Dissertations / Theses on the topic 'VECTOR DE VIRUS'

Doctor of Philosophy
Division of Biology
Kristin Michel
The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

Foamy viruses (FVs) are unique ancient retroviruses that infect all non-human primates, but do not cause disease. We aimed to understand the FV pre-integration complex by isolating it from infected cells and characterising its protein constituents. Using a PCR to quantify integration in infected cells, we determined that integration occurs from 10 hours post-transduction. In synchronised cells, the peak of integration correlated well with cells passing through mitosis. However, we were unable to detect in vitro strand-transfer activity to indicate that active pre-integration complexes had been isolated. We conclude that FV pre-integration complexes are likely to be inactive in the conditions tested. A further aim was to optimise FV vectors for use in mesenchymal stem cells and test this vector in mouse models of sphingolipidoses, namely metachromatic leukodystrophy and Krabbe disease. We permitted transduction of cells at a high multiplicity of infection by exchanging the envelope from the prototype FV to that of the macaque. We tested various FV vectors in mesenchymal stem cells and determined that the non-toxic macaque envelope increased transduction efficiency from under 65% to over 95% in a single round of transduction. We achieved high and sustained transgene expression using the phosphoglycerate kinase promoter. Transduced MSCs delivered to the brains of the mouse model for metachromatic leukodystrophy caused only a modest improvement in sulphatide storage, the primary biochemical output for efficacy, although results are inconclusive. In the mouse model for Krabbe disease, transduced MSCs delivered to the brain or the peritoneum had no effect on disease progression. In conclusion, FV vectors are suited to gene therapy of MSCs since they offer the highest transduction efficiency from a single round of transduction, while MSC based gene therapy strategies for Krabbe disease or metachromatic leukodystrophy are unlikely to offer clinical benefit.

Includes bibliographical references (leaves 131-137).
Of the many vaccine trials which have taken place, the most promising results have been obtained from the recent phase 3 clinical trial which tested the ability of a dual protein and Canarypox virus recombinant to protect humans against HIV-1 infections. Because poxviruses are being developed as vaccine vectors against a number of diseases, it is important to continue the search for novel poxvirus vectors, in particular, those that do not cross-neutralize one another. This thesis describes the preliminary work performed on the development of Penguinpox virus (PEPV) as a vaccine vector.

Reverse genetics technology has facilitated the genetic manipulation of many nonsegmented negative strand RNA viruses (NNSV), including Sendai virus (SeV). It has provided the means to exploit SeV as a vector for vaccines or gene therapy, via insertion of extra-numeral transcription units (ENTU) encoding heterologous genes. SeV contains six contiguous genes flanked by the leader (Ld) and trailer (Tr) promoter sequences (3 'Ld-N-P-M-F-HN-L-Tr 5 '). Characterisation of an infectious clone containing the entire antigenome of SeV confirmed that it contained all elements necessary for recombinant (r)SeV rescue. We inserted a heterologous gene encoding respiratory syncytial virus (RSV) fusion (F) protein between the Nand P genes of SeV. A recombinant virus (rSeV/RSV F) was successfully rescued and expressed functional RSV F protein. Importantly, it induced protective immunity in a BALB/c mouse model, demonstrating its vaccine potential against RSV. A common consequence of ENTU insertion is growth attenuation. Therefore, we hypothesised that a rSey containing a bicistronic gene, in which the second cistron encodes a heterologous gene, would circumvent this limitation. To address this, we used a 9nucleotide sequence with known Internal Ribosome Entry Site (IRES) activity. Furthermore, . multiple linked repeats of this IRES increased the expression efficiency of the second cistron. We inserted the Renilla luciferase (rLlle) ORF, preceded by 1, 3 or 7 synthetic IRES copies within the SeV N gene 5' untranslated region. We successfully rescued the corresponding rSeVs, thereby confirming the feasibility of generating bicistronic NNSVs. We confirmed luciferase expression in infected cells and that the number of IRES copies influenced expression efficiency. However, luciferase activity was invariably lower than that from rSeV expressing rLuc from an ENTU. Importantly, and in contrast to rSeV ENTU constructs, our data demonstrated that bicistronic rSeVs were not growth attenuated. Therefore, our approach offers a novel way to express heterologous genes from NNSVs.

As an arbovirus, bluetongue virus (BTV) needs to replicate in its vector, biting midges of the genus Culicoides, in order to be transmitted between susceptible vertebrate hosts, which include wild and domestic ruminants. However, the latest identified serotypes of BTV, namely BTV-25, BTV 26 and BTV-27, have shown an inability to effectively amplify in BTV-susceptible Culicoides sonorensis derived KC cells, or in adult C. sonorensis midges. They also show a ‘non-conventional’ phenotype, specificity for small ruminants, where particularly in goats, longer viraemia, direct contact transmission and asymptomatic infection is observed. This, together with the failure of most earlier serological and RT PCR based assays to detect these ‘novel’, genetically distinct BTV serotypes, may help to explain why these viruses went unnoticed until recently. However, with the improvement of molecular diagnostic assays, increased awareness and surveillance, it appears that the current circulation of these non conventional viruses is more common than initially thought, and their number is expected to increase with reports of additional novel virus strains from Mongolia. The work presented in this thesis is designed to further characterise virus isolate KUW2010/02, a BTV strain belonging to the non-conventional BTV serotype 26 (BTV 26), with particular interest in the viral determinants that restrict vector competence. A reverse genetics system for BTV, established in the Arbovirus Molecular Research Group at the Pirbright Institute, was used to generate specific reassortant viruses in mammalian BSR cells, containing genome segments derived from the BTV-1 reference strain and from BTV-26. The replication of these ‘engineered’ viruses was studied in KC cells, as a model for BTV vector interactions. Four genome segments from BTV-26 were identified that are associated with a restriction of BTV replication in both Culicoides cells (in vitro), and in adult Culicoides (in vivo). These include: genome segment 1 (Seg-1) encoding the viral RNA-dependent-RNA-polymerase VP1; Seg-2, encoding outer capsid protein VP2; Seg-3, encoding the sub-core shell protein VP3; and Seg 7, encoding core surface protein VP7. Further investigations in vitro, revealed an inability of BTV-26, or the reassortant virus containing outer-capsid protein VP2 from BTV-26, to bind to the outer surface of KC cells even though they did bind efficiently to BSR cells. This indicates the absence of a suitable binding receptor for VP2 of BTV-26 on the surface of C. sonorensis cells. The polymerase (VP1) and sub-core (VP3) proteins of BTV-26 were shown to be functional, at least during the initial stages of infection, in a Culicoides cell environment at 27 °C, with detection of low levels of transcription and non structural protein synthesis in these cells. However, a block at an unknown post transcriptional stage(s), restricts the successful completion/amplification of replication and dissemination to other cells, by the BTV viruses containing these genes (Seg 1/VP1, Seg-3/VP3). The core surface protein (VP7) of BTV-26 caused only a partial restriction, detected as a delay in BTV replication in KC cells, with virus titres eventually reaching the same levels as for BTV-1. Mutagenesis studies of VP7 revealed that the lower domain of VP7 of BTV 26 was responsible for this delayed growth phenotype in KC cells. Reassortment (exchange) of segments can occur between BTV-26 and a conventional isolate (BTV-1), confirming the classification of BTV-26 [KUW2010/02] as a member of the species Bluetongue virus. This also indicates the importance of providing a better understanding of these non-conventional viruses (such as BTV-26) since reassortment with co-circulating conventional strains could occur in the field, potentially generating progeny virus strains with novel and unpredictable phenotypes (e.g. with both higher pathogenesis and efficient direct contact transmission). This work excludes adult C. sonorensis midges as a potential vector for BTV 26 strongly suggesting that it is a non vector borne virus. In general, the results obtained shed some light on BTV-Culicoides interactions, a field of research that has not been extensively studied, and demonstrate that these non-conventional viruses are powerful models for the further study of BTV replication in Culicoides systems.

Doctor of Philosophy
Department of Chemical Engineering
Peter Czermak
Peter H. Pfromm
Large-scale insect cell culture utilizing the baculovirus expression vector system (BEVS) can be used to produce biopharmaceuticals such as vaccines and therapeutic proteins. Biopharmaceutical production processes are generally complex and sensitive to many process parameters and changes, but on-line monitoring in this area is relatively limited and the fundamental understanding of the intricate relationships between significant process parameters and the process outcome, especially on the multi-liter or multi-m3 scale, is rarely conclusive. Dielectric spectroscopy (DS), which is based on the frequency dependent measurement of the passive dielectric properties of materials, was applied to large-scale insect cell cultures infected with a baculovirus under low multiplicity of infection conditions to produce a recombinant protein of the virus-like particle class. DS not only allowed the qualitative monitoring of the infection and recombinant protein production process within the culture in real-time but also the detection of important culture events, e.g. the peak in baculovirus production/concentration. Additionally, DS seemed to be able to serve as a predictive tool for the overall recombinant protein yield early in the process. Partial Least Square models were successfully developed allowing monitoring of the cultures progress in terms of cell density, size, and even nutrient concentration replacing the need for discrete sampling and therefore reducing contamination risks. In summary, DS has been demonstrated to have the potential to increase bioprocess understanding and the repeatability of recombinant protein production in the BEVS but ultimately also to satisfy the increased requirements for process monitoring as delineated recently in the Process Analytical Technology initiative by the Food and Drug Administration.

Human norovirus (NoV) is the leading cause of acute non-bacterial gastroenteritis worldwide. Despite the significant health, emotional, and economic burden caused by human NoV, there are no vaccines or therapeutic interventions for this virus. This is due in major part to the lack of a cell culture system and an animal model for human NoV infection.

Thus, a vector-based vaccine may be ideal for controlling this disease. The major capsid gene (VP1) of a human NoV was inserted into the VSV genome at the glycoprotein (G) and large (L) polymerase gene junction. Recombinant VSV expressing VP1 protein (rVSV-VP1) was recovered from an infectious cDNA clone of VSV. Expression of the capsid protein by VSV resulted in the formation of human NoV virus-like particles (VLPs) that are morphologically and antigenically identical to the native virions. Recombinant rVSV-VP1 was attenuated in cultured mammalian cells as well as in mice. Mice inoculated with a single dose of rVSV-VP1 stimulated a significantly stronger humoral and cellular immune response compared to baculovirus-expressed VLP vaccination. These results demonstrated that that the VSV-based human NoV vaccine induced strong humoral, cellular, and mucosal immunity in a mouse model.

To further improve the safety and efficacy of the VSV-based human NoV vaccine, the gene for the 72kDa heat shock protein (HSP70) was inserted into rVSV and rVSV-VP1 vectors as an adjuvant, which resulted in construction of recombinant VSV expressing HSP70 (rVSV-HSP70) and VSV co-expressing human NoV VP1 protein and HSP70 (rVSV-HPS70-VP1), respectively. At the same inoculation dose, both rVSV-HSP70-VP1 and rVSV-VP1 triggered similar levels of specific immunity, even though VP1 expression by rVSV-HSP70-VP1 was approximately five-fold less than that of rVSV-VP1. To compensate for the reduced VP1 expression levels, the inoculation dose of rVSV-HSP70-VP1 was increased five-fold or same dosage of rVSV-VP1 and rVSV-HSP70 was combined vaccinated. Mice immunized with five does of rVSV-HSP70-VP1 or those receiving combined vaccination generated significantly higher mucosal and/or T cell immunity than those immunized with rVSV-VP1 alone (P<0.05). Therefore, this data indicates that insertion of HSP70 into the VSV vector further attenuates the VSV-based vaccine and HSP70 enhances the human NoV-specific immunities.

To determine whether the VSV-based human NoV vaccine confers protection from human NoV challenge, a gnotobiotic pig model was developed. Newborn gnotobiotic piglets vaccinated intranasally with rVSV-based vaccine (rVSV-VP1) produced high levels of specific serum IgG and fecal and vaginal IgA antibody. Three weeks after vaccination, piglets were orally challenged with human NoV. All three piglets in the unvaccinated challenged group developed histopathologic lesions typical of human NoV infection in the duodenum and proximal jejunum on day 5 post-challenge. However, only one of five vaccinated piglets exhibited focal epithelium loss and villous atrophy, and mild edema in the small intestines. Immunofluorescent assay showed that a large amount of human NoV antigens were detected in duodenum, jejunum, and ileum of the challenge control group but not vaccinated group. These results demonstrate that the rVSV-based human NoV vaccine triggered partially protective immunity in swine and protected gnotobiotic pigs from challenge by human NoV.

Oncolytic virotherapy is emerging as a new treatment option for cancer patients. At present, there are relatively few oncolytic virus clinical trials that are underway or have been conducted, however one virus that shows promise in pre-clinical models is Vesicular Stomatitis Virus (VSV). VSV is a naturally occurring oncolytic rhabdovirus that has the ability to preferentially replicate in and kill malignant versus normal cells. VSV also has a low seroprevalence, minimal associated morbidity and mortality in humans, and simple non-integrating genome that can be genetically manipulated, making it an optimal oncolytic vector. Currently, many labs are using a variety of different strategies including inserting trans genes that can modulate the innate and adaptive immune response. VSV can also be retargeted by altering its surface glycoprotein (G) or be made replication incompetent by deleting the G protein. Currently, our lab has engineered a series of new recombinant VSVs, incorporating either the murine p53 (mp53), IPS-1, or TRIF transgene. mp53, IPS-1 and TRIF were incorporated into the normal VSV-XN2 genome and mp53 was also incorporated into the mutated VSV-ΔM vector generating VSV-mp53, VSV-IPS-1, VSV-TRIF and VSV-ΔM-mp53. Our data using these new viruses indicate that these viruses preferentially replicate in and kill transformed versus non-transformed cells and efficiently express the transgene. However, despite the ability for VSV-IPS-1 and VSV-TRIF to induce a robust type 1 IFN response, VSV-ΔM-mp53 was the only construct that had reduced toxicity and elicited an increased anti-tumor response against a syngeneic metastatic mammary tumor model. VSV- ΔM-mp53 treatment lead to a reduction in IL-6 and IP-10 production, an increase in tumor specific CD8+ T cells, and immunologic memory against the tumor. Collectively these studies highlight the necessity for additional VSV construct development and the generation of new clinically relevant treatment schema.

L' ENGROGUIMENT DE LES CUCURBITÀCIES: DIAGNÓSTIC I MICROSCOPIA DE LES RELACIONS VIRUS-PLANTA I VIRUS-VECTOR

La malaltia de l'engroguiment de les cucurbitàcies, de gran importància, en els cultius del sud-est espanyol, està produïda per dos Closteroviridae transmesos per mosques blanques, el virus del fals engroguiment de la remolatxa (BPYV) i el virus de l'engroguiment enanitzant de les cucurbitàcies (CYSDV). BPYV és transmès específicament i de forma semipersistent per Trialeurodes vaporariorum (Westwood) i CYSDV per Bemisia tabaci (Gennadius). En els darrers anys, encara que s'ha produït un gran avenç en el coneixement a escala molecular, alguns dels aspectes de la relació virus-planta i virus-vector són encara desconeguts. L' objecte d'aquest treball ha estat l'estudi de les relacions que BPYV i CYSDV estableixen amb les cucurbitàcies i les mosques blanques per ajustar-lo a un dels models virus-planta-vector descrit.

Es varen establir tres grups d'estudis: (i) en el primer, destinat a l'establiment d'una tècnica de diagnòstic i a la propagació de BPYV i CYSDV, durant dos anys es varen realitzar mostreigs dirigits a plantes amb símptomes d'engroguiment en cultius protegits de meló i cogombre del sud-est espanyol i en cultius de meló a l'aire lliure de la zona de Lleida, varen evaluar-se distintes tècniques de diagnòstic i es realitzaren assaigs de transmissió amb els vectors, (ii) en el segon grup varen abordar-se les realcions virus-planta, mitjançant estudis citopatològics i de localització en els que va assajar-se immunomarcatge amb or coloidal (IGL) i hibridació in situ (ISH) amb sondes d'ARN i (iii) en el tercer varen estudiar-se les relacions virus-vector, mitjançant comparació de l'anatomia interna de T.vaporariorum i de B. tabaci relacionada amb la transmissió, varen assajar-se dues tècniques d'inclusió en resina per IGL i es realitzaren proves de IGL i ISH.

En el sud-est espanyol, l'engroguiment està associat amb BPYV i CYSDV, detectant-se aquest darrer en un major nombre de mostres. En canvi, a l'àrea de Lleida, aquests virus no es detecten i únicament trobem virus transmesos per pugons,essent el virus del mosaic del cogombre (CMV) el més abundant, juntament amb el virus del mosaic de la sindria soca 2 (WMV-2) i el virus del mosaic groc del carbassó (ZYMV). En aquesta zona, els casos d'engroguiment no associats a virus són majors quan les mostres són recollides al final del cultiu.

Els estudis citopatològics realitzats amb BPYV i CYSDV mostraren la presència d'alteracions a les cèl.lules del floema, a les que es va observar la proliferació de vesícules membranoses citoplasmàtiques amb distinta morfologia, grans agregats de partícules virals, hipertròfia en mitocòndries i la desorganització de les membranes dels cloroplasts. Cap dels virus estudiats produeix la formació de dipòsits cònics de material electrodens a la zona del plasmalema característics del virus de l'engroguiment infecciós de l'enciam (LIYV), membre tipus dels Crinivirus. Els resultats de les proves de IGL i IHS varen confirmar la limitació d'aquests virus al floema. Les proteïnes de coberta de CYSDV i LIYV es varen detectar en el citoplasma de les cél.lulas acompanyants del floema; i mitjançant ISH, BPYV i CYSDV varen localitzar-se en el citoplasma d'aquestes cèl.lules.

El canal alimentari de T. vaporariorum presenta unes característiques anatòmiques semblants a les descrites i observades a B. tabaci, de manera que les diferències en el seu comportament com a insectes vectors no poden explicar-se amb aquest tipus d'observacions. Les proves d' IGL mostraren la localització tant de CYSDV com de LIYV en els trams inicials del canal alimentari i de l'intestí anterior del seu insecte vector, B. tabaci, descartant-se la possible circulació d'aquests virus a l'interior del cos de l' insecte.

En base als resultats obtinguts, el model de transmissió que més s'ajusta a la transmissió semipersistent dels Crinivirus per mosques blanques és el de ingestió-regurgitació.
EL AMARILLEO DE LAS CUCURBITÁCEAS: DIAGNÓSTICO Y MICROSCOPÍA DE LAS RELACIONES VIRUS-PLANTA Y VIRUS-VECTOR


La enfermedad del amarilleo de las cucurbitáceas, de gran importancia en los cultivos del sudeste español, está producida por dos Closteroviridae transmitidos por moscas blancas, el virus del falso amarilleo de la remolacha (BPYV) y el virus del amarilleo enanizante de las cucurbitáceas (CYSDV). BPYV es transmitido específicamente y de modo semipersistente por Trialeurodes vaporariorum (Westwood) y CYSDV por Bemisia tabaci (Gennadius). En los últimos años, aunque se ha producido un gran avance en el conocimiento a escala molecular, algunos de los aspectos de la relación virus-planta y virus-vector son todavía desconocidos. El objeto de este trabajo ha sido el estudio de las relaciones que BPYV y CYSDV establecen con las cucurbitáceas y las moscas blancas para ajustarlo a uno de los modelos virus-planta-vector descrito.

Se abordaron tres grupos de estudios: (i) en el primero, destinado al establecimiento de una técnica de diagnóstico y a la propagación de BPYV y CYSDV, durante dos años se realizaron muestreos dirigidos a plantas con síntomas de amarilleo en cultivos protegidos de melón y pepino del sudeste español y en cultivos de melón al aire libre de la zona de Lleida, se evaluaron distintas técnicas de diagnóstico y se realizaron ensayos de transmisión con los vectores, (II) en el segundo se abordaron las relaciones virus-planta, mediante estudios citopatológicos y de localización en los que se ensayó inmunomarcaje con oro coloidal (IGL) e hibridación in situ (ISH) con sondas de ARN; y (III) en el tercero se estudiaron las relaciones virus-vector, se realizó un estudio comparativo de la anatomía interna de T.vaporariorum y de B. tabaci relacionada con la transmisión, se ensayaron dos técnicas de inclusión para IGL y se realizaron pruebas de IGL e ISH.

En el sudeste español, el amarilleo está asociado con BPYV y CYSDV, detectándose este último en un mayor número de muestras. En cambio, en el área de Lleida, éstos virus no se detectan y únicamente están presentes virus transmitidos por pulgones, siendo el virus del mosaico del pepino (CMV) el más abundante, junto con el virus del mosaico de la sandía cepa 2 (WMV-2) y el virus del mosaico amarillo del calabacín (ZYMV). En esta zona, los casos de amarilleo no asociados a virus son mayores cuando las muestras son recogidas al final del cultivo.

Los estudios citopatológicos realizados con BPYV y CYSDV mostraron la presencia de alteraciones en las células del floema, en las que se observó la proliferación de vesículas membranosas citoplasmáticas con distinta morfología, grandes agregados de partículas virales, hipertrofia en mitocondrias y la desorganización de las membranas de los cloroplastos. Ninguno de los virus estudiados produce la formación de depósitos cónicos de material electrodenso en la zona del plasmalema característicos del virus del amarilleo infeccioso de la lechuga (LIYV), miembro tipo de los Crinivirus. Los resultados de las pruebas de IGL e IHS confirmaron la limitación de estos virus al floema. Las proteínas de cubierta de CYSDV y LIYV se detectaron en el citoplasma de las células acompañantes del floema; y mediante ISH, BPYV y CYSDV se localizaron en el citoplasma de estas células.

El canal alimentario de T. vaporariorum presenta unas características anatómicas semejantes a las de B. tabaci, por lo que las diferencias en su comportamiento como insectos vectores no se pueden explicar con este tipo de observaciones. Las pruebas de IGL mostraron la localización tanto de CYSDV como de LIYV en los tramos iniciales del canal alimentario y del intestino anterior de su insecto vector, B. tabaci, descartándose la posible circulación de estos virus en el interior del cuerpo del insecto.

En base a los resultados obtenidos, el modelo de transmisión que más se ajusta a la transmisión semipersistente de los Crinivirus por moscas blancas es el de ingestión-regurgitación.
CUCURBITS YELLOWING: DIAGNOSIS AND MICROSCOPY OF VIRUS-HOST AND VIRUS-VECTOR RELATIONSHIPS


Cucurbit yellowing disease, specially important in souteastern Spain, is caused by two Closteroviridae transmitted by whiteflies, Beet pseudo-yellows virus (BPYV) and Cucurbit yellow stunting disorder virus (CYSDV). BPYV is transmitted specifically in a semipersistent manner by Trialeurodes vaporariorum (Westwood) (Homoptera:Aleyrodidae) and CYSDV by Bemisia tabaci (Gennadius). In recent years, despite much progress in their knowledge at molecular level, some aspects of the relationships between viruses and their host, and vectors remain still unknown. The objective of this work was to study the relationships of BPYV and CYSDV with cucurbits and with whiteflies to adjust a described virus-host-vector model.

Three groups of experiences were envisaged. (I) In the first one, with the goal to find a suitable procedure for detection of BPYV and CYSDV and to propagate these viruses, cucumber and melon plants with yellowing symptoms from southeastern Spain greenhouses and from melon crops growing open field in Lleida basin were collected for two years; different detection techniques were tested and vector transmission assays were made. (II) In the second one, virus-host relations were envisaged through cytopathological and localisation, by immunogold labelling (IGL) and in situ hibridisation (ISH) with ARN probes, studies. (III) And in the third group, virus-vector relationships were studied, a comparative study of the alimentary canal, that is related with virus transmission, of T. vaporariorum and B. tabaci was made; two embedding protocols for IGL were compared; and IGL and ISH were made.

In southeastern Spain, yellowing symptoms are associated with BPYV and CYSDV infections. The number of CYSDV infected samples was larger than BPYV ones. In Lledia basin, BPYV and CYSDV were not detected, although aphid-transmitted viruses are present. Cucumber mosaic virus (CMV), Watermelon mosaic virus-2 (WMV-2) and Zucchini yellows mosaic virus (ZYMV) were the most abundant viruses found. Herein, the number of yellowing plants testing negative was higher when surveys were conducted at the end of the growing season.

Ultrastructural changes induced by BPYV and CYSDV were detected in phloem cells, exhibiting formation of membranous cytoplasmic vesicles with different morphology, accumulation of virus particles in large masses, hypertrophied mitochondrion and degeneration of chloroplasts. No conical electrondense plasmalemma deposits, typical of Lettuce infectious yellows virus (LIYV) -the type member of the genus Crinivirus-; were observed in BPYV and CYSDV infections. IGL and ISH analyses confirmed that BPYV and CYSDV are phloem-limited. CYSDV and LIYV coat proteins were detected in the cytoplasm of companion cells cytoplasm; and by ISH, BPYV and CYSDV were localised in the cytoplasm.

The alimentary canal of T. vaporariorum shows anatomical characteristics similars to those of B. tabaci, so their different vector behaviour could not be explained with this kind of observations. CYSDV and LIYV are detected in the initial part of the alimentary canal and foregut of B. tabaci, thus excluding the possible circulation of these viruses in the vector's body.

The data obtained suggest that the most adapted transmission model to whiteflies transmitted Crinivirus in a semipersistent manner is the ingestion-regurgitation model, although some factors involved in this process are still unknown.

[EN] Plant viruses are obligate intracellular parasites which were used to develop recombinant plant virus vectors to express heterologous proteins and to modify endogenous metabolic pathways of natural products in plants. The main limitation of many plant virus-based systems is the difficulty to co-express various heterologous proteins in the same cell with proper subcellular localization, which is a crucial question in metabolic engineering. This work provides a solution to overcome this problem by using a potyvirus-based vector system. Potyviruses (genus Potyvirus, family Potyviridae) are plus-strand single-stranded RNA viruses, which have a genome expression strategy that allows the equimolar production of most viral proteins. On the basis of an infectious clone of Tobacco etch virus (TEV), Bedoya et al. (2010) developed an expression system in which the RNA-dependent RNA polymerase (NIb) gene was replaced by an expression cassette, harboring several heterologous proteins. This viral vector was able to express three fluorescent proteins with nucleocytoplasmic localization in equimolar amounts in transgenic tobacco plants in which NIb was supplemented in trans. Despite of the apparent simplicity of potyvirus genome expression strategy, foreign cDNA insertion is a complicated task. Thus, our first goal was to analyze the effect of gene insertion on TEV genome stability. As a result of this work, a novel insertion position was discovered at the amino-terminal end of the potyvirus polyprotein, which opened the possibility to explore new questions of recombinant protein expression. Since metabolic pathways are highly compartmentalized, proper subcellular targeting of enzymes is an essential task. Thus, our second objective centralized on the subcellular targeting of expressed proteins from the TEV-based viral vector. cDNAs coding for the green fluorescent protein (GFP) fused to chloroplast, nucleus and mitochondria targeting signal sequences were inserted into the newly described amino-terminal insertion position or into an internal site, replacing the NIb cistron. Our results showed that for protein delivery to chloroplasts and mitochondria, foreign genes have to be inserted at the amino-terminal site of the viral vector, but for nuclear delivery, both insertion positions are suitable. The last objective of this work was to investigate whether the potyvirus-based vector was able to express an entire heterologous multistep biosynthetic pathway in plant cells. For this aim we purposed to produce lycopene, a plant pigment with health promoting properties. To do so, we inserted cDNAs coding for the enzymes of a three-step metabolic pathway of bacterial origin into the potyvirus-based vector. Infected tobacco plants developed orange symptoms indicating lycopene accumulation, which was confirmed by high-performance liquid chromatography analysis and microscopy observations. Our results also illustrated that the sole expression of Pantoea ananatis phytoene synthase, crtB, is enough to induce carotenoid accumulation, conferring yellow coloration to the infected tissue and serves as reporter system to visually track viral infection in several plant species.
[ES] Los virus de plantas son parásitos intracelulares obligados que han sido utilizados para desarrollar vectores virales y expresar proteínas heterólogas y modificar rutas metabólicas endógenas de productos naturales. La principal limitación de muchos sistemas basados en virus de plantas es la dificultad de coexpresar diversas proteínas heterólogas en la misma célula con la localización subcelular apropiada, lo cual es una cuestión crucial en ingeniería metabólica. Este trabajo presenta una solución para superar este problema mediante el uso de un vector viral basado en un potyvirus. Los potyvirus (género Potyvirus, familia Potyviridae) son virus de RNA de cadena positiva simple que tienen una estrategia de expresión génica que permite la producción de la mayoría de las proteínas virales en cantidades equimolares. Basado en un clon infeccioso del virus del grabado del tabaco (Tobacco etch virus, TEV) Bedoya et al. (2010) desarrollaron un sistema de expresión en el que el gen de la RNA polimerasa dependiente de RNA (NIb) fue sustituido por un casete de expresión, que albergaba varias proteínas heterólogas. Este vector viral fue capaz de expresar tres proteínas fluorescentes con localización nucleocitoplásmica en cantidades equimolares en plantas de tabaco transgénicas que complementaban el cistron NIb en trans. A pesar de la aparente simplicidad de la estrategia de expresión génica de los potyvirus, la inserción de un cDNA foráneo es una tarea complicada. Por lo tanto, nuestro primer objetivo fue analizar el efecto de la inserción en la estabilidad del genoma de TEV. Como resultado de este trabajo, descubrimos una nueva posición de inserción en el extremo amino-terminal de la poliproteína viral que nos permitió explorar otras cuestiones sobre la expresión de proteínas recombinantes. Dado que las vías metabólicas son muy compartimentalizadas, la adecuada localización subcelular de enzimas es una tarea esencial en ingeniería metabólica. Por eso, nuestro segundo objetivo se centró en la distribución de las proteínas heterológas expresadas con el vector viral a diferentes orgánulos subcelulares. cDNAs que codificaban la proteína fluorescente verde (green fluorescent protein, GFP) fusionada a péptidos señal se insertaron en la nueva posición amino-terminal y en un sitio interno, sustituyendo el cistrón NIb, para enviarla al cloroplasto, núcleo y a la mitocondria. Nuestros resultados mostraron que para la distribución de proteínas al cloroplasto y mitocondria, los genes foráneos deben ser insertados en el sitio amino-terminal del vector viral, pero para la distribución nuclear, ambas posiciones son adecuadas. El último objetivo de este trabajo fue estudiar si el vector viral basado en potyvirus es capaz de expresar una ruta biosíntética de múltiples pasos en células vegetales. Para ello nos propusimos producir licopeno, un pigmento vegetal con propiedades beneficiosas para la salud humana. Para ello, insertamos un cDNA que codificaba las enzimas de una ruta metabólica de tres pasos de origen bacteriano en el vector viral. Las plantas de tabaco infectadas con el vector viral desarrollaron síntomas de color naranja indicando la acumulación de licopeno, que fue confirmado por análisis de cromatografía líquida de alta eficacia y observaciones de microscopía. Nuestros resultados también ilustraron que la sola expresión de la fitoeno sintasa de Pantonea ananatis, crtB, es suficiente para inducir la acumulación de carotenoides que confieren una coloración amarilla al tejido infectado y sirve como sistema reportero visual en varias especies de plantas.
[CAT] Els virus de plantes són paràsits intracel·lulars obligats que han estat utilitzats per a desenvolupar vectors virals i expressar proteïnes heteròlogues y modificar rutes metabòliques endògenes de productes naturals silenciant certs gens o expressant factors de transcripció i enzims metabòlics. La principal limitació de molts sistemes basats en virus de plantes és la dificultat de coexpressar diverses proteïnes heteròlogues en la mateixa cèl·lula amb la localització subcel·lular apropiada, cosa que és una qüestió crucial en enginyeria metabòlica. Aquest treball presenta una solució per a superar aquest problema mitjançant l'ús d'un vector viral basat en un potyvirus. Els potyvirus (gènere Potyvirus, família Potyviridae) són virus d'RNA de cadena positiva simple que tenen una estratègia d'expressió gènica que permet la producció de la majoria de les proteïnes virals en quantitats equimolars. Basat en un clon infecciós del virus del gravat del tabac (Tobacco etch virus, TEV) Bedoya et al. (2010) van desenvolupar un sistema d'expressió en el qual el gen de l'RNA polimerasa depenent d'RNA (NIb) va ser substituït per un casset d'expressió, que albergava diverses proteïnes heteròlogues. Aquest vector viral va ser capaç d'expressar tres proteïnes fluorescents amb localització nucleocitoplàsmica en quantitats equimolars en plantes de tabac transgèniques que complementaven el cistró NIb en trans. Malgrat l'aparent simplicitat de l'estratègia d'expressió gènica dels potyvirus, la inserció d'un cDNA forà és una tasca complicada. Per tant, el nostre primer objectiu va ser analitzar l'efecte de la inserció en l'estabilitat del genoma de TEV. Com a resultat d'aquest treball, hem descobert una nova posició d'inserció en l'extrem amino terminal de la poliproteïna viral que ens va permetre explorar altres qüestions sobre l'expressió de proteïnes recombinants. Atès que les vies metabòliques són molt compartimentalitzades, l'adequada localització subcel·lular d'enzims és una tasca essencial en enginyeria metabòlica. Per açò, el nostre segon objectiu es va centrar en la distribució de les proteïnes heteròlogues expressades amb el vector viral a diferents orgànuls subcelul·lars. cDNAs que codificaven la proteïna fluorescent verda (green fluorescent protein, GFP) fusionada a pèptids senyal es van inserir en la nova posició amino terminal i en un lloc intern, substituint el cistró NIb, per a enviar-la al cloroplast, nucli i al mitocondri. Els nostres resultats van mostrar que per a la distribució de proteïnes al cloroplast i mitocondri, els gens forans han de ser inserits en el lloc amino terminal del vector viral, però per a la distribució nuclear, ambdues posicions són adequades. El lloc amino terminal va resultar ser més adequat per a produir quantitats més grans de proteïnes recombinants, però el lloc d'inserció intern va demostrar ser més estable. Sobre la base d'aquests resultats, hem sigut capaços de distribuir dues proteïnes fluorescents diferents als cloroplasts i nuclis des d'un únic vector viral. L'últim objectiu d'aquest treball va ser estudiar si el vector viral basat en potyvirus és capaç d'expressar una ruta biosintètica de múltiples passos en cèl·lules vegetals. Per açò ens vam proposar produir licopè, un pigment vegetal amb propietats beneficioses per a la salut humana. Per això inserírem un cDNA que codificaba els tres enzims de una ruta metabòlica de tres passos d'origen bacterià en el vector viral. Les plantes de tabac infectades amb el vector viral van desenvolupar símptomes de color taronja indicant l'acumulació de licopè, que va ser confirmat per anàlisi de cromatografia líquida d'alta eficàcia i observacions de microscòpia. Els nostres resultats també van il·lustrar que la sola expressió de fitoè sintasa de Pantonea ananatis, crtB, és suficient per a induir l'acumulació de carotenoides que confereixen una colora
Majer, E. (2016). Metabolic engineering of plants using a disarmed potyvirus vector [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/68477
TESIS

Prototypic foamy virus (PFV)-based vectors are currently made by transient transfection. Continuous vector production by cells in which PFV proteins are stably led would allow rapid, reproducible generation of large quantities of vector. Previous attempts at constructing packaging cell lines have resulted in very low titre production. Here, we utilise a method described to generate a stable HIV-1 producing cell-line in an attempt to produce high titres of PFV vector. PFV gag-pol or env genes were stably transduced into a variety of cell lines using an MLV-based delivery system. The resultant cell lines were positive for stable DNA integration and for high levels of protein expression, and produced between 10³ and 10⁵ infectious units of PFV vector. However the cells gradually reduced protein production which finally ceased completely after 4-5 weeks, possibly due to cytotoxicity.

Gene therapy is the introduction or alteration of genetic material with the intention to treat disease. To support this aim, viruses have been modified, with elements linked to viral pathogenicity removed from their genome and replaced by the genetic material to be delivered. Gene therapy vectors based on lentiviruses have many advantages, such as the ability to transduce non-dividing cells and to target specific cell types via pseudotyping. They have been successfully used in ex vivo clinical trials for several haematopoietic stem cell disorders. Lentiviral vectors, however, suffer from substantially lower titres than the more popular adeno-associated virus (AAV)-based vectors and therefore have limited applicability for in vivo gene therapy which requires much greater quantities of virus. The main aim of this thesis was to investigate strategies to improve lentiviral vector productivity during manufacture, in order to increase the likelihood of lentiviruses being adopted for disease treatment. Initial experiments were based on the lentiviral vector manufacturing process currently being developed by the United Kingdom Cystic Fibrosis Gene Therapy Consortium for the generation of highly concentrated, purified lentivirus for clinical use. Supplementation of FreeStyle 293 Expression Medium used during upstream processing was attempted, but none of the assessed supplements led to significant increases in lentiviral vector production. Investigation into intrinsic immunity to viral infection indicated that over-expression of the protein kinase RNA-activated (PKR) led to lower production titres, but over-expression of its inhibitors was not successful at increasing titres. The focus then shifted to reducing, or 'knocking-down', inhibitory factors present in the host cells, which could adversely affect viral titres. Investigation of the published HIV-1 literature revealed a possible 152 candidate inhibitory factors described as having a negative impact on HIV-1 replication in the late stages of the life cycle of the virus. A novel siRNA screen was developed to assess the effect of ‘knock-down' of inhibitory factors on lentiviral vector titre. Application of the screen to 89 candidate inhibitory factors identified nine genes which, when knocked-down, resulted in increased lentiviral vector production by more than 40%. Further work will be necessary to understand the role of the inhibitory factors in lentiviral vector production, but novel cell lines in which genes encoding these factors have been permanently deleted from producer cells could lead to higher titres, reducing costs in the manufacture of lentiviral vectors and making in vivo gene therapy more feasible from a health economics perspective.

Mosquito-borne diseases continue to be a burden to global health. The viruses that cause these diseases are maintained in nature through a biological transmission cycle involving susceptible vertebrate and mosquito hosts. While knowledge of the interactions occurring between mosquito-borne viruses and vertebrates is considerable, much less is known about the interactions of these viruses with their disease vectors. Studies with Drosophila melanogaster have been important in understanding how insects respond to viral infections. However, mosquitoes and the viruses they vector have co-evolved during a long period of time. Unfortunately, many of the genetic advantages of a fly model are not available when working with mosquitoes. Nevertheless, a sequenced genome, and molecular tools such as high-throughput sequencing and RNAi knockdown are helping to bridge these gaps. Here we describe several additional tools for the study of virus-vector interactions in the mosquito.
Ph. D.

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第13073号
論農博第2843号
新制||農||1046(附属図書館)
学位論文||H29||N5029(農学部図書室)
33224
京都大学大学院農学研究科農学専攻
(主査)教授 北島 宣, 教授 土井 元章, 教授 田尾 龍太郎
学位規則第4条第2項該当

In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels.
Ph. D.

Mosquito species of the Culex genus are the enzootic vectors for several bird-associated viruses that cause disease in humans. In Europe, these viruses include Sindbis (SINV), West Nile and Usutu viruses. The morphologically similar females of Cx. torrentium and Cx. pipiens are potential vectors of these viruses, but difficulties in correctly identifying the mosquito species have caused confusion regarding their respective distribution, abundance, ecology, and consequently their importance as vectors. Species-specific knowledge from correctly identified field material is however of crucial importance since previous research shows that the relatively unknown Cx. torrentium is a far more efficient SINV vector than the widely recognized Cx. pipiens. The latter is involved in the transmission of several other viruses, but its potential importance for SINV transmission is debated. In this thesis I describe the development of a molecular method for species identification, based on reliably identified males of Cx. torrentium and Cx. pipiens. This identification method was then used in consecutive studies on the distribution and relative abundance of the two species in Sweden and 12 other European countries, SINV field infection rates in mosquitoes identified to species level, and evaluation of potential trap bias associated with common sampling techniques. The results showed that Cx. torrentium is a far more common species in Europe than previously assumed. In Sweden and Finland, it is the dominant species, accounting for 89% of the sampled Culex population. In central Europe, it is equally common to Cx. pipiens, while Cx. pipiens dominates south of the Alps Mountain range. Larvae of both species are often found together in both artificial containers (e.g. car tires) and natural sites. Also, a trapping bias against Cx. torrentium was revealed for CDC-traps. For the first time, SINV was isolated from species-identified Cx. torrentium and Cx. pipiens mosquitoes caught in the field, with Cx. torrentium being superior in infection rates (36/1,000 vs. 8.2/1,000). Future studies on SINV, as well as other mosquito-borne bird viruses in Europe, can hopefully gain from the baseline information provided here, and from principles of vector discrimination discussed in the thesis.

Virus infection of mammalian cells induces several stress mechanisms, including autophagy and type-I interferon (IFN). Autophagy, a cellular homeostatic mechanism in which intracellular materials are sequestered into double-membrane vesicles and targeted to lysosomes for degradation, is also activated in response to virus infection. Most positive single-stranded RNA viruses studied to date utilise autophagy to increase virus replication. IFN is a potent anti-viral mechanism, which can be divided into two parts: (i) induction and secretion of IFN and (ii) IFN signalling and priming of uninfected cells for a rapid response upon infection and induction of an anti-viral state in infected cells. Alphaviruses are medically important RNA viruses. Semliki Forest virus (SFV) provides a well-characterised model for studying alphavirus infection. A number of strains have been identified, which differ in virulence in adult mice. In this thesis three hypotheses were investigated: (i) that SFV infection induces autophagy in cell culture and utilises this response to enhance virus replication, (ii) that the quality, quantity and/or protective efficacy of the IFN response differ between virus strains and between human and murine cells and (iii) that non-structural protein (nsP)-2 and/or nsP3 antagonise the IFN response. SFV4, SFV L10 and SFV A7(74) infection induced autophagy in Huh7 cells as early as one hour post-infection. Pharmacological induction or inhibition of autophagy had no affect on SFV4 replication, except at a very low multiplicity of infection. NsP3, capsid and dsRNA rarely colocalised with the autophagosome marker LC3. Taken together these results indicate that SFV does not use autophagosomes for replication and autophagy is not important in controlling SFV4 infection at a high MOI, at least in Huh7 cells. However, autophagy may be important in controlling SFV4 spread at a low MOI. An IFN bioassay was established. In fibroblasts, SFV4, SFV L10 and SFV A7(74) induced relatively little IFN in comparison to that induced by Sendai virus. In human fibroblasts, similar levels of IFN were induced by all three virus strains. In mouse fibroblasts, SFV4 induced more IFN than SFV L10. Treatment of fibroblasts with IFN prior to infection greatly reduced, but did not abolish, the replication and spread of all three strains. Therefore, SFV is sensitive to IFN. Analysis of IFN signalling demonstrated that all three strains of SFV inhibited STAT1 phosphorylation during infection of fibroblasts. The growth and viability of SFV infected cells varied between human and mouse cells. The complete genetic sequences of SFV L10 and SFV A7(74) were determined using Solexa (Illumina) sequencing and compared to the sequence of SFV4. The sequences of SFV L10 and SFV4 were extremely similar; only seven differences were identified. Multiple amino acid substitutions were identified in SFV A7(74) compared to SFV4, these mostly mapped to nsP3. To investigate the hypothesis that nsP2 and or nsP3 antagonise the IFN response, two virus mutants were studied: SFV4nsP2RDR and SFV4nsP3Δ50. SFV4nsP2RDR encodes a point mutation in the nuclear localisation signal of nsP2, which largely restricts nsP2 to the cell cytoplasm. SFV4nsP3Δ50 contains a deletion of 50 amino acids in the C-terminus hyperphosphorylated region of nsP3. Neither mutant inhibited STAT1 phosphorylation as efficiently as WT SFV4; SFV4nsP2RDR was particularly poor at inhibiting STAT1 phosphorylation. Both mutants induced more IFN in fibroblasts than SFV4. In summary, autophagy had a limited affect on SFV replication. In contrast, strains of SFV were highly sensitive to IFN, but antagonised this response through the nsP2 protein inhibiting STAT1 phosphorylation.

La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population.
Ph. D.

Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis.
Master of Science
Usutu virus (USUV) is an emerging mosquito-borne virus that was first isolated from a mosquito in 1959 in South Africa, and since then, has become a major problem throughout Africa and Europe causing acute to severe infection in dozens of patients. Additionally, this virus is causing massive die-offs in Eurasian blackbird populations. This is particularly problematic because birds play a critical role in ecosystems as they act as forms of pest control, pollinators, and seed dispersers. Depletion of these species could lead to an imbalance and, eventually, collapse of our natural ecosystem. Additionally, there is a growing concern of USUV making its way into the United States, following a similar track of emergence to WNV's introduction in New York in 1999 and its subsequent spread throughout the states. WNV's introduction to the United States was detrimental to native bird populations and humans, and has caused tens of thousands of infections and thousands of deaths since this introduction. Research has shown USUV causes similar disease symptoms to WNV. The self-limiting illness from these viruses typically includes fever and rashes but some infections can result in more severe cases causing inflammation of the brain and surrounding areas. Like many other prominent mosquito-borne viruses, there is no specific treatment or vaccine for WNV or USUV. Because USUV is so closely related to WNV, and their similar characteristics may point towards similar emergence in the United States, it is essential to garner more information on USUV. The overall goal of this thesis was to establish a reliable tool(s) for further characterization of USUV and demonstrate the potential for USUV emergence in the United States. We first developed molecular tools, known as viral clones, that are valuable to the scientific community which allows the manipulation of USUV genetic material to perform further downstream studies. Our objective for this initial study was to create a molecular tool that would behave similarly to their natural, or "parental", virus. The results from this study suggest we have successfully produced these tools. Furthermore, we sought to determine the potential for field-caught mosquitoes from Southwest Virginia, USA to transmit a recently isolated strain of USUV. These data suggest that while these mosquitoes do have the ability to become infected with USUV, they have a limited potential to transmit this virus to animal hosts. Altogether, these studies have allowed us to expand our knowledge on USUV's potential emergence in the United States and develop powerful tools to continue this essential research.

Tick-borne pathogens represent a large threat to the UK and International Public Health authorities. Due to recent changes in legislation, an increase in animal & human movements and changing climate, the UK may now be at an increased risk of importing exotic tick species and their associated pathogens. It is vital to assess the susceptibility of UK tick species to these highly fatal tick-borne viral zoonoses. To date, studies investigating the interaction of many pathogens with their vectors have been hindered due to the lack of a suitable tick transmission model at high containment. This thesis investigates the intrinsic ability of Ixodes ricinus, the most widely distributed tick in Europe and the UK, to acquire, replicate and transmit both Hazara virus (HAZV, a hazard group 2 surrogate for CCHFV) and Crimean–Congo haemorrhagic fever virus (a hazard group 4 pathogen) addressing their potential to act as competent vectors. During the last decade CCHFV has emerged in new areas within Europe and the principle tick vector of CCHFV has been detected within the UK. The development of methods for use with ticks and highly pathogenic viruses within ticks was an essential part of this work. Firstly techniques for the handling, extraction and storage of RNA obtained from I. ricinus ticks were optimised and different endogenous controls were assessed for their ability to amplify mRNA transcripts for use as endogenous controls. The use of the immersion technique for use with I. ricinus nymphs was optimised and working procedures and protocols for handling ticks at containment level 2 and 4 were established. Ixodes ricinus nymphs are susceptible to infection with HAZV with 100% becoming infected 13 days post-immersion. HAZV was able to establish itself within the key target organs of the tick midgut and salivary glands, produce infectious virus particles and transmit virus to 38% of mice. This artificial method of inoculation was optimised for use within the CL4 environment and was used to show that I. ricinus nymphs were not susceptible to CCHFV via immersion. In addition to horizontal transmission, I. ricinus ticks also demonstrated vertical transmission of HAZV through to the adult stage. This is the first time I. ricinus ticks have been assessed for their susceptibility to HAZV and CCHFV and their use in establishing the first high containment In vivo tick feeding model in Europe.

Herpes simplex virus (HSV) is a neurovirulent virus that in the course of natural infection of man predominantly infects sensory neurons. The aim of this project was to develop a safe, nonvirulent HSV, capable of expressing exogenous genes which altered the binding characteristics of the virus so that tropism was directed predominantly to motor nerves. It was envisaged that these viruses could then act as prototypes for gene therapy vectors targeted to the treatment of motor nerve diseases. To achieve this, two mutant viruses were created, RFa and RFb. These contained deletions of the main HSV glycoprotein involved in cellular binding (glycoprotein C). Gene fusions were created of truncated portions of gC (amino acids 377-511(RFa) and amino acids 477-511 (RFb)) to E. coli heat-labile enterotoxin B-subunit (LTB). The gene fusions were inserted in the RL1 gene thereby abolishing expression of the virulence factor ICP34.5. LTB is a ligand which binds to several gangliosides, including GM1 and GM2 which are motor neuron markers. It was hoped that by deletion of the main viral protein involved in adsorption to cells and replacing it with an LTB-containing fusion protein, the tropism of the mutant viruses could be altered to promote an increase in motor neuron infection. RFb was constructed. RFa constructed but could not be purified to homogeneity. This was thought to be due to poor adsorption/penetration or cell-to-cell spread, brought about by expression of the LTB fusion protein. RFb was analysed to determine the effect of expression of the novel LTB fusion protein within the context of the HSV genome. Western blot analysis using antibodies directed against LTB failed to detect expression of the LTB-gC fusion protein. In vitro replication studies showed that the RFb was non-virulent as demonstrated by its inability to replicate in growth arrested 3T6 cells, a phenotype characteristic of HSV which fails to produce ICP34.5. However no marked difference in virus replication kinetics was seen between RFb and wild type HSV (17+) on two motor neuron-like cell lines (NSC-19 and NSC-34).

The present thesis explored ethological and geographical approaches for the investigation of vector-borne parasites. In the first part, the role of associative learning on vector preferences for hosts was investigated through a comprehensive series of behavioural experiments using the vector of dengue and yellow fever diseases, the mosquito Aedes aegypti. To this end, the possibility that the mosquitoes were able to associate unconditional stimuli with particular odours and visual patterns to which they were responsive was explored, but no evidence supporting the hypothesis that associative learning abilities are present in adults of this species was found. A critical review of the literature on learning in mosquitoes conducted afterward allowed the reinterpretation of findings in the field, narrowing the scope of evidence suggesting the existence of these cognitive abilities in some species. In the second part of the thesis, the distribution and evolution of mosquito-borne viruses was investigated with the use of geo-coded environmental data and spatial statistics. Initially, the eco-climates associated with the distribution of Japanese encephalitis virus were described and modelled, allowing the production of a worldwide predictive map defining the probability of each region to develop this disease in the future. Predominating amongst those areas shown to be under high risk were the equatorial regions of South America and Africa. The methodology used to infer such patterns – non-linear discriminant analysis – was subsequently explored with a number of simulations. Overall, differences in the choice of parameters required for the analysis were shown to lead to differences in the final outputs produced, basically in those cases where the environmental range for which predictions are generated is not rigorously limited. Finally, eco-climate surrogates for the evolution of the Japanese encephalitis serocomplex were investigated, but the current environmental distances between the viruses did not seem to be associated with the events leading to their speciation.

Influenza Viren stellen eine große Bedrohung der öffentlichen Gesundheit dar. Die saisonale Grippeschutzimpfung induziert Antikörper gegen den Kopfbereich des viralen Oberflächenproteins Hämagglutinin (HA), in dem verstärkt Antigendrift auftritt. Dadurch wird die Effektivität der saisonalen Grippeimpfung auf den Impfstamm beschränkt und es besteht kein ausreichender Schutz gegen virale Driftvarianten. Eine universellere Grippeimpfung wird dringend benötigt. Die Entdeckung breit reaktiver Antikörper gegen den konservierten HA-Stammbereich hat die Erforschung neuartiger Impfstrategien vorangetrieben. Mit Chimären oder Headless HA kann eine Fokussierung der Immunantwort auf immunsubdominante Bereiche im HA-Stammbereich erzielt werden. Auch innovative Impfstoffplattformen wie Adeno-assoziierte Virus (AAV)-Vektoren bergen ein immenses Potenzial, da sie zum einen für die Verwendung im Menschen zugelassen sind und zum anderen die Immunogenität des Antigens positiv beeinflusst. Die Immunisierung mit AAV-Vektoren, die wildtypisches HA, Chimäre HA oder Nukleoprotein exprimieren, führte in dieser Arbeit in Mäusen zur Induktion breit reaktiver Antikörper, nicht aber die Immunisierung mit AAV-Headless HA oder inaktiviertem Grippeimpfstoff. Die AAV-Vektor Impfstoffe führten zur robusten Induktion Fc-Gamma-Rezeptor-aktivierender Antikörper, die beispielsweise Antikörper-vermittelte zelluläre Zytotoxizität auslösen können. Nicht nur die Impfung mit AAV-Chimären HA, sondern auch mit AAV-wildtypischem HA induzierte Antikörper gegen den HA-Stammbereich. Somit kann anscheinend allein durch eine AAV-Vektor vermittelte Expression des Antigens die Immundominanz des HA-Kopfbereiches abgemildert werden. Abschließend konnte zum ersten Mal die Schutzwirkung einer AAV-Vektor Immunisierung gegen HA im Frettchen demonstriert werden. Die in dieser Arbeit beschriebenen Ergebnisse zeigen somit das große Potenzial von AAV-Vektoren als Impfvehikel für eine breit reaktive Grippeschutzimpfung auf.
Influenza viruses represent a severe threat to public health. A seasonal vaccine is available, which readily leads to the induction of antibodies against the head domain of the viral surface protein hemagglutinin (HA), which is prone to antigenic drift. Thus, seasonal vaccination induces only strain specific protection, while it is not effective against drifted virus strains. Hence, there is an urgent need for a universal influenza vaccine. The discovery of broadly reactive antibodies against the highly conserved HA-stalk domain has prompted great interest into research on vaccination strategies to induce broadly protective HA antibodies. Chimeric and headless HA have shown promising results with respect to re-focusing immunity towards immunosubdominant epitopes in the HA-stalk to induce protective HA-stalk antibodies. Also, innovative vaccine delivery platforms such as Adeno-associated virus (AAV)-vectors offer an attractive developmental perspective. AAV-vectors are licensed for use in humans and the AAV-vectored antigen expression positively influences its immunogenicity. In this thesis, immunization with AAV-vectors expressing wildtype HA, chimeric HA or nucleoprotein induced broad protection in mice, but not vaccination with AAV-vectors expressing headless HA or an inactivated influenza vaccine. Protection was associated with the ability of the AAV-vectored vaccines to induce Fc-gamma-receptor-activating antibodies, which might activate antibody-dependent cellular cytotoxicity. Not only chimeric HA but also wildtype HA induced antibodies against the HA-stalk, suggesting that AAV-vectored antigen expression can mitigate the immunodominance of virus strain-specific epitopes in the HA-head. Importantly, for the first time a protective effect AAV-vectored immunization towards HA could be shown in ferrets. Thus, results described in this thesis suggest a large potential for the development of AAV-vectors as carriers for a broadly protective influenza vaccine.

Bibliography: pages 190-214.
This thesis describes investigations into the potential use of the Subgroup I geminivirus, maize streak virus (MSV), as a gene vector. These involved testing MSV-based replicons in transgenic cell lines, in transient expression assays in maize cells and in an infectious gene expression system in plants. MSV vectors which contained three different versions of a bar (bialaphos resistance) expression cassette in place of the viral movement and coat protein genes were used to generate transformed maize cell lines. A high proportion of these contained MSV-based episomes at high copy number. However, embryogenic maize tissue of the Hill line was not regenerable when an MSV-based replicon was present, possibly due to toxicity of the viral replication associated protein. In non-regenerable Black Mexican sweetcorn cell lines some of the MSV-bar episomes, which ranged in size from 3.15 kb to 4.78 kb, replicated for periods of over two years, and appeared structurally stable. The cellular levels of the bar gene product, phosphinothricin acetyl transferase (PAT), were significantly enhanced in lines where the gene was amplified by linkage to an MSV replicon in comparison with lines where the same gene was not amplified. Northern blot analysis also showed that higher levels of bar mRNA were produced in lines where the gene was amplified. However, the 3- to 5-fold enhancement in gene expression was less than was anticipated, based on results from similar Subgroup ill geminivirus-based transgene amplification systems. Several mutants of the MSV genome were generated to investigate the extent to which genome amplification contributes to the expression of the viral coat protein gene. The introduction of an Ncol restriction site at the start of the coat protein gene facilitated fusion of the gus marker gene with the coat protein upstream transcription and translation regulatory sequences. In one viral construct the plus strand origin of replication was inactivated by insertion of a short oligonucleotide; in another, the viral rep gene was inactivated by a frameshift mutation. These constructs were used to show that the MSV coat protein promoter has low, but measurable constitutive activity in the absence of genome amplification, but that viral replication enhances coat protein expression about 45-fold. I found no evidence for Rep-mediated transactivation of the coat protein promoter.

Cystic fibrosis (CF) is an autosomal recessive genetic disorder of which lung disease is the leading cause of morbidity and mortality. One attractive strategy for the treatment of CF lung disease is to directly deliver CF transmembrane conductance regulator gene to airway epithelia. Although promising results have been reported, barriers present in the lung make successful gene transfer to the respiratory tract difficult. In order to improve gene transfer strategies in the intrapulmonary airways, we need to identify the bottlenecks of transduction for the vector system. A previous study reported that feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways in mice than the intrapulmonary airways (Sinn, P.L. et al. 2008, J. Viol). Our first goal was to identify barriers to lentiviral gene transfer in the murine airways. We demonstrate that host immune response is not the major barrier preventing efficient FIV-mediated transduction in the intrapulmonary airways. We show that the FIV vector transduces murine primary nasal epithelial cell cultures with greater efficiency than murine primary tracheal epithelial cell cultures. In addition, GP64 pseudotyped vesicular stomatitis virus (VSV) transduces better in nasal epithelia compared to intrapulmonary airways in mice. On the other hand, we observed that VSVG glycoprotein-pseudotyped VSV transduces the intrapulmonary airway as well as nasal epithelia in mice with similar efficiency. Our results suggest that differentially expressed cellular factor(s) specific for GP64 or FIV vector may be the major barrier(s) for FIV vector-mediated gene transfer in the murine intrapulmonary airways. The recent development of CF porcine models prompted us to investigate possible barriers for lentiviral vector-mediated gene transfer in porcine cells. Our preliminary results showed that HIV transduction was restricted in porcine but not human lung-derived cell lines. Porcine TRIM5 has sequences similar to restrictive bovine TRIM5 orthologs. Therefore, our second goal was to investigate the possible restriction of lentiviral vectors by porcine TRIM5. We demonstrate that transient overexpression or knockdown of endogenously expressed porcine TRIM5 does not affect HIV or FIV transduction. Lastly, we characterized a mucin domain-deleted EBOV (EBOVΔO) glycoprotein mutant with increased transduction. This EBOVΔO 5-mer mutant was generated based on mutants with an increased transduction as identified by alanine scanning mutagenesis (Brindlay, M.A. et al. 2007. J. Viol). We show that VSV pseudotyped with the 5-mer mutant increased transduction both in vitro and in mice when compared to the wild-type EBOVΔO. Structural analysis demonstrated that 5 mutations were located proximal to the GP1-GP2 interface. Enhanced transduction likely results from a lower energy metastable state of the glycoprotein. FIV pseudotyped with 5-mer also shows increased transduction in multiple cell lines. Identification of barriers in intrapulmonary airways and improvements of vector systems will help the advancement of gene therapy for CF.

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.

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.