Mammarenaviral Infection Is Dependent on Directional Exposure to and Release from Polarized Intestinal Epithelia

Warner, Nikole L.; Jokinen, Jenny D.; Beier, Juliane I.; Sokoloski, Kevin J.; Lukashevich, Igor S.

Since the gastrointestinal tract likely plays an essential role in the arenavirus rodent-to-human transmission, we used the human adenocarcinoma Caco-2 cell line [35] to establish an in vitro model of the intestinal epithelia lining of the gut ( Figure 1A) . The formation of a polarized monolayer was assessed by TEER and by detection of the tight junction protein Zonal-Occludin-3 (ZO-3), a partition marker of the apical and basolateral sides of the cells, was readily detected in between sister cells ( Figure 1B) . Furthermore, polarized Caco-2 were stained with antibody against α-DG, a principal cell receptor for OW arenaviruses ( Figure 1B ). Since the gastrointestinal tract likely plays an essential role in the arenavirus rodent-to-human transmission, we used the human adenocarcinoma Caco-2 cell line [35] to establish an in vitro model of the intestinal epithelia lining of the gut ( Figure 1A) . The formation of a polarized monolayer was assessed by TEER and by detection of the tight junction protein Zonal-Occludin-3 (ZO-3), a partition marker of the apical and basolateral sides of the cells, was readily detected in between sister cells ( Figure 1B) . Furthermore, polarized Caco-2 were stained with antibody against α-DG, a principal cell receptor for OW arenaviruses ( Figure 1B ). In general, the arenavirus species used in this study are not associated with cytopathic effects. Nonetheless, it was essential to the utility of our model to confirm that the apical and basolateral separations were intact during infection. In line with their non-cytopathic nature, the OW arenaviruses used in this study did not negatively affect electric resistance of epithelial monolayers during the 5-day observation period, suggesting that integrity of monolayers was preserved during the infection ( Figure 1C ). In contrast, the alphavirus Venezuelan equine encephalitis (VEEV) strain TC-83, which is known to be highly cytopathic, readily disrupted the integrity of polarized Caco-2 cells. Analysis of the mRNA and protein levels of tight junction proteins was tested and no significant In general, the arenavirus species used in this study are not associated with cytopathic effects. Nonetheless, it was essential to the utility of our model to confirm that the apical and basolateral separations were intact during infection. In line with their non-cytopathic nature, the OW arenaviruses Viruses 2018, 10, 75 6 of 15 used in this study did not negatively affect electric resistance of epithelial monolayers during the 5-day observation period, suggesting that integrity of monolayers was preserved during the infection ( Figure 1C ). In contrast, the alphavirus Venezuelan equine encephalitis (VEEV) strain TC-83, which is known to be highly cytopathic, readily disrupted the integrity of polarized Caco-2 cells. Analysis of the mRNA and protein levels of tight junction proteins was tested and no significant change in quantity of tight junction proteins was observed in infected cells as compared to mock infected.