Abstract
The course of infection upon virus entry into the cell depends not only on the biological characteristics of the cells and of the virus itself, but also on the intensity of the cell infection by the virus, i.e., on the multiplicity of infection. The purpose of our work was to perform a comparative study of the responses of two human cell lines, the lung carcinoma cell line A-549 and the endothelium cell line ECV-304, to the infection with the influenza virus A at different multiplicities of infection. At the first passage, both cell lines responded by enhancement of proliferation and apoptosis induction only to the low doses of influenza virus (ID 1–10). In A-49 cells, the stimulatory effect of the low virus doses was observed 1–2 days earlier than in ECV-304 cells. Enhanced proliferation was observed in both cell lines from the second to the fourth passages, when cells were infected with higher virus doses (ID 100 and 1000). In addition, the response of the A-549 cells to low doses of the H3N2 strain of the influenza virus A depended on the virus propagation conditions—namely, no enhancement of cell proliferation was observed in response to the infection with the virus propagating in chicken embryonated eggs, in contrast to infection with the virus that propagated in cell culture. Immunocytochemistry of A-549 cells has demonstrated that, on the third day after infection, there could be observed a change (in the dose-dependent manner) in the intracellular localization of p53 and cyclin A, proteins involved in the cell cycle progression. At the low virus dose, cyclin A was predominantly detected in the nuclei (63%), while at the high virus dose it was p53 (54%), which was predominantly detected in this cellular compartment, this observation confirming that stimulation of cell proliferation in the case of very low multiplicity of infection and cell division arrest takes place in the case of high multiplicity of influenza virus infection. The study of the influenza virus A reproduction in A-549 and ECV-304 cells using a whole number of virology techniques showed low sensitivity of these cells to the influenza virus, which manifested in the gradual decrease in the viral RNA expression and the impairment of mature viral particles assembly during several passages. Therefore, the decrease in the multiplicity of infection is associated in the A-549 and ECV-304 cells with impairment of production of mature virus particles or certain virus protein synthesis, which is accompanied by cell proliferation enhancement and apoptosis induction. As a result of the comparative study of the two cell lines (A-549 and ECV-304) upon infection with different doses of influenza virus A, we have revealed common principles and specific features indicating the effects of the biological properties of the viruses and cells, as well as of the multiplicity of infection on the course of virus infection.
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Abbreviations
- PC:
-
percent of control
- AI:
-
apoptosis index
- ID:
-
infectious dose
- mAbs:
-
monoclonal antibodies
- TCID50 :
-
50% tissue culture infectious dose, the virus titer able to produce cytopathic effects in 50% of cells in a monolayer
- CPE:
-
cytopathic effect
- TNF:
-
tumor necrosis factor
- FS:
-
fetal serum
- НА:
-
hemagglutinin
- NА:
-
neuraminidase
References
Brown, J., Reading, S., Jones, S., Fitchet, C., Howl, J., Martin, A., Longland, C., Michelangeli, F., Dubrova, Y., and Brown, C., Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell lines T24/83, Lab. Investig., 2000, vol. 80, pp. 37–45.
Chirathaworn, C., Pongpanich, A., and Poovorawon, Y., Herpes simplex 1 induced LOX-1 expression in an endothelial cell lines ECV304, Viral Immunol., 2004, vol. 17, pp. 308–314.
Durnova, A.O., Kadyirova, R.A., Danilenko, D.M., Djukov, M.I., Martyntseva, V.A., Smirnova, T.D., Eropkin, M.Yu., and Kiselev, O.I., Study of the reproduction of influenza A virus in human endometrial cells, Akush. Zhensk. Bol., 2013, vol. 12, no. 2, pp. 23–28.
Fujimoto, A., Onodera, H., Mori, A., Nagayama, S., Yonenaga, Y., and Tachibana, T., Tumour plasticity and extravascular circulation in ECV304 human bladder carcinoma cells, Anticancer Res., 2006, vol. 26, pp. 59–70.
He, Y., Xu, K., Keiner, B., Zhou, J., Czudai, V., Li, T., Chen, Z., Lui, J., Klenk, H.-D., Shu, Y.L., and Sun, B., Influenza A virus replication induces cell cycle arrest in G0/G1 phase, J. Virol., 2010, vol. 84, pp. 12832–12840.
Heath-Engel, H., and Lindgwood, C., Verotoxin sensitivity of ECV304 cells in vitro and in vivo in a xenograft tumour model: VT1 as a tumour neovascular marker, Angiogenesis, 2003, vol. 6, pp. 129–141.
Kido, H., Okumura, Y., Takahashi, E., Pan, H.-Y., Wang, S., Yao, D., Yao, M., Chida, J., and Yano, M., Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure, Biochim. Biophys. Acta, 2012, vol. 1824, pp. 186–194.
Kiselev, O.I., Genom pandemicheskogo virusa grippa A/H1N1-2009 (Genome of Pandemic Influenza Virus A/H1N1-2009), Moscow: Dmitreyd Grafik Grupp, 2011.
Klenk, H.-D., Wagner, R., Heuer, D., and Wolff, T., Importance of hemagglutinin glycosylation for the biological functions of influenza virus, Virus Res., 2002, vol. 82, pp. 73–78.
Liew, K. and Chow, V., Differential display RT-PCR analysis of ECV304 decanalizationwith Denge virus type 2 reveals messenger RNA expression profiles of multiple human genes involved in known and novel roles, J. Med. Virol., 2004, vol. 72, pp. 597–609.
Lin, Y., Xiong, X., Wharton, S., Martin, S., Coombs, P., Vachieri, S., Christodoulou, E., Walker, P., Lui, J., Skehel, J., Gambian, S., Hay, A., Daniels, R., and McCauley, J., Evolution of the receptor binding properties of the influenza A (H3N2) hemagglutinin, Proc. Natl. Acad. Sci. U. S. A., 2012, vol. 109, pp. 21474–21479.
Mo, X., Ma, W., Zang, Y., Zhao, H., Deng, Y., Yuan, W., Wang, Y., Li, Y., Zhu, C., Liu, M., and Wu, X., Microarray analyses of differentially expressed human genes and biological processes in ECV304 cells infected with rubella virus, J. Med. Virol., 2007, vol. 79, pp. 1783–1791.
Mochalova, L., Gambaryan, A., Romanova, J., Tuzikov, A., Chinarev, A., Katinger, D., Katinger, H., Egorov, A., and Bovin, N., Receptor-binding properties of modern human influenza viruses primarily isolated in vero and MDCK cells and chicken embryonated eggs, Virology, 2003, vol. 313, pp. 473–480.
Ocana-Macchi, M., Bel, M., Guzylack-Piriou, L., Ruggli, N., Liniger, M., McCullough, K., Sakoda, Y., Isoda, N., Matrosovich, M., and Summerfield, A., Hemagglutinin-dependent tropism of H5N1 avian influenza virus for human endothelial cells, J. Virol., 2009, vol. 83, pp. 12947–12955.
Short, K., Veldhuis Kroeze, E., Reperant, L., Richard, M., and Kuiken, Th., Influenza virus and endothelial cells: a specific relationship, Microbiology, 2014, vol. 5, pp. 1–11.
Shwetank Date, O., Kim, K., and Manjunath, R., Infection of human endothelial cells by japanese encephalitis virus; increased expression and release of soluble HLA-E, PLoS One, 2013, vol. 8, p. e79197.
Smirnova, T.D., Gudkova, T.M., Kuznetsova, I.K., and Ryzhak, G.A., Development of the model of interaction of influenza A virus with human lymphoblastoid cell lines for the study of biological features of viruses and determination of activity of antivirals, Klet. Kul’tury. Inform. Byul., 2009, vol. 24, pp. 25–34.
Smirnova, T.D., Danilenko, D.M., Gudkova, T.M., Pisareva, M.M., Kadyrova, R.A., and Slita, A.V., Influence of different doses of infectious influenza A viruses on proliferation of human monolayer cell cultures, Klet. Kul’tury. Inform. Byul., 2011a, vol. 27, pp. 3–12.
Smirnova, T.D., Danilenko, D.M., Eropkin, M.Yu., Deeva, E.G., and Kiselev, O.I., Influence of Rimantadine, Ribavirin, and Triazavirine on influenza virus replication in human monolayer and limphoblastoid cell lines, Antibiot. Khimioter., 2011b, vol. 56, nos. 11–12, pp. 11–16.
Smirnova, T.D., Danilenko, D.M., Plotnikova, M.A., Kadyrova, R.A., Slita, A.V., and Eropkin, M.Yu., Influence of different subtypes of influenza A virus in the presence of antiviral agents on proliferation and induction of tumor necrosis factor in human cell lines A-549 and ECV-304, Klet. Kul’tury. Inform. Byul., 2012, vol. 28, pp. 37–49.
Smirnova, T.D., Danilenko, D.M., Il’inskaya, E.V., Smirnova, S.S., and Eropkin, M.Yu., Impact of various multiplicity of infection of influenza A virus on proliferation and apoptosis induction in cultured cell lines of lymphocytic and monocytic origin (Jurkat, NC-37, THP-1, U-937), Tsitologiia, 2015, vol. 57, no. 7, pp. 526–532.
Suda, K., Rothen-Rutishauser, B., Gunthert, M., and Wunderli-Andenspach, H., Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features, In Vitro Cell Dev. Biol. Animal, 2001, vol. 37, pp. 505–514.
Sun, X., and Whittaker, G., Role of actin cytoskeleton during influenza virus internalization into polarized epithelial cells, Cell Microbiol., 2007, vol. 9, pp. 1672–1682.
Takahashi, K., Sawasaki, Y., Hata, J., Mukai, K., and Goto, T., Spontaneous transformation and immortalization of human endothelial cells, In Vitro Cell Dev. Biol., 1990, vol. 26, pp. 265–274.
Teijaro, J., Walsh, K., Cahalan, S., Fremgen, D., Roberts, E., Scott, F., Martinborough, E., Peach, R., Oldstone, M., and Rosen, H., Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection, Cell, 2011, vol. 146, pp. 980–991.
Ueda, M., Yamate, M. A., Daidoji, T., Okuno, Y., Ikuta, K., and Nakaya, T., Maturation efficiency of viral glycoproteins in the ERimpacts the production of influenza A virus, Virus Res., 2008, vol. 136, pp. 91–97.
van der Brand, J., Stitelaar, K., van Amarongen, G., Rimmelzwaan, G., Simon, J., de Wit, E., Munstre, V., Bestebroer, T., Fouchier, R., Kuiken, T., and Osterhaus, A., Severity of pneumonia due to new H1N1 influenza virus in ferrets is intermediate between that due seasonal H1N1 virus and highly pathogenic avian influenza H5N1 virus, J. Infect. Dis., 2010, vol. 201, pp. 993–999.
Veckman, V., Osterland, P., Fagerland, R., Melen, K., Matikainen, S., and Julkinen, I., TNF-α and IFN-α enhance influenza-A-virus-induced chemokine gene expression in human A-549 lung epithelial cells, Virology, 2006, vol. 345, pp. 96–104.
Vester, D., Rapp, E., Gade, D., Genzel, Y., and Reichi, U., Quantitative analysis of proteome alterations in human influenza A virus-infected mammalian lines, Proteomics, 2009, vol. 9, pp. 3316–3327.
Yartseva, N.M. and Fedortseva, R.F., Characterization of the spontaneously transformed human endothelial cell line ECV304. 1. Multiple chromosomal rearrangements in ECV304 cells, Cell Tissue Biol., 2008, vol. 2, no. 4, pp. 428–435.
Zhang, X., Tang, Q., and Xu, L., Herpes simplex virus 2 infects human endothelial ECV304 cells and induces apoptosis synergistically with ox-LDL, J. Toxicol. Sci., 2014, vol. 39, pp. 909–917.
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Original Russian Text © D.M. Danilenko, S.S. Smirnova, T.D. Smirnova, M.M. Pisareva, M.A. Plotnikova, A.O. Drobintseva (Durnova), M.Yu. Eropkin, 2016, published in Tsitologiya, 2016, Vol. 58, No. 3, pp. 192–200.
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Danilenko, D.M., Smirnova, S.S., Smirnova, T.D. et al. Human lung carcinoma (A-549) continuing cell line and human endothelial (ECV-304) continuing cell line responses to the influenza virus at different multiplicities of infection. Cell Tiss. Biol. 10, 305–313 (2016). https://doi.org/10.1134/S1990519X16040040
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DOI: https://doi.org/10.1134/S1990519X16040040