Emergence of a novel recombinant USA/GBI29/2015-like strain of Seneca Valley virus in Guangdong Province, 2018

Abstract

Since June 2017, several outbreaks of a Seneca Valley virus (SVV) USA/GBI29/2015-like strain have emerged in pigs in China. In our study, we successfully isolated the SVV strain CH-GDZQ-2018, confirmed by immunofluorescence and Western blot assays. Phylogenetic and recombinant analyses showed that the USA/GBI29/2015-like CH-GDZQ-2018 strain was the result of recombination between epidemic strains local to Guangdong, showing that SVV has undergone evolution in China.

Seneca Valley virus (SVV) is a non-enveloped, single-stranded RNA virus with a genome length of 7.3 kb belonging to the genus Senecavirus in the family Picornaviridae. Infecting pigs causes a disease characterized by vesicles, coronary band hyperemia, and lameness (1). The virus possesses a large open reading frame that encodes a large polyprotein, which is further cleaved into 12 viral proteins comprising 1 leader protein, 4 structural proteins (VP1, VP2, VP3, and VP4), and 7 non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). The structural proteins are responsible for viral invasion (2). The non-structural proteins are related to the innate immune response and apoptosis (3–5).

Recently, many countries have reported SVV infection associated with porcine idiopathic vesicular disease and the outbreak of SVV has affected the productivity and economics of the pork industry to some extent (6–9). In China, since the first SVV strain was isolated from swine in Guangdong Province in 2015 (6), many provinces have reported outbreaks of SVV. Because of environmental changes, the virus might mutate to adapt to the environment (10–12). Currently, studies have indicated that 2 recombinant SVV strains have emerged in China (3,13). Here, we report a novel and recombinant SVV strain, CH-GDZQ-2018, isolated from Guangdong Province. This novel strain is genetically closely related to the USA/GBI29/2015 strain isolated in the United States (7) but is distinct from the previously isolated USA/GBI29/2015-like strains in Guangdong (14), indicating the evolution of the USA/GBI29/2015-like strain in China.

In October 2018, vesicular lesions emerged in sows of an intensive pig farm in Zhaoqing City, Guangdong Province. The sows showed ulcerative lesions on the coronary band and fluid-filled vesicles on the snout. The sick sows also demonstrated depression and anorexia. The disease lasted for approximately 10 d and then the sick sows began to recover.

Vesicle fluid samples were collected from some of the infected pigs according to the animal ethics regulations of the National Engineering Center for the Swine Breeding Industry (NECSBI 2015–16). Total RNA was extracted from vesicle fluid samples using TRIzol reagent (TaKaRa, Kusatsu, Japan) in accordance with the manufacturer’s instructions. Using the extracted RNA, reverse transcription polymerase chain reaction (RT-PCR) assays were performed for SVV, foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV) (10,15). The results showed that the samples were positive for SVV but negative for FMDV, SVDV, VSV, and VESV (Figure 1A). To isolate SVV, 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (ThermoFisher, Waltham, Massachusetts, USA). Vesicle fluid samples were filtered and added to DMEM with cultured cells. Then, the cells were maintained at 37°C with 5% CO₂ and monitored daily for cytopathic effects (CPE). When CPE appeared in 70% of cells, the virus was harvested. Mouse anti-SVV VP1 polyclonal antibody was prepared in our lab using recombinant SVV VP1 protein. Identification of SVV was performed using immunofluorescence assay (IFA) and Western blotting. The 293T cells inoculated with vesicle fluid for 24 h showed typical CPE characterized by rounding, shrinkage, and degeneration (Figure 1B). In Figure 1C, IFA showed that the SVV-infected cells demonstrated specific green fluorescence, indicating that SVV was isolated successfully. This was confirmed by Western blot assay, which demonstrated a virus-specific band in the SVV-infected cells (Figure 1D).

Figure 1.

Identification of Seneca Valley virus (SVV) CH-GDZQ-2018 strain. A — Detection of vesicular disease-associated pathogens [foot-and-mouth disease virus (FMDV), vesicular exanthema of swine virus (VESV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV)] by reverse transcription polymerase chain reaction. B — The cytopathic effect of 293T cells infected with SVV CH-GDZQ-2018 strain at 8 h post-infection. C — Immunofluorescence assay of 293T cells infected with SVV CH-GDZQ-2018 strain at 8 h post-infection. D — Western blot identification of SVV VP1 protein in 293T cells infected with third-passage SVV CH-GDZQ-2018 strain at 8 h post-infection.

Complete genome sequencing was performed using 7 pairs of overlapping primers as described previously (6). The PCR products were amplified from the isolated virus, purified, and cloned into pMD19-T vectors and sequenced by Sangon Biotech (Shanghai, China). Sequence data were assembled using SeqMan, DNASTAR’s software application (Madison, Wisconsin, USA), to generate the complete genome sequence. Finally, full genomic fragments of SVV were amplified and the isolated strain was designated as CH-GDZQ-2018 (GenBank accession no. MN423333). The genome of CH-GDZQ-2018 comprised 7262 nucleotides (nt), excluding its poly(A) tail. Compared with other representative strains, the genome sequence of CH-GDZQ-2018 had the highest nucleotide identity (99%) with CH-GD-2017-1; 98.5% to 98.8% nucleotide identity with USA/GBI29/2015-like strains CH-GDDLZ01-2017, CH-GDDLZ02-2017, CH-GDQC-2017, CH-GDYD-2017, CH-GDYS01-2017, CH-GDYS02-2017, and CH-GD-2-17-2; 98.1% nucleotide identity with USA-GBI-29-2015; 95.3% nucleotide identity with prototype strain 11-55930-3, and only 93.2% nucleotide identity with SVV-001, the first SVV isolated from the United States in 2002. Compared with other SVV strains isolated in China, CH-GDZQ-2018 shared 95.7 to 97.3% complete genome identity. Comparisons of nucleotide and amino acid similarities between CH-GDZQ-2018 and other SVV strains were also conducted. We found that VP4 and 3B were conserved, having 100% amino acid similarity. Viral proteins L, VP2, and 2C were relatively conserved. Except for the smallest protein 2A (88.9 to 100% identity) having only 9 amino acids, 3A was the most variable protein across all SVV strains (90.0 to 97.8% identity). As shown in Table I, unlike the comparison with CH-01-2015, CH-GDZQ-2018 had fewer amino acid mutations than USA/GBI29/2015. Despite this, there were several novel amino acid mutations in 2C (S74D) and 3D (V108D and F143I) between CH-GDZQ-2018 and USA/GBI29/2015. Whether these mutations affect virus virulence and pathogenicity should be explored in the future.

Table I.

Mutation analysis of the amino acid sequences of CH-GDZQ-2018 compared to SVV-001 and SVV CH-01-2015 strains.

Protein	SVV-001	SVV CH-01-2015	USA/GBI29/2015
2B	D42N, V57I, A108V	A108V	A108V
2C	K13T, S74G, N292S, T304A	K13T, S74G	S74D
3A	S19T, T31A, E33D, R66K, A73T, Q75R, G82E, S83P, V84T	Q32P, E33D, R66K, A73T, Q75R, A84T	R75Q, T84A
3B	—	—	—
3C	V18A, V81L, M86V, S120A, E141D, L177M, K201R	V18A, A39T, I81L, M86V, E141D, L177M, K201R	V18A, A39T, M86V, K201R
3D	I10V, I119V, N120S, V131A, A137P, A138T, V141M, D342N, K395R, L400V, A455T	I119V, V131A, A137P, A138T, D342N, K395R, L400V, A455T	V108D, I119V, A138T, F143I, D342N, K395R, A455T
L	R56K, V75I	R56K, V75I	V75I
VP1	Q62A, E63T, A93V, G97D, F161Y, A172T, V221I, I239V, S250N	T65A, S94N, A172T, S250N	S250N
VP2	T15I, N218S, Y276F	T15I, A278T	—
VP3	L18I, E57G, P60S, V63E, T68A, V75I, T77A, R82K, T141A, V168I, V169I	P60S, V75I, T77A, T141A, V168I	V75I, V168I
VP4	—	—	—

To analyze the evolution of the isolated SVV strain, representative strains of complete genome sequences available in GenBank were collected and used for phylogenetic analyses. The phylogenetic tree of the whole genome was constructed by the neighbor-joining method with 1000 bootstrap replicates using Molecular Evolutionary Genetics Analysis software (MEGA version 6.0; Pennsylvania State University, University Park, Pennsylvania, USA). The phylogenetic analysis showed that CH-GDZQ-2018 was clustered together with the 2017 Chinese isolates belonging to USA-GBI-29-2015-like strains (Figure 2). These results showed that SVV USA-GBI-29-2015-like strains in China have been mutating and becoming more complex.

Figure 2.

Phylogenetic analysis and recombination analysis of SVV CH-GDZQ-2018 complete genomic sequences. Phylogenetic trees were constructed using the neighbor-joining method with 1000 bootstrap replicates using MEGA 6.0 software. The newly isolated SVV CH-GDZQ-2018 strain is marked as a black circle.

Genetic recombination is a vital strategy by which viruses adapt to their environment. To detect probable recombination events, the genomic sequence was scanned for possible recombination events with the software package SimPlot (v3.5.1; Johns Hopkins University School of Medicine, Baltimore, Maryland, USA) using CH-YS01-2017 and CH-GD2017-2 as the parent viruses and the CH-GDZQ-2018 genomic sequences as the query. A window of 200 bp and a step size of 20 bp were applied. The results indicated that CH-GDZQ-2018 resulted from a recombination between CH-GD-2017-2 and CH-GDYS01-2017. Two recombination break-points were found at 3241 nt and 4700 nt of the CH-GDZQ-2018 genome and they were located in the VP1 and 2C regions, thus separating the genome into 3 regions. The fragments (regions 1 to 3240 nt and 4701 to 7286 nt) originated from CH-GD2017-2. The other fragment arose from CH-GDYS01-2017 (Figure 3).

Figure 3.

Recombination analysis of SVV CH-GDZQ-2018. Reference strains CH-GDYS01-2017 (red) and CH-GD-2017-2 (blue) were used as putative parental strains. The X-axis indicates the location of the query sequence and the Y-axis indicates the percentage of identity.

HeN-1/2018 was the first reported recombinant SVV strain with recombinant fragments comprising structural protein regions (parts of VP4 and VP2 and most of VP3). It was recombined between non-Chinese strains. CH-GDFS-2018 is also a recombinant SVV strain with recombinant fragments similarly comprising structural protein regions (part of VP2 and part of VP3). However, it was recombined between a local epidemic strain and a foreign strain. Surprisingly, the recombinant fragments of CH-GDJY-2018 were longer, including parts of 2C, 3A, 3B, 3C, and 3D. Unlike HeN-1/2018 and CH-GDFS-2018, it was recombined between local epidemic strains. As with CH-GDJY-2018, our isolated strain CH-GDZQ-2018 was also recombined between local epidemic strains. However, its recombinant fragments included parts of VP1, 2A, 2B, and most of 2C. These results indicated that the recombination of SVV could occur in almost any coding region of the virus and that SVV in China is becoming increasingly complicated. In the future, the recombination of SVV between domestic epidemic strains might be common.

In summary, a novel recombinant USA/GBI29/2015-like strain of CH-GDZQ-2018 was identified and isolated from sick pigs in Guangdong Province. To prevent this disease, further research on the pathogenic mechanisms of SVV and methods of prevention and control need to be conducted.