In general, antibiotics are not rated as substances that inhibit or support influenza virus replication. because of the limited availability of high-quality eggs, buy 1242137-16-1 especially in the case of an impending pandemic. In addition, some of the human strains of influenza virus do not replicate in eggs and require adaptation passages and/or a reassortment with a well-adjusted donor virus for improved buy 1242137-16-1 growth. Moreover, the cultivation of human-derived influenza viruses in a host such as CE might lead to the selection of host range mutant variants that are characterized by structural changes in the hemagglutinin (HA) molecule, which, in turn, might have a negative effect on receptor specificity and the immunogenicity of egg-derived vaccines (1, 9, 25, 28). Alternative approaches involving the use of accredited continuous cell lines have been pursued by several vaccine manufacturers in recent years. Madin-Darby canine kidney (MDCK), African green monkey kidney (Vero), or PER.C6 cells have been used successfully to produce influenza viruses (3, 13, 23, 32). Vero cells are well characterized and widely accepted by regulatory authorities. They have been used to produce human virus vaccines, including those against poliomyelitis and rabies, for more than 30 years. Recently, they were also approved for use in vaccines against smallpox, rotavirus infection (7), and influenza (3). Increased yields of influenza virus in Vero cells could be achieved by implementing a serum-free medium cultivation (17) and multiple additions of trypsin to the medium (18). The primate origin of Vero cells may have a positive impact on the retention of the biological properties of human influenza viruses. Compared to MDCK and egg-derived isolates, Vero-derived influenza virus strains proved to be more similar to human original viruses in terms of their receptor specificity and glycosylation pattern (21, 26). These characteristics would suggest the use of Vero cells for virus isolation and surveillance purposes, but despite all advantages, the isolation rates obtained in Vero cells still tend to be lower than those in MDCK cells. We show here that the sensitivity of Vero cells to influenza virus buy 1242137-16-1 infection can Rabbit Polyclonal to MMP-9 be dramatically improved by supplementing with the polyene antimycotic-antibiotic amphotericin B (AmB). We found that AmB is able to promote the initial steps of viral infection, including the enhancement of viral endocytosis and fusion with endosomal membranes. Improved infectivity in Vero cells resulted in better viral recovery from clinical samples. MATERIALS AND METHODS Reagents. The antibiotic-antimycotic mix was obtained from Sigma-Aldrich. AmB was used in its solubilized form (ca. 45% AmB, 35% sodium deoxycholate, and the rest sodium phosphate and chloride). It was obtained in either liquid (Fungizone; Sigma-Aldrich) or pulverized (AmB BMS; Bristol-Myers Squibb) form. Cells. The Vero (African green monkey kidney) cells were obtained from the European Collection of Cell Cultures (ECACC; no. 88020401) or the American Type Culture Collection (ATCC; CCL-81) and were cultivated in serum-free conditions using OptiPRO SFM supplemented with 4 mM l-glutamine (SFM; both from Gibco/Invitrogen). The MDCK cells were obtained from ATCC (CCL-34) and cultured in ATCC-formulated Eagle minimum essential medium (Gibco/Invitrogen), supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen). Human nasal buy 1242137-16-1 epithelial (HNE) cells (primary culture) were obtained from Provitro, Germany, and were cultured according to the manufacturer’s instructions. Human bronchial epithelial (16HBE14o?) cells were kindly provided by J. Seipelt (Vienna, Austria). The cells were cultivated in minimal essential medium.