Gelatin-methacryloyl hydrogels containing turnip mosaic virus for fabrication of nanostructured materials for tissue engineering.

Current tissue engineering techniques frequently rely on hydrogels to support cell growth, as these materials strongly mimic the extracellular matrix. However, hydrogels often need ad hoc customization to generate specific tissue constructs. One popular strategy for hydrogel functionalization is to add nanoparticles to them. Here, we present a plant viral nanoparticle the turnip mosaic virus (TuMV), as a promising additive for gelatin methacryloyl (GelMA) hydrogels for the engineering of mammalian tissues. TuMV is a flexuous, elongated, tubular protein nanoparticle (700-750 nm long and 12-15 nm wide) and is incapable of infecting mammalian cells. These flexuous nanoparticles spontaneously form entangled nanomeshes in aqueous environments, and we hypothesized that this nanomesh structure could serve as a nanoscaffold for cells. Human fibroblasts loaded into GelMA-TuMV hydrogels exhibited similar metabolic activity to that of cells loaded in pristine GelMA hydrogels. However, cells cultured in GelMA-TuMV formed clusters and assumed an elongated morphology in contrast to the homogeneous and confluent cultures seen on GelMA surfaces, suggesting that the nanoscaffold material per se did not favor cell adhesion. We also covalently conjugated TuMV particles with epidermal growth factor (EGF) using a straightforward reaction scheme based on a Staudinger reaction. BJ cells cultured on the functionalized scaffolds increased their confluency by approximately 30% compared to growth with unconjugated EGF. We also provide examples of the use of GelMA-TuMV hydrogels in different biofabrication scenarios, include casting, flow-based-manufacture of filaments, and bioprinting. We envision TuMV as a versatile nanobiomaterial that can be useful for tissue engineering.

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests.

BACKGROUND: The fight against the current coronavirus disease 2019 (COVID-19) pandemic has created a huge demand of biotechnological, pharmaceutical, research and sanitary materials at unprecedented scales. One of the most urgent demands affects the diagnostic tests. The growing need for rapid and accurate laboratory diagnostic tests requires the development of biotechnological processes aimed at producing reagents able to cope with this demand in a scalable, cost-effective manner, with rapid turnaround times. This is particularly applicable to the antigens employed in serological tests. Recombinant protein expression using plants as biofactories is particularly suitable for mass production of protein antigens useful in serological diagnosis, with a neat advantage in economic terms. METHODS: We expressed a large portion of the nucleoprotein (N) derived from SARS-CoV-2 in Nicotiana benthamiana plants. After purification, the recombinant N protein obtained was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to SARS-CoV-2 in human sera. To validate the ELISA, a panel of 416 sera from exposed personnel at essential services in Madrid City Council were tested, and the results compared to those obtained by another ELISA, already validated, used as reference. Furthermore, a subset of samples for which RT-PCR results were available were used to confirm sensitivity and specificity of the test. RESULTS: The performance of the N protein expressed in plants as antigen in serologic test for SARS-CoV-2 antibody detection was shown to be highly satisfactory, with calculated diagnostic sensitivity of 96.41% (95% CI: 93.05-98.44) and diagnostic specificity of 96.37 (95% CI: 93.05-98.44) as compared to the reference ELISA, with a kappa (K) value of 0.928 (95% CI:0.892-0.964). Furthermore, the ELISA developed with plant-derived N antigen detected SARS-CoV-2 antibodies in 84 out of 93 sera from individuals showing RT-PCR positive results (86/93 for the reference ELISA). CONCLUSION: This study demonstrates that the N protein part derived from SARS-CoV-2 expressed in plants performs as a perfectly valid antigen for use in COVID-19 diagnosis. Furthermore, our results support the use of this plant platform for expression of recombinant proteins as reagents for COVID-19 diagnosis. This platform stands out as a convenient and advantageous production system, fit-for-purpose to cope with the current demand of this type of biologicals in a cost-effective manner, making diagnostic kits more affordable.

Viral Strain-Specific Differential Alterations in Arabidopsis Developmental Patterns.

Turnip mosaic virus (TuMV) infections affect many Arabidopsis developmental traits. This paper analyzes, at different levels, the development-related differential alterations induced by different strains of TuMV, represented by isolates UK 1 and JPN 1. The genomic sequence of JPN 1 TuMV isolate revealed highest divergence in the P1 and P3 viral cistrons, upon comparison with the UK 1 sequence. Infectious viral chimeras covering the whole viral genome uncovered the P3 cistron as a major viral determinant of development alterations, excluding the involvement of the PIPO open reading frame. However, constitutive transgenic expression of P3 in Arabidopsis did not induce developmental alterations nor modulate the strong effects induced by the transgenic RNA silencing suppressor HC-Pro from either strain. This highlights the importance of studying viral determinants within the context of actual viral infections. Transcriptomic and interactomic analyses at different stages of plant development revealed large differences in the number of genes affected by the different infections at medium infection times but no significant differences at very early times. Biological functions affected by UK 1 (the most severe strain) included mainly stress response and transport. Most cellular components affected cell-wall transport or metabolism. Hubs in the interactome were affected upon infection.

High-level production of active human TFPI-2 Kunitz domain in plant.

Plants are an attractive production system alternative to cell bioreactor not only because of its lower production costs, but also due to its lack of mammalian pathogens and contaminants, plant capacity to generate appropriate eukaryotic folding and in many cases correct post-translational modifications. In recent years, several recombinant proteins and antibodies have been introduced in the biopharmaceutical market, in particular in cancer therapeutics. Kunitz domain 1 (KD1), a domain of Human Tissue Factor Pathway Inhibitor-2 (TFPI-2), has an outstanding potential in cancer treatment because it is a potent inhibitor of extracellular serine proteinases involved in tumor progression and angiogenesis. We present here the expression and purification of active human KD1 in different Nicotiana species as hosts and its stability during the infection process using a construct derived from a Tobacco mosaic virus (TMV) vector. Our purification protocol allows to recover over 100mg of active human KD1 per batch of 1 kg of plant tissue at about 97% purity. The yields are reproducible, being N. benthamiana the best system where higher levels of KD1 are obtained. Recombinant KD1 was also used to produce a high-sensitivity polyclonal antibody able to detect not only KD1 but also full-length TFPI-2. Finally, we show that this platform is a valuable alternative for the large scale production of KD1.

Plant viral elongated nanoparticles modified for log-increases of foreign peptide immunogenicity and specific antibody detection.

Elongated and flexuous recombinant nanoparticles were derived from Turnip mosaic virus to be used as bioscaffolds for increased peptide immunogenicity and peptide-specific antibody sensing. For this purpose, a 20-amino acid peptide derived from human vascular endothelial growth factor receptor 3 (VEGFR-3) was fused to the N-terminal region of Turnip mosaic virus coat protein (CP) by genetic insertion. The insertion was between codons corresponding to the first and second amino acids of the CP in two versions of a previously reported virus-derived vector. Systemic infections of two genetic constructs were achieved in two different plant hosts. The construct proved stable upon successive passages and generated virus nanoparticles identifiable under the electron microscope. The chimeric structures held the VEGFR-3 peptide. Purified VER3 nanoparticles were used to immunize mice, whose sera showed log increases of antibodies against the VEGFR-3 peptide when compared with mice immunized with peptide alone, thus providing the first quantitative data on the potential of elongated flexuous viruses for peptide immunogenicity increases. Purified VER3 nanoparticles also showed log increases in their ability to detect VER3 antibodies in sera, when used as reagents in ELISA assays, an application also used here for the first time.

Genomic heterogeneity and host recovery of isolates of Malva vein clearing virus.

Malva vein clearing virus (MVCV), a tentative species of the genus Potyvirus, was identified as the causal agent of viral symptoms in Malva sp. weed plants. Amplified viral genomic fragments corresponding to approximately 20% of the 3' terminal region of its genome were obtained using non-species specific, genus-specific reagents. The sequences of the PCR fragments were determined. BLAST and phylogenetic analyses of the deduced amino acid sequence indicated that MVCV is a distinct species of the genus Potyvirus and close to Pea seed-borne mosaic virus (PSbMV) with which it forms a new phylogenetic cluster within the genus. The results show that MVCV is a definitive member of the Potyvirus genus. Specific MVCV PCR primers were designed and validated as diagnostic tools, and used to assess the variability of the species. Much variation was found and this was not correlated with either the geographical origin of the isolates, or the severity of the symptoms. Recovery from viral symptoms was observed in natural and experimental hosts. Tests in experimental hosts showed that it was a true viral recovery, in that the virus was absent and the recovered tissues could not be infected. This is the first reported example of true viral recovery of a potyvirus in a natural system.

A biomarker for the identification of four Phaeoacremonium species using the beta-tubulin gene as the target sequence.

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt-R1bt) with homology to the beta-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines.

A developmentally linked, dramatic, and transient loss of virus from roots of Arabidopsis thaliana plants infected by either of two RNA viruses.

Possible effects of host developmental stage on the amount of virus present in systemically infected plant tissues hitherto have received little attention. In this study, the pattern of virus accumulation over the plant lifespan has been examined in systemically invaded tissues of Arabidopsis thaliana infected by either of two distinct (+)RNA viruses: Turnip mosaic virus, a member of Potyvirus, and Oilseed rape mosaic virus, a member of Tobamovirus. Quantitative analyses of virus coat protein and virus genomic RNA in roots versus aerial plant parts revealed generally sinusoidal temporal patterns of virus accumulation. In noninoculated leaves, a time period was found during which no virus accumulation was detected. This period was coincident with the approximately 7 days of inflorescence bud formation and differentiation. In roots, virion content reached high levels a few days after inoculation, dropping dramatically during the period of bud formation and quickly recovering after it. These results, together with electron microscopy observations, are consistent with loss of virions due to disassembly. Fluorescence observations of green fluorescent protein-tagged virus-infected root tissue also were consistent with a net loss of virus-specified proteins. Inoculations performed after the emergence of the inflorescence and on A. thaliana flowering-time mutants support the temporal link between observed changes in virus content and inflorescence bud formation. Different host-involving biochemical processes can be invoked to provide mechanistic clues, but no one of them alone seems sufficient to explain the complex patterns of tight temporal regulation of virus accumulation observed in these experiments.

Ultra-sensitive detection of two garlic potyviruses using a real-time fluorescent (Taqman) RT-PCR assay.

A method for the detection of Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), the two most prevalent garlic potyviruses, has been developed that combines IC-RT-PCR/RT-PCR with the use of TaqMan probes. Comparisons with ELISA results obtained with identical OYDV and LYSV infected samples showed sensitivity in detecting these viruses increased up to 10(6)-fold. OYDV and LYSV were detected using different fluorochromes in the probe, thus allowing unequivocal diagnosis for each of them. The polyvalence of the designed virus-specific primers and probes was shown through their application to the detection of three isolates from very different geographical areas and from different hosts. A second version of the method avoids the need for an immunocapture step through the performance of a TaqMan RT-PCR assay directly on extracts of garlic cloves. This modification on the proposed basic method allows the analysis of bulb samples in 3-4h but did not give reproducible results with leaves. Both versions of the new diagnostic method bear great potential for their implementation in virus-free certification schemes in garlic, a vegetatively propagated crop for which such a certification is critical for a high-quality product.

Variations of Leek yellow stripe virus Concentration in Garlic and Its Incidence in Argentina.

The purpose of this work was to determine variations in titer of Leek yellow stripe virus (LYSV) throughout the crop cycle and bulb storage, and to evaluate the incidence of infected plants in the main garlic-production regions of Argentina. One hundred plants with LYSV from each of five cultivars were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in six different vegetative stages in White- and Red-type garlic cultivars and seven stages in cv. Rosado Paraguayo, throughout the year. In two White-type garlic cultivars, LYSV showed peaks of viral concentration in May, at the beginning of the crop cycle, and in November, just before harvest. In two Red-type garlic selections, an increase was detected in November (period of bulbing). The highest virus titers for these four garlic cultivars were detected in devernalized clove. In Rosado Paraguayo, the peak virus concentration occurred in September prior to harvesting. In a survey at 14 different localities in Argentina, 3,066 random samples were analyzed. LYSV was found in 80 to 98% of the plants from all regions, except in Santa Cruz, where 34% of plants were infected. The importance of this study is that it allows us to recommend the most suitable moment of the year to make the analysis with DAS-ELISA.