A propolis sample from Coatepec, Veracruz was used (provided by Raquel Zepeda, INANA A. C.). This town is located at 19˚27' north, 96˚58' west, at an altitude of 1200 m.a.s.l., with a regular template-humid weather and an average temperature of 19.2˚C [15]. The ecosystem is that of a mountain mesophile forest, that in México is characterized because its canopy consists predominantly of template climate deciduous trees (liquidambar, oaks, beeches and pine trees), while the understory is conformed mainly of evergreen tropical species (bushes from the families: Acanthaceae, Rubiaceae and Myrsinaceae). Epiphytes like orchids, bromeliads, peppers, and aroids abound on treetops. Arborescent ferns and the plenitude of epiphytes like bromeliads, ferns, orchids, and moss, are some of the characteristic elements of these forests [16] [17].

In the laboratory there are 27 samples from the same geographical area and harvested in April 2019, these samples are divided, number in parentheses, among the following species: Scaptotrigona mexicana (8), Nannotrigona perilampoides (5), Melipona beecheii (3), Scaptotrigona pectoralis (3), Partamona orizabensis (2), Plebeia melánica (1), Plebeia frontalis (2) and Plebeia sp (3). To carry out this study, the sample with the highest content of phenolic and flavonoid compounds was chosen, so one of the propolis from Plebeia frontalis was used. Propolis ethanolic extract (PEE) was obtained from 50 grams of raw propolis, which was previously crushed and cleaned of impurities (wood shavings, net remains, dead bees, among other things), 100 ml of 70% ethanol were added and the resultant mixture was subjected to extraction by means of a ultrasonicator (Branson, CPX1800H, Danbury, USA), the resultant product was then filtrated using a vacuum filtration system. Finally, the obtained filtrate was concentrated using a rotary evaporator (Science MED, SM100-PRO, Finland) and was left to dry by vacuum pump. The dry PEE was then protected from light and refrigerated at 4˚C until use [18].

Determination of phenolic compounds was accomplished using a Folin-Ciocalteu assay. Used reagents and solvents were acquired from Sigma-Aldrich. Samples that were going to be evaluated were prepared at a concentration of 0.05 mg/mL. For determination of phenolic compounds, a calibration curve was made at a concentration range of 6.25 - 200 µg/mL, using gallic acid as the standard (Sigma-Aldrich). For this 6 ml of distilled water and 500 µL of Folin-Ciocalteu reagent were added to 1 ml of PEE, the mix was allowed to react for 5 minutes; 1.5 ml of 20% Na₂CO₃ were added and then distilled water, until 10 ml were reached. The mixture was left to react for 2 hours and the absorbances of the samples were measured at a wavelength of 760 nm by UV-VIS absorption spectrophotometry (DLAB, SP-UV1000, China). Phenolic compounds were expressed as a percentage (%) [18].

Flavonoid content was determined by means of the aluminum chloride (AlCl₃) colorimetric assay. Used reagents and solvents were acquired from Sigma-Aldrich. In order to determine flavonoid content, a calibration curve was made, with a concentration range of 1 - 90 µg/mL, using quercetin as the standard reference. For this 1 ml of reagent-graded methanol and 1 ml of 2% AlCl₃ were added to 1 ml of the PEE solution (0.05 mg/mL), The mixture was allowed to react for 10 minutes and the absorbance was determined at a wavelength of 415 nm by UV-VIS absorption spectrophotometry (DLAB, SP-UV1000, China). Results were expressed as a percentage (%) [18].

Monolayers of Vero cells (African green monkey kidney) were kept in DMEM (Dulbecco’s Modified Eagle Medium), the medium was supplemented with 7% bovine fetal serum and antibiotic (penicillin-streptomycin), and was kept at 37˚C in a humidified atmosphere, 95% air and 5% CO₂. The Buzzel strain of Canine Distemper Virus, kept at −20˚C, was used to infect the cells, using a viral concentration of 10^12.16 (TCID₅₀ = 10^12.16/mL) [19] [20].

Vero cells were grown on sterile coverslips inside 60 mm Petri dishes, two groups were formed; negative control (uninfected) and positive control, the latter was inoculated with 100 µL of the viral solution; both groups were incubated for 72 hours, at which time the medium was discarded and the cells were fixed using 10% formalin prepared in PBS for 20 minutes, washes were performed with PBS. At the end, cultures were observed by inverted microscopy and photographed using a digital camera. After this, the necessary steps to complete the immunofluorescent assay were preformed; for this purpose, cells were permeabilizated using Triton X-100 (0.5%) for 5 minutes. Next, PBS washes took place and the cells were blocked using bovine serum albumin (1% in PBS) for 20 minutes, followed by another wash with PBS, and then the cells were incubated with the viral antibody, Canine Distemper Virus Mab (VMRD, cat. CJ-F-CDV-MAB-1ML), for 12 hours at 4˚C. Once this time had elapsed, washes were performed, first with PBS and, finally, with double distilled water. Coverslips were mounted (UltraCruz Mounting Medium for fluorescence with DAPI, Santa Cruz Biotechnology, cat. sc-24941), edges were sealed and then kept in refrigeration. Finally, they were observed by fluorescence microscopy (Carl Zeiss, mod. axioscope) [20] [21].

Mean cytotoxic concentration was determined by means of tetrazolium dye (MTT) colorimetric assay; for this, VERO cells were seeded at a density of 2.4 × 10⁴ cells/well, in 96-well plates, the plates were then incubated for 24 hours, and then PEE diluted in DMEM-acetone (1 mL/30 µL, respectively) was added at different concentrations (2, 1, 0.5, 0.25, 0.125 mg/mL). Cells were incubated for another 72 hours, then a viability colorimetric assay was performed; for this, 10 µL of MTT per well were added, followed by a 4 hours incubation period, at which time the medium was discarded and 100 µL of dimethyl sulfoxide were added; 15 minutes after, readings were made using an ELISA reader (595 nm). Obtained values were plotted so as to determine the mean cytotoxic concentration (CC₅₀), as well to determine the concentration to be used in the infection assays, so that a cellular viability of about 80% could be guaranteed [22].

PEE antiviral activity was evaluated in vitro using VERO-cell monolayers in 96-well ELISA plates. For infection, 30 µL of the viral suspension were put into each well and the treatment consisted of 100 µL of propolis (0.3 mg/mL of DMEM). The treatment was administered at two points; one hour before infection and at the same time of infection. In both cases, cultures were evaluated 72 hours post infection by a viability colorimetric assay (MTT), which has been already described [12].

Absorbances obtained were analyzed for each of the treatments and controls to evaluate the antiviral effect of PEE. The data obtained were processed by program Minitab (18 version, Spanish) by an ANOVA test (0.05 significance).

The results of Folin-Ciocalteau and aluminum chloride colorimetric assays showed that the propolis that was used for the present study has a total phenol concentration of 5.34%, and a flavonoid concentration of 3.5%. All of the analytical tests implemented during the study were based according to the parameters established by the Mexican Official Norm: Propolis, production and specifications of its processing: NOM-003-SAG/GAN-2017 [18].

VERO cell cultures were exposed to increasing concentrations of PEE to determine its level of toxicity. Through the colorimetric MTT assay, a cellular viability curve was made, and the median cytotoxic concentration (CC₅₀) was determined to be at 0.5 mg/ml. The concentration to be used during infection trials was also determined, this being 0.3 mg/mL, such concentration guarantees an 80% cellular viability.

Infected Vero cell cultures were evaluated at 72 hours post infection (hpi) under the inverted microscope. In the non-infected culture, cells displayed normal cellular morphology and growth, observing a monolayer with a confluence of above 80% (Figure 1(a)). On the other hand, when analyzing the infected cultures, cellular debris was observed, as well as loss of adhesion and a decreased cellular density, being this the result of the cellular lysis caused by the virus (Figure 1(b)).

The presence of CDV in infected cultures was determined by a direct immunofluorescence assay. Observation of infected cultures by fluorescence microscopy allowed us to document areas of green color (Figure 2(a)), these areas were the result of the presence of anti-CDV antibodies that were marked with fluorescein; in contrast, in the uninfected cellular culture, no color emission was appreciated (Figure 2(c)). To confirm the presence of actual cells in both preparations, nuclei were marked by DAPI (blue color) fluorescent stain (Figure 2(b) and Figure 2(d)). In both preparations, we can point out the presence of cells, as shown by the stained nuclei; the nuclei are pleomorphic in the infected cells due to the cellular damage caused by CDV.

The antiviral activity of the treatments used was determined with the colorimetric MTT assay. Obtained data are the result of multiple repetitions (n = 20); for noninfected cells a median of 0.426 was obtained, and for infected cells the obtained value was 0.156; whereas the values obtained for the treatments where: a value of 0.288 was obtained when the treatment was administered an hour

before infection, and a value of 0.258 when the treatment was administered at the same time of infection. The values were analyzed by an ANOVA test (p = 0.05), the infected culture presented statistically significant differences between treatments and the uninfected culture. From this, we can point out that the implemented treatments exhibit a statistically significant antiviral effect (Figure 3).