Simultaneous Detection of RIG-1, MDA5, and IFIT-1 Expression Is a Convenient Tool for Evaluation of the Interferon-Mediated Response.

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.

A novel method for multiplex protein biomarker analysis of human serum using quantitative MALDI mass spectrometry.

In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4-5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established.