Plants and extracts
All the plants were grown in the botanical garden of Vytautas Magnus University (Lithuania). Sixteen ethanol extracts were prepared from medicinal plants. The content of biologically active compounds is dependent on the edaphoclimatic conditions of the plant’s cultivation, the vegetation phase, the phenotype and the method of preparation of the raw material. Previous studies comparing the dried raw and fresh material of medicinal plants and different sample preparation methods have shown that drying and other conditions affect the qualitative and quantitative composition of the raw material [35, 36]. In many cases, drying is used as a standardised preparation of raw material of medicinal plants since it reduces water content and the risk of microbiological spoilage of raw material. In the present study, drying was used to collect all the samples of different plants during the intensive blooming vegetation phase for the simultaneous determination of biological activities, in consideration of the fact that different medicinal plants differ in dynamics of the accumulation of biologically active compounds, vegetation, and therefore in the harvesting of raw material [37]. The potentially antiviral plants were selected for extraction depending on the accumulated compounds in herbs, leaves and roots.
Preparation of plant extracts
The solvent ethanol was diluted with sterile bidistilled water to 40% (vol.) concentration. Dried plant material from each plant (500 μg) was extracted with 10 ml solvent. The extraction was performed in an orbital shaker for 24 h at room temperature (20 °C). Each extract was filtrated using a paper filter and then polyvinyl difluoride membrane filter with 0.22-μm pore size. The concentration of the extracts was 50 mg/ml, with reference to the starting material. All the prepared plant extracts were stored in a refrigerator at 4 °C.
Cell line
Vero cells (ATCC CCL-81) were provided by Dr. I. Jacevičienė from the Department of Virus Research at the National Food and Veterinary Risk Assessment Institute in Lithuania. The cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS) at 37 °C in 5% CO2 incubator. Nystatin (100 units/ml) and gentamycin (50 μg/ml) were used to prevent microbial contamination.
Virus
The Vero-adapted Beaudette IBV strain was used. The virus was provided by Dr. M. H. Verheije of Utrecht University in The Netherlands. The virus stocks were prepared and stored at − 80 °C in aliquots.
Cytotoxicity assay
The cytotoxic concentration (CC50) was determined for each extract on Vero cells using MTT assay [38]. First, cells were seeded at a concentration of 1 × 104 cells/well in a 96-well plate and grown at 37 °C for 24 h. Each extract was tested in octuplicate once. After 72 h MTT reagent (10 μl, 5 mg/ml, Sigma-Aldrich) was added and incubated for 4 h at 37 °C. Then 100 μl dimethyl sulphoxide (DMSO) (Carl Roth, Germany) was added to each well and the plates were placed on the shaker for 5 min. The absorbance of each well was measured at 620 nm in a microplate reader (Multiskan™ FC Microplate Photometer) and the percentage of cell survival was calculated. Finally, dose-response curves were plotted to enable the calculation of CC50 that causes lysis and death in 50% of cells.
Screening extracts for antiviral activity
For determination of antiviral properties, one-day-old Vero cells (seeded 1 × 104 cells/well) in a 96-well plate were used. The virus was used at a multiplicity of infection (MOI) of 0.05. Each extract was serially diluted twofold to 1:128 in DMEM and assessed for the ability to inhibit IBV replication using four mechanisms. Every sample of extract was tested twice in quadruplicate. In addition, controls of cells, the virus and extracts were included. An inverted microscope (Leica, Germany) was used to observe cells after all procedures.
In the first method, the virus was treated with the diluted extract for 1 h in a separate 96-well plate and then poured onto the cells. The mixtures were discarded after incubation for 1 h at 37 C in 5% CO2 and then the cells were washed twice with PBS. After washing, DMEM containing 2% of FBS was added. Observation by microscopy for inhibition of cytopathic effect (CPE) was performed after incubation for 72 h at 37 °C in 5% CO2.
In the second method, the mixtures of virus and the diluted extract were poured onto the cells immediately. The mixtures were discarded after incubation for 1 h at 37 °C in 5% CO2 and then the cells were washed twice with PBS. After washing, DMEM containing 2% of FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 °C in 5% CO2.
In the third method, the cells were inoculated with the virus and then treated with extract. First the cells were inoculated with the virus and incubated for 1 h at 37 °C in 5% CO2. Then the unadsorbed virus was discarded and the cells were washed twice with PBS. After washing, the cells were treated with the diluted extracts for 1 h at 37 °C in 5% CO2. After washing the cells twice with PBS, DMEM containing 2% of FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 °C in 5% CO2.
In the fourth method, the cells were treated with extract prior to inoculation. First the cells were treated with the diluted extracts for 1 h at 37 °C in 5% CO2. Then the cells were washed twice with PBS and inoculated with the virus. After incubation for 1 h at 37 °C in 5% CO2 the cells were washed twice with PBS, and DMEM containing 2% of FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 °C in 5% CO2.
The most promising plant extracts were selected for determination of EC50 and selectivity index (SI) based on the results of the antiviral effect assay.
Determination of EC50 and SI
Eight out of 16 plant extracts were chosen for the determination of EC50 and SI using the first method. Extracts were titrated from 1 to 1:128 CC50 and used for virus treatment. After 72 h the MTT assay was performed as outlined above. The 50% effective concentrations (EC50) were calculated from the plot of percentages of cell viability against extract concentrations.
Plaque reduction assay
Concentrations of extracts from 1 CC50 to 0.125 CC50 and 25,000 plaque-forming units (PFU) of IBV were mixed and incubated at room temperature for 1 h.
Plant extracts were diluted with DMEM to prepare four concentrations equivalent to 1 CC50, 0.5 CC50, 0.25 CC50, and 0.125 CC50, as calculated by cytotoxicity assay. These dilutions were then mixed with 25,000 plaque-forming units (PFU) of IBV and incubated at room temperature for 1 h. After incubation, a confluent monolayer of Vero cells in 6-well plates was inoculated with 1 ml of virus (MOI 0.05) and plant extract mixtures for 1 h at 37 °C in 5% CO2 and then discarded. The agarose 0.4% in maintenance medium was added to cells, and the plates were stored at room temperature for 15 min and incubated at 37 °C and 5% CO2. After 72 h the plates were microscopically examined for detection of CPE and then 0.2 ml MTT (5 mg/ml) was used for staining. Plaques were counted after incubation at 37 °C in 5% CO2 for 4 h. The number of plaques was expressed as log10 and the reduction rate was calculated as follows:
$$ \frac{\mathrm{PFU}\ \mathrm{number}\ \mathrm{of}\ \mathrm{virus}\ \mathrm{control}\hbox{-} \mathrm{PFU}\ \mathrm{number}\ \mathrm{after}\ \mathrm{the}\ \mathrm{treatment}\ \mathrm{with}\ \mathrm{plant}\ \mathrm{extract}}{\mathrm{PFU}\ \mathrm{number}\ \mathrm{of}\ \mathrm{virus}\ \mathrm{control}}\times 100\% $$
Virus yield reduction
A virus yield reduction was evaluated by means of virus titration and real-time reverse transcriptase polymerase chain reaction (RT-PCR). The cells were inoculated as outlined above in the plaque reduction assay section. Plant extracts were diluted with DMEM to prepare four concentrations equivalent to 1 CC50, 0.5 CC50, 0.25 CC50, and 0.125 CC50 as calculated by cytotoxicity assay. These dilutions were then mixed with 25,000 plaque-forming units (PFU) of IBV and incubated at room temperature for 1 h. After incubation a confluent monolayer of Vero cells in 6-well plates was inoculated with 1 ml of virus (MOI 0.05) and plant extract mixtures for 1 h at 37 °C in 5% CO2, and then discarded. After inoculation DMEM containing 2% of FBS was added and the cells were incubated for 24 h. CPE of the virus was evaluated using light microscopy. After that, the plates were frozen and thawed three times and the aliquots of the virus were prepared by centrifugation for 15 min at 2000 RPM. The prepared mixtures were used for quantification of both treated and untreated virus and viral nucleic acids by means of TCID50 assay and quantitative real-time RT-PCR respectively.
TCID50 assay
Determination of TCID50 of the control virus and the treated one was performed in 96-well plates. CPE was evaluated after 7 days. Virus titres were calculated using the Kärber method (Kärber, 1931).
Real-time RT-PCR assay
Ribonucleic acid (RNA) used in the real-time RT-PCR was extracted by means of TRIzol Reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Real-time RT-PCR was performed as described by Meir [39]. Briefly, a conserved region of 336 base pairs located at nucleotide position 741–1077 of the H120 strain N gene sequence (GenBank accession no. AM260960) was used to design primers and probe for the real-time RT-PCR assay. A downstream primer IBV-f (5-ATGCTCAACCTTGTCCCTAGCA-3) located at nucleotide position 811–832, an upstream primer IBV-r (5-TCAA-ACTGCGGATCATCACGT-3) located at nucleotide position 921–941, and a TaqMan® probe IBV-TM (FAM-TTGGAAGTAGAGTGACGCCCAAACTTCA-BHQ1) located at nucleotide position 848–875 were used to amplify a 130-bp fragment. Both the primers and the probe were synthesised by Applied Biosystems, UK. The 25 μl real-time RT-PCR reaction contained 12.5 μl 2 × RT-PCR buffer mix (AgPath™ One-Step RT-PCR kit, Applied Biosystems), 1 μl 25 × RT-PCR enzyme mix (Applied Biosystems), primers to a final concentration of 400 nM, probe to a final concentration of 120 nM, 2 μl RNA template, and nuclease-free water. The reaction was carried out in StepOne™ Plus real-time PCR system (Applied Biosystems) at 45 °C for 10 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 45 s. Amplification plots were recorded and analysed, and the threshold cycle (Ct) was determined with the Mastercycler RealPlex2 (Eppendorf).
The real-time RT-PCR was repeated four times, and then delta Ct values were calculated by subtracting the Ct values of virus control from Ct values of virus samples treated with plant extracts. Means and standard deviations of delta Ct values were then calculated to evaluate the effect of plant extracts on viral replication.
Statistical and data analysis
The differences between the methods and extracts were evaluated by Fisher’s criteria and the Student’s t-test. The data were regarded as significant when P < 0.05. Hierarchical clusterisation and multidimensional scaling (MDS) were performed using the software R-Studio. For hierarchical clusterisation, the Euclidean method was used. For MDS, the Euclidean method was used projecting all dimensions to two dimensions.
