The affected pigs exhibited the clinical signs of vesicular disease. Thus the agents of pig vesicular disease, including FMDV, SVV, SVDV, VESV and VSV were tested. All the viruses were tested negative, except for SVV. The filtered specimens were inoculated onto the confluent monolayer BHK21 cells to isolate the virus. At 12 h postinfection, CPE, characterized by cell rounding, disruption and focal detachment, was observed in virus-infected cells, but not in mock-infected cells (Supplementary Figure S1A). The virus specimens were serially propagated in cultured BHK21 cells, collected, and subjected to RT-PCR detection with primers specific to the SVV VP1 gene. As shown in Supplementary Figure S1B, the amplified products were 975 bp in length. The amplified fragments were subsequently cloned into the pMD-20T vector, and the constructed plasmids were sent to BGI Company for sequencing. Sequence analysis was carried out using the BLAST program of GenBank (NCBI). The analyses indicated that the two amplified VP1 gene sequences showed 98.9% and 99.1% nucleotide similarity with the SVV USA/GBI29/2015 strain (KT827251). Therefore, we successfully isolated two SVV strains and named them SVV CH-GD-2017-1 and SVV CH-GD-2017-2.
The viral genome sequences of CH-GD-2017-1 and CH-GD-2017-2 were further determined to evaluate the characteristics of the newly isolated viruses. Eight pairs of primers were designed with Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA, USA) and used for amplification of the different regions of the SVV genome. The expected fragments were amplified by PCR and subjected to sequencing and analysis (Supplementary Figure S2). The sequences were assembled using DNAstar software. The complete genome sequences were submitted to GenBank. The results showed that the lengths of the two SVV were 7285 nt (SVV CH-GD-2017-1) and 7284 nt (SVV CH-GD-2017-2). The accession numbers were SVV CH-GD-2017-1 (MF189000) and SVV CH-GD-2017-2 (MF189001), and the viral genome both contained the same genome organization: 5'-UTR-L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'-UTR, without insertions or deletions. The length of each gene of the viral genome was shown in Table 1.
Table 1. The genome organization of SVV CH-GD-2017-1 and SVV CH-GD-2017-2, as well as the sequence identity of each gene with other strains available in GenBank.
The nucleotide sequences of the two isolated strains were compared to the other 52 SVV strains (available in the GenBank). We determined that both CH-GD-2017-1 and CH-GD-2017-2 showed high genomic nucleotide similarity with the SVV strains isolated from the US in 2015–2016, and had the highest similarity with the US strain USA/ IA39812/2015_P1 (KU95408). CH-GD-2017-1 showed 98.6% nucleotide similarity with USA/IA39812/2015_P1, and CH-GD-2017-2 showed 98.8% with USA/IA39812/ 2015_P1. The two isolates showed 97.7%–98.8% nucleotide similarity with other recent US strains, and shared 96.1%–98.5% nucleotide similarity with other Chinese strains. Both CH-GD-2017-1 and CH-GD-2017-2 were more closely related to the US strains than previous Chinese strains.
Comparisons of the amino acid similarities of different genes from CH-GD-2017-1 and CH-GD-2017-2 to other SVV strains were also performed (Table 1). We found that the VP4 proteins of all other strains had 100% amino acid similarity except for the two Thailand strains, CH-GD-2017-1 and CH-GD-2017-2. Compared to the other SVV strains, the VP4 of CH-GD-2017-1 and CH-GD-2017-2 included a unique mutation at amino acid 48 (N48S, from asparagine to serine). The two Thailand strains included a unique mutation at amino acid 65 (N65S). Thus, new mutations might have appeared in the recent SVV strains isolated in China and Thailand. In addition, we observed that CH-GD-2017-1 and CH-GD-2017-2 shared 100% amino acid similarity in 3B with all other SVV strains except SVA CH/GXI09/2016. Alignment of the amino acid sequences of all SVV strains suggested that the VP4, VP2, 2B, and 2C were relatively conservative. The 2C protein was the most highly conserved protein in all SVV strains (98.8%–100% similarity), while 3A was the most variable protein in all SVV strains (90%–98.9% similarity). Therefore, the 3A gene could be used as a target gene to evaluate SVV variation and diversity.
A phylogenetic tree for the SVV strains was constructed using complete genome sequences. The results indicated that CH-GD-2017-1 and CH-GD-2017-2 were clustered in a branch with all US SVV strains, as well as the Chinese strains isolated in 2017 (also included one strain isolated in December 2016) (Fig. 1). The CH-GD-2017-1 and CH-GD-2017-2 were most closely related to the US USA/ IA39812/2015_P1 strain, and showed a distant relationship to Chinese strains isolated before December 2016, as well as all previous Guangdong strains. The Chinese strains isolated before 2017 except the SVA/HLJ/CHA/2016 strain (Isolated in December 2016) (Wang et al. 2017) were more closely related to the Brazilian or Canadian strains.
We also compared the amino acids mutations in CH-GD-2017-1 and CH-GD-2017-2 to the first identified SVV strain, SVV-001, and the first reported Chinese strain, SVV CH-01-2015. As shown in Table 2, there were 55 mutated amino acids between the new strains and SVV-001, and 28 mutated amino acids between the new strains and SVV CH-01-2015. The 3A, VP3 and 3D proteins included more mutated amino acids than the other viral proteins, with 3A being the most variable viral protein.
Table 2. Mutation analysis of the amino acid sequences of CH-GD-2017-1 and CH-GD-2017-2, compared to SVV CH-01-2015 and SVV-001 strains.
SVV was first identified in 2002. In 2007, SVV was detected in swine. SVV was only reported in the US and Canada before 2014, while it was reported in additional countries after 2015. We analyzed reports of SVV strains from different countries based on the genome sequences available in GenBank at the time of writing this article. At present, 20 strains have been reported in the US, 16 in China, 12 in Canada, three in Brazil, one in Thailand, and one in Colombia (Fig. 2). The US and China have reported most of the SVV cases, and no cases have been reported in European and African countries. In China, SVV cases mainly emerged in the eastern and central regions, with no cases being reported in inner China (Fig. 3). Based on the isolation date recorded in GenBank, SVV outbreaks mainly occurred during the winter and spring.
SVV Isolation, Identification and Detection
Genomic Sequencing of the Two SVV Strains
Genetic Analysis of the Two SVV Strains
Amino Acid Mutations in CH-GD-2017-1 and CH-GD-2017-2, Compared to SVV-001 and SVV CH-01-2015
Global Geographical Distribution of SVVs
Table Supplementary Table S1. The primers used in this study.
Figure Supplementary Figure S1. Identification of two SVV strains from Guangdong Province in 2017. A. BHK-21 cells were mock-infected or infected with CH-GD-2017-1 and SVV CH-GD-2017-2. The CPE was observed at 12 hours postinfection. B. Amplification of VP1 genes from CH-GD-2017-1 and SVV CH-GD-2017-2 isolated from the virus-infected BHK-21 cell cultures.
Figure Supplementary Figure S2. Amplification of different regions of viral genomes of CH-GD-2017-1 and SVV CH-GD-2017-2. M represented DNA ladder. Lanes 1¬8 represented the eight fragments amplified by the eight pairs of primers listed in Supplementary Table S1.