Biological Clones Research Articles

Complete sequences of pIJ101-based Streptomyces-Escherichia coli shuttle vectors.

High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces-Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.

Setup and Applications of Modular Protein Expression Toolboxes (MoPET) for Mammalian Systems.

The design and generation of an optimal protein expression construct is the first and essential step in the characterization of any protein of interest. However, the exchange and modification of the coding and/or noncoding elements to analyze their effect on protein function or generating the optimal result can be a tedious and time-consuming process using standard molecular biology cloning methods. To streamline the process to generate defined expression constructs or libraries of otherwise difficult to express proteins, the Modular Protein Expression Toolbox (MoPET) has been developed (Weber E, PloS One 12(5):e0176314, 2017). The system applies Golden Gate cloning as an assembly method and follows the standardized modular cloning (MoClo) principle (Weber E, PloS One 6(2):e16765, 2011). This cloning platform allows highly efficient DNA assembly of pre-defined, standardized functional DNA modules effecting protein expression with a focus on minimizing the cloning burden in coding regions. The original MoPET system consists of 53 defined DNA modules divided into eight functional main classes and can be flexibly expanded dependent on the need of the experimenter and expression host. However, already with a limited set of only 53 modules, 792,000 different constructs can be rationally designed or used to generate combinatorial expression optimization libraries. We provide here a detailed protocol for the (1) design and generation of level 0 basic parts, (2) generation of defined expressions constructs, and (3) generation of combinatorial expression libraries.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Extracellular Vesicle Molecular Profiling for Diagnostic Purposes: An Application of Phage Display Technology.

Phage display is a molecular biology cloning technique that allows the expression of genes of interest along with the phage surface protein. The technique described for the following method used a genomic library for the expression of peptides composed of 12 amino acids, with the objective of selecting peptides which presented specific affinity to the molecules of interest. As a target, purified extracellular vesicles from cell cultures of cells 5637 and RT4 were chosen, which in turn have enormous application and can help to understand the functioning of bladder cancer, allowing the development of new vaccines, drugs, therapies, and diagnoses.

انسانی کلوننگ کا شرعی و تحقیقی مطالعہ

This paper explores the diverse social, cultural and scientific facets of coloning as an alternate means of human reproduction. We review the various types, and implications of coloning as practiced in the modern world, specifically as a means of child birth for infertile parents. We also present the Islamic injunctions and worldview on different types of cloning. We assert that although biological cloning can be an productive technological intervention to promote agriculture yield, however it’s application for human reproduction is inappropriate, and finds no support in Islamic injunctions. Therefore, our paper suggest that human cloning should be prohibited acknowledging its socioeconomic demerits.

An Overview of Current Knowledge on in vitro Babesia Cultivation for Production of Live Attenuated Vaccines for Bovine Babesiosis in Mexico.

The instrumentation of the in vitro culture system has allowed researchers to learn more about the metabolic and growth behavior of Babesia spp. The various applications for in vitro cultivation of Babesia include obtaining attenuated strains for vaccination or pre-munition, the selection of pure lines with different degrees of virulence, studies on biological cloning, ultrastructure, antigen production for diagnostics, drug sensitivity assessments, and different aspects of parasite biology. Although there are different types of vaccines that have been tested against bovine babesiosis, so far, the only procedure that has offered favorable results in terms of protection and safety has been the use of live attenuated vaccines. In countries, such as Australia, Argentina, Brazil, Uruguay and Israel, this type of vaccine has been produced and used. The alternative to live vaccines other than splenectomized calf-derived biological material, has been the in vitro cultivation of Babesia bovis and B. bigemina. The development of in vitro culture of Babesia spp. strains in a defined medium has been the basis for the initiation of a source of parasites and exoantigens for a variety of studies on the biochemistry and immunology of babesiosis. The use of live immunogens from attenuated strains derived from in vitro culture is highlighted, which has been proposed as an alternative to control bovine babesiosis. In several studies performed in Mexico, this type of immunogen applied to susceptible cattle has shown the induction of protection against the experimental heterologous strain challenge with both, Babesia-infected blood and animal exposure to confrontations on tick vector-infested farms. The combination of transfection technologies and the in vitro culture system as integrated methodologies would eventually give rise to the generation of genetically modified live vaccines. However, a greater challenge faced now by researchers is the large-scale cultivation of Babesia parasites for mass production and vaccine distribution.

Establishment of Murine Infection Models with Biological Clones of Dengue Viruses Derived from a Single Clinical Viral Isolate.

Dengue virus (DENV) is a single-stranded RNA virus transmitted by mosquitoes in tropical and subtropical regions. It causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in patients. Each year, 390 million people are estimated to be infected by four serotypes of dengue virus, creating a great burden on global public health and local economy. So far, no antiviral drug is available for dengue disease, and the newly licensed vaccine is far from satisfactory. One large obstacle for dengue vaccine and drug development is the lack of suitable small animal models. Although some DENV infection models have been developed, only a small number of viral strains can infect immunodeficient mice. In this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of DENV infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in Balb/c mice with transient blockage of type I IFN responses. This study not only offers new small animal models of dengue viral infection, but also provides new viral variants for further investigations on dengue viral pathogenesis.

Growth, fruiting peculiarities and efficiency of cultivation of Ukrainian apple clones (Malus domestica Borkh.) of the ‘Jonagold’ variety

Purpose. On the basis of a comprehensive study of the characteristics of growth, development, fruiting and quality of the fruits of spontaneous mutants of the apple tree (Malus domestica Borkh.) of ‘Jonagold’ variety, select clones which differ from the original variety in tree growth strength and are superior in yield, marketability, fruit keeping quality and economic production efficiency of the latter.Methods. Field, laboratory, comparison, generalization and mathematical statistics.Results. The results of studying the growth and fruiting characteristics of 20 Ukrainian spontaneous clones of ‘Jonagold’ variety in the conditions of the Western Forest-Steppe of Ukraine are presented. It is revealed that plants of the studied clones did not differ in terms of beginning and duration of the main phenological phases and vegetation period from the varieties widely distributed and well adapted to Podillia conditions. The harvesting ripeness of the clone fruits came in the second or third decades of September with a difference of seven days. Trees of most clones (DP-4, DP-5, DP-9, DP-10, DP-13, DP-14 and DP-17) were medium-growing. The yield of five to eight-year-old clone plantations was 12.1–32.3 t/ha, which provided cost-effective cultivation in the conditions of Podillia, the level of production profitability was 66–242%. All spontaneous mutants formed fruits larger than average size (except for DP-20, forming large ones), which are beautiful, elegant and vary in the intensity and character of the skin color depending on the clone. The clones DP-1, DP-3, DP-17, DP-18 and DP-19 were distinguished by a high yield of fruits of the highest and first grade.The clones DP-1, DP-3, DP-17, DP-18 and DP-19 were marked by the highest yield of apples of the higher and the first commodity grade, and this property was significantly superior to the yield of the control variety. The tested clones are divided into three groups according to the fruit ripening dates: early winter, winter, and late winter.Conclusions. A long-term assessment of the objects of study revealed that the biological properties Ukrainian clones of ‘Jonagold’ variety correspond to the climatic conditions of the Western Forest-Steppe ofUkraine. Most of them belong to the medium-growth group (DP-4, DP-5, DP-9, DP-10, DP-13, DP-14 and DP-17) and are characterized by a mixed type of fruiting. Clones DP-1, DP-3, DP-17, DP-18 and DP-19 were distinguished by a high yield of fruits of the highest and first commodity grade. According to the main indicators of fruit quality, clones DP-3, DP-8, DP-13, DP-14, DP-16, DP-17, DP-18 and DP-20 were recognized as the best; the highest economic efficiency of apple production in Podillia was provided by clones DP-14, DP-17, DP-18 and DP-19. The latter ones are recommended for registration in “The State Register of plant varieties suitable for dissemination inUkraine”, as promising clones for introduction into planting for the production of fruits of universal purpose in the zone of the Western Forest-Steppe ofUkraine.

Unequal distribution of genes and chromosomes refers to nuclear diversification in the binucleated Giardia intestinalis

The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5′ chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.

Metabolic Network Modelling of Chinese Hamster Ovary (CHO) Culture Bioreactors Operated as Microbial Cell Factories

Chinese hamster ovary (CHO) epithelial cells are one of the most used therapeutic medical lines for the production of different biopharmaceutical drugs. They have a high consumption rate with a fast duplication cycle that makes them an ideal biological clone. The higher accumulated amounts of toxic intracellular intermediates may lead to lower organism viability, protein productivity and manufactured biosimilar, so a careful optimal balance of medium, bioreactor operational parameters and bioprocess is needed. A precise phenomenological knowledge of metabolism's chemical transformations can predict problems that may arise during batch, semi-continuous fed batch and continuous reactor operation. For a better detailed understanding (and relations), future performance optimization and scaling, mechanistic model systems have been built. In this specific work, the main metabolic pathways in mammalian structured CHO cultures are reviewed. It starts with organic biochemical background, controlling associated phenomena and kinetics, which govern the sustaining conversion routes of biology. Then, individual turnover paths are described, overviewing standard mathematical formulations that are commonly applied in engineering. These are the core of black box modeling, which relates the substrates/products in a simplified relationship manner. Moreover, metabolic flux analysis (MFA)/flux balance analysis (FBA), that are traditionally characterizing mechanisms, are presented to a larger portion extent. Finally, similarities are discussed, illustrating the approaches for their structural design. Stated variables' equations, employed for the description of the growth in the controllable environmental conditions of a vessel, the researched reaction series of proliferating dividing CHO population, joint with the values of maximal enzymatic activity, and solutions are outlined. Processes are listed in a way so that a reader can integrate the state-of-the-art. Our particular contribution is also denoted.

P27 - Genome characterization of HIV-2 biological clones with divergent replication capacities

P27 - Genome characterization of HIV-2 biological clones with divergent replication capacities

An Overview of Filamentous Bacteriophages for Phage display and its po-tential clinical applications

Viruses are a transition between living and non-living organisms. Their biological machinery is tailored in collaborating with cellular organisms to very effectively reproduce themselves. This inherent potential of hi jacking the transcription and translation machinery of the cell, and picking up and moving about DNA from the organisms with which they interact is exploited by the biologists. Bacteriophage is a kingdom of viruses that infect bacteria, and filamentous bacteriophages which are made up of long, thin filaments containing a circular single-stranded DNA genome which reproduce without killing the host, belong to genus Inovirus. This review article is an overview of filamentous bacteriophages, various vectors developed for use in the molecular biology and gene cloning. However, the major emphasis of the review is on their use in phage display technology. Although selection of proteins is the main feature of this technology, it has been modified to be utilized for diverse applications. A comprehensive detail of all the applications is beyond the scope of this review, so recent developments using phage display in cancer diagnosis and therapy are discussed in this article.

Biological and molecular characterization of citrus tatter leaf virus in Taiwan

Citrus tatter leaf virus (CTLV) causes chlorotic leaves with bud union incompatibility in citrus scion and its current incidence in Taiwan is 65%. However, the burden of infection has long been underestimated due to its common latency, and the serious threat of tatter leaf disease should be investigated and addressed without delay. Here, the biological characteristics and genome sequences of Taiwanese isolates are evaluated. Full‐length sequencing and phylogenetic analysis of three local isolates revealed similarities with isolates from neighbouring countries, indicating their possible origins. Two mild isolates obtained from Liuchen sweet orange (LCd‐NA‐1) and kumquat (Kq‐6‐2‐46) were inoculated on indicator plants and exhibited differences in foliar symptom expression and temperature tolerance compared with foreign isolates causing severe disease. Improved quantitative PCR‐based detection methods using redesigned primer pairs indicated high sensitivity and specificity for both local and foreign isolates. Similar but delayed systemic symptoms were observed when wildtype virus in vitro transcripts generated from a full‐length infectious clone of the Ponkan mandarin isolate (pCTLV‐Pk‐8) were inoculated on Chenopodium quinoa. These symptoms were unevenly distributed and highly concentrated on the inoculated sides of leaves and top leaves. A single codon mutation (U5642C) in the coat protein transcription start site in the pCTLV‐Pk‐8 sequence resulted in defective systemic infection, indicating a role for the coat protein in viral movement. The combination of biological assays, improved detection and infectious clone construction provides comprehensive information and a foundation for citrus virus management and further research.

Peptide and Recombinant Allergen Vaccines for Food Allergy.

Food allergy is a significant public health problem, with no suitable treatments available for patients. Currently, patients are limited to avoidance and the use of readily available emergency medications. Immunotherapy is an appealing therapeutic strategy for inducing tolerance. Studies with whole native allergens have demonstrated the efficacy of immunotherapy for food allergy; however, the risk of IgE-mediated reactions with such treatment is significant. Advances in molecular biology techniques, including purification, sequencing, and cloning, have allowed researchers to identify specific allergen components and T cell binding epitopes. Support for the use of recombinant and peptide vaccines for food allergy comes from prior studies involving aeroallergens and hymenoptera venom. By manipulating allergen structure and IgE binding, allergenicity can be reduced, thereby reducing systemic reactions, making recombinant and peptide vaccines a safe and effective form of immunotherapy. Pre-clinical studies using in vitro and murine models demonstrated a more tolerant state following the use of these therapies. Studies with human subjects will be necessary to characterize the effects of recombinant and peptide food allergy vaccines and to demonstrate a safe treatment option for patients.

Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle.

Propagation of genetic information is a fundamental property of living organisms. Escherichia coli has a 4.6 Mb circular chromosome with a replication origin, oriC. While the oriC replication has been reconstituted in vitro more than 30 years ago, continuous repetition of the replication cycle has not yet been achieved. Here, we reconstituted the entire replication cycle with 14 purified enzymes (25 polypeptides) that catalyze initiation at oriC, bidirectional fork progression, Okazaki-fragment maturation and decatenation of the replicated circular products. Because decatenation provides covalently closed supercoiled monomers that are competent for the next round of replication initiation, the replication cycle repeats autonomously and continuously in an isothermal condition. This replication-cycle reaction (RCR) propagates ∼10 kb circular DNA exponentially as intact covalently closed molecules, even from a single DNA molecule, with a doubling time of ∼8 min and extremely high fidelity. Very large DNA up to 0.2 Mb is successfully propagated within 3 h. We further demonstrate a cell-free cloning in which RCR selectively propagates circular molecules constructed by a multi-fragment assembly reaction. Our results define the minimum element necessary for the repetition of the chromosome-replication cycle, and also provide a powerful in vitro tool to generate large circular DNA molecules without relying on conventional biological cloning.

PROPRIÉTÉS PHYSIQUES ET MÉCANIQUES DU BOIS DE TECK DE PROVENANCES TANZANIENNE ET LOCALE AU BÉNIN

Le teck (Tectona grandis L. f.) est une espèce ligneuse tropicale dont le bois est très demandé. Au Bénin, la provenance tanzanienne et la provenance locale sont les deux variétés de cette espèce les plus plantées. Pour contribuer à la caractérisa- tion du bois de teck du Bénin, nous avons déterminé et comparé, pour le bois de chaque provenance, les modules d’élasti- cité et de cisaillement, la longueur du fût, la circonférence et l’épaisseur d’écorce à hauteur d’abattage, le pourcentage de bois de cœur, la circonférence à une hau- teur de 1,30 m du sol et la masse volu- mique (ou la densité) à 12 % de teneur en eau. Les échantillons ont été préle- vés dans les plantations de la Lama sur des arbres de 20 à 25 ans (six billes de provenance locale et quatre billes de provenance tanzanienne). Les résultats obtenus sur la base de cet échantillon indiquent que, pour cette classe d’âge, les valeurs de ces propriétés pour le bois de teck de provenance locale sont supé- rieures à celles de la provenance tanza- nienne, sauf pour le pourcentage de bois de cœur. Sur la base de notre échantil- lonnage, le teck de provenance locale semble donc d’une certaine qualité, qu’il faut tendre à valoriser par la sélection bio- logique ou clonale des meilleurs sujets, aptes à s’adapter non seulement au chan- gement climatique mais aussi aux struc- tures des sols. Cela permettrait de favori- ser la production du bois de teck sur toute l’étendue du territoire national, voire de la sous-région. Toutefois, la mise en place d’une variété hybride conciliant les atouts des deux provenances pourrait aussi être une grande avancée dans l’amélioration des essences exploitées au Bénin.

Polyphasic insights into the microbiomes of the Takamatsuzuka Tumulus and Kitora Tumulus.

Microbial outbreaks and related biodeterioration problems have affected the 1300-year-old multicolor (polychrome) mural paintings of the special historic sites Takamatsuzuka Tumulus (TT) and Kitora Tumulus (KT). Those of TT are designated as a national treasure. The microbiomes of these tumuli, both located in Asuka village, Nara, Japan, are critically reviewed as the central subject of this report. Using culture-dependent methods (conventional isolation and cultivation), we conducted polyphasic studies of the these microbial communities and identified the major microbial colonizers (Fusarium spp., Trichoderma spp., Penicillium spp., dark Acremonium spp., novel Candida yeast spp., Bacillus spp., Ochrobactrum spp., Stenotrophomonas tumulicola, and a few actinobacterial genera) and noteworthy microbial members (Kendrickiella phycomyces, Cephalotrichum verrucisporum (≡Doratomyces verrucisporus), Sagenomella striatispora, Sagenomella griseoviridis, two novel Cladophialophora spp., Burgoa anomala, one novel species Prototheca tumulicola, five novel Gluconacetobacter spp., three novel Bordetella spp., and one novel genus and species Krasilnikoviella muralis) involved in the biodeterioration of mural paintings, plaster walls, and stone chamber interiors. In addition, we generated microbial community data from TT and KT samples using culture-independent methods (molecular biological methods, including PCR-DGGE, clone libraries, and pyrosequence analysis). These data are comprehensively presented, in contrast to those derived from culture-dependent methods. Furthermore, the microbial communities detected using both methods are analytically compared, and, as a result, the complementary roles of these methods and approaches are highlighted. In related contexts, knowledge of similar biodeterioration problems affecting other prehistoric cave paintings, mainly at Lascaux in France and Altamira in Spain, are referred to and commented upon. Based on substrate preferences (or ecological grouping) and mapping (plotting detection sites of isolates), we speculate on the possible origins and invasion routes whereby the major microbial colonizers invaded the TT stone chamber interior. Finally, concluding remarks, lessons, and future perspectives based on our microbiological surveys of these ancient tumuli, and similar treasures outside of Japan, are briefly presented. A list of the microbial taxa that have been identified and fully or briefly described by us as known and novel taxa for TT and KT isolates since 2008 is presented in Supplementary Materials.

Barrier-Independent, Fitness-Associated Differences in Sofosbuvir Efficacy against Hepatitis C Virus.

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvir-specific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvir-specific resistance mutations, linked to in vivo fitness of pretreatment viral populations.

Influence of mutation and recombination on HIV-1 in vitro fitness recovery.

The understanding of the evolutionary processes underlying HIV-1 fitness recovery is fundamental for HIV-1 pathogenesis, antiretroviral treatment and vaccine design. It is known that HIV-1 can present very high mutation and recombination rates, however the specific contribution of these evolutionary forces in the "in vitro" viral fitness recovery has not been simultaneously quantified. To this aim, we analyzed substitution, recombination and molecular adaptation rates in a variety of HIV-1 biological clones derived from a viral isolate after severe population bottlenecks and a number of large population cell culture passages. These clones presented an overall but uneven fitness gain, mean of 3-fold, respect to the initial passage values. We found a significant relationship between the fitness increase and the appearance and fixation of mutations. In addition, these fixed mutations presented molecular signatures of positive selection through the accumulation of non-synonymous substitutions. Interestingly, viral recombination correlated with fitness recovery in most of studied viral quasispecies. The genetic diversity generated by these evolutionary processes was positively correlated with the viral fitness. We conclude that HIV-1 fitness recovery can be derived from the genetic heterogeneity generated through both mutation and recombination, and under diversifying molecular adaptation. The findings also suggest nonrandom evolutionary pathways for in vitro fitness recovery.

A severe symptom phenotype in pepper cultivars carrying the Tsw resistance gene is caused by a mixed infection between resistance-breaking and non-resistance-breaking isolates of Tomato spotted wilt virus

Pepper (Capsicum annuum) plants with the Tsw resistance gene showing unusually severe symptoms consisting of local lesions, chlorosis, stunting and systemic necrosis on the apical leaves were found in a commercial field in north eastern Spain in 2009. The presence of Tomato spotted wilt virus (TSWV) was confirmed in all diseased plants. After mechanical inoculation of Nicotiana glutinosa with infected field samples, biological clones of the virus were isolated from individual local lesions. These biological clones produced two different types of symptoms after inoculation on Tsw resistant pepper plants: (i) typical symptoms caused by resistance-breaking (RB) isolates characterized by chlorosis and stunting, and (ii) severe symptoms as observed in the field plants. Similar symptoms in pepper plants carrying the Tsw resistance gene were reproduced under controlled conditions, after simultaneous inoculation of RB and non-resistance-breaking (NRB) isolates. The NRB isolate was detected in a low proportion in the apical uninoculated leaves, whereas NRB isolates could not infect resistant pepper when inoculated alone. Co-infection by NRB and RB isolates induced disease synergism with systemic necrosis on the apical leaves. To our knowledge, this is the first case in which a synergic interaction between isolates of the same virus has been described, which has the ability to overcome a natural genetic resistance. This finding could have epidemiological implications for the management of TSWV.