Penicillin

Acanthamoeba polyphaga mimivirus, a prototype of the Mimiviridae family, and APMV M4, a strain derived from APMV after 150 passages in amoeba culture (Boyer et al., 2011 (link)), were kindly provided by Dr. Didier Raoult (Aix Marseille Université, France). The Brazilian mimivirus isolates Kroon virus, Oyster virus, and Samba virus were produced and purified as previously described (La Scola et al., 2003 (link)). Briefly, A. castellanii (ATCC 30010) cells were grown in 75-cm² cell culture flasks (Nunc, USA) in peptone-yeast extract-glucose (PYG) medium (Visvesvara and Balamuth, 1975 (link)) supplemented with 7% fetal calf serum (FCS, Cultilab, Brazil), 25 mg/mL fungizone (amphotericin B, Cristalia, Brazil), 500 U/mL penicillin, and 50 mg/mL gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebas were infected and incubated at 32°C until cytopathic effects were observed. Supernatants from the infected amoebas were collected and filtered through a 0.8-μm filter to remove cell debris. The viruses were purified by centrifugation through a sucrose cushion (24%), suspended in phosphate-buffered saline (PBS), and stored at -80°C. For the gene expression experiments, A. castellanii was maintained in PYG medium in the absence or in the presence of 7% FCS at 32°C or in Page’s amoeba saline (PAS) at 32°C to induce starvation.

APMV particles were isolated and purified from infected amoebae as previously described [16] (link). Briefly, Acanthamoeba castellanii (ATCC 30234) were grown in 75 cm² cell culture flasks (Nunc, USA) in PYG (peptone-yeast extract-glucose) medium supplemented with 7% fetal calf serum (FCS, Cultilab, Brazil), 25 mg/ml Fungizone (amphotericin B, Cristalia, São Paulo, Brazil), 500 U/ml penicillin and 50 mg/ml gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebas were infected with APMV and incubated at 37°C until the appearance of cytopathic effects. APMV-rich supernatants from the infected amoeba were collected and filtered through a 0.8-micron (Millipore, USA) filter to remove amoeba debris. The viruses were then purified using a Gastrografin gradient (45–36–28%) [5] (link), suspended in PBS and stored at −80°C.

Tupanvirus soda lake (TPVsl) was isolated from a soda lake sample from the Pantanal region in Brazil and was produced and purified as previously described (Abrahão et al., 2018 (link)). Briefly, A. castellanii (ATCC 30010) cells were grown in 75 cm² cell culture flasks (Nunc, United States) in peptone–yeast extract–glucose (PYG) medium (Visvesvara and Balamuth, 1975 (link)) supplemented with 25 mg/mL fungizone (Amphotericin B, Cristalia, Brazil), 500 U/mL penicillin, and 50 mg/mL gentamicin (Schering-Plough, Brazil). After reaching confluence, the amoebae were infected at a multiplicity of infection (m.o.i) of 0.1 and incubated at 32°C until cytopathic effects (CPE) were observed. Supernatants from the infected amoebae were collected and filtered through a 0.8 μm filter to remove cell debris. The viruses were purified by centrifugation through a sucrose cushion (22%), suspended in phosphate-buffered saline (PBS), and stored at -80°C.