Virus goes viral: an educational kit for virology classes

TPV, CVG, PDV, and Niemeyer virus (NYMV) were propagated individually using Acanthamoeba castellanii (American Type Culture Collection [ATCC] 30010) cultured at 32 °C in 175 cm² cell culture flasks with 50 mL peptone-yeast extract with glucose (PYG) medium supplemented with 25 mg/ml amphotericin B (Fungizone; Cristalia, São Paulo, Brazil) and 500 U/ml penicillin (Schering-Plough, Brazil). The amoebae were infected with at a multiplicity of infection (MOI) of 0.01. Cells were incubated until the expected CPEs appeared in culture, and the media was collected by two centrifugation steps. A. castellanii cells and cellular debris were first removed by centrifugation (400 x g for 10 min), and the particles were purified by centrifugation (36,000 x g for 1 h) through a sucrose cushion (40–50%), suspended in PAS, and stored at − 80 °C. KAUV, OBRV, and FTSV were propagated individually in Vermamoeba vermiformis (ATCC50237) cultivated at 32 °C in PYG medium with a MOI of 0.01. After the appearance of CPEs, the cells and supernatants were collected, with sterile serological pipettes, stored in sterile conical tubes, and the viruses were purified through ultracentrifugation with a sucrose cushion.

African green monkey kidney BSC-40 cells (ATCC CRL-2761) and Vero cells (ATCC CCL-81) were maintained in an atmosphere with 5% CO₂ at 37 °C in Eagle’s minimum essential medium (MEM; Gibco BRL, Invitrogen, Carlsbad, CA, United States), supplemented with 5% fetal bovine serum (FBS; Cultilab, Brazil), 2.5 μg/mL amphotericin B, 500 U/mL penicillin (Cristalia), and 50 μg/mL streptomycin (Schering-Plough, São Paulo, Brazil).

YFV (vaccine strain 17DD), MAYV (Strain BeAr20290), CHIKV (Genotype ECSA – Strain BHI3762/H804917, kindly provided by Dr. Maurício Lacerda Nogueira), VACV group I (Isolate Caragola eye I), VACV group II (Isolate Caragola eye II), and CPXV (strain Brighton Red) were individually multiplied in Vero cells in MEM that contained 1–2.5% FBS, 0.25 μg/mL amphotericin B, 100 U/mL penicillin 100 U/mL (Schering-Plough, Brazil), and 100 μg/mL streptomycin at 37 °C in an atmosphere with 5% CO₂ in a large 175 cm² bottle. Subsequently, for YFV, MAYV, and CHIKV, the cell infection supernatant was transferred to tubes and centrifuged at 3,000 x g for 5 min. The clarified supernatants from these centrifugations were stored at − 70 °C. VACV and CPXV were purified on a sucrose gradient as previously described [25] and stored at − 70 °C.

The slides produced with the purified viral particles stained with crystal violet allowed a simple evaluation of the distinct morphology and particle sizes for each isolate (Fig. 1). CVG, OBRV, and PDV particles were ovoid and measured ~ 1 μm in size and ~ 0.5 μm in diameter (Fig. 1). NYMV particles were ~ 0.6 μm and appeared spherical when viewed with optical microscopy. TPV particles were large and strongly stained with crystal violet. They appeared to have a spherical head and a cylindrical tail under optical microscopy (Fig. 1).

Viral factories were visualized after staining the cells with the hemacolor kit. The Mimivirus viral factory, such as for NYMV, is very identifiable, and was represented by a light halo with a strongly marked dark purple center located in the cell cytoplasm (Fig. 1, blue arrow). The nucleus was stained dark purple (Fig. 1, yellow arrow).

Two cell lines were infected with different viruses to produce CPE slides (Figs. 2 and 3). BSC-40 cells assays provided visibly distinct lysis plaques after infection with different viruses. CPXV generated large circular plaques of clear destruction with very stained round cell remains (Fig. 2). The presence of A-type eosinophilic inclusion bodies was also observed in CPXV-infected BSC-40 cells (Fig. 2). These inclusion bodies appeared as large pink circles in the host cell cytoplasm (Fig. 2, black arrow). Both VACV groups presented a distinct plaque phenotype; group I showed small lysis plaques, while group II showed large plaques with very stained and stretched cells that formed a web (Fig. 2). There were lysis plaques generated by arbovirus infection in the Vero cell monolayers. YFV generated largely undefined edge lysis plaques that included numerous stretched cells (Fig. 3). CHIKV presented large circular plaques, while MAYV showed small undefined edge lysis plaques (Fig. 3).

The slides generated in previous sections were used to compose the “Virus Goes Viral” kit. The kit has a total of 15 labeled slides: five viral particle slides, nine CPE slides, and one viral factory slide (Fig. 4). The kit includes associated teaching materials on a CD-ROM, including an infographic material, which aims to assist in understanding what is being observed (Additional file 3) and also high-resolution model images of each slide and CPE images from several giant viruses in A. castellanii and V. vermiformis that were not fixed and stained in slide form (Additional file 1: Figure S1 and Additional file 2: Figure S2). These CPEs include: cell rounding for CVG, Marseillevirus, PDV, TPV, and KAUV; cell stretching for OBRV; cell bunches for TPV; lysis plaques for FSTV (Additional file 1: Figure S1 and Additional file 2: Figure S2). The “Virus Goes Viral” kit and its attached material encompass 13 different viral species and 17 miscellaneous effects in host cells. This kit has been used in practical virology classes for the Biological Sciences course (Universidade Federal de Minas Gerais [UFMG], Brazil). The students seemed very receptive to the proposal and many were excited about the practice. Some expressed interest in starting research with giant viruses and initiated contact with our laboratory.