Ultrastructural organization and reproduction of virions in Vero (E6) cells in influenza A/H1N1 pmd09 virus monoinfection and coinfection with SARS-CoV-2 (Delta and Omicron strains)

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Abstract

Introduction. RNA-containing viruses, especially influenza viruses, are of high epidemiological significance. The manifestation of COVID-19 has led to the registration of coinfection cases, the pathogenesis of which is poorly studied. The Vero (E6) cell line is widely used to study the morphogenesis of various viruses, including influenza and coronavirus.

The aim of the work is to study the ultrastructure of Vero (E6) cells and the reproduction of viral particles during monoinfection with the influenza A virus and coinfection of this virus with two SARS-CoV-2 genovariants in dynamics 6, 18 and 24 hours after inoculation.

Materials and methods. The Vero (E6) cell line model was used for in vitro study of the viral infection effects and an analysis of the dynamics of changes in the number of intracellular viral particles. The study involved 4 experimental groups: Vero (E6) cells mono-infected with the influenza virus strain A/H1N1 pmd09 at a dose of 0.1 MOI; Vero (E6) cells co-infected with the influenza virus strain A/H1N1 pmd09 and Delta strain of SARS-CoV-2 at a total dose of 0.1 MOI; Vero (E6) cells co-infected with the influenza virus strain A/H1N1 pmd09 and Omicron strain of SARS-CoV-2 at a total dose of 0.1 MOI. In each study group, cells were monitored at time points of 6, 18, and 24 hours.

Results. After 6 h, no pathological structures were detected in all groups, except for virus-containing transport vesicles. After 18 h, vacuolization of the ER of varying degree was noted in all the studied groups. After 24 h, ultrastructural changes, namely vacuolization of organelles and/or compaction of the cytoplasm, were encountered in all groups comparatively more frequently than at 6 h and 18 h time points. . The dynamics of the number of viral particles increased significantly by 24 h time point in the monoinfection group. However, none of the coinfection groups demonstrated a tendency for the number of viral particles to change, since no statistically significant differences were found between the 6 h, 18 h, and 24 h stages.

Conclusion. The results obtained suggested that the interaction between A/H1N1 pmd09 and SARS-CoV-2 viruses contributed to an overall decrease in the formation of new virions in Vero (E6) cells in both cases of coinfection.

About the authors

Ksenia F. Emtsova

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: k.emtsova@g.nsu.ru
ORCID iD: 0009-0003-5165-5357

Trainee Researcher, Department of Microscopic Research

Russian Federation, 630559, Novosibirsk Region, Koltsovo

Ekaterina V. Spiridonova

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: spiridonova_ev@vector.nsc.ru
ORCID iD: 0009-0006-8655-6713

Trainee Researcher, Department of Microscopic Research

Russian Federation, 630559, Novosibirsk Region, Koltsovo

Vladimir V. Omigov

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Author for correspondence.
Email: omigov_vv@vector.nsc.ru
ORCID iD: 0000-0002-2028-6099

PhD (Medicine), Leading Researcher

Russian Federation, 630559, Novosibirsk Region, Koltsovo

Anastasia A. Moiseeva

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: moiseeva_aa@vector.nsc.ru
ORCID iD: 0000-0001-7048-2357

junior researcher Department of Zoonotic Infections and Influenza

Russian Federation, 630559, Novosibirsk Region, Koltsovo

Elena I. Danilenko

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: danilenko_ev@vector.nsc.ru
ORCID iD: 0009-0007-8106-7037

junior researcher Department of Zoonotic Infections and Influenza

Russian Federation, 630559, Novosibirsk Region, Koltsovo

Oleg S. Taranov

State Scientific Center for Virology and Biotechnology «Vector» of the Federal Service for Surveillance in the Sphere of Consumer Rights Protection and Human Welfare (Rospotrebnadzor)

Email: taranov@vector.nsc.ru
ORCID iD: 0000-0002-6746-8092

Head of Department of Microscopic Research

Russian Federation, 630559, Novosibirsk Region, Koltsovo

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2. Fig. 1. Dynamics of ultrastructural changes in Vero (E6) cells in influenza A/H1N1 pmd09 virus monoinfection. Electron diffraction patterns А, B, and C correspond to the 6h stage, D and E to the 18h stage, and F and G to the 24h stage. А – a section of the cell cytoplasm with optically empty zones representing ER cell profiles with focal vacuolization (arrows). Scale bar is 1 μm; B – a section of the cell cytoplasm with virus-containing transport vesicles (arrows). Scale bar is 500 nm; C – a section of the cell cytoplasm with vacuolated ER cavities (arrows). Multiple adherent viral particles are localized along the plasma membrane. Scale bar is 2 μm; D – a section of the cell cytoplasm with vesicles (V) and a nucleus (N) with signs of invagination of the nuclear membranes (arrow). Electron diffraction pattern. Scale bar is 1 μm; E – a section of the cell cytoplasm containing A/H1N1 pmd09 viral particles (arrows). Scale bar is 500 nm; F – a section of a cell with sharply cleared hyaloplasm, a large number of membrane structures and free viral particles (arrows). Scale bar is 1 μm; G – a fragment of the cell cytoplasm with a nucleus (N). The karyoplasm is cleared relative to the hyaloplasm. Scale bar is 1 μm; H – a «Bar-plot» graph reflecting the dynamics of changes in the number of viral particles per cell at stages 6, 18 and 24 h.

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3. Fig. 2. Dynamics of ultrastructural changes in Vero (E6) cells coinfected with the A/H1N1 pmd09 influenza virus and the Delta strain of SARS-CoV-2. Electron diffraction patterns А, B, and C correspond to the 6-h stage, D and E to the 18-h stage, and F and G to the 24-h stage. А – a cell region demonstrating high synthetic activity: multiple lumens of the Golgi apparatus and rER (arrows) are visible, with signs of vacuolization in places. Scale bar is 2 μm; B – fragments of the membrane with microvilli and viral particles in the adhesion state (arrows). Scale bar is 500 nm; C – sections of two preserved cells. The cell in the upper part of the electronogram has a comparatively denser hyaloplasm, in which vesicles containing electron-dense substance are localized (arrow). N – nucleus. Scale bar is 2 μm; D – a section of the cell cytoplasm with high synthetic activity, expressed in vacuolization of the ER profiles (arrows). Scale bar is 2 μm; E – a section of the cell hyaloplasm with virus-containing transport vesicles (arrows) near the nucleus (N). Scale bar is 500 nm; F – a fragment of the cell cytoplasm with viral particles localized along the periphery of the vesicular membrane (arrow). Scale bar is 1 μm; G – a section of the cell nucleus (N) with signs of invagination of the nuclear membranes (arrow). Scale bar is 1 μm; H – a «Bar-plot» graph reflecting the dynamics of changes in the number of viral particles per cell at stages 6, 18 and 24 h.

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4. Fig. 3. Dynamics of ultrastructural changes in Vero (E6) cells coinfected with the A/H1N1 pmd09 influenza virus and the Omicron strain of SARS-CoV-2. Electron diffraction pattern A corresponds to the 6-h stage, B – to the 18-h stage, and C – to the 24-h stage. А – a section of the cell cytoplasm with organelles in a state of vacuolation (arrows). Scale bar is 500 nm; В – a section of the cell hyaloplasm with profiles of the Golgi complex (GC), some of which have a double membrane (solid arrows) and vesicles near to it (V); free viral particles are localized in the hyaloplasm (dashed arrows). Scale bar is 500 nm; C – a general electronogram of a cell in a state of destruction. The hyaloplasm (H) has an increased density. The nucleus (N) contains a nucleolus (Ns), the chromatin is compacted. Scale bar is 1 μm; D – a «Bar-plot» graph reflecting the dynamics of changes in the number of viral particles per cell at stages 6, 18 and 24 h.

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Copyright (c) 2025 Emtsova K.F., Spiridonova E.V., Omigov V.V., Moiseeva A.A., Danilenko E.I., Taranov O.S.

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This work is licensed under a Creative Commons Attribution 4.0 International License.

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