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Application Of Virus Induced Gene Silencing Of Brachypodium Distachyon, A Model Organism For CropsDemircan, Turan 01 June 2009 (has links) (PDF)
Grass family is most important family in plant kingdom due to intensive usage of crops in agriculture. To date, molecular biology researches on grass family have had limitations because of inappropriate characteristics of barley and wheat to conduct experiments on them. Brachypodium distachyon that belongs to grass family has recently emerged as a model organism for crops. It shares common characteristics for a model plant due to its small genome, small physical plant size, a short lifecycle, and less demanding growth requirements / as other model organisms / Arabidopsis thaliana, Oryza sativa, and Zea mays (Draper et al. 2001). Especially after appreciating, the genetic distance of O. sativa to grasses (Garvin et al. 2008), it become a key organism to understand complicated genomic organization of agriculturally valuable grasses. Virus-induced gene silencing (VIGS) is one of the revolutionary methods allowing a rapid and effective loss of a gene function through RNA interference (Holzberg et al. 2002 / Liu et al. 2008). Barley stripe mosaic virus (BSMV) is still the most effective vector used in monocot gene silencing. It has a tripartite RNA genome having a wide range of infection ability for monocots including barley, oat, wheat, and maize as host (Holzberg et al. 2002 / Scofield 2005). In this thesis, Phytoene desaturase (PDS) gene of Brachypodium distachyon was silenced via BSMV mediated VIGS. Additionally, with Green fluorescence protein (GFP) bearing BSMV transcripts, GFP expression was observed under fluorescent microscope. To our knowledge, this is the first report demonstrating a VIGS via BSMV in Brachypodium distachyon. The success of virus induced gene silencing method in Brachypodium distachyon, will be a new convenient tool for evaluating functions of crop genes in this model organism.
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Application of crispr/cas9-based reverse genetics in leishmania braziliensis: Conserved roles for hsp100 and hsp23Adaui, Vanessa, Kröber-Boncardo, Constanze, Brinker, Christine, Zirpel, Henner, Sellau, Julie, Arévalo, Jorge, Dujardin, Jean Claude, Clos, Joachim 01 October 2020 (has links)
The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis. / Alexander von Humboldt-Stiftung / Revisión por pares
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Hemagglutinin reassortment dynamics of the zoonotic H9N2 avian influenza virusMannsverk, Steinar S January 2020 (has links)
The H9N2 avian influenza virus (AIV) has emerged, spread and established itself in poultry globally, in just under 30 years. During this time, multiple reassortants of H9N2 with increased zoonotic potential have been isolated in poultry and humans, causing a major threat to the economy and global health. Curiously, H9N2 appears to be compatible with multiple Hemagglutinin (HA) and Neuraminidase subtypes, in nature. Here, the aim was to investigate the HA reassortment dynamics of the poultry adapted H9N2 AIV, in a laboratory setting. Firstly, HA subtypes from wild bird isolates were cloned, before being co-transfected with the backbone of a chicken H9N2 AIV. The rescued H9N2 reassortants were titred on cells before the replication kinetics of a subset of the HA reassortants was assessed. The cDNA sequence of seven HA subtypes induced extensive recombination in E. coli, but ultimately ten out of eleven available HA subtypes were successfully cloned. Further, the chicken H9N2 AIV was compatible with all ten HA subtypes, producing infectious viral particles after co-transfection. However, all HA reassortants displayed decreased replicative fitness in MDCK-2 cells, compared to the wild-type virus. Interestingly, HA subtypes with similar genotypes cluster into distinct HA clades and groups, but these HA clades did not correlate with the replicative fitness of the reassortants. This study suggests that poultry adapted H9N2 AIV is compatible with many HA subtypes, highlighting the importance of reducing its spread in poultry, to reduce reassortment opportunities.
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Molecular requirements of influenza virus hemagglutinin for site-specific S-acylation and virus replicationBrett, Katharina 04 August 2015 (has links)
Das Hämagglutinin (HA) des Influenzavirus ist post-translational durch S-Acylierung von drei Cysteinen modifiziert. Zwei davon befinden sich in seiner zytoplasmatischen Domäne (CD) und enthalten Palmitat und eines am Cytosol-zugewandten Ende der Transmembranregion (TMR) wird bevorzugt mit Stearat acyliert. Es wird vermutet, dass entweder die Aminosäureumgebung der Acylierungsstelle oder dessen Lage relativ zur Membran bestimmt welcher Fettsäuretyp angeheftet wird. Diese Acylierungstellen sind zudem essentiell für die Virusreplikation. Ob auch andere Aminosäuren der CD essentiell sind, ist nicht bekannt. Nach einem umfangreichen Sequenzvergleich zur Identifikation konservierter Aminosäuren wurden rekombinante Viren mit Aminosäureaustauschen in der Nähe der drei Acylierungstellen hergestellt. Diese Austausche enthielten Punktmutationen, Verschieben des TMR Cysteins in die CD sowie die Deletion der gesamten CD. Viren ohne CD und ein Austausch neben einem acylierten Cystein verhinderten die Virusreplikation. Eine konservative Substitution derselben Position, andere Austausche in TMR und CD sowie das Schieben des TMR-Cysteins in die CD dagegen beeinflussten das Viruswachstum nur schwach. Einige der mutierten Codons revertierten zur ursprünglichen oder einer neuen Aminosäure. Rekombinante Viren wurden in MDCK-Zellen und embryonierten Hühnereiern vermehrt und mittels Massenspektrometrie analysiert. Es wurden keine unteracylierten Peptide detektiert, und selbst die zwei Letalmutationen behielten die Acylierung. Punktmutationen beeinträchtigten nur mäßig den Stearat-Gehalt, wogegen die Verlagerung des TMR-Cysteins in die CD die Stearylierung praktisch eliminierte. Mehr Stearat wurde angeheftet, wenn humane Viren in Säugerzellen im Vergleich zu aviären Zellen angezüchtet wurden. Die Position einer Acylierungsstelle repräsentiert relativ zur TMR-Spanne das Hauptsignal der Stearylierung während der Sequenzkontext und der Zelltyp das Fettsäuremuster modulieren. / Influenza virus’s hemagglutinin (HA) is post-translationally modified by S-acylation of three cysteines. Two are located in its cytoplasmic tail (CT) and contain palmitate and one at the end of the transmembrane region (TMR) is acylated primarily with stearate. It is hypothesized that either the acylation site’s amino acid environment or its location relative to the membrane determines which type of fatty acid is attached. Additionally, these acylation sites are essential for virus replication. Whether other amino acids in the CT are required for virus replication, is not known. Based on a comprehensive sequence comparison to identify conserved amino acids, recombinant viruses with amino acid substitutions in the vicinity of HA’s acylation sites were created. These substitutions included point mutations, shifting of a TMR cysteine to the CT and the deletion of the entire tail. The truncated tail mutation and a substitution adjacent to an acylated cysteine disabled virus replication. In contrast, a conservative substitution at this position, other exchanges in TMR and CT and moving the TMR cysteine to the CT had only subtle effects on virus growth. Yet, some of the mutated codons reverted to the original or other amino acids. Recombinant viruses were propagated in MDCK cells and embryonated chicken eggs and analyzed by mass spectrometry. No under-acylated peptides were detected, even the two lethal mutations did not abolish acylation. Point mutations only moderately affected the stearate content, while relocating the TMR cysteine to the CT virtually eliminated attachment of stearate. More stearate was attached if human viruses were grown in mammalian compared to avian cells. Hence, the location of an acylation site relative to the TMR represents the principal signal for stearate attachment, while the sequence context and the cell type modulate the fatty acid pattern.
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Étude de l'infection par le métapneumovirus humain : facteurs de virulence et développement de vaccins vivants atténués / Study of hMPV infection and virulence factors for live-attenuated vaccines developmentDubois, Julia 31 January 2018 (has links)
Le métapneumovirus humain (hMPV) est un virus responsable d'infections aiguës des voies respiratoires telles que des bronchiolites, des bronchites ou des pneumonies, principalement chez les populations à risques que sont les jeunes enfants de moins de 5 ans, ainsi que les personnes âgées ou immunodéprimées. Découvert en 2001, ce virus et sa pathogénèse ne restent encore aujourd'hui que partiellement caractérisés. De ce fait et malgré les besoins, il n'y a aucun vaccin ou traitement thérapeutique spécifique et efficace contre le HMPV disponible sur le marché. Dans ce contexte, mon projet de thèse s'est articulé autour de deux axes principaux : (i) L'étude de la protéine de fusion F du virus hMPV, protéine majeure antigénique de surface et responsable de l'entrée du virus dans la cellule cible. Elle a pour particularité d'induire de manière autonome la fusion membranaire in vitro et d'être associée à des effets cytopathiques variable selon les souches virales. De par son rôle clé pour le virus hMPV, la protéine F a déjà fait l'objet de plusieurs études structurales et fonctionnelles mais les déterminants de cette activité fusogénique ne sont pas encore entièrement caractérisés. Nous nous sommes donc intéressés à l'identification de déterminants du phénotype viral hyperfusogénique, localisés dans les domaines heptad repeats de la protéine F du hMPV. (ii) L'atténuation de deux souches virales cliniques (CAN98-75 et C-85473) par délétion de gènes accessoires dans le but de développer des candidats vaccinaux adaptés aux enfants en bas âge. Différents virus ont été générés par génétique inverse et les délétions des gènes accessoires SH et G dans les deux fonds génétiques viraux ont été étudiées pour leur impact sur l'infectivité, la réplication et la pathogénèse virale in vitro et in vivo ainsi que leur contribution pour le développement de virus atténués candidats vaccinaux / Human metapneumovirus (hMPV) is a major pathogen responsible of acute respiratory tract infections, such as bronchiolitis or pneumonia, affecting especially infants, under five years old, elderly individuals and immunocompromised adults. Identified since 2001, this virus and its pathogenesis still remain largely unknown and no licensed vaccines or specific antivirals against hMPV are currently available. In this context, my research project was built over two main subjects: (i) The study of the fusion F glycoprotein which is the major antigenic protein of hMPV and is responsible of viral entry into host cell. By its crucial role for the virus, the F protein has already been characterized in several structural and/or functional studies. Thus, it has been described that the hMPV F protein induces membrane fusion autonomously, resulting in variable cytopathic effects in vitro, in a strain-dependent manner. However, as the determinants of the hMPV fusogenic activity are not well characterized yet, we focused on identification of some of these, located in heptad repeats domains of the protein. (ii) The evaluation of hMPV SH and G gene deletion for viral attenuation. Liveattenuated hMPV vaccine candidates for infants’ immunization has been constructed thank to this deletion approach at the beginning of hMPV vaccine development efforts. Despite encouraging results, these candidates have not been further characterized and the importance of the viral background has not been evaluated
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Preparatory investigations for developing a transcript-based rotavirus reverse genetics system / Luwanika MleraMlera, Luwanika January 2012 (has links)
Reverse genetics systems that are based on either viral transcripts or cDNA genome
segments cloned in plasmids have recently been reported for some of the dsRNA
viruses of the Reoviridae family, namely African horsesickness virus, bluetongue
virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only
allow the manipulation of a single genome segment have been described. These
rotavirus single genome segment reverse genetics systems are not true stand-alone
systems because they require a helper virus and a recombinant virus selection step.
A true selection-free, plasmid- only or transcript-based reverse genetics system for
rotaviruses is lacking.
This study sought to identify and characterise the factors that need to be understood
and overcome for the development of a rotavirus reverse genetics system using
mRNA derived from the in vitro transcription of a consensus nucleotide sequence as
well as from double-layered particles. The consensus whole genome sequence of
the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent
whole genome amplification and 454® pyrosequencing. For the
rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at
position 397 in a hydrophobic region of VP4. NSP1 contained seven additional
amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus
nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'-
terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10
(NSP4) of the DS-1 strain were determined in this study. The consensus genome
segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously
reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known
passage history revealed a mixed infection with two SA11 strains. One of the strains
was a reassortant which contained genome segment 8 (NSP2) from the bovine
rotavirus O agent. The other ten consensus genome segments of the two strains
could not be differentiated. Novel minor population variants of genome segments 4
(VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic
analyses of the rotavirus SA11 genomes showed that the two SA11 strains were
closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were
purchased and used to generate exact capped transcripts by in vitro transcription
with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in
vitro transcription using purified rotavirus SA11 double-layered particles. The purified
rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104
cells. Work on MA104 cells was discontinued due their very low transfection efficacy.
In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death.
However, no viable rotavirus was recovered following attempts to infect MA104 cells
with the BSR and COS-7 transfected cell lysates. The cell death was determined to
be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1
genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR
and COS-7 cells. Based on visual inspection, the translation seemed to be higher in
the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells.
This suggested that the transfection of rotavirus transcripts induced an innate
immune response which could lead to the development of an antiviral state.
Therefore, the innate immune response to rotavirus transcripts was investigated in
HEK 293H cells using qRT-PCR and western blot analyses. Results of this
investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in
transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of
cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other
cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly
induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction
of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not
abrogated. The importance of a consensus sequence and the insights gained in the
current study regarding the role of the innate immune response after transfection of
rotavirus transcripts into cells in culture, should aid the development of a true
rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013
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Preparatory investigations for developing a transcript-based rotavirus reverse genetics system / Luwanika MleraMlera, Luwanika January 2012 (has links)
Reverse genetics systems that are based on either viral transcripts or cDNA genome
segments cloned in plasmids have recently been reported for some of the dsRNA
viruses of the Reoviridae family, namely African horsesickness virus, bluetongue
virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only
allow the manipulation of a single genome segment have been described. These
rotavirus single genome segment reverse genetics systems are not true stand-alone
systems because they require a helper virus and a recombinant virus selection step.
A true selection-free, plasmid- only or transcript-based reverse genetics system for
rotaviruses is lacking.
This study sought to identify and characterise the factors that need to be understood
and overcome for the development of a rotavirus reverse genetics system using
mRNA derived from the in vitro transcription of a consensus nucleotide sequence as
well as from double-layered particles. The consensus whole genome sequence of
the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent
whole genome amplification and 454® pyrosequencing. For the
rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at
position 397 in a hydrophobic region of VP4. NSP1 contained seven additional
amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus
nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'-
terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10
(NSP4) of the DS-1 strain were determined in this study. The consensus genome
segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously
reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known
passage history revealed a mixed infection with two SA11 strains. One of the strains
was a reassortant which contained genome segment 8 (NSP2) from the bovine
rotavirus O agent. The other ten consensus genome segments of the two strains
could not be differentiated. Novel minor population variants of genome segments 4
(VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic
analyses of the rotavirus SA11 genomes showed that the two SA11 strains were
closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were
purchased and used to generate exact capped transcripts by in vitro transcription
with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in
vitro transcription using purified rotavirus SA11 double-layered particles. The purified
rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104
cells. Work on MA104 cells was discontinued due their very low transfection efficacy.
In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death.
However, no viable rotavirus was recovered following attempts to infect MA104 cells
with the BSR and COS-7 transfected cell lysates. The cell death was determined to
be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1
genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR
and COS-7 cells. Based on visual inspection, the translation seemed to be higher in
the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells.
This suggested that the transfection of rotavirus transcripts induced an innate
immune response which could lead to the development of an antiviral state.
Therefore, the innate immune response to rotavirus transcripts was investigated in
HEK 293H cells using qRT-PCR and western blot analyses. Results of this
investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in
transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of
cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other
cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly
induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction
of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not
abrogated. The importance of a consensus sequence and the insights gained in the
current study regarding the role of the innate immune response after transfection of
rotavirus transcripts into cells in culture, should aid the development of a true
rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013
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Combinaison d’approches classiques et de génétique inverse en vue d'une meilleure compréhension du tropisme et de l'activité oncolytique du réovirus de mammifèresSandekian, Véronique 12 1900 (has links)
No description available.
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Construção e manipulação de clone infeccioso de uma amostra brasileira do vírus da diarreia viral bovina / Construction and manipulation of infectious clone from a brazilian bovine viral diarrhea virus isolateArenhart, Sandra 29 March 2012 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bovine viral diarrhea virus (BVDV) is a worldwide pathogen associated with
important losses to livestock production. Most of these losses come from reproductive
disorders and from the ability of the virus to produce persistent infections following in utero
infection of the fetus. A number of reverse genetics methodologies have been used for BVDV
in order to better understand the biology of the virus, which allowed the elucidation of a
number of biological features including virus replication, host-virus interaction, immune
response, and the pathogenesis of fetal infection. The present study describes the construction,
characterization and manipulation of an infectious clone out of a non-cytophatic Brazilian
BVDV strain IBSP4-ncp. The cDNA recombinant clone was constructed by yeast
homologous recombination with a low-copy vector, from three genomic fragments
comprising the open reading frame (ORF). The two untranslated regions (5' and 3' UTR) were
replaced by the respective UTRs of the reference strain NADL. The constructed vector was
transcribed in vitro and the resulting RNA was transfected on MDBK cells to rescue
infectious virus. The rescued viruses (IC-pBSC_IBSP4-ncp#2 and #3) were maintained for
ten passages in tissue culture and characterized in vitro, showing replication dynamics, focus
size and morphology similar to those of the parental IBSP-4. Genomic analysis revealed five
point mutations in the gene coding for Npro protein, resulting in amino acid changes. These
mutations probably reflect an adaptation of the virus to the heterologous UTRs. The infectious
clone IC-pBSC_IBSP4-ncp#2 was further used for the construction of a recombinant virus
expressing the Gaussia luciferase (Gluc) reporter gene. The reporter gene was inserted
between the Npro and Core genes, being flanked by an upstream linker and a downstream
sequence of the Foot and Mouth Disease virus protease (FMDV2Apro) for accurate protein
processing. The recombinant vector was in vitro transcribed and the RNA was transfected on
MDBK cells. Recombinant infectious viruses were rescued (IC-pBSC_IBSP4-ncpGluc#3 and
#4) and characterized in vitro, showing replication dynamics, focus size and morphology
similar to those of the parental IBSP-4 clone. The Gluc reporter gene was accurately
expressed and processed by the recombinant virus during 15 passages in tissue culture. These
studies revealed that the infectious clone constructed herein can be easily manipulated and is
able to carry in its genome heterologous genes up to 555 base pairs in length in a stable
fashion and without interference with its replication efficiency. Thus, the constructed clone
may be very useful for genetic manipulation towards studying different aspects of the BVDV
biology and its interactions with the host, and for the development of vaccine strains with
attenuated phenotype and/or with antigenic markers. / O vírus da diarreia viral bovina (BVDV) é um patógeno de bovinos distribuído
mundialmente, associado com importantes perdas econômicas. As maiores perdas devem-se
aos problemas reprodutivos causados pela infecção, e pela capacidade do vírus de causar
persistência após infecção fetal no terço inicial da gestação. Para entender melhor a biologia
desse vírus, sistemas de genética reversa foram desenvolvidos e tem permitido a elucidação
de vários aspectos da replicação viral, interação vírus hospedeiro, resposta imune e
patogenia da infecção fetal. O presente estudo relata a construção, caracterização e
manipulação de um clone infeccioso, a partir da cepa brasileira não-citopática IBSP4-ncp. O
clone de DNA recombinante foi construído pela técnica de recombinação homóloga em
levedura, utilizando um vetor de baixo número de cópias, construído a partir de três
fragmentos genômicos, que compreendiam a fase aberta de leitura (open reading frame, ORF)
do vírus. As duas regiões não traduzidas (5 e 3 UTR) foram substituídas pelas respectivas
UTRs da cepa de referência NADL. O vetor construído foi transcrito in vitro e o RNA obtido
foi transfectado em células MDBK para recuperação de vírus infecciosos. Os vírus
recuperados (CI-pBSC_IBSP4-ncp#2 e #3) foram mantidos por 10 passagens em cultivo
celular e caracterizados in vitro, apresentando dinâmica de replicação, tamanho e morfologia
de focos similares ao vírus parental IBSP-4. A análise do genoma por sequenciamento revelou
cinco mutações pontuais no gene Npro, com trocas de aminoácidos, provavelmente refletindo
uma adaptação do vírus às UTRs heterólogas. O clone infeccioso construído CIpBSC_
IBSP4-ncp#2, foi então utilizado para a construção de um vírus recombinante
expressando o gene repórter Gaussia luciferase (Gluc). O gene repórter foi inserido entre os
genes Npro e Core do vírus. Para o processamento da proteína repórter, uma sequência ligante
foi adicionada anteriormente ao gene, e a sequência da protease do vírus da Febre Aftosa
(FMDV2Apro) foi inserida após o gene. O vetor recombinante construído foi transcrito in vitro
e o RNA obtido foi transfectado em células MDBK. Vírus recombinantes infecciosos foram
recuperados (CI-pBSC_IBSP4-ncpGluc#3 e #4) e caracterizados in vitro, apresentando
dinâmica de replicação, tamanho e morfologia de focos similares ao vírus obtido do clone
infecioso. O gene repórter Gluc foi corretamente expresso e processado pelo vírus
recombinante durante 15 passagens em cultivo celular. Com os resultados obtidos nestes
estudos, conclui-se que o clone infeccioso construído pode ser facilmente manipulado e é
capaz de carrear em seu genoma, e expressar de forma estável, genes heterólogos com até 555
pares de base, que parecem não interferir com sua capacidade replicativa. Dessa forma, o
clone obtido pode ser muito útil para manipulação genética visando estudar diferentes
aspectos da biologia do BVDV e de suas interações com o hospedeiro, assim como para a
produção de cepas vacinais com fenótipo atenuado e/ou com marcadores antigênicos.
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Recherche de signaux d'empaquetage spécifiques du génome des virus influenza A / Identification of specific packaging signals in influenza A viruses genomeGerber, Marie 27 September 2016 (has links)
Les huit ARN viraux (ARNv) génomiques des influenzavirus de type A sont sélectivement empaquetés dans les virions, vraisemblablement sous la forme d’un complexe supramoléculaire maintenu par des appariements ARN/ARN entre signaux d’empaquetage. Afin d’améliorer la compréhension des règles qui gouvernent ce mécanisme, nous avons déterminé le réseau d’interactions formé entre les ARNv de la souche modèle de laboratoire A/Puerto Rico/8/34 (H1N1). Nous avons ensuite défini, au nucléotide près et par deux approches in vitro, les séquences des ARNv impliquées dans la formation de certaines interactions. Le rôle fonctionnel de deux d’entre elles a été testé en contexte infectieux. Nous avons également poursuivi l’étude d’une interaction identifiée au laboratoire entre deux ARNv de la souche A/Moscou/10/99 (H3N2) circulante. Enfin, nous avons collaboré avec le Dr L. Brown (Australie) sur l’étude du rôle d’une interaction entre deux ARNv de la souche A/Udorn/307/72 (H3N2). / The genome of influenza A viruses comprises eight viral RNAs (vRNAs) likely to be selectively packaged into progeny virions as organized supramolecular complexes where vRNAs are held together by base pairing within the packaging signals. To better understand the rules governing this mechanism, we investigated the vRNA/vRNA interaction network of the A/Puerto Rico/8/34 (H1N1) strain. We then identified, at the nucleotide level, using two in vitro approaches, the vRNA sequences involved in several of the interactions. Two of them were functionally tested in an infectious context. We also studied an interaction previously identified in the laboratory between two vRNAs belonging to the circulating A/Moscow/10/99 (H3N2) strain. Finally, we collaborated with Dr L. Brown (Australia) in order to assess the role of an interaction between two vRNAs of the A/Udorn/307/72 (H3N2) strain.
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