Clinical samples were collected from 28 animals (26 epithelium and two vesicular fluid samples); each sample in the study is a representative for each animal. (S1-Additional file 1).
Criteria for selection of suspected animals depended mainly on the appearance of FMD clinical symptoms (salivation, tongue and mouth vesicles appearance, depression, and loss of appetite). Epithelial tissue was collected from an unruptured or recently ruptured vesicle, from the tongue or buccal mucosa, and was placed in a viral transport medium with added antibiotics. Vesicular fluid was withdrawn from the unruptured vesicle using a sterile needle and was submitted in sterile sample container. All collected samples were submitted in ice container containing ice packs to maintain cold chain temperature during transport of the samples to the laboratory. The samples were collected between October and December 2020 from clinically diagnosed cattle and buffaloes (showing symptoms suspected to be FMD) from five governorates in Egypt; Behera (n = two), Cairo (n = seven), Minya (n = five), Port Said (n = six) and Qalyubia (n = eight). The clinical disease was reported in both vaccinated and non-vaccinated animals however, diseased animals in Port Said and Qalyubia were vaccinated with a local polyvalent inactivated vaccine manufactured by the Veterinary Serum and Vaccine Research Institute (VSVRI, Cairo, Egypt).
Baby Hamster Kidney (BHK-21) cells were used for FMD virus isolation as was previously described . Approximately, cells of ninety percentage of confluent monolayer were prepared in 25 cm3 cell culture flasks containing minimum essential medium (MEM) with Earl’s salts supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin. The growth media supernatant was decanted from the flask and then the cell monolayer was washed three times with sterile phosphate buffer saline and 0.5 ml of the prepared viral sample was inoculated gently onto the cell monolayer. Flasks were incubated for 1 h at 37°C in the presence of 5% CO2. This was followed by the addition of MEM and cells were incubated for 24-72 hr at 37°C in the presence of 5% CO2. Inoculated cells were harvested when cytopathic effects (CPE) were observed and cells were frozen and thawed three times and were passaged for three successive blind passages in the same way.
Viral RNA extraction
Viral RNA was extracted using EasyPure viral RNA kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Briefly, FMDV RNA was isolated from 200 μL sample as starting materials and RNA was eluted in a final volume of 30 µl RNase-free water. RNA extracts were either used directly after extraction or kept at − 20 °C for further analysis.
Real-time RT-PCR (qRT-PCR) and one-step RT-PCR
Extracted samples were screened for the presence of FMDV RNA using primers and probe directed toward the conserved 3D gene (Table 1). TransScript® Probe One-Step qRT-PCR SuperMix (TransGen, Beijing, China) was used according to the manufacture’s instruction for the amplification of 3D gene. The PCR cycles consisted of 45 °C for 5 min of reverse transcription, then initial denaturation at 94 °C for 2 min, 40 cycles of 94 °C for 5 s and 60 °C for 30 s with fluorescence data collection in this step each cycle. Samples with cycle threshold (Ct) lower than 30 were used for the conventional one-step RT-PCR. The complete VP1 gene was amplified using the EasyScript One-Step RT-PCR SuperMix (TransGen, Beijing, China). Briefly, a total of 5 μL of extracted viral RNA was used in the RT-PCR which consisted of 12.5 μL of reaction mix, 0.4 μL of enzyme mix, and 3.1 μL of RNase-free water were mixed with 2 μL of each primer (10 μM) into a final volume of 25 µl. One-Step RT-PCR was performed using the following conditions: 45 °C for 25 min. (reverse transcription step), 94 °C for 5 min, followed by 40 cycles of 94 °C for 45 s and 60 °C for 1 min. except for SAT2 specific detection primer at 50 °C for 45 s. and 72 °C for 45 s followed by a final extension at 72 °C for 10 min. The primers targeting the variable region in the 1D gene of viral RNA are shown in Table 1.
Following the amplification of RT-PCR products in the programmable thermal cycler T100 (BioRad, USA), the PCR products were analyzed on 1.2% agarose gel electrophoresis system using Tris Borate EDTA buffer (1X) stained with ethidium bromide. A 5 μL of the RT-PCR products, as well as the DNA ladder, were loaded into the preformed wells. DNA fragments of positive samples were excised from the agarose gel and the amplified RT-PCR products were purified from the gel using QIAquick Gel Extraction Kit (Qiagen, USA) according to the manufacturer’s instructions and DNA was eluted in a final volume of 30 µl. Determination of DNA concentration was performed using Qubit™ dsDNA HS assay kit (Molecular Probes, Life technologies, USA) using the Qubit® 2.0 fluorometer for accurate DNA quantification.
The purified PCR products were sequenced in both directions using the dideoxy chain termination method using the same primers (Table 1). Sequencing reaction was prepared using BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher, USA) with 3.2 picomol concentrations of both the forward and reverse primers for each positive sample in two different reactions.
Thermal cycling conditions for sequencing were at 96 °C for 1 min, followed by 25 cycles at 96 °C for 10 s. 50 °C for 5 s and 60 °C for 2 min using T-100TM Thermal Cycler (Bio-Rad, USA). Sequencing reaction product was purified with Centri-Sep™ Spin Columns (Thermo Fisher, USA), followed by electrokinetic injection on capillary electrophoresis systems 3500 Genetic analyzer (Applied Biosystems, USA).
Computational and bioinformatics analyses were used to determine the substitution rates and construct a phylogenetic tree [18, 19]. Nucleic acid and amino acid sequence similarities were determined using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) available at NCBI using default parameters. Results produced from local alignment were used to determine the sequence to be used in multiple alignment analysis. Multiple sequence alignment (MSA) was conducted using ClustalW/BioEdit software—version 7.1 . Results of MSA were trimmed and stripped from columns containing gaps and phylogenetic tree was generated using the neighbor-joining method with bootstrapping over 1000 replicates using MEGA software version X [19, 25]
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