausi yra paukščių gripo H5 ir H7 viruso potipiai. Didžiausią susirūpinimą ir nerimą kelia H5N1 paukščių gripo virusas, kuris priklauso Influenzavirus A viruso atmainai. Svarbiausias antigenas, kuris sukelia apsauginių antikūnų produkciją, o taip pat svarbus viruso prisijungimui prie ląstelių pirmosiose infekcijos stadijose, yra viriono apvalkalo glikoproteinas - hemagliutininas. Pagal hemagliutinino antigenines savybes virusas skirstomas į 16 potipių (H1-H16). Žmonės serga H1, H2 ir H3 potipiais, tačiau visada išlieka galimybė, kad įvykus geno mutacijoms, poslinkiams ar pasikeitus genetinei informacijai pavojingi taps ir likę potipiai. Todėl yra būtina stebėti, kokie A tipo gripo viruso potipiai vyrauja. Darbe yra apžvelgta didžiųjų ančių (Anas platyrhynchos) bendra charakteristika, literatūriniai duomenys apie paukščių gripo virusą, aprašytas polimerazinės grandininės reakcijos metodas, tikro laiko ir atvirkštinės transkripcijos polimerazinės grandininės reakcijos eiga. Skyriuje „Medžiagos ir metodai“ aprašyta, kokia medžiaga buvo naudota tyrimuose, aprašytas RNR išskyrimas ir gryninimas, naudojami rinkiniai. Aprašyta paukščių gripo A viruso H5 ir H7 potipių nustatymas vieno žingsnio realaus laiko atvirkštinės transkripcijos PGR būdu. Iš viso 949 vandens paukščių mėginiai buvo surinkti Lietuvoje... [toliau žr. visą tekstą] / Avian influenza is a viral infection that is easily spread through a dangerous contagious disease of birds that disease sick in wild and domestic birds. The most dangerous and pathogenicity are avian influenza H5 and H7 virus subtypes. The biggest concern and disturbing of the H5N1 avian influenza virus, which belongs to Influenza A Virus species. The aim was to analyze the mallards which was infected by avian influenza virus in Lithuania At work is reviewed mallards (Anas platyrhynchos) general characteristics, the literature results of the avian influenza virus, described polymerase chain reaction method and real time reverse transcriptase polymerase chain reaction process. In chapter „Materials and Methods” describes what material was used in the research described in RNA isolation and purification for use in kits. Described by avian influenza A virus of the H5 and H7 subtypes of setting a one-step real-time reverse transcription PCR. 319 influenza A virus samples from which 22 samples were positive for influenza A virus. Also during the year 2007 to get 630 influenza A virus samples from 43 of the positive samples. After the 2007 and 2011 data analysis reveals that the majority of the positive influenza A samples of migratory birds have been obtained during the cold season. Haemagglutinin subtypes used successfully to identify by the Blast search GenBank database. Avian influenza viruses positive samples were identified exclusively from mallards, hunted in various... [to full text]

The current work reports on structural studies of ribonucleoprotein complexes, Escherichia coli and Thermomyces lanuginosus ribosomes, and Pariacoto virus (PaV) using molecular modeling. Molecular modeling is the integration and representation of the structural data of molecules as models. Integrating high-resolution crystal structures available for the E. coli ribosome and the cryo-EM density maps for the PRE- and POST- accommodation states of the translational cycle, I generated two all-atom models for the ribosome in two functional states of the cycle. A program for flexible fitting of the crystal structures into low-resolution maps, YUP.scx, was used to generate the models. Based on these models, we hypothesize that the kinking of the tRNA plays a major role in cognate tRNA selection during accommodation. Secondly, we proposed all-atom models for the eukaryotic ribosomal RNA. This is part of a collaboration between Joachim Frank, Andrej Sali, and our lab to generate an all-atom model for the eukaryotic ribosome based on a cryo-EM density map of T. lanuginosus available at 8.9Å resolution. Homology modeling and ab initio RNA modeling were used to generate the rRNA components. Finally, we propose a first-order model for a T=3, icosahedral, RNA virus called Pariacoto virus. We used the structure available from x-ray crystallography as the starting model and modeled all the unresolved RNA and protein residues. Only 35% of the total RNA genome and 88% of the protein were resolved in the crystal structure. The generated models for the virus helped us determine the location of the missing N-terminal protein tails. The models were used to propose a new viral assembly pathway for small RNA viruses. We propose that the basic N-terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into a mature virus. This process is reminiscent of DNA condensation by positively charged ions.

Bibliography: leaves 104-130. / vi, 130, [25] leaves, [13] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Copies of author's previously published articles inserted. / Includes corrigendum. / Bibliography: leaves 97-106. / xiii, 106, [69] leaves, [31] leaves of plates : ill. (some col.), maps ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis reports an investigation of the etiology of ScSMD (sugarcane striate mosaic disease) by biological and microscopic studies and by nucleic acid studies. Studies of ScSMD affected sugarcane are followed by the detection, isolation, cloning and partial sequencing of a disease specific dsRNA, and the tentative classification of the putative viral agent from the partial sequence. This study also describes the ScSMD associated virion and an improved protocol for the purification of dsRNA. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1997

Bibliography: leaves 116-117 / viii, 131, [82] leaves, [20] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1983

Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997. / Bibliography: leaves 104-130.