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1
Afsharifar, Alireza. "Characterisation of minor RNAs associated with plants infected with cucumber mosaic virus." Title page, table of contents and abstract only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09pha2584.pdf.
Full textAbstract:
Bibliography: leaves 127-138. This thesis studies the minor double stranded RNAs (dsRNA) and single stranded RNAs (ssRNA) which are consistently associated with plants infected with Q strain of cucumber mosaic virus (Q-CMV). The investigations are focused on the structural elucidation of new RNAs which have been observed in single stranded and double stranded RNA profiles of Q strain of CMV.
2
Upton, John H. "The role of RNA secondary structure in replication of Nodamura virus RNA2." To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2009. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
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3
Wahyuni, Wiwiek Sri. "Variation among cucumber mosaic virus (CMV) isolates and their interaction with plants." Title page, contents and summary only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phw137.pdf.
Full textAbstract:
Includes appendix containing journal publications co-authored by the author. Includes bibliographical references (leaves 130-151). Eighteen strains of Cucumber mosaic virus, including forteen from Australia, two from the USA, and two from Japan were used in this study.
4
Williams, Rhys Harold Verdon George. "Further studies on the structure and function of the cucumber mosaic virus genome : a thesis submitted to the University of Adelaide, South Australia for the degree of Doctor of Philosophy." 1988, 1988. http://web4.library.adelaide.edu.au/theses/09PH/09phw7261.pdf.
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5
Chen, Baoshan. "Encapsidation of nucleic acids by cucumovirus coat proteins /." Title page, contents and summary only, 1991. http://web4.library.adelaide.edu.au/theses/09PH/09phc5183.pdf.
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6
Shi, Bu-Jun. "Expression and function of cucumoviral genomes." Title page, contents and summary only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phs5546.pdf.
Full textAbstract:
Bibliography: leaves 104-130. 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.
7
Ligat, Julio S. "Pathology and distribution in the host of pea seed-borne mosaic virus." Title page, contents and summary only, 1993. http://web4.library.adelaide.edu.au/theses/09PH/09phl723.pdf.
Full textAbstract:
Includes bibliographical references (leaves 82-92). Five isolates of pea seed-borne mosaic virus were compared by host range and symptomatology on 16 pisum sativum cultivars lines, 21 lines of Lathyrus and Lens spp. and several indicator species
8
Hajimorad, Mohammad Reza. "Variation in alfalfa mosaic virus with special reference to its immunochemical properties." Title page, contents and summary only, 1990. http://web4.library.adelaide.edu.au/theses/09PH/09phh154.pdf.
Full textAbstract:
Includes Appendix listing other publications by the author. Includes bibliographical references (leaves 134-181). Alfalfa mosaic virus was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms. Experiments showed that each isolate was biologically distinct and that the host range and symptomatology of each isolate was affected by the environmental condition.
9
Wakeford, Laura 1956. "COMPLEMENTATION BETWEEN TEMPERATURE-SENSITIVE MUTANTS OF POLIOVIRUS." Thesis, The University of Arizona, 1987. http://hdl.handle.net/10150/276556.
Full textAbstract:
Conditional lethal mutants of poliovirus type 1 (Mahoney) were generated by treatment with the mutagen hydroxylamine. Temperature-sensitive mutants were selected by the replica plating technique at temperatures of 33°C (permissive) and 39°C (restrictive). New mutants were generated to achieve a larger population of mutants and also to generate additional RNA- mutants in this population. These mutants were characterized by two criteria: RNA synthesis and thermal stability. RNA synthesis is measured by the accumulation of labeled uridine incorporation into trichloroacetic acid (TCA) insoluble material. The thermal stability is determined by the difference in plaque forming units before and after treatment of the virion at 45°C. Complementation co-infections (5 MOI for each virus stock) were analyzed for the presence of the 150S virion particle of poliovirus after sedimentation through a linear sucrose gradient. Complementation is observed between RNA(+) mutants v.s. RNA(-) mutants, and between two RNA(-) mutants, but not between two RNA(+) mutants. Although reciprocal complementation has not been documented in this study some speculation on complementation is presented in this thesis.
10
Sheldon, Candice Claire. "Hammerhead mediated self-cleavage of plant pathogenic RNAs /." Title page, contents and summary only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phs544.pdf.
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11
Geering, Andrew D. W. "The epidemiology of cucumber mosaic virus in narrow-leafed lupins (Lupinus angustifolius) in South Australia." Title page, table of contents and summary only, 1992. http://web4.library.adelaide.edu.au/theses/09PH/09phg298.pdf.
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12
Du, Preez Jacques. "The construction of an infectious clone of grapevine virus A (GV A)." Thesis, Link to the online version, 2005. http://hdl.handle.net/10019/1012.
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13
Fosu-Nyarko, John. "Studies on Subterranean clover mottle virus towards development of a gene silencing vector." Thesis, Fosu-Nyarko, John (2005) Studies on Subterranean clover mottle virus towards development of a gene silencing vector. PhD thesis, Murdoch University, 2005. https://researchrepository.murdoch.edu.au/id/eprint/54/.
Full textAbstract:
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV.
To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants.
Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea.
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows:
* pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene
* pFLP1gfp, pFL with gj27 gene fused to the 3' end of the ORF 1,
* pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable and gene cloned in tandem between the CP gene and the 3' NCR of SCMoV,
* pFLCPVsgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and
* pFLREPsgprogfp, pFL with the ORF2b, a putative sgPro sequence and a translatable gfP gene cloned in tandem between a truncated CP gene and the 3' NCR
These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.
14
Fosu-Nyarko, John. "Studies on Subterranean clover mottle virus towards development of a gene silencing vector." Fosu-Nyarko, John (2005) Studies on Subterranean clover mottle virus towards development of a gene silencing vector. PhD thesis, Murdoch University, 2005. http://researchrepository.murdoch.edu.au/54/.
Full textAbstract:
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV.
To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants.
Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea.
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows:
* pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene
* pFLP1gfp, pFL with gj27 gene fused to the 3' end of the ORF 1,
* pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable and gene cloned in tandem between the CP gene and the 3' NCR of SCMoV,
* pFLCPVsgprogfp, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and
* pFLREPsgprogfp, pFL with the ORF2b, a putative sgPro sequence and a translatable gfP gene cloned in tandem between a truncated CP gene and the 3' NCR
These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.
15
Dutta, Ranendra Nath. "Experimental Test of Solitary Wave Theory in Viral Populations." University of Toledo Health Science Campus / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=mco1226950654.
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16
Aubry, Fabien. "Development and applications of a new reverse genetics method for the generation of single-stranded positive-sense RNA viruses." Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM5066.
Full textAbstract:
La génétique inverse est devenue une méthode clé pour la production de virus à ARN génétiquement modifiés et pour comprendre les propriétés cellulaires et biologiques des virus. Cependant les méthodes les plus fréquemment utilisées, basées sur le clonage de génomes viraux complets dans des plasmides, sont laborieuses et imprévisibles. La première partie de cette thèse présente des études sur la mise au point d'un nouveau système de génétique inverse, appelé méthode ISA (Amplicons-Sous génomique-Infectieux), qui permet la génération, en quelques jours, de virus infectieux sauvages et génétiquement modifiés appartenant à trois familles différentes de virus à ARN simple brin de polarité positive, avec une grande maîtrise des séquences virales. Dans la deuxième partie de cette thèse, nous avons appliqué pour la première fois à un arbovirus (CHIKV), le ré-encodage des codons - une méthode développée récemment et très excitante pour le développement de vaccins vivants atténués. En utilisant une approche aléatoire de ré-encodage des codons qui attribue au hasard des codons sur la base de la séquence en acides aminés correspondante, nous avons mis en évidence des pertes importantes de fitness réplicatif sur des cellules de primates et d'arthropodes. La diminution du fitness réplicatif est en corrélation avec le degré de ré-encodage, une observation qui peut aider à la modulation de l'atténuation virale. En utilisant l'expérience acquise avec le CHIKV, nous avons transposé avec succès ce mécanisme d'atténuation au JEV et amélioré notre maîtrise du processus d'atténuation en utilisant une combinaison de la synthèse de novo et de la méthode ISA<br>Reverse genetics has become a key methodology for producing genetically modified RNA viruses and deciphering cellular and viral biological properties, but the most commonly used methods, based on the preparation of plasmid-based complete viral genomes, are laborious and unpredictable. The first part of this thesis presents studies relating to the development of a new reverse genetics system, designated the ISA (Infectious-Subgenomic-Amplicons) method, which enabled the generation of both wild-type and genetically modified infectious viruses belonging to three different families of positive, single stranded RNA viruses within days with great control of the viral sequences. In the second part of this thesis, we applied for the first time to an arbovirus (CHIKV), codon re-encoding - a recently developed and very exciting method for the development of live attenuated vaccines. Using a random codon re-encoding approach which randomly attributed nucleotide codons based on their corresponding amino acid sequence, we identified major fitness losses of CHIKV in both primate and arthropod cells. The decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. Detailed analysis of these observed replicative fitness losses indicated that they are the consequence of several independent re-encoding induced events. Using the experience acquired on the CHIKV, we successfully transposed this attenuation mechanism to JEV and improved our control of the attenuation process by using a combination of de novo synthesis and the ISA method
17
au, J. Fosu@murdoch edu, and John Fosu-Nyarko. "Studies on Subterranean clover mottle virus towards development of a gene silencing vector." Murdoch University, 2005. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050421.123457.
Full textAbstract:
Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that
infects subterranean clover (Trifolium subterraneum) and a number of related legume species.
The ultimate aim of this research was to investigate aspects of SCMoV that would support its
use as a gene silencing vector for legume species, since RNA (gene) silencing is now a
potential tool for studylng gene function. The ability of viruses to induce an antiviral defense
system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral
genomes are used as vectors to introduce genes or gene ii-agments to understand the function of
endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects
of SCMoV host range and molecular biology needed to be studied.
A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this
work involved study of the host range and symptom development of SCMoV in a range of
leguminous and non-leguminous plants. The aim of this work was to identify new and most
suitable hosts among economically important crop and model legumes for functional genomic
studies, and also to study symptom development in these hosts for comparison with host
responses to any SCMoV-based viral vectors that might be used in later infection studies. A
total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7
families were examined for their response to SCMoV infection, including established and new
crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator
plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae,
Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for
symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3
weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera
of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were
infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and
one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This
work considerably expanded knowledge of the host range of SCMoV.
To provide the information needed to modify the SCMoV genome to develop gene vectors,
the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL,
SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and
molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a
complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence
of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and
the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames
(ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids
with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by
four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of
63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b
is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The
transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of
RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes
the 27.3 kDa coat protein (CP).
Nucleotide variation between the complete sequences of the isolates was two to three orders of
magnitude larger than base misincoporation rates of the polymerases used in RT-PCR.
Molecular relationship analysis between all five isolates, undertaken with the complete
nucleotide sequences, clearly separated them into three groups. These groups reflect similar
significantly diverse groupings based on the symptoms and their severity in subterranean
clover. Intra-isolate sequence variability is therefore a possible cause of the differences in
symptom severity. The analysis also showed that there were more nucleotide substitutions at
the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the
higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio)
estimated for the ORF1, compared to the near conservation of sequences of the other ORFs.
These results, together with functional and comparative sequence analysis with other
sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a
severity determinant or as a viral suppressor of RNA silencing in plants.
Because more information on SCMoV genome function was required, the possible involvement
of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of
grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish
(Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for
in planta synthesis of reporter gene constructs was developed. The vector was based on
pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and
nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple
subcloning site. A custom-made particle inflow gun was used to introduce the constructs into
plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated
particles. Transient gene expression of GFP was monitored 24-96 hours after particle
bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in
the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was
limited spread of these fusion proteins from one cell to another 36-48 hours after
transformation. These results indicate that the P 1 and CP cannot move independently from cell
to cell. Other viral/cellular components might be needed to form a complex with these proteins
to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of
SCMoV were identified by sequence comparison. These could be tested by mutagenesis using
full-length infectious clones.
To determine the possibility of gene expression of vectors based on SCMoV, three forms of a
full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous
transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with
35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and
subterranean clover seedlings were inoculated by particle bombardment using gold particles
coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR
after two weeks in grasspea but not in subterranean clover and chickpea.
Experiments were undertaken towards developing the SCMoV genome into a VIGS vector.
Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA
clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be
derived. The rationale used in developing these constructs was gene insertion andlor
replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of
SCMoV. The major constructs were as follows:
pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene,
pFLP 1 gfp, pFL with gj27 gene fused to the 3 ' end of the ORF 1,
pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable & gene cloned
in tandem between the CP gene and the 3' NCR of SCMoV,
pFLCPVsgprogf$, pFL with a putative sgPro sequence and a translatable gfp gene
cloned in tandem between a truncated CP gene and the 3' NCR and
pFLREPsgprog@, pFL with the ORF2b, a putative sgPro sequence and a translatable
&fP gene cloned in tandem between a truncated CP gene and the 3' NCR
These constructs were all made, but a detailed assessment of their vector potential could not be
done because there was a delay of about one year whilst the Office of the Gene Technology
Regulator processed the application for permission for glasshouse testing. Although additional
work needs to be undertaken to complete development of a final RNA silencing vector, this
study has contributed to new knowledge in terms of extending understanding of SCMoV host
range, symptoms, sequence variation and control of gene expression. The constructs made
have also laid the groundwork for development of a legume gene silencing vector based on
SCMoV.
18
Lee, Se Il. "Statistical thermodynamics of virus assembly." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33900.
Full textAbstract:
Experiments show that MgSO4 salt has a non-monotonic effect as a function of MgSO4 concentration
on the ejection of DNA from bacteriophage lambda.
There is a concentration, N0, at which the minimum amount of DNA is ejected.
At lower or higher concentrations, more DNA is ejected. We propose that this non-monotonic behavior
is due to the overcharging of DNA at high concentration of Mg⁺² counterions.
As the Mg⁺² concentration increases from zero, the net charge of ejected DNA changes its sign from negative to positive.
N0 corresponds to the concentration at which DNA is neutral.
Our theory fits experimental data well.
The DNA-DNA electrostatic attraction is found to be -0.004 kBT/nucleotide.
Simulations of DNA-DNA interaction of a hexagonal DNA bundle support our theory.
They also show the non-monotonic DNA-DNA interaction and reentrant behavior of DNA condensation by divalent counterions.
Three problems in understanding the capsid assembly for a retrovirus are studied:
First, the way in which the viral membrane affects the structure of in vivo assembled HIV-1 capsid is studied.
We show that conical and cylindrical capsids have similar energy at high surface tension of the viral membrane,
which leads to the various shapes of HIV-1 capsids. Secondly, the problem of RNA genome packaging inside spherical viruses
is studied using RNA condensation theory. For weak adsorption strength of capsid protein, most RNA genomes are located at the center
of the capsid. For strong adsorption strength, RNA genomes peak near the capsid surface and the amount of RNA packaged is proportional to the capsid area instead its volume. Theory fits experimental data reasonably well.
Thirdly, the condensation of RNA molecules by nucleocapsid (NC) protein is studied.
The interaction between RNA molecules and NC proteins is important for the reverse transcription of viral RNA which relates to the viral infectivity.
For strong adsorption strength of the NC protein, there is a screening effect by RNA molecules around a single NC protein.
19
Terenius, Olle. "Anti-parasitic and anti-viral immune responses in insects." Doctoral thesis, Stockholm : Institutionen för genetik, mikrobiologi och toxikologi, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-224.
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Carneiro, Bruno Moreira [UNESP]. "Estudo in vitro do efeito da interferência por RNA (RNAi) na replicação do vírus da hepatite C." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/108909.
Full textAbstract:
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000778124.pdf: 2188391 bytes, checksum: fd262617e1148f5350687c3217aafda3 (MD5)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>A Hepatite C é a inflamação do fígado causada pela infecção pelo vírus da hepatite C (HCV). Essa inflamação ocorre na maioria das pessoas infectadas pelo vírus e, dependendo da intensidade e tempo de duração, pode levar à cirrose e câncer do fígado. No momento não existem vacinas eficazes e o tratamento convencional com peg-IFN e ribavirina tem eficácia bastante limitada e efeitos colaterais graves. Terapias moleculares utilizando a RNAi demonstraram ser eficientes na inibição deste vírus in vitro. O objetivo deste trabalho foi desenvolver diferentes metodologias para inibição da replicação do HCV utilizando-se da técnica de RNAi. Foram desenvolvidas 5 moléculas de dicer substrate siRNA, que em sua maioria foram capazes de reduzir a replicação viral em até 90% em relação ao controle negativo. Ainda, não foi observada a seleção de mutantes resistentes do vírus após o tratamento com os DsiRNAs por 21 dias. Também foram desenvolvidos 3 vetores lentivirais contendo sequencias codificantes de shRNA contra o HCV. Com a utilização destas partículas lentivirais foi possível reduzir a replicação do HCV em aproximadamente 99% em relação ao controle negativo. Neste estudo foi demonstrado que o HCV pode ser inibido eficientemente utilizando tecnologias distintas de RNAi. Os DsiRNAs são mais potentes que os tradicionais siRNAs e com menor probabilidade de selecionar mutantes resistentes. Os vetores lentivirais são eficientes na entrega dos genes contendo os shRNAs e potentes na inibição do HCV. Ambas as tecnologias no futuro poderão ser utilizadas na terapia alternativa de pacientes crônicos ou no caso dos DsiRNAs de forma preventiva à infecção<br>Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis, and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. No vaccine is available for HCV and the current standard of care, which consists of pegylated interferon-? and ribavirin, has limited efficacy against certain HCV genotypes, and also produces significant adverse effects. Molecular therapies have proven to be effective in the inhibition of the virus in vitro. Molecular therapies based on RNAi has shown good efficiency on knockdown of HCV in vitro.The aim of this study was to develop different methodologies for inhibiting HCV replication using the RNAi technique. We developed five dicer substrate siRNA molecules, which mostly were able to reduce viral replication by 90% compared to the negative control. Still, there was no selection of resistant mutants of the virus after treatment with DsiRNAs for 21 days. In addition, we developed lentiviral vectors containing sequences encoding shRNA against HCV. With the use of these lentiviral particles, it was possible to reduce HCV replication by approximately 99% compared to negative control. In this study, it was demonstrated that HCV could be effectively inhibited using different RNAi technologies. The DsiRNAs are more powerful than traditional siRNAs and less likely to select resistant mutants. The lentiviral vectors are efficient in delivery of genes containing shRNAs and potent in the inhibition of HCV. Both technologies in the future may be used in alternative therapy for chronic patients or in case of DsiRNAs preventively to infection
21
O'Brien, Siobhan. "Regulation of Cellular and HIV-1 Gene Expression by Positive Transcription Elongation Factor B: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/528.
Full textAbstract:
RNA polymerase II-mediated transcription of HIV-1 genes depends on positive transcription elongation factor b (P-TEFb), the complex of cyclin T1 and CDK9. Recent evidence suggests that regulation of transcription by P-TEFb involves chromatin binding and modifying factors. To determine how P-TEFb may connect chromatin remodeling to transcription, we investigated the relationship between P-TEFb and histone H1. We show that P-TEFb interacts with H1 and that H1 phosphorylation in cell culture correlates with P-TEFb activity. Importantly, P-TEFb also directs H1 phosphorylation during Tat transactivation and wild type HIV-1 infection. Our results also show that P-TEFb phosphorylates histone H1.1 at a specific C-terminal site. Expression of a mutant H1.1 that cannot be phosphorylated by P-TEFb disrupts Tat transactivation as well as transcription of the c-fos and hsp70 genes in HeLa cells. P-TEFb phosphorylation of H1 also plays a role in the expression of muscle differentiation marker genes in the skeletal myoblast cell line C2C12. Additionally, ChIP experiments demonstrate that H1 dissociates from the HIV-1 LTR in MAGI cells, stress-activated genes in HeLa cells, and muscle differentiation marker genes in C2C12 cells under active P-TEFb conditions. Our results overall suggest a new role for P-TEFb in both cellular and HIV-1 transcription through chromatin.
22
Choudhury, Md Abu Hasnat Zamil. "Population Dynamics of RNA viruses." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/60866/1/Md._Choudhury_Thesis.pdf.
Full textAbstract:
Between 50 and 100 million people are infected with dengue viruses each year and more than 100,000 of these die. Dr Choudhury has demonstrated that populations of dengue viruses in individual patients are genetically and functionally very diverse and that this diversity changes significantly at the time of major outbreaks of disease. The results of his studies may inform strategies which will make dengue vaccines far more effective.
23
Rosskopf, John J. "CIS-acting signals for replication of Nodamura virus RNA1." To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2009. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
Full text
24
Turner, David Richard. "Protein-RNA interactions in tobacco mosaic virus assembly." Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328799.
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Lamprecht, Renate Luise. "Molecular characterisation of South African isolates of grapevine fanleaf virus and a new, associated satellite RNA." Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85600.
Full textAbstract:
Thesis (PhD)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Grapevine fanleaf virus (GFLV) is one of the oldest, most widespread and devastating
viruses infecting grapevine, and occurs globally where Vitis vinifera is grown. In South
Africa (SA) GFLV is predominant in the Breede River Valley, one of the highest wine
producing regions in SA. To date, only three GFLV isolates have been completely
sequenced internationally, and limited sequence information is available for SA GFLV
isolates. In this study, the first full-length GFLV genome sequence from a South African
isolate, GFLV-SAPCS3, was determined. Full-length sequences were used for
phylogenetic analysis and revealed that the SA isolates are separate from other
sequenced GFLV isolates. Full-length sequences were also used to investigate putative
intra- and interspecies recombination events involving GFLV-SAPCS3 RNA1 and RNA2
between GFLV and Arabis mosaic virus (ArMV) isolates. Using two different
recombination analysis software packages, the most notable of the putative
recombination events involving GFLV-SAPCS3 indicated that the GFLV-SAPCS3 RNA2
5’ UTR might have evolved from an interspecies recombination event between GFLVF13-
type and ArMV Ta-type isolates. The presence of satellite RNAs (satRNA)
associated with South African GFLV isolates was also investigated. In a collaborative
study (see Chapter 4 for details), more than a 100 GFLV- infected grapevine plants
were screened for satRNAs. SatRNAs were present in only two plants, containing
isolates GFLV-SACH44 and GFLV-SACH47. The full-length nucleotide sequences of
the GFLV-SACH44 genomic RNAs 1 and 2, and the associated satRNA were
determined. No significant sequence variation could be detected between the GFLV
isolates that had the presence of a satRNA and those that had not. The GFLV-SACH44
RNA2 5’ UTR also had the same conserved sequence that was found in GFLVSAPCS3,
which suggests that GFLV-SACH44, like GFLV-SAPCS3, may have arisen
from a common ancestor, which may have originated from an interspecies
recombination event. The GFLV-SACH44 satRNA was found to be more closely related
to the ArMV large satRNA than to the satRNA associated with GFLV-F13. A full-length
cDNA clone of GFLV-SACH44 satRNA was constructed and its replication and systemic spread in herbaceous hosts, when mechanically co-inoculated with two GFLV isolates
as helper viruses, was demonstrated. Replication of the GFLV-SACH44 satRNA cDNA
clone was however abolished when co-inoculated with an ArMV helper virus, even
though it is phylogenetically more closely related to ArMV satRNAs. The full-length
satRNA clones were modified to be used as vectors for expression and/or silencing of
foreign genes, by inserting the green fluorescence protein (GFP) full-length or partial
sequences downstream of the open reading frame of the satRNA. These constructs
were cloned into a binary vector to allow for agro-infiltration into plants. Full-length
cDNA clones of GFLV-SAPCS3 RNA1 and RNA2 were constructed to be used in
conjunction with modified GFLV-SACH44 satRNA full-length clones. The full length
GFLV-SAPCS3 RNA1 and RNA2 clones were however not infectious in Nicotiana
benthamiana after agro-infiltration and therefore the evaluation of the modified satRNA
expression and silencing constructs had to be aborted. Attempts to understand this
failure revealed that, among other point mutations, four frameshifts had occurred in the
RNA1 full-length clone, rendering the transcripts untranslatable, and hence noninfectious.
Strategies to correct the mutations are discussed. Once these mutations
have been corrected this study can continue in evaluating the use of the satRNA
component for expression and silencing analysis.<br>AFRIKAANSE OPSOMMING: Grapevine fanleaf virus (GFLV) is een van die oudste, mees wydverspreide en mees
verwoestende virusse wat wingerd affekteer en word wêreldwyd waar Vitis vinifera
verbou word, gevind. In Suid Afrika (SA) kom GFLV veral in die Breederivier vallei, een
van die mees produktiewe wyn-produserende areas in SA, voor. Tot dusver is daar net
drie GFLV isolate waarvan die volledige nukleïensuurvolgorde internasionaal bepaal is.
Die nukleïensuurvolgorde informasie vir SA GFLV isolate is redelik beperk. In hierdie
studie was die eerste volledige nukleïensuurvolgorde van ‘n SA GFLV isolaat, GFLVSAPCS3,
bepaal. Die volledige nukleïensuurvolgordes was vir filogenetiese analise
gebruik, en vermeende intra- en interspesie rekombinasie gebeurtenisse, wat GFLVSAPCS3
RNA1 en RNA2 betrek, tussen GFLV en Arabis mosaic virus (ArMV) isolate
was ondersoek. Twee verskillende rekombinasie-analise sagteware programme was
gebruik en die noemenswaardigste van die vermeende rekombinasie gebeurtenisse,
met betrekking tot GFLV-SAPCS3, het aangedui dat die GFLV-SAPCS3 RNA2 5’
ontransleerde streek (UTR) waarskynlik van ‘n interspesie rekombinasie gebeurtenis
tussen ‘n GFLV-F13-tipe en ‘n ArMV-Ta-tipe isolaat ontwikkel het. Die teenwoordigheid
van satelliet RNAs (satRNAs), wat met SA GFLV isolate geassosieer is, was ook
ondersoek. Meer as ‘n 100 GFLV ge-infekteerde wingerd plante was in ‘n
samewerkingsprojek (sien Hoofstuk 4 vir besonderhede) getoets vir die
teenwoordigheid van satRNAs. SatRNAs was net in twee plante teenwoordig, in isolate
GFLV-SACH44 en GFLV-SACH47. Die vollengte nukleïensuurvolgordes van GFLVSACH44
RNA1, RNA2 en geassosieerde satRNA was bepaal. Geen beduidende
volgorde variasie tussen die GFLV isolate wat satRNAs bevat het, en die GFLV isolate
sonder satRNA was waargeneem nie. Die GFLV-SACH44 RNA2 5’ UTR het ook die
gekonserveerde volgorde, wat in GFLV-SAPCS3 teenwoordig was, gehad en dit dui
daarop dat GFLV-SACH44, soos GFLV-SAPCS3, van dieselfde stamvader, wat tydens
‘n vorige rekombinasie gebeurtenis ontstaan het, mag ontwikkel het. Die GFLVSACH44
satRNA was meer naverwant aan die ArMV satRNAs as aan die satRNA, wat
met GFLV-F13. ‘n Vollengte cDNA kloon van die GFLV-SACH44 satRNA was ontwikkel en die replisering en sistemiese verspreiding in sagte plante, nadat dit met twee GFLV
isolate as helper virusse saam ge-inokuleer was, was gedemonstreer. Replisering van
die GFLV-SACH44 satRNA cDNA kloon was egter ontwrig toe dit saam met ‘n ArMV
helper virus saam ge-inokuleer was, al is dit filogeneties meer verwant aan ArMV
satRNAs. Die vol-lengte satRNA klone was gemodifiseer om as vektore vir uitdrukking
en/of uitdowing van transgene te dien, deur om vol-lengte of gedeeltelike groen
fluoressensie proteïen (GFP) nukleïensuurvolgordes aan die einde van die satRNA
leesraam te koppel. Hierdie konstrukte was in ‘n binêre vektor gekloon om agroinfiltrasie
in plante toe te laat. Vol-lengte cDNA klone van GFLV-SAPCS3 RNA1 en
RNA2 was ontwikkel om in samewerking met die gemodifiseerde GFLV-SACH44
satRNA konstrukte gebruik te word. Die vol-lengte GFLV-SAPCS3 RNA1 en RNA2
klone het egter nie in Nicotiana benthamiana gerepliseer na agro-infiltrasie nie, daarom
was die evaluasie van die gemodifiseerde satRNA konstrukte gestaak. Pogings om die
mislukking te verstaan, het daarop gewys dat, behalwe punt mutasies, vier leesraam
versteurings in die RNA1 vollengte kloon voorgekom het, wat ontransleerbare
transkripte, en dus nie-repliserende konstrukte tot gevolg gehad het. Strategieë om die
mutasies te korrigeer is bespreek. Sodra die mutasies gekorrigeer is, kan die studie
voortgaan om te evalueer of die satRNA komponent vir uitdrukking en uitdowing analise
gebruik kan word.
26
Visser, Marike. "Small RNA profiling of virus-infected apple plants." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/95828.
Full textAbstract:
Thesis (PhD)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Apple stem grooving virus (ASGV) is globally associated with latent infection in commercial apple trees. Little
is known about this plant-‐virus interaction. This study made use of
next-‐generation sequencing to investigate the effect of virus-‐infection on the expression of the different small RNA (sRNA) species
namely, miRNAs, nat-‐siRNAs, phasiRNAs, rasiRNAs, tRNA-‐derived
sRNAs and vsiRNAs. Broad and narrow size-‐range datasets were generated using sRNA libraries prepared from total and size-‐selected
RNA, respectively. Through bioinformatic data analyses, 130 genomic
loci were predicted to give rise to miRNAs, 85 of which were novel
MIR genes. Targets were predicted for the majority of miRNAs, a
few of which could be validated with a publicly available degradome
dataset. Cis-‐ and trans-‐natural antisense transcripts (NATs) were
identified, of which only the latter were highly enriched for sRNAs
in their overlapping regions. Transcript as well as genomic regions
were also identified that can give rise to phasiRNAs. For 25 of these
loci an in-‐phase miRNA target site was identified, half of which could
be validated with the degradome dataset. Nearly all apple repeat sequences in Repbase were associated with sRNA synthesis. sRNAs
derived from both ends of mature tRNAs were identified. These
sRNAs corresponded to tRFs and tRNA-‐halves. Reads associated
with tRNA-‐halves were prominent in the broad range datasets.
sRNAs, originating from the central regions of tRNAs, were also
observed. Analysis of the vsiRNAs suggested the presence of two
ASGV genetic variants in two of the samples, while the third sample
was infected with only one variant. Comparison of the vsiRNA profiles
generated from the two datasets highlighted the influence of library
preparation on the interpretation of results. Differential expression
analysis of the identified apple sRNA species showed no variation
between healthy and infected plants, except for the tRNA-‐derived
sRNAs, which did show altered expression levels. Taken together,
the various sRNA species characterised in this study significantly
extended the existing knowledge of apple sRNAs and provide a
broad platform for future functional studies in apple. This study
also presents the first and most comprehensive report on sRNAs
involved in ASGV infection in apple.<br>AFRIKAANSE OPSOMMING: Appel gleufstam virus (ASGV) word wêreldwyd geassosieer met latente infeksie in kommersiële appelbome. Min inligting oor hierdie plant-‐virus interaksie is beskikbaar. Hierdie studie het van volgende-‐generasie volgordebepaling gebruik gemaak om die effek van virusinfeksie
op die uitdrukking van verskillende klein RNA (sRNA) spesies, nl.
miRNAs, nat-‐siRNAs, phasiRNAs, rasiRNAs, tRNA-‐afkomstige sRNAs
en vsiRNAs, te ondersoek. Datastelle met breë en smal grootte-‐verspreiding is gegenereer m.b.v. sRNA biblioteke wat onderskeidelik
voorberei is vanaf totale RNA en RNA van ‘n bepaalde grootte.
Deur middel van bioinformatiese data-‐ontleding is 130 genomiese
loci voorspel wat aanleiding kan gee tot miRNAs, waarvan 85 nuwe
MIR gene is. Teikens is voorspel vir die meerderheid van die miRNAs
en 'n aantal daarvan kon bevestig word m.b.v. 'n publiek-‐beskikbare
degradoom datastel. Cis-‐ en trans-‐natuurlike antisense transkripte
(NATs) is geïdentifiseer, waarvan slegs die laasgenoemde verryk
was vir sRNAs in hul oorvleuelende areas. Transkrip sowel as
genomiese areas, wat aanleiding kan gee tot phasiRNAs, is ook
geïdentifiseer. Vir 25 van hierdie loci is 'n in-‐fase miRNA teiken
geïdentifiseer, waarvan die helfte bevestig kon word met die degradoom datastel. Byna al die appel herhalende volgordes in
Repbase was geassosieer met sRNA sintese. sRNAs afkomstig van
beide kante van volwasse tRNAs is geïdentifiseer. Hierdie sRNAs
het ooreengestem met tRFs en tRNA-‐helftes. Volgordes geassosieer
met tRNA-‐helftes was prominent in die breë grootte-‐verspreiding
datastelle. sRNAs, afkomstig van die sentrale dele van tRNAs, is
ook waargeneem. Ontleding van die vsiRNAs het die teenwoordigheid
van twee ASGV genetiese variante in twee van die monsters aangetoon, terwyl die derde monster met slegs een variant geïnfekteer was. Die vergelyking van vsiRNA profiele, gegenereer
vanaf die twee datasteltipes, beklemtoon die invloed van biblioteek
voorbereiding op die interpretasie van resultate. Ontleding van die
differensiële uitdrukking van die geïdentifiseerde appel sRNA spesies
het geen verskil tussen gesonde en geïnfekteerde plante getoon
nie, behalwe vir die tRNA-‐afkomstige sRNAs, wat wel verandering die
vlak van uitdrukking getoon het. Die verskillende sRNA spesies wat
in hierdie studie geïdentifiseer is, het die bestaande kennis van appel
sRNAs aansienlik uitgebrei en bied 'n breë platform vir toekomstige
funksionele studies in appel. Hierdie studie bied ook die eerste, en
mees omvattende verslag oor sRNAs betrokke in ASGV infeksie in
appel.
27
Gnädig, Nina Franziska. "Role of genetic diversity of RNA viruses in viral evolution and pathogenicity." Paris 7, 2012. http://www.theses.fr/2012PA077095.
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Le taux de fréquence de mutation des virus à ARN est très élevé, en raison des erreurs commises par TARN polymerase dépendante de TARN. Par conséquent, une population virale génétiquement diverse est générée, sur la base d'une séquence consensus. Cette population, ou quasi-espèce, se comportent comme une entité unique en terme d'évolution et de sélection. Cet aspect permet aux virus de s'adapter rapidement à de nouveaux environnements. Pendant le première partie de mon travail, je me suis concentrée sur l'étude d'un variant haute fidélité (A372V) et d'un variant basse fidélité (S299T) de la polymerase du CVB3. Il a été montré que ces mutants sont résistants aux composés amilorides, utilisés dans le traitement de l'hypertension. Notre étude apporte les premières preuves de l'effet mutagène de l'amiloride et montre que les deux variants résistent via deux mécanismes différents. La deuxième partie de mon travail a consisté à caractériser de nouveaux variants de basse fidélité de la RdRp du CVB3 in vivo. Le panel de virus à basse fidélité présente des fréquences de mutation qui s'étendent de 5,6 mutations par 10⁴ nucléotides à 11,2, comparées aux 4,3 mutations pour le virus sauvage. Ils présentent une atténuation in vivo qui correspond à leur fréquence de mutation. Les variants les plus atténués étaient incapables d'établir une infection dans le cœur et à long terme dans la rate. Nos données montrent que les variants qui génèrent des populations génétiquement plus diverses produisent trop de génomes défectueux et pas assez de génomes viables pour assurer la survie de la population virale, lorsque la taille est significativement réduite<br>RNA viruses have the highest mutation frequencies in nature, mainly due to their error prone RNA-dependent RNA polymerase (RdRp). The result is a highly diverse RNA virus population of genetically similar yet not identical genomes (mutant spectrum or quasispecies). This very important aspect of RNA virus biology enables these viruses to adapt quickly to new environments and escape immune responses of the host. During the first half of my PhD thesis, I focused on studying a higher fidelity polymerase (A372V) and a lower fidelity polymerase (S299T) of CVB3. These mutants were previously found to be resistant to amiloride compounds. In our study, we provided the first evidence of a mutagenic activity of amiloride and show that the two fidelity variants resist through two different mechanisms. The second part of my thesis project consisted on characterizing several new low fidelity CVB3 RdRp variants in vivo. These variants were selected by the rational targeting of residues based on polymerase structure. The panel of low fidelity viruses, whose mutation frequencies range from 5. 6 mutations per 10 nucleotides to 11. 2 compared to 4. 3 mutations for wild type, presented a range of attenuation that correlated with their mutation frequencies. The most attenuated variants were unable to establish a productive infection in the heart or a long-term infection of the spleen. Our data also show that the variants that generate genetically more diverse populations harbor too many mutated or defective genomes, and not enough viable genomes, to ensure survival of the viral population when its size is significantly reduced as it occurs during a bottleneck event
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Sleat, D. E. "Packaging and expression of recombinant RNAs." Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.382121.
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Fedewa, Gregory. "Quantifying Nucleotide Variation in RNA Virus Populations by Next-generation Sequencing." Thesis, University of California, San Francisco, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10936274.
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<p> RNA viruses include several notable human pathogens including HIV, hepatitis C virus, West Nile virus, influenza, and Ebola virus. This group of viruses includes viruses with incredibly diverse genome structures, such as single-stranded genomes, double-stranded genomes, multipart genomes, negative-stranded genomes, and positive-stranded genomes. They also exist as heterogeneous populations that can mutate and rapidly evolve due to their error-prone polymerases. These errors then accumulate as they are passed down through generation. They can, therefore, be used as a historical marker for genetic relationships. If these errors result in a change of fitness for the virus they can then be used to locate areas in the genome that are undergoing selection pressures.</p><p> In this work, I use these principles to examine what changes are necessary for Ebola virus to infect boa constrictor cells and how high priority RNA viruses mutate as a function of routine viral passaging and propagation. In <i> Chapter 2</i>, I show that Ebola virus requires no additional mutations in order to replicate efficiently in boa constrictor cells. In <i>Chapter 3</i>, I show that SNV analysis can be used to track the identity and passage history of different RNA viruses.</p><p>
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Collins, Fergal M. "Investigation of the morbillivirus large protein by reverse genetics." Thesis, Queen's University Belfast, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326404.
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Cheung, Mei-sze, and 張美思. "The application of an Epstein-Barr Virus specific antisense ribozyme for the in vitro suppression of EBNA-1 and LMP-1 expression." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2002. http://hub.hku.hk/bib/B42577056.
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Carneiro, Bruno Moreira. "Estudo in vitro do efeito da interferência por RNA (RNAi) na replicação do vírus da hepatite C /." São José do Rio Preto, 2013. http://hdl.handle.net/11449/108909.
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Orientador: Paula Rahal<br>Banca: Clarice Arns<br>Banca: João Pessoa Araújo Junior<br>Banca: Maurício Lacerda Nogueira<br>Banca: Cristiane Damas Gil<br>Resumo: A Hepatite C é a inflamação do fígado causada pela infecção pelo vírus da hepatite C (HCV). Essa inflamação ocorre na maioria das pessoas infectadas pelo vírus e, dependendo da intensidade e tempo de duração, pode levar à cirrose e câncer do fígado. No momento não existem vacinas eficazes e o tratamento convencional com peg-IFN e ribavirina tem eficácia bastante limitada e efeitos colaterais graves. Terapias moleculares utilizando a RNAi demonstraram ser eficientes na inibição deste vírus in vitro. O objetivo deste trabalho foi desenvolver diferentes metodologias para inibição da replicação do HCV utilizando-se da técnica de RNAi. Foram desenvolvidas 5 moléculas de dicer substrate siRNA, que em sua maioria foram capazes de reduzir a replicação viral em até 90% em relação ao controle negativo. Ainda, não foi observada a seleção de mutantes resistentes do vírus após o tratamento com os DsiRNAs por 21 dias. Também foram desenvolvidos 3 vetores lentivirais contendo sequencias codificantes de shRNA contra o HCV. Com a utilização destas partículas lentivirais foi possível reduzir a replicação do HCV em aproximadamente 99% em relação ao controle negativo. Neste estudo foi demonstrado que o HCV pode ser inibido eficientemente utilizando tecnologias distintas de RNAi. Os DsiRNAs são mais potentes que os tradicionais siRNAs e com menor probabilidade de selecionar mutantes resistentes. Os vetores lentivirais são eficientes na entrega dos genes contendo os shRNAs e potentes na inibição do HCV. Ambas as tecnologias no futuro poderão ser utilizadas na terapia alternativa de pacientes crônicos ou no caso dos DsiRNAs de forma preventiva à infecção<br>Abstract: Hepatitis C virus (HCV) frequently establishes persistent infections in the liver, leading to the development of chronic hepatitis, and, potentially, to liver cirrhosis and hepatocellular carcinoma at later stages. No vaccine is available for HCV and the current standard of care, which consists of pegylated interferon-α and ribavirin, has limited efficacy against certain HCV genotypes, and also produces significant adverse effects. Molecular therapies have proven to be effective in the inhibition of the virus in vitro. Molecular therapies based on RNAi has shown good efficiency on knockdown of HCV in vitro.The aim of this study was to develop different methodologies for inhibiting HCV replication using the RNAi technique. We developed five dicer substrate siRNA molecules, which mostly were able to reduce viral replication by 90% compared to the negative control. Still, there was no selection of resistant mutants of the virus after treatment with DsiRNAs for 21 days. In addition, we developed lentiviral vectors containing sequences encoding shRNA against HCV. With the use of these lentiviral particles, it was possible to reduce HCV replication by approximately 99% compared to negative control. In this study, it was demonstrated that HCV could be effectively inhibited using different RNAi technologies. The DsiRNAs are more powerful than traditional siRNAs and less likely to select resistant mutants. The lentiviral vectors are efficient in delivery of genes containing shRNAs and potent in the inhibition of HCV. Both technologies in the future may be used in alternative therapy for chronic patients or in case of DsiRNAs preventively to infection<br>Doutor
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Dunn, Ewan Frazer. "Development of a reverse genetics system to investigate Bunyamwera virus RNA synthesis." Thesis, University of Glasgow, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343641.
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McCulley, Anna. "Primer selection of E. coli tRNALys,3 by human immunodeficiency virus type-1." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2007. https://www.mhsl.uab.edu/dt/2008r/mcculley.pdf.
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Tanov, Emil Pavlov. "The identification of biologically important secondary structures in disease-causing RNA viruses." University of the Western Cape, 2012. http://hdl.handle.net/11394/4562.
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Magister Scientiae - MSc<br>Viral genomes consist of either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The viral RNA molecules are responsible for two functions, firstly, their sequences contain the genetic code, which encodes the viral proteins, and secondly, they may form structural elements important in the regulation of the viral life-cycle. Using a host of computational and bioinformatics techniques we investigated how predicted secondary structure may influence the evolutionary dynamics of a group of single-stranded RNA viruses from the Picornaviridae family. We detected significant and marginally significant correlations between regions predicted to be structured and synonymous substitution constraints in these regions, suggesting that selection may be acting on those sites to maintain the integrity of certain structures. Additionally, coevolution analysis showed that nucleotides predicted to be base paired, tended to co-evolve with one another in a complimentary fashion in four out of the eleven species examined. Our analyses were then focused on individual structural elements within the genome-wide predicted structures. We ranked the predicted secondary structural elements according to their degree of evolutionary conservation, their associated synonymous substitution rates and the degree to which nucleotides predicted to be base paired coevolved with one another. Top ranking structures coincided with well characterized secondary structures that have been previously described in the literature. We also assessed the impact that genomic secondary structures had on the recombinational dynamics of picornavirus genomes, observing a strong tendency for recombination breakpoints to occur in non-coding regions. However, convincing evidence for the association between the distribution of predicted RNA structural elements and breakpoint clustering was not detected.
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Chong, Jean-Leon. "Functional characterization of Ded1p, a DExD/H box RNA helicase, in saccharomyces cerevisiae." The Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=osu1092284292.
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Palmer, William Hunt. "Evolution and genetics of antiviral immunity in Drosophila." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31557.
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Virus-host interactions determine virus transmissibility and virulence, and underlie coevolution that shapes interesting biological phenomena such as the genetic architecture of host resistance and host range. Characterization of the virus factors that exert selective pressure on the host, and the host genes which underlie resistance and adaptation against viruses will help to define the mechanistic pathways embroiled in host-virus coevolution. In this thesis, I describe the viral causes and host consequences of host-virus coevolution. These include genomic signatures consistent with antagonistic coevolution in antiviral RNA interference pathway genes such as high rates of positive selection and polymorphism, loci that underlie genetic variation in resistance to virus infection, and apparent conflict between NF-κB signalling and DNA virus infection. The RNA interference (RNAi) pathway is the most general innate immune pathway in insects, underlined by the observation that many viruses encode suppressors of RNAi (VSRs). The relationship between RNAi and VSRs has garnered attention as a plausible battleground for host-virus antagonistic coevolution, and genomic patterns in Drosophila support this hypothesis. However, genomic patterns in the N-terminal domain of the key RNAi effector gene, Argonaute-2, have not been described. In Chapter 2, I sequence the Argonaute-2 N-terminal domain using PacBio long-read sequencing technology to describe variation within and across Drosophila species, and test whether this variation is associated with resistance to Drosophila C Virus. The RNAi pathway evolves adaptively in Drosophila, but this has not been formally extended across invertebrate species. In Chapter 3, I quantify rates of adaptive protein evolution and describe evidence for selective sweeps in RNAi pathway genes using population genomic data from 8 insect and nematode species. These analyses indicate that RNAi genes involved in suppression of transposable elements and defence against viruses evolve rapidly across invertebrates, and I identify genes with signatures of elevated adaptation in multiple insect species. Host genes that underlie host-virus interactions have been described in RNA virus infection of Drosophila, however substantially less attention has focussed on the host response to DNA viruses, primarily because no DNA viruses have been isolated from Drosophila. In Chapter 4, I describe the isolation of Kallithea virus, a Drosophila dsDNA nudivirus, and characterise the host response to infection and genetic variation in resistance. I find that Kallithea virus infection causes early male-specific lethality, a cessation of oogenesis, and induction of undescribed virus-responsive genes. Further, I describe genetic variation in resistance and tolerance to Kallithea virus infection, and identify a potential causal variant for virus-induced mortality in Cip4. Insect viruses commonly encode viral suppressors of RNAi, however there are a multitude of antiviral immune mechanisms besides RNAi which may select for viral-encoded inhibitors. In Chapter 5, I describe the requirement for RNAi and NF-κB in immunity against Kallithea virus, and map gp83 as a virus-encoded inhibitor of NF-κB signalling. I find that gp83 inhibits Toll signalling at the level of, or downstream of NF-κB transcription factors, and that this immunosuppressive function is conserved in other nudiviruses.
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Pickett, Brett E. "The contribution of different mechanisms of viral sequence variation to the evolution of positive-sense single-stranded RNA viruses." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2010. https://www.mhsl.uab.edu/dt/2010p/pickett.pdf.
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Hampson, J. "A study of the genetics and growth of a tripartite RNA virus using ts mutants." Thesis, University of Warwick, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380288.
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Bhatt, Samir. "Statistical analysis of natural selection in RNA virus populations." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:64341c38-f09e-48ed-84e8-7ab9f171a753.
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A key goal of modern evolutionary biology is the identification of genes or genome regions that have been targeted by natural selection. Methods for detecting natural selection utilise the information sampled in contemporary gene sequences and test for deviation from the null hypothesis of neutrality. One such method is the McDonald Kreitman test (MK test), which detects the the molecular 'footprint' left by natural selection by considering the frequency of observed mutations within the sampled population. In this thesis I investigate the applicability of the MK test to viral populations and develop several new methods based on the original MK test. In chapter 2, I use a combination of simulation and methodological improvements to show that the MK test can have low error when applied to analysis of RNA virus populations. Then, in chapter 3, I develop an extension of the MK test with the purpose of estimating rates of adaptive fixation for all genes of the human influenza A virus subtypes H1N1 and H3N2. My results are consistent with previous studies on selection in influenza virus populations, and provide a new perspective on the evolutionary dynamics of human influenza virus. In chapter 4 I develop a formal statistical framework based, on the MK test, for calculating the number of non neutral sites at any frequency range in the site frequency spectrum. In this framework, I introduce a new method for reconstructing the site frequency spectrum that incorporates sampling error and allows for the inclusion of prior knowledge. Using this new framework I show that the majority of nucleotide sites in hepatitis C virus sequences sampled during chronic infection represent deleterious mutations. Finally, in chapter 5 I use the generalised framework introduced in chapter 4 to develop a statistic for evaluating the deleterious mutation load of a population. I apply this test sequences that represent 96 RNA virus genes and show that my approach has comparable power to equivalent phylogenetic methods. In this thesis I have developed computationally efficient methods for analysis of genetic data from virus populations. It is my hope that these methods will become useful given the explosion in sequence data that has accompanied recent improvements in sequencing technology.
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Clinch, Barry F. "A study of methods in reverse genetics for the rescue of influenza virus subgenomic RNA." Thesis, University of Reading, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244944.
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Stenbak, Carolyn Rinke. "Foamy virus polymerase : enzymatic activities and assembly /." Thesis, Connect to this title online; UW restricted, 2003. http://hdl.handle.net/1773/11510.
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Song, Rujun. "Dimerization of human immunodeficiency virus type 1 genome : dimer maturation process and role of the 5' untranslated region in dimerization." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111918.
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Human Immunodeficiency Virus type I genome consists of two identical RNA molecules that are non-covalently linked to form a dimer. HIV-1 immature and mature genomic RNA (gRNA) dimers were found in protease defective (PR -) and wild type virions, respectively, and the 5'untranslated region (5' UTR) was shown to play key roles during the genome dimerization process; but the dimerization mechanism still remains to be clarified My research project is to characterize the dimerization process and the role of 5' UTR in genome dimerization in virions produced by tissue culture cells. I'll firstly show the dimer maturation processes of HIV-1 gRNA isolated from newly released to grown-up (≥10h old) wild type, PR-, and SL1 defective (DeltaIDS) virions respectively. The results showed that HIV-1 gRNA dimer maturation process was protease-dependent and involved multiple steps: from low to high dimerization level and dimer thermostability, and from low dimer mobility to intermediate and high mobility. PR- virions did not freeze gRNA conformation in the primordial nascent state and gRNA changed from monomeric in newly released virions to half dimeric in grown-up virions, which showed that genome was packaged in the form of monomeric RNA or fragile dimers, more thermolabile than immature dimers in grown-up PR- virions. DeltaDIS inhibited gRNA dimerization by about 50% in newly released virions, though grown-up DeltaDIS gRNA was fully dimeric, which indicated that the DIS played the initiation role in gRNA dimerization in HIV-1 virions. The gRNA dimerization rate in PR- or DeltaDIS virions was much slower than that in wild type virions. These results show for the first time the whole process of dimer maturation after virion release, the gRNA conformation rearrangement in PR- virions, and the initiation role of the DIS in HIV-1 virions. Next, I'll provide a rather systematic search for the contribution of different regions in 5' UTR to HIV-1 gRNA dimerization by studying selected mutations singly or together with defective SL1. The results showed that the 5'trans-activation response element (5'TAR) was directly involved in gRNA dimerization, and a long distance base-pairing interaction between a sequence in U5 region (nts105-1l5) and another around the initiation codon of the gag gene (nts334-344) was structurally contributive to gRNA dimerization. Deletions of sequences around the 3'end of Primer Binding Site (PBS) stem-loop moderately decreased gRNA dimerization level. Other sequences in 5' UTR except DIS/SL1, which was previously known to play important roles, didn't show any systematic role. Here the results suggested that the absence of inhibition on gRNA dimerization level with defective DIS might be the compensation of the direct role of 5'TAR; and wild type-like dimerization level of DeltaTAR must be the direct contribution of the DIS.
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Palmer, Matthew T. "The influence of retroviral codon usage on the acquisition of the tRNA used to prime reverse transcription." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2006. https://www.mhsl.uab.edu/dt/2007r/palmer.pdf.
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Wadsworth, Santiago. "Genetic and biochemical study of P15, the RNA silencing suppressor of Peanut clump virus." Strasbourg, 2010. http://www.theses.fr/2010STRA6233.
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Le RNA silencing est un mécanisme de régulation négative de l’expression des gènes, retrouvé chez la plupart des organismes eucaryotes et basé sur des interactions d’ARN séquence-spécifiques. Il est déclenché par de l’ARN double brin qui est découpé par Dicer en de petits ARNs de 21 ‡ 24nt, nommés short-interfering (si)RNAs ou microRNAs. Un brin du duplex est incorporé dans un complexe appelé RNA-induced silencing complex (RISC), pour guider le clivage endonucléolytique ou l’inhibition traductionnelle d’ARNm présentant une homologie de séquence. Chez les plantes, le RNA silencing joue un rôle important dans la défense antivirale, le développement et l’adaptation aux stress biotiques et abiotiques. Pour contrer ce mécanisme de défense, les virus de plantes ont élaboré des protéines dites suppresseurs de RNA silencing. Au cours de cette thèse, nous avons analysé les interactions entre le RNA silencing et le Peanut clump virus (PCV). Nous avons démontré que les siRNA dérivés de PCV sont produits par Dicer-like 4 (DCL4) et dans une moindre mesure par DCL2, se traduisant par l’accumulation de siRNA de 21 et 22nt respectivement. La P15, suppresseur de RNA silencing du PCV, provoque une réduction drastique des niveaux de siRNA provenant des molécules d’ARN exogène de type tige-boucle ; et stabilise le brin non incorporé dans RISC. Dans une deuxième partie, nous avons étudié les interactions de P15 et ces protéines cibles. Nous avons ainsi identifié DCL1 parmi les protéines co-immunoprécipitées par P15 et montré que P15 affecte le niveau d’accumulation de DCL1, suggèrant ainsi que P15 pourrait déclencher une régulation négative DCL1-dépendante du système antiviral<br>RNA silencing refers to a set of RNA-based eukaryotic processes that regulate gene expression in a sequence-specific manner. It initiates with the processing of dsRNA by Dicer (RNase type III) proteins into small molecules of dsRNA of 21- to 24-nt in length, generally referred as short-interfering (si)RNAs or microRNAs, depending on their origin. One brand of the small duplex is incorporated into an effector complex, the RNA-Induced Silencing Complex, to guide the regulation of gene expression through different mechanisms, including endonucleolytic cleavage, translational repression or histone- and DNA-modifications. In plants, siRNAs and miRNAs are involved in viral defense, development and adaptation to biotic and abiotic stresses. As natural targets of RNA silencing, plant viruses have produced highly diverse proteins called silencing suppressors. First, we have studied the genetic requirements of Peanut clump virus (PCV) viral-si(vs)RNA production in Arabidopsis and shown that PCV RNA is primary processed by Dicer-like 4 (DCL4) to produce 21-nt vsRNAs, with DCL2 playing a minor role in the production of 22-nt PCV viRNAs. We also observed that P15, the PCV-encoded silencing suppressor, drastically reduces the accumulation of siRNAs derived only from transgenic hairpins of perfect dsRNA and stabilizes the non-incorporated brand of the miRNA duplex. Another part of this work consisted in the identification of plant proteins that may interact with P15. Among others, we identified DCL1 as P15 co-immunoprecipitated protein and showed that P15 alters the level of DCL1, suggesting that P15 may mediate a DCL1-dependent negative regulation of the antiviral plant defense
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Phan, Ngoc Minh Hien. "Perusing peripatetic pathogenic viruses: Hepatitis B virus and severe acute respiratory syndrome coronavirus 2." Thesis, Queensland University of Technology, 2021. https://eprints.qut.edu.au/226107/1/Ngoc%20Minh%20Hien_Phan_Thesis.pdf.
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This project characterised the genetic diversity of hepatitis B virus (HBV) and SARS-CoV-2, which reflects viral evolution, and drives transmission, viral pathogenesis, and clinical outcomes. Genomes and coding genes of these viruses were analysed using next-generation sequencing, bioinformatic tools and publicly available viral sequences. HBV nucleotide diversity was identified amongst HBV sequences from Australian and non-Australian blood donors. In addition, the frequency of HBV mutants circulating in Southeast Asia, Australia and New Zealand was identified. Europe and East Asia were shown as major sources of Australian SARS-CoV-2 during the beginning of the pandemic in 2020.
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Chen, Kuang-Yu. "Mechanistic study and prediction of influenza A virus genetic reassortment." Thesis, Université de Paris (2019-....), 2019. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=4762&f=29825.
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La nature segmentée du génome des virus de la grippe A (IAV) permet une évolution rapide par réassortiment génétique. Bien que le nombre théorique de génotypes issus d'un réassortiment entre deux virus soit de 256 (28), la panoplie complète des différents génotypes n'a jamais été observée et certains gènes ont tendance à co-ségréger, suggérant que le réassortiment génétique est biaisé. Cependant, à ce jour, les contraintes qui façonnent le réassortiment génétique restent largement méconnues. L'objectif de mon projet est de progresser dans la compréhension des règles sous-jacentes au réassortiment génétique afin d'améliorer notre capacité à prédire le réassortiment entre les IAV circulant dans la nature.Nous avons dans un premier temps étudié l’incompatibilité entre sous-unités hétérologues de la polymérase virale (FluPol) réunies suite à un réassortiment génétique. En effet, nous avons observé qu'un virus réassortant dont le segment PB2 dérive du virus A/WSN/33 (WSN) dans un fond génétique A/PR/8/34 (PR8) était atténué, malgré un degré d’identité de 97% entre les protéines PR8-PB2 et WSN-PB2. Des passages en série indépendants ont conduit à la sélection de révertants phénotypiques portant des mutations secondaires distinctes sur les sous-unités PA, PB1 et PB2. L’impact de ces mutations a été étudié par génétique inverse et à l’aide de tests d’activité sur les polymérase virales. Pour chaque virus révertant, au moins une mutation a été localisée à l'interface de dimérisation de FluPol et s'est avérée réguler son taux de dimérisation. La mutation PA-E349K en particulier joue un rôle majeur dans la correction d'un défaut initial de réplication virale (ARNc -> ARNv). Nos résultats montrent que les sous-unités de la FluPol co-évoluent non seulement pour assurer des interactions optimales entre sous-unités, mais également des niveaux appropriés de dimérisation, indispensables à une réplication efficace. Ainsi, la dimérisation de la FluPol pourrait être l’un des facteurs limitant l’issue du réassortiment génétique.Parallèlement, afin d’étudier le réassortiment génétique de manière exhaustive et avec une puissance statistique suffisante, nous avons cherché à adapter un système déjà éprouvé de microfluidique en goutte pour un séquençage ciblé, à haut débit et massivement parallélisé, de > 105 IAV issus d’un réassortiment entre deux IAVs. Pour établir la faisabilité du système deux souches virales saisonnières circulantes ont été choisies et des amorces ciblant les huit segments d’ARNv de chaque virus ont été conçues, testées et optimisées. Une expérience contrôle préliminaire réalisée sur des cellules uniques infectées individuellement, montre que les informations sont correctement préservées au niveau de la cellule unique mais que la détection des segments et des souches était déséquilibrée. De nouvelles amorces ont été conçues et des stratégies d'amplification alternatives mises en œuvre et optimisées. Après analyse du réassortiment entre les deux souches saisonnières et validation des données par comparaison avec les données de surveillance, notre système sera appliqué au réassortiment génétique entre les virus saisonniers humains et les virus animaux d’intérêt zoonotique.À long terme, les données générées via notre plateforme devraient aider à la compréhension des mécanismes de réassortiment génétique entre virus influenza. Notre plateforme pourrait également devenir un outil prédictif s’ajoutant aux outils d'évaluation du risque de pandémie grippale ainsi qu’un outil de surveillance<br>The segmented nature of the genome of influenza A viruses (IAVs) allows rapid evolution by genetic reassortment. Although the theoretical number of genotypes that can emerge from reassortment between two viruses is 256 (28), the full panel of different genotypes was never observed and certain genes tend to co-segregate, suggesting that genetic reassortment is biased. However, to date, the constraints that shape genetic reassortment remain largely unknown. The objective of my project is to make progress in understanding the rules underlying genetic reassortment in order to improve our capacity to predict reassortment among co-circulating IAVs.First, we investigated the incompatibility between non-cognate subunits of the influenza polymerase complex (FluPol) brought together by genetic reassortment. Indeed, we observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone was attenuated, despite a 97% identity between the PR8- and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and PB2. The constellation of mutations present on the revertant viruses was studied using reverse genetics and cell-based reconstitution of the viral polymerase. For each revertant virus, at least one mutation was located at the FluPol dimerization interface and was found to regulate the levels of FluPol dimer. For one of them, PA-E349K, a major role in correcting an initial defect in viral replication (cRNA -> vRNA) was demonstrated. Hence, our results show that the FluPol subunits co-evolve not only to ensure optimal inter-subunit interactions but also proper levels of dimerization of the heterotrimer, essential for efficient viral RNA replication. Thus, we suggest that FluPol dimerization is one of the factors that can restrict the outcome of genetic reassortment.In parallel, in order to study the outcome of genetic reassortment comprehensively and achieve adequate statistical power, we aimed at adapting a proven droplet-based microfluidic single-cell RNA-seq system for customized high-throughput massively parallelized targeted sequencing of > 105 reassortant IAVs. For a proof-of-concept, two circulating seasonal viral strains were chosen and gene specific primers targeting their eight segments were designed, tested and optimized. From a preliminary compartimentalized control experiment, we found that single cell information was well preserved but that segment and strain detection were imbalanced. New primers were designed and alternative amplification strategies were implemented and optimized. A new control experiment will be performed prior to analysis of reassortment between the two seasonal strains and validation of the data by comparison with surveillance data. Once validated, our system will be applied to genetic reassortment between human seasonal viruses and animal viruses of zoonotic interest. In the long term, the data generated through our platform should help understanding the mechanism of IAV genetic reassortment and become a valuable predictive tool added to the Pandemic Influenza Risk Assessment Tools for pandemic preparedness
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Neuman, Benjamin. "Use of defective-RNAs containing reporter genes to investigate targetted recombination in avian infectious bronchitis virus." Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367694.
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Khidr, Yehia. "Development of a strategy to induce RNA-silencing in squash against virus diseases by genetic transformation." [S.l. : s.n.], 2007. http://nbn-resolving.de/urn:nbn:de:bsz:100-opus-1963.
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