Amphotericin b

The NSC-34 motor neuron cell line (CELLutions Biosystems Inc., Ontario) was maintained in growth media consisting of DMEM supplemented with 10% FBS, 4 mM I-glutamine (Gibco), 2.5 µg/ml amphotericin B (Fisher Scientific) and 100 U/ml penicillin/100 µg/ml streptomycin (Gibco). Cells were sub-cultured every 3–4 days prior to reaching confluence. To differentiate, the cells cultures were switched to differentiation media consisting of Neurobasal (Gibco), 4 mM I-glutamine (Gibco), 2.5 µg/ml amphotericin B (Fisher Scientific), 100 U/ml penicillin/100 µg/ml streptomycin (Gibco).

A549 were used in this study. A549 cells were obtained from (American Type Culture Collection, Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle medium nutrient mixture/F-12 medium (DMEM-F12) (Fisher Scientific, Grand Island, NY, USA) containing L-glutamine supplemented with 10% Corning regular fetal bovine serum (FBS) (Fisher Scientific, Grand Island, NY, USA), 1% penicillin/streptomycin (Fisher Scientific, Grand Island, NY, USA), and 0.2% amphotericin-B (0.5 μg/mL) (Fisher Scientific, Grand Island, NY, USA). For virus infection, DMEM exosome-free media was prepared with exosome-depleted FBS using DMEM/F12 medium containing L-glutamine supplemented with 2% exosome-free Corning FBS, 1% penicillin/streptomycin, and 0.2% amphotericin-B (0.5 μg/mL) (Fisher Scientific, Grand Island, NY, USA). Cells were cultured at 37°C in a humidified atmosphere supplemented with 5% CO₂.

For virus isolation, Vero cells (ATCC CCL-81) and Aedes albopictus clone C6/36 cells (ATCC CRL-1660) were used. Heart and brain samples from partridge no. 7, which showed the lower Ct values (20.4 and 21.52, respectively), were chosen for virus isolation. Tissue samples were homogenized at 20% (w/v) in PBS containing penicillin, streptomycin and amphotericin B (antibiotic-antimycotic), used according to the manufacturer’s instructions (Gibco, Waltham, MA, USA). Following centrifugation (3000× g, 10 min, 4 °C), the supernatant was filtered through a 0.45-µm-pore-size filter (Millipore Express, Darmstad, Germany) and used to inoculate sub-confluent (70%) Vero and C6/36 cells, maintained in Eagle’s medium supplemented with 5% FBS, penicillin, streptomycin, and amphotericin B (antibiotic-antimycotic used at 1:100, Gibco), and 50 µg/mL gentamicin (Gibco), incubated, respectively, at 37 °C and 28 °C in a humidified atmosphere with 5% CO₂ and observed daily for cytopathic effect (CPE) in a phase-contrast microscope. Three passages were done for both samples.

Isolation of herpesvirus was attempted from organs of hares coinfected with MYXV and LeHV-5, namely from liver and spleen, penile and vulva samples. In addition, liver and spleen samples from two hares with single herpesvirus infection, were also used.
Samples were homogenized at 20% in phosphate-buffered saline containing penicillin, streptomycin and amphotericin B (antibiotic-antimycotic), used according to the manufacturer (Gibco, Life Technologies Corporation). Following centrifugation, the supernatant was filtered through a 0.45-μm-pore-size filter (Millipore Express) and used to inoculate sub 70% confluent Candrel R Feline Kidney (CRFK) epithelial cells (ATCC-CCL-94), Vero cells (ATCC No. CRL-1986), Rabbit Kidney (RK13) cells (ATCC-CCL-37) and Hella cells (ATCCNo. CRM-CCL-2)- RK13 cells grown in Eagle medium and the others in Dulbecco's modified Eagle's Media was supplemented with 10% foetal calf serum (Gibco), penicillin, streptomycin and amphotericin B (antibiotic-antimycotic used at 1:100), 50μg/ml gentamicin (Gibco). Cells were maintained at 37°C under humidified atmosphere with 5% CO₂ and observed daily for cytopathic effect (CPE) by phase contrast microscopy. Three passages were carried out. The supernatant and cell pellet of each passage were tested for the presence of herpesvirus by PCR [17 (link)].

Isolation of MYXV from the rabbits’ tissues (15758PT20, 20545PT20 and 22660PT20) was achieved separately from eyelid, genitalia and lung. Samples were homogenised at 20% (w/v) in PBS containing penicillin, streptomycin and amphotericin B (antibiotic-antimycotic), used according to the manufacturer (Gibco, Massachusetts, EUA). Following centrifugation (3000× g, 10 min), the supernatant was filtered through a 0.45-μm-pore-size filter (Millipore Express, Darmstad, Germany) and used to inoculate sub confluent (70%) Rabbit Kidney (RK13) cells (ATCC-CCL-37). RK13 cells were grown in Eagle´s medium supplemented with 5% foetal calf serum (Gibco), penicillin, streptomycin and amphotericin B (antibiotic-antimycotic used at 1:100, Gibco) and 50 μg/mL gentamicin (Gibco). Cells were maintained at 37 °C under humidified atmosphere with 5% CO₂ and observed daily for cytopathic effect (CPE) by phase contrast microscopy. The supernatant and cell pellet of each passage were tested for the presence of MYXV by qPCR [17 (link)].
The isolation of the virus allowed verifying its viability in the rabbit tissues, inferred by the cytopathic effect and in-house immunofluorescence protocol using MYXV positive hare serum (protocol available on request). The photographs were taken using an Inverted research microscope, Nikon Eclipse Ti (Nikon Instruments Europe, Amsterdam, Netherlands).