Plum Pox Virus Genome-Based Vector Enables the Expression of Different Heterologous Polypeptides in Nicotiana benthamiana Plants

Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.