Lack of Effect of Lamivudine on Ebola Virus Replication

Services Laboratories, Ames, IA, USA), which was initially passaged 5 times in Vero cells, was used to infect the MK-DIECs.After adsorption for 1 h, the cells were cultured in serum-free Dulbecco modified Eagle medium supplemented with 0.02% yeast extract, 0.3% tryptose phosphate broth, and 1% penicillin/streptomycin (infection medium).To examine the requirement of trypsin (Sigma, St. Louis, MO, USA) for PEDV replication in MK-DIECs, we added trypsin (0, 2.5, 5, or 10 µg/mL) to the infection medium.We also cultured MK-DIECs in 96-well plates and similarly infected them with PEDV for the detection of PEDV nucleoprotein (NP) by IFA using fluorescein isothiocyanate-labeled mouse PEDV NP monoclonal antibody (SD-1F; Medgene Labs, Brookings, SD, USA).At 12, 24, and 36 h after infection, released virus in infected cells was quantified by virus titration in Vero cells by inoculating 10fold serial dilutions.After 24 h, viral NP was detected by IFA staining.The virus titer was calculated according to the Reed-Muench method and expressed as the 50% tissue culture infectious dose/mL.We detected PEDV NP in MK-DIECs 24 hours after infection in medium with and without trypsin (data not shown).However, the numbers of cells positive for PEDV NP was larger in cells cultured with trypsin (2.5 µg/mL and 5 µg/mL) than without trypsin (Figure , panel A).PEDV also induced cytopathic effects (CPEs) in these cells, which were characterized by rounding of cells, syncytium formation, and cell detachment.The CPEs were more pronounced in cells infected with added trypsin; as little as 2.5 µg/mL of trypsin in infection medium was sufficient to induce substantial CPEs in infected cells (Figure, panel B).No signs of CPEs were observed in uninfected control cells, and cells did not display any trypsin-mediated toxicity.The virus titers were detectable in PEDV-infected cells 12 h after infection.The titers further increased at 24 h, reaching a peak at 36 h after infection.Infected cells in infection medium with 10 µg /mL added trypsin had the highest titers (online Technical Appendix Figure 2).Coronaviruses are RNA viruses that are prone to high levels of mutation resulting in novel reassortants.Birds and bats are considered reservoirs of coronaviruses.However, reserviors of PEDV are not yet known.In conclusion, we have demonstrated that PEDV replicates in MK-DIECs.Availability of a cell line of intestinal origin that supports PEDV replication may be of value for studying mechanisms of virus-cell interactions and for developing live attenuated and killed vaccines.