Baculovirus-Free SARS-CoV-2 Virus-like Particle Production in Insect Cells for Rapid Neutralization Assessment.

Virus-like particles (VLPs) resemble authentic virus while not containing any genomic information. Here, we present a fast and powerful method for the production of SARS-CoV-2 VLP in insect cells and the application of these VLPs to evaluate the inhibition capacity of monoclonal antibodies and sera of vaccinated donors. Our method avoids the baculovirus-based approaches commonly used in insect cells by employing direct plasmid transfection to co-express SARS-CoV-2 envelope, membrane, and spike protein that self-assemble into VLPs. After optimization of the expression plasmids and vector ratios, VLPs with an ~145 nm diameter and the typical "Corona" aura were obtained, as confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Fusion of the membrane protein to GFP allowed direct quantification of binding inhibition to angiotensin II-converting enzyme 2 (ACE2) on cells by therapeutic antibody candidates or sera from vaccinated individuals. Neither VLP purification nor fluorescent labeling by secondary antibodies are required to perform these flow cytometric assays.

High immature platelet fraction with reduced platelet count on hospital admission. Can it be useful for COVID-19 diagnosis?

INTRODUCTION: Health professions are heavily engaged facing the current threat of SARS-CoV-2 (COVID-19). Although there are many diagnostic tools, an accurate and rapid laboratory procedure for diagnosing COVID-19 is recommended. We focused on platelet parameters as the additional biomarkers for clinical diagnosis in patients presenting to the emergency department (ED). MATERIALS AND METHODS: Five hundred and sixty-one patients from February to April 2020 have been recruited. Patients were divided into three groups: (N = 50) COVID-19 positive and (N = 21) COVID-19 negative with molecular testing, (N = 490) as reference population without molecular testing. A Multiplex rRT-PCR from samples collected by nasopharyngeal swabs was performed and the hematological data collected. RESULTS: We detected a mild anemia in COVID-19 group and lymphopenia against reference population: hemoglobin (g/dL) 13.0 (11.5-14.8) versus 13.9 (12.8-15.0) (P = .0135); lymphocytes (109 /L) 1.24 (0.94-1.73) versus 1.99 (1.49-2.64) (P < .0001). In addition, abnormal platelet parameters as follows (COVID group vs reference population): PLT (×109 /L) 209 (160-258) vs 236 (193-279) (P = .0239). IPF (%) 4.05 (2.5-5.9) versus 3.4 (2.2-4.9) (P = .0576); H-IPF (%) 1.25 (0.8-2.2) versus 0.95 (0.6-1.5) (P = .0171) were identified. In particular, COVID positive group had a high H-IPF/IPF Ratio compared to reference population [0.32 (0.29-0.36) versus 0.29 (0.26-0.32), respectively, (P = .0003)]. Finally, a PLT difference of nearly 50 × 109 /L between pre/postCOVID-19 sampling for each patient was found (N = 42) (P = .0194). CONCLUSIONS: COVID-19 group results highlighted higher IPF and H-IPF values, with increased H-IPF/IPF Ratio, associated to PLT count reduction. These findings shall be adopted for a timely diagnosis of patients upon hospital admission.

Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients.

Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.