Comparative analysis of MxA, OAS1, PKR gene expression levels in leukocytes of patients with influenza and coronavirus infection

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Abstract

BACKGROUND: The innate immune response, particularly the interferon system, plays a crucial role in defending the host against viral pathogens. Interferon signaling induces the expression of specific antiviral proteins known as interferon-stimulated genes, which inhibit viral replication through various mechanisms.

AIM: This study aimed to develop a quantitative PCR system to assess the molecular regulation of human interferon-stimulated genes MxA, OAS1, and PKR, and to determine their expression in blood leukocytes in response to RNA-containing viruses.

MATERIALS AND METHODS: Leukocytes were isolated from patients with laboratory-confirmed influenza and COVID-19 infections 3–4 days after symptom onset. Ex vivo viral infection was induced using influenza viruses A/California/07/09pdm (H1N1pdm09), B/Malaysia/2506/04 (Vic), strain A2 respiratory syncytial virus, and SARS-CoV-2 HCoV-19/Russia/SPE-RII-3524V/2020.

RESULTS: A multiplex qPCR assay was developed for analyzing human MxA, OAS1, and PKR gene expression, with high amplification efficiency. The test system was used to study the molecular regulation of these genes in leukocytes in influenza and COVID-19 patients. The expression levels of MxA, OAS1, and PKR genes were significantly increased in blood leukocytes of hospitalized patients 3–4 days after symptom onset. Stimulation of leukocytes by influenza A, influenza B, and respiratory syncytial virus led to increased mRNA levels of these genes, while stimulation by SARS-CoV-2 did not result in changes in gene expression.

CONCLUSIONS: The multiplex test system can be used to characterize the expression of antiviral effector interferon-stimulated genes, aiding in the study of virus evasion from the innate immune response.

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About the authors

Sergey A. Klotchenko

Smorodintsev Research Institute of Influenza

Email: fosfatik@mail.ru
ORCID iD: 0000-0003-0289-6560
SPIN-code: 2632-6195

Cand. Sci. (Biol.), Senior Research Associate at the Influenza Vaccine Laboratory

Russian Federation, Saint Petersburg

Ekaterina A. Romanovskaya-Romanko

Smorodintsev Research Institute of Influenza

Email: ekaterina.romanovskaya@influenza.spb.ru
ORCID iD: 0000-0001-7560-398X
SPIN-code: 1012-8043

Cand. Sci. (Biol.), Leading Research Associate at the Vector Vaccine Laboratory

Russian Federation, Saint Petersburg

Veronika A. Oleynik

Smorodintsev Research Institute of Influenza; Saint Petersburg State Institute of Technology (Technical University)

Email: working.lyutik@gmail.com

Research Laboratory Assistant at the Influenza Vaccine Laboratory, student

Russian Federation, Saint Petersburg; Saint Petersburg

Marya A. Egorova

Smorodintsev Research Institute of Influenza

Email: sci-work_maria@mail.ru
ORCID iD: 0000-0003-1408-8413
SPIN-code: 6055-1423

Research Associate at the Systemic Virology Laboratory

Russian Federation, Saint Petersburg

Varvara S. Monakhova

Smorodintsev Research Institute of Influenza

Email: varvara.bio@gmail.com
SPIN-code: 2111-8493

Research Associate at the Laboratory of Genetic Engineering and Expression of Recombinant Proteins

Russian Federation, Saint Petersburg

Evgeny V. Venev

Smorodintsev Research Institute of Influenza; Botkin Clinical Infectious Diseases Hospital

Email: imberbis@gmail.com

Senior Lecturer, infectious disease doctor

Russian Federation, Saint Petersburg; Saint Petersburg

Marina A. Plotnikova

Smorodintsev Research Institute of Influenza

Author for correspondence.
Email: biomalinka@mail.ru
ORCID iD: 0000-0001-8196-3156
SPIN-code: 2986-9850

Cand. Sci. (Biol.), Senior Research Associate at the Vector Vaccine Laboratory

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. Determination of PCR efficiencies in monoplex and multiplex approaches: a — multiplex amplification of MxA, OAS1, and PKR genes (simultaneous detection in one tube); b — separation of PCR products using agarose gel electrophoresis: 1 — OAS1, 2 — PKR, 3 — MxA, 4 — simultaneous amplification of three genes in multiplex PCR, 5 — DNA Ladder, 100 bp (Fermentas); c — standard curve for MxA gene in monoplex approach; d — standard curve for the OAS1 gene in monoplex approach; d — standard curve for the PKR gene in monoplex approach

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3. Fig. 2. Relative expression of MxA, OAS1, and PKR genes in leukocytes of patients with influenza infection А (IVA), coronavirus disease (COVID-19), and in healthy volunteers (HV). Statistical significance was determined for groups of infected people compared with a group of healthy volunteers by Kruskal–Wallis test (with pairwise Dunnett’s multiple comparisons test): * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

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4. Fig. 3. Patterns expression of MxA (a), OAS1 (b) and PKR (c) genes in leukocytes (from healthy volunteers) in response to in vitro stimulation of leukocytes by influenza viruses А (IVA), B (IVB), SARS-CoV-2, and respiratory syncytial virus (RSV) compared to uninfected cells (CC). Statistical significance was determined using single-factor analysis of variance (ANOVA) for paired samples with Holm–Sidak correction for groups stimulated by viruses relative to control cells group: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS is non-significant, the differences are not reliable

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5. Fig. 1. MxA gene amplification using different fluorophores and quenchers

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6. Fig. 2. Growth curves of fluorescence obtained for the dilution series of samples (from –9 to –2) during real-time PCR detection on channel FAM for MxA detection (a); ROX for OAS1 detection (b); Cy5 for PKR detection (c)

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