An Overview of the Application of Viruses to Biotechnology.

Viruses may cause devastating diseases in several organisms; however, they are simple systems that can be manipulated to be beneficial and useful for many purposes in different areas. In medicine, viruses have been used for a long time in vaccines and are now being used as vectors to carry materials for the treatment of diseases, such as cancer, being able to target specific cells. In agriculture, viruses are being studied to introduce desirable characteristics in plants or render resistance to biotic and abiotic stresses. Viruses have been exploited in nanotechnology for the deposition of specific metals and have been shown to be of great benefit to nanomaterial production. They can also be used for different applications in pharmacology, cosmetics, electronics, and other industries. Thus, viruses are no longer only seen as enemies. They have shown enormous potential, covering several important areas in our lives, and they are making our lives easier and better. Although viruses have already proven their potential, there is still a long road ahead. This prompt us to propose this theme in the Special Issue "The application of viruses to biotechnology". We believe that the articles gathered here highlight recent significant advances in the use of viruses in several fields, contributing to the current knowledge on virus applications.

Establishment of a Sensitive qPCR Methodology for Detection of the Olive-Infecting Viruses in Portuguese and Tunisian Orchards.

Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have eventual negative effects on olive oil quality. The work presented here describes the development and application of a new SYBR® Green-based real-time quantitative PCR (qPCR) analysis for specific and reliable quantification of highly spread olive tree viruses: Olive latent virus 1 (OLV-1), Tobacco necrosis virus D (TNV-D), Olive mild mosaic virus (OMMV), and Olive leaf yellowing-associated virus (OLYaV). qPCR methodology revealed high specificity and sensitivity, estimated in the range of 0.8-8 copies of the virus genome, for the studied viruses. For validation of the method, total RNA and double strand RNA (dsRNA) from naturally infected trees were used. In a first trial, dsRNAs from trees of cv. "Galega vulgar" from a Portuguese orchard, were subjected to qPCR and from the 30 samples tested, 26 were TNV-D and/or OMMV-positive and 25 were OLV-1 positive. In a second trial, total RNA from trees of different cultivars from Tunisian orchards, were here tested by qPCR and all viruses were detected. From the 33 samples studied, the most prevalent virus detected in Tunisia orchards was OLV-1 (31 samples diagnosed), followed by OLYaV (20 samples diagnosed), and finally the combination in last TNV-D and/or OMMV (12 samples diagnosed). In both trials, qPCR demonstrated to be effective and sensitive, even when using total RNA as template. qPCR through the use of a SYBR® Green methodology enabled, for the first time, a reliable, sensitive, and reproducible estimation of virus accumulation in infected olive trees, in which viruses are usually in low titres, that will allow gaining new insights in virus biology essential for disease control and give an important contribution for establishment of sanitary certification of olive propagative material.

Olive Mild Mosaic Virus Coat Protein and P6 Are Suppressors of RNA Silencing, and Their Silencing Confers Resistance against OMMV.

RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the Olive mild mosaic virus (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana 16C plants. CP and p6 showed suppressor activity, with CP showing significantly higher activity than p6, yet activity that was lower than the full OMMV, suggesting a complementary action of CP and p6. These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the Tombusviridae family.