Volume: 1, 2022
1st International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland:
ENVIRONMENT – PLANT – ANIMAL – PRODUCT
Abstract number: A001
DOI: https://doi.org/10.24326/ICDSUPL1.A001
Published online: 26 April 2022
ICDSUPL, 1, A001 (2022)
Preliminary confirmation of alternative cellular receptor in neurons for mouse coronavirus (MHV-JHM)
Michalina Bartak1, Anna Słońska1, Marcin W. Bańbura1, Joanna Cymerys1
1 Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8, 02-786, Warsaw, Poland
* Corresponding author: michalina_bartak@sggw.edu.pl
Abstract
Mouse coronavirus (mouse hepatitis virus; MHV) has a broad spectrum of tissue tropism, causing respiratory, gastrointestinal, and CNS diseases. Mechanisms of coronavirus neurotropism, including MHV-JHM strain, ranging from the leading receptor used during adsorption to cells or the regulation of cytoskeleton motility during intracellular transport, are still not well understood. According to the literature, MHV enters hepatocytes mainly through CEACAM1 receptors. However, in the nervous system there may be potential other receptors – PSG16, that can be utilized during entry.In order to understand the mechanisms of MHV-JHM neuroinfection, the cellular receptor and the role of the actin cytoskeleton during virus replication in neurons in vitro were determined. The study was performed simultaneously on a mouse hepatocyte cell line, which constitute the target cells for MHV. In this study, primary neuronal cultures obtained from BALB/c mouse fetuses and a mouse hepatocyte cell line [ATCC CCL-9.1] infected with MHV-JHM were used. Viral antigen was detected by indirect immunofluorescence using monoclonal antibodies against spike S2 protein, and visualized using Alexa Fluor 488. Cell receptors were detected using anti-PSG16 or anti-CEACAM1 antibodies, and Alexa Fluor 647 were used for visualization. Actin filaments were visualized using TRITC-phalloidin conjugate. For immunofluorescence analysis Olympus FV10i confocal microscope and ImageJ software were used. Our research showed that the MHV-JHM strain utilized the actin cytoskeleton of neurons during infection (15, 30 min. pi and 1, 2, 24, 48, 72 hpi). It was observed: (i) condensation of actin filaments in the cortical layer of the cytoplasm (2 and 24 hpi); (ii) presence of viral antigens along stress fibers; (iii) formation of long actin structures used by MHV-JHM for intercellular transport (24, 48, and 72 hpi). Immunofluorescence analysis confirmed that CEACAM1 was the predominant receptor in hepatocytes, the target cells for this coronavirus. In neurons, we have observed that MHV-JHM utilize both cellular receptors CEACAM1 and PSG16; however, colocalization analysis performed by ImageJ revealed a dominant role of PSG16 during neuronal infection. To confirm obtained results, quantitative Real-Time PCR analysis is planned to be performed in MHV-JHM infected neurons with silenced CEACAM1 coreceptors.
How to cite
M. Bartak, A. Słońska, M.W. Bańbura, J. Cymerys, 2022. Preliminary confirmation of alternative cellular receptor in neurons for mouse coronavirus (MHV-JHM). In: 1st International PhD Student’s Conference at the University of Life Sciences in Lublin, Poland: Environment – Plant – Animal – Product. https://doi.org/10.24326/ICDSUPL1/A001