2.4 Sequencing and phylogenetic analyses
The SVV-CH-09-2018 whole genome was divided into seven overlapping
fragments. The fragments were PCR-amplified using Prime STAR®HS (TaKaRa
Bio, Dalian, China). The primers are listed in Table 1. The PCR product
was purified and cloned into the pEASY®-Blunt Simple clone vector
(Transgene, China, and the cloned products were sequenced by Sangon
Biotech (Shanghai, China). The SeqMan II program in the DNASTAR software
package (DNASTAR, Madison, WI, USA) was used to assemble the seven
overlapping fragment sequences into a complete genomic sequence, using
the 5’-full RACE core kit and 3’-full RACE core kit (TaKaRa Bio).
Specific primers were used to detect 5’- and 3’-untranslated regions
(UTRs) (Table 1). The sequences targeted by the primers were designed
using the existing SVV sequence with the PCR product sequence.
To construct the gene development tree, we first analyzed the
SVV-CH-09-2018 gene sequence by using BLAST(Leme, Alfieri, & Alfieri,
2017). After obtaining and filtering similar sequences, we screened SVV
genes from viruses isolated from several countries, including the United
States, China, Canada, Vietnam, Colombia, and Brazil(Hales et al., 2008;
Leme et al., 2015). MEGA7.0 and OMICSTUDIO evolutionary tree software
were used for the phylogenetic genome analysis, after aligning the
selected block by Clustal, using the Neighbor-Joining method. In the
phylogeny test, we used the Bootstrap method with 1000 replications.
Evolutionary tree processing was continued in OMICStudio after the
preliminary evolutionary tree was obtained.