Joua ! f ral V!ro!gY.!1996!.,.77,.25qT2.S!.4:.Fr!nte d i.r Grea! Br!tain . Virus entry into a polarized epithelial cell line (MDCK): similarities and dissimilarities between influenza C virus and bovine coronavirus Beate Schultze, Gert Zimmer and Georg Herder Institut for Virologie, Philipps-Universit Herrler et al., 1988). FoIowing the adsorption step, the glycoproteins induce fusion between the viral envelope and the cellular membrane and thus influence the introduction of the viraI genome into the cytoplasm. In the case of influenza C virus, fusion is an acid- dependent event. The fusion reaction is triggered by the acidic environment encountered within endosomes after endocytotic uptake (reviewed by Herrler Herrler Vlasak eta., 1988; Schultze pore size 04 ,m; diameter 245 mm) placed in a the apical and basal media were replaced daily. Electrical resistance was measured using a Millicell ERS apparatus. Only cell monolayers with a resistance higher than I000 2 cm 2 were used for experiments. Viruses. Strain Johannesburg/I/66 of influenza C virus was grown in 8-day-old embryonated eggs by allantoic inoculation. The allantoic fluid was harvested after incubation of the eggs for 3 days at 33 C (Herrler 2 mg/ml) for 40 min at 37 C, cells were infected for 20 min at room temperature with either BCV or influenza C virus. After three washes with PBS, cells were incubated with MEM to allow the virus infection to proceed. Results Inactivation of cell surface receptors by sialidase Studies with MDCK I cells cultured on plastic Petri dishes have indicated that both influenza C virus (Herrler i ii ii i! iiii iiii N iii i!i i iiii ii i iii !i iiiiii!iiii i i 256 64 . 16 4 1024 256 64 , 16 4 .-.-., -, .-.-,. x-, .-.-., x .,NR Apical Basolateral Infection 2 1000 cm 2. Tight junctions were opened by incubation in medium deficient in calcium ions for 24 h (hatched bars). Control cells were incubated in medium containing calcium ions (cross-hatched bars). After incubation with BCV from either the apical or basolateral domain, cells were maintained in Ca2+-containing medium. The virus yield in the apical medium was determined 24 h p,i, by measuring the haemagglutinating activity. Table 1. Ability of BBG to serve as receptors for influenza C virus and BCV on MDCK I and MDCK II cells Asialo cells were obtained by treatment of monolayers of the two cell types with sialidase. Ganglioside- treated cells were obtained by incubation of asialo cells with BBG as described in Methods. Native, asialo and BBG-treated cells were infected with influenza C virus or BCV. The yield of virus released from the cells was determined 20 h p.i. by measuring the haemagglutinating activity of the supernatant. Virus yield (HA units/ml) MDCK I MDCK II Native Asialo BBG-treated Native Asialo BBG-treated BCV 256 2 32 2 2 2 Influenza C virus I28 2 128 2 2 512 indicates that both viruses are released only from the apical membrane of MDCK cells, which has previously been demonstrated for influenza C virus by electron microscopy (Herrler et al., i981). As shown in Fig. 3, infection of MDCK I cells with BCV from the apical surface resulted in a high virus yield, whereas no virus production was detectable after incubation with virus from the basolateral side. On the other hand, influenza C virus was able to infect filter-grown cells from both sides. Virus was also detected in the supernatant when the inoculum was applied to the basolateral domain of the plasma membrane. Thus, although BCV and influenza C virus use the same receptor, they differ in their ability to initiate an infection from the basolateral surface. Infection of cells after disruption of tight junctions The inefficient basolateral infection by BCV may be due to a surface component present on the apical, but absent from the basolateral, domain of the plasma membrane. Alternatively, it might reflect the difficulties of the virus in passing through the pores of the filter membrane. To prove that the filter membrane itself is no hindrance to virus infection from the basolateral surface, cells were incubated in calcium-deficient medium for !51C iiiiiiiiiiiiiiiii!ili i iiii !iii i iiii i iiii iii iiii!ii!iiiiiiiiiiii!iiiiiiiiiiiiii!iiiiiiiiiiiii 24 h. The lack of calcium ions resulted in a loss of electrical resistance, indicating that tight junctions were leaky. After incubation of either the apical or the basolateral surface of the cells with BCV, the inoculum was removed and replaced with medium containing calcium ions. After incubation at 37 C for 24 h, the amount of virus released into the medium was determined by measuring the haemagglutinating activity of the virus in the apical chamber. With cells kept in calcium-free medium, inoculation from the basolateral surface resulted in