RESUMO
Plants of Senna multiglandulosa (family Fabaceae), an ornamental shrub, growing adjacent to tomato and chrysanthemum greenhouses located in San Diego, Texcoco, Estado de Mexico, had leaves with putative virus symptoms, consisting of annular or irregular chlorotic spots of different sizes (Supplementary Fig. 1a). To investigate the presence of a virus, high-throughput sequencing (HTS) was performed. Total RNA was extracted from symptomatic leaves of S. multiglandulosa plants using the SV Total RNA Isolation System Kit (Promega, USA). A portion of the RNA was sent to BGI Genomics (China) for cDNA library construction and sequencing on the DNBSEQ platform (BGI Genomics). HTS yielded 14,673,469 clean paired reads (150x2), which were assembled de novo into 91,879 contigs using SPAdes v3.15 software (Prjibelski et al. 2020). The contigs ranged from 78 to 14,534 nucleotides (nts), which were subjected to BLASTx and BLASTn analyses. A single viral contig of 9,501 nts was detected (average coverage: 56,716x per nt) representing the nearly complete genome of tobacco etch virus (TEV). The highest identity was 79.26% at the nt level (92% query coverage) with TEV isolate TEV7DA (GenBank: DQ986288; length: 9,539 nts) from the USA, and 86.67% at the amino acid (aa) level considering the polyprotein, which are higher than the species demarcation threshold (<76% nt and <82% aa) for the genus Potyvirus (Inoue-Nagata et al. 2022). Additionally, the sequence obtained from S. multiglandulosa revealed 79.21-79.37% nt identities with different TEV isolates from Solanaceae plants (Capsicum annuum, MW748496; Solanum lycopersicum, OM471966.1; Nicotiana tabacum, OL311684.1). The new TEV genome was deposited in GenBank under accession number ON110203. The results obtained by HTS were confirmed by RT-PCR with the original isolated RNA using a pair of specific primers designed from the TEV sequence (TEV-NIb-F, 5'- GCGCTTAAATGCAGACTCGG-3' and TEV-NIb-R, 5'-GTGAAAGTTCAGCAGCAAGCGCA-3') that amplify a 550-bp fragment of the RNA-dependent RNA polymerase. The obtained amplicon was sequenced by the Sanger method, and was 100% identical to the sequence generated by HTS. Subsequently, N. tabacum and N. glutinosa plants were mechanically inoculated using TEV-positive S. multiglandulosa leaves as the inoculum source. Twenty days after inoculation, light chlorotic spots and necrotic lesions were observed on N. tabacum and mosaic on N. glutinosa (Supplementary Fig. 1b-c). RT-PCR analysis confirmed the presence of TEV infection in these indicator plants. To determine the incidence of S. multiglandulosa plants showing TEV-infection symptoms, a survey (n=16) was carried out on two farms in Texcoco; the survey showed a 100% incidence of symptoms. Five survey samples were randomly selected, and the presence of TEV was confirmed by RT-PCR. The discovery of Tobacco etch virus (family Potyviridae: genus Potyvirus) in tobacco was reported in Kentucky, USA in 1928 (Valleau and Johnson, 1928), one of the most common and damaging viruses for the chili crop in Mexico (Delgado, 1974). TEV causes heavy yield loss in several Solanaceae plants and infects more than 120 species in 19 families of dicotyledons (Holmes, 1946). S. obtusifolia (originally Cassia obtusifolia) was the first legume reported as a natural host of TEV in Florida, USA (Anderson, 1954). To our knowledge, this is the first report of the natural infection of S. multiglandulosa by TEV in Mexico and the first TEV genome isolated and sequenced from a legume. S. multiglandulosa is widely distributed in 16 states in Mexico, both cultivated and naturalized, however, it is not considered native to the country (Rzedowski and Calderón, 1997). The occurrence of TEV in S. multiglandulosa represents an alternative reservoir of the virus, with an important role in the epidemiology of the disease.
RESUMO
U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.
RESUMO
BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.