Un paradigme actuel en virologie est que les virus de plantes ne peuvent pas affecter les animaux vertébrés et ne sont pas pathogènes pour l'homme. Cependant, plusieurs éléments remettent en question ce paradigme. L'objectif de cette Thèse a été d'étudier si les tobamovirus peuvent être pathogènes chez l'homme. Ce manuscrit comprend :1) une revue de la littérature portant sur la présence et l'impact potentiel des virus de plantes chez les animaux dont l'homme. 2) une étude sur l'exposition humaine aux tobamovirus et les effets possibles Dans la région de Marseille, nous avons montré la présence du PMMoV dans 7.2% des selles de patients et dans 57 % des produits alimentaires à base de piment. De plus, la présence du PMMoV dans les selles de patients a pu être corrélée des signes cliniques. De plus, nous avons montré que le TMV infectieux était présent dans le tabac de toutes les cigarettes testées mais aussi dans 45 % des salives de fumeurs testées. 3) une étude in vivo. Suite à une inoculation intra-trachéal des souris avec du TMV, nous avons constaté que le virus persiste jusqu'à 14 jours dans le tissu pulmonaire et les macrophages pulmonaires. De plus, nous avons détecté du TMV dans le cytoplasme des macrophages murins jusqu'à 15 jours après inoculation. 4) une étude sur le mode de transmission du TMV à l'homme. Le TMV détecté dans la salive des fumeurs ne serait pas véhiculé par la fumée de cigarette, mais par contact avec du tabac infecté. Nos résultats suggèrent que la frontière entre virus de plantes et virus d'animaux n'est pas aussi stricte qu'il est communément admis et incitent à réévaluer l'éventuelle pathogénicité des phytovirus pour l'homme. / A current paradigm in virology is that plant viruses cannot affect vertebrates and are not pathogenic for humans. However, several recent findings challenge this paradigm. The aim of this thesis was to investigate whether tobamoviruses may be pathogenic in humans. This manuscript contains: 1) A review of the literature of the reasons why plant viruses may cross the border from plants to vertebrates and the possible impact of plant viruses in animals including humans. 2) A study on exposure to tobamovirus and possible effects of these viruses for humans. In the Marseille geographical area, we showed the presence PMMoV in 7.2% of stools from patients tested and in 57% of food products containing hot peppers. In addition, the presence of PMMoV in the stools of patients could be significantly correlated with clinical symptoms. Then, we showed that infectious TMV was present in the tobacco of all cigarettes tested and TMV RNA was detected in 45% of smokers' saliva tested. 3) In vivo study: mice intra-tracheal inoculation with TMV. The results showed the persistence until 14 days of viruses in the lung tissue and in lung alveolar macrophages of mice. In addition, we detected TMV in the cytoplasm of murine macrophages up to 15 days after inoculation. 4) A study of TMV transmission to humans through cigarette smoking. TMV does not seem to be vehicled by cigarette smoke to smoker's saliva but by direct contact with infected tobacco. Our results suggest that the boundary between plant viruses and animal viruses is not as strict as it is commonly accepted and prompt to reassess the potential pathogenicity of these plant viruses for humans.

The Tobacco mosaic virus (TMV) is a positive sense single stranded RNA virus and is found across the world. TMV can impact the overall yield and quality of the crop resulting in an economic loss. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, mottling, necrotic lesions and stunted growth. Historically, TMV has caused controversy on whether this economically significant virus is seedborne or seed transmitted. The objective of this study is to track TMV infection from infected seeds to seedlings to determine the percentage of seed transmission. This experiment used three pods from three different TMV infected cultivar K 326 flue-cured tobacco plants. Seeds from each pod were germinated in a growth chamber for approximately ten days. Samples were separated into seed coat, root and leaves after germination. Total RNA was extracted from each part and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample. Endpoint RT-PCR was used to determine a conservative threshold value from the RT-qPCR results. These results demonstrated that TMV influenced percent germination with a range from 94% to 50%. Seed coats had a significantly higher virus titer concentration (P < 0.05) when compared to the roots and leaves. Statistical analysis revealed highly significant (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Results demonstrate that TMV is seed transmitted in flue-cured tobacco. / Master of Science / The Tobacco mosaic virus (TMV) is an RNA virus that occurs globally in areas where tobacco is grown. TMV is a tobamovirus and infects over 350 plant species. TMV can reduce the yield and quality of the crop which will result in an economic loss for the grower. Plants that are infected with TMV show a variety of symptoms such as mosaic pattern, necrotic lesions, and stunted growth, and there are no effective ways to eradicate the virus. There has been controversy on whether to categorize TMV as a seedborne virus or a seed-transmitted virus because the location of the virus within a seed is unknown. This study examined seeds from three pods grown on three different TMV-infected flue-cured tobacco plants of cultivar K 326 to track TMV infection from infected seeds to seedlings. Seeds from each pod were germinated in a growth chamber for ten days and samples were separated into leaves, root and seed coat. Each sample had total RNA extracted and synthesized into cDNA for analysis. A quantitative real-time PCR (RT-qPCR) assay was used to determine TMV concentration of each sample since this technology can detect small amounts of virus. Endpoint RT-PCR was used to conservatively determine an infection threshold value from the RT-qPCR results. Percent germination of TMV infected seeds ranged from 94% to 50%. Seed coats had a significantly higher virus titer (P < 0.05) when compared to the roots and leaves in each pod. Statistical analysis showed (P < 0.0001) differences among pods for virus titer and there is a highly significant plant by pod interaction (P < 0.0001). Endpoint RT-PCR confirmed TMV infection in leaves, roots and seed coats. Percent infection in iv leaves ranged from 2% to 24% and percent infection for roots ranged from 8% to 40%. Therefore, results show that TMV is seed-transmitted in flue-cured tobacco.

[ES] Las plantas están emergiendo como una alternativa atractiva a los sistemas convencionales de producción heteróloga, como bacterias, cultivos celulares de mamíferos, levaduras u hongos, para la síntesis de productos de alto valor, como metabolitos secundarios y proteínas. La demanda de estos productos a menudo se ve limitada por la capacidad de producción y los costos asociados. Sin embargo, utilizar las plantas como plataformas de producción ofrece ventajas en términos de accesibilidad económica, sostenibilidad, escalabilidad y la ausencia de patógenos humanos y de animales, lo que las convierte en una opción cada vez más atractiva. A pesar de las limitaciones en la capacidad de carga, la expresión génica transitoria utilizando vectores virales proporciona un método eficiente y reproducible para la producción de proteínas recombinantes en plantas, a diferencia de la transformación estable, que requiere más mano de obra y tiempo. Los vectores derivados del virus del mosaico del tabaco (TMV), particularmente aquellos en los que el gen de la proteína de la cubierta viral (CP) se reemplaza principalmente por el gen de interés, son clásicos en la biotecnología vegetal y se utilizan con frecuencia para la producción a gran escala de proteínas recombinantes. En este trabajo, nuestro primer objetivo fue optimizar un vector de TMV para mejorar la producción. La fusión traduccional del extremo amino-terminal de la CP del TMV con la proteína recombinante de interés mostró un aumento en la acumulación de la proteína verde fluorescente, interferón alfa-2a y un nanobody contra la proteína Spike del SARS-CoV-2 en hojas de Nicotiana benthamiana. La inserción de un sitio de clivaje específico, basado en las proteasas de inclusión nuclear (NIaPro) de dos potyvirus, junto con la expresión de las proteasas correspondientes, además de la producción de la proteína recombinante madura, aumentó aún más la acumulación de las proteínas recombinantes mencionadas anteriormente. En segundo lugar, nuestro objetivo fue establecer estrategias para producir proteínas recombinantes de interés industrial y farmacéutico en N. benthamiana utilizando un vector de TMV. Logramos producir grandes cantidades de una xilanasa termofílica, activa en condiciones extremas de temperatura y pH alcalino, en N. benthamiana utilizando un vector de TMV. La enzima que se acumuló rápidamente en los tejidos de la planta se dirigió al apoplasto, lo que facilitó enormemente la purificación y evitó cualquier efecto adverso en el crecimiento de la planta. Se demostró que esta enzima producida en planta es útil para la producción de xilooligosacáridos probióticos. También produjimos grandes cantidades de una glucosa oxidasa (GOX) modificada en hojas de N. benthamiana utilizando un vector de TMV. La GOX producida en planta, que también se purificó fácilmente a partir de fluidos apoplásticos, exhibió potentes propiedades antimicrobianas contra Staphylococcus aureus y Escherichia coli. / [CA] Les plantes están emergint com una alternativa atractiva als sistemes convencionals de producció heteròloga, com ara bacteries, cultius cel·lulars de mamífers, llevats o fongs, per a la síntesi de productes d'alt valor, com son metabòlits secundaris i proteïnes d'interés industrial i farmacéutic. La demanda d'aquests productes sovint es veu limitada per la capacitat de producció i els costos associats. No obstant això, utilitzar les plantes com a plataformes de producció ofereix avantatges en termes d'accessibilitat econòmica, sostenibilitat, escalabilitat i l'absència de patògens humans i animals, la qual cosa les converteix en una opció cada vegada més atractiva. Malgrat les limitacions en la capacitat de càrrega, l'expressió gènica transitoria mitjançant vectors virals proporciona un mètode eficient i reproducible per a la producció de proteïnes recombinants en plantes, a diferència de la transformació estable, que requereix més mà d'obra i temps. Els vectors derivats del virus del mosaic del tabac (TMV), particularment aquells en els quals el gen de la proteïna de la coberta viral (CP) és principalment reemplaçat pel gen d'interès, són clàssics en la biotecnologia vegetal i s'utilitzen sovint per a la producció a gran escala de proteïnes recombinants. En aquest treball, el nostre primer objectiu va ser optimitzar un vector de TMV per a millorar la producció. La fusió traduccional de l'extrem amino-terminal de la CP del TMV amb la proteïna recombinant d'interès va mostrar un augment en l'acumulació de la proteïna verda fluorescent, interferó alfa-2a i un nanobody contra la proteïna Spike del SARS-CoV-2 en fulles de Nicotiana benthamiana. La inserció d'un lloc de tall específic, basat en les proteases d'inclusió nuclear (NIaPro) de dos potivirus, juntament amb l'expressió de les proteases corresponents, a més de la producció de la proteïna recombinant madura, va augmentar encara més l'acumulació de les proteïnes recombinants esmentades anteriorment. En segon lloc, el nostre objectiu va ser establir estratègies per a produir proteïnes recombinants d'interés industrial i farmacèutic en N. benthamiana utilitzant un vector de TMV. Vam aconseguir produir grans quantitats d'una xilanasa termofílica, activa en condicions extremes de temperatura i pH alcalí, en N. benthamiana utilitzant un vector de TMV. El enzim que es va acumular ràpidament en els teixits de la planta es va dirigir a l'apoplast, la qual cosa va facilitar enormement la purificació i va evitar qualsevol efecte advers en el creixement de la planta. Es va demostrar que aquesta enzima produïda en planta és útil per a la producció de xilooligosacàrids probiòtics. També vam produir grans quantitats d'una glucosa oxidasa (GOX) modificada en fulles de N. benthamiana utilitzant un vector de TMV. La GOX produïda en planta, que també es va purificar fàcilment a partir de fluids apoplàstics, va exhibir potents propietats antimicrobianes contra Staphylococcus aureus i Escherichia coli. / [EN] Plants are emerging as an attractive alternative to conventional heterologous production systems, including bacteria, mammalian cell cultures, yeast, or fungi, for the synthesis of high-value products, such as secondary metabolites and proteins. The demand for these products is often limited by production capacity and associated costs. However, using plants as production platforms offers advantages in terms of affordability, sustainability, scalability, and the absence of human and livestock pathogens, making them an increasingly appealing choice. Despite limitations in cargo capacity, transient gene expression using viral vectors provides an efficient and reproducible method for producing recombinant proteins in plants, unlike stable transformation, which is more labor- intensive and time-consuming. Vectors derived from tobacco mosaic virus (TMV), particularly those in which the viral coat protein (CP) gene is mostly replaced with the gene of interest, are classic in plant biotechnology and frequently use for large-scale production of recombinant proteins. In this work, we first aimed to optimize a TMV vector to improve production. Translational fusion of the amino- terminal end of TMV CP to the recombinant protein of interest led to increased accumulation of the green fluorescent protein, interferon alfa-2a and a nanobody against the Spike protein of SARS-CoV-2 in Nicotiana benthamiana leaves. Insertion of a specific cleavage site, based on the nuclear inclusion a proteases (NIaPro) form two potyviruses, along expression of the cognate proteases led to, in addition to production of the mature recombinant protein, a further increase in the accumulation of the aforementioned recombinant proteins. Second, we aimed to set up strategies to produce recombinant proteins of industrial and pharmaceutical interest in N. benthamiana using a TMV vector. We successfully produced large amounts of a thermophilic xylanase, active under extreme temperature and alkaline pH conditions, in N.benthamiana using a TMV vector. The enzyme that accumulated rapidly in plant tissues was targeted the apoplast, which enormously facilitated purification, and avoided any adverse effect on plant growth. This plant-made enzyme was shown to be useful for the production of probiotic xylooligosaccharides. We also produced large amounts of an engineered glucose oxidase (GOX) in N. benthamiana leaves using a TMV vector. The plant-made GOX that was also easily purified from apoplastic fluids exhibited potent antimicrobial properties against Staphylococcus aureus and Escherichia coli. / This work was supported Generalitat Valenciana, grant INNEST/2021/7 from Agència Valenciana de la Innovació, and by grant PID2020-114691RB-I00 from the Spanish Ministerio de Ciencia e Innovación, through the Agencia Estatal de Investigación (co-financed European Regional Development Fund), and by the Bio Based Industries Joint Undertaking, under the European Union’s Horizon 2020 research and innovation program (Project WOODZYMES, Grant Agreement H2020-BBI- JU-792070). M.N.-S. is the recipient of a predoctoral contract from the Spanish Ministerio de Ciencia e Innovación (PRE2018-084771). / Nicolau Sanus, M. (2023). Production of Recombinant Proteins with Pharmaceutical and Industrial Applications in Plants Using a Tobacco Mosaic Virus-Derived Vector [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/200381