Viral RNA was extracted from a 250 μL sample using the TRIzol Reagent (Takara, Shiga, Japan). The PEDV cDNA was synthesized by means of the PrimeScript High Fidelity RT-PCR Kit (Takara). Full-length PEDV genome amplification was performed with primers described previously [14 (link)]. The 5′ and 3′ end sequences were determined with the 5′ and 3′ RACE kit (Takara). For each amplicon, more than three independent clones were sequenced to determine the consensus sequence of a given genomic region.
The ClustalX (ver.1.81) software was used to align the full-length genome sequences. Phylogenetic analyses based on the S and open reading frame 3 (ORF3) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where 1000 bootstrap replicates were implemented in the MEGA6.0 software [15 ].
The ClustalX (ver.1.81) software was used to align the full-length genome sequences. Phylogenetic analyses based on the S and open reading frame 3 (ORF3) genes or the entire genome were performed by the maximum-likelihood method with the general time-reversible nucleotide substitution model, where 1000 bootstrap replicates were implemented in the MEGA6.0 software [15 ].