Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA.

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12-316) and antigen N (nucleocapsid residues 37-402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449-711 and nucleocapsid protein residues 160-406) and QCoV7 truncated antigen (nucleocapsid residues 37-402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

A novel highly specific biotinylated MAC-ELISA for detection of anti-SARS-CoV-2 nucleocapsid antigen IgM antibodies during the acute phase of COVID-19.

The gold standard for diagnosing COVID-19 in the acute phase is RT-qPCR. However, this molecular technique can yield false-negative results when nasopharyngeal swab collection is not conducted during viremia. To mitigate this challenge, the enzyme-linked immunosorbent assay (ELISA) identifies anti-SARS-CoV-2 IgM antibodies in the initial weeks after symptom onset, facilitating early COVID-19 diagnosis. This study introduces a novel and highly specific IgM antibody capture ELISA (MAC-ELISA), which utilizes biotinylated recombinant SARS-CoV-2 nucleocapsid (N) antigen produced in plants. Our biotinylated approach streamlines the procedure by eliminating the requirement for an anti-N-conjugated antibody, circumventing the need for peroxidase-labeled antigens, and preventing cross-reactivity with IgM autoantibodies such as rheumatoid factor. Performance evaluation of the assay involved assessing sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy using 682 RT-qPCR-positive samples, categorized by weeks relative to symptoms onset. Negative controls included 205 pre-pandemic serum samples and 46 serum samples from patients diagnosed with other diseases. Based on a cut-off of 0.087 and ROC curve analysis, the highest sensitivity of 81.2% was observed in the 8-14 days post-symptom (dps) group (2nd week), followed by sensitivities of 73.8% and 68.37% for the 1-7 dps (1st week) and 15-21 dps groups (3rd week), respectively. Specificity was consistently 100% across all groups. This newly developed biotinylated N-MAC-ELISA offers a more streamlined and cost-effective alternative to molecular diagnostics. It enables simultaneous testing of multiple samples and effectively identifies individuals with false-negative results.

Metagenomic analyses of plant virus sequences in sewage water for plant viruses monitoring.

Frequent monitoring of emerging viruses of agricultural crops is one of the most important missions for plant virologists. A fast and precise identification of potential harmful viruses may prevent the occurrence of serious epidemics. Nowadays, high-throughput sequencing (HTS) technologies became an accessible and powerful tool for this purpose. The major discussion of this strategy resides in the process of sample collection, which is usually laborious, costly and nonrepresentative. In this study, we assessed the use of sewage water samples for monitoring the widespread, numerous, and stable plant viruses using HTS analysis and RT-qPCR. Plant viruses belonged to 12 virus families were found, from which Virgaviridae, Solemoviridae, Tymoviridae, Alphaflexiviridae, Betaflexiviridae, Closteroviridae and Secoviridae were the most abundant ones with more than 20 species. Additionally, we detected one quarantine virus in Brazil and a new tobamovirus species. To assess the importance of the processed foods as virus release origins to sewage, we selected two viruses, the tobamovirus pepper mild mottle virus (PMMoV) and the carlavirus garlic common latent virus (GarCLV), to detect in processed food materials by RT-qPCR. PMMoV was detected in large amount in pepper-based processed foods and in sewage samples, while GarCLV was less frequent in dried and fresh garlic samples, and in the sewage samples. This suggested a high correlation of virus abundance in sewage and processed food sources. The potential use of sewage for a virus survey is discussed in this study. Supplementary Information: The online version contains supplementary material available at 10.1007/s40858-023-00575-8.