Viral extraction from the samples was performed with a PureLink™ Viral RNA/DNA Mini Kit (Invitrogen, USA) according to the manufacturer’s instructions. The full S1 segment (~1600 bp) was amplified with the IBVS1-F (5′-AACTCTGCACGCAAATTA-3′) and IBVS1-R (5′-TGTTGTCGCAAACAGGACC-3′) primers described by Zhang et al. [23 (link)].
The RT-PCR was performed using a SuperScript® III One-Step kit (Invitrogen, USA) with the following parameters: 30 min at 55 °C, 2 min at 94 °C, 40 cycles of 15 s at 94 °C, 30 s at 49 °C, finally an extension step at 68 °C for 5 min was added. The RT-PCR products were visualized in an agarose gel then purified using a PureLink™ Quick Gel Extraction Kit according to the manufacturer’s instructions. Finally, an ABI 3730XL genetic analyzer was used in the sequencing process.
The alignment calculation for the nucleotide sequences was performed with Mafft 7.2 software [24 (link)] using the L-INS-i method. The nucleotide model substitution was then identified using Jmodeltest software v2.1.7 [25 (link)], and a maximum-likelihood tree was constructed with the online platform PhyML 3.0 [26 (link)] using the dataset previously published by Valastro et al. [14 (link)]. Finally, the amino-acid sequences were aligned using Bioedit v7.2.5 [27 ].
The RT-PCR was performed using a SuperScript® III One-Step kit (Invitrogen, USA) with the following parameters: 30 min at 55 °C, 2 min at 94 °C, 40 cycles of 15 s at 94 °C, 30 s at 49 °C, finally an extension step at 68 °C for 5 min was added. The RT-PCR products were visualized in an agarose gel then purified using a PureLink™ Quick Gel Extraction Kit according to the manufacturer’s instructions. Finally, an ABI 3730XL genetic analyzer was used in the sequencing process.
The alignment calculation for the nucleotide sequences was performed with Mafft 7.2 software [24 (link)] using the L-INS-i method. The nucleotide model substitution was then identified using Jmodeltest software v2.1.7 [25 (link)], and a maximum-likelihood tree was constructed with the online platform PhyML 3.0 [26 (link)] using the dataset previously published by Valastro et al. [14 (link)]. Finally, the amino-acid sequences were aligned using Bioedit v7.2.5 [27 ].
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