Pathogenesis of a senecavirus a isolate from swine in shandong Province, China

Journal Pre-proof Pathogenesis of a Senecavirus A isolate from swine in Shandong Province, China Juan Bai, Hui Fan, Erxuan Zhou, Liang Li, Shihai Li, Junfang Yan, Ping Jiang

PII:

S0378-1135(19)31300-8

DOI:

https://doi.org/10.1016/j.vetmic.2020.108606

Reference:

VETMIC 108606

To appear in:

Veterinary Microbiology

Received Date:

6 November 2019

Revised Date:

1 February 2020

Accepted Date:

4 February 2020

Please cite this article as: Bai J, Fan H, Zhou E, Li L, Li S, Yan J, Jiang P, Pathogenesis of a Senecavirus A isolate from swine in Shandong Province, China, Veterinary Microbiology (2020), doi: https://doi.org/10.1016/j.vetmic.2020.108606

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Pathogenesis of a Senecavirus A isolate from swine in Shandong Province, China

Juan Bai1,2*, Hui Fan1, Erxuan Zhou1, Liang Li1, Shihai Li1, Junfang Yan1, Ping

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Jiang1,2*

Key Laboratory of Animal Diseases Diagnostic and Immunology, Ministry of

Agriculture, MOE International Joint Collaborative Research Laboratory for Animal

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Health & Food Safety, College of Veterinary Medicine, Nanjing Agricultural

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University, Nanjing 210095, China

Jiangsu Co-innovation Center for Prevention and Control of Important Animal

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Infectious Diseases and Zoonoses, Yangzhou, China

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*Corresponding author:

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China. Email address: [email protected] (Juan Bai); [email protected] (Ping Jiang)

Highlights

 A new virulent SVA strain was isolated and identified in Shandong province in China. 

The SVV-CH-SD strain can infect 30–100 day-old pigs, but only cause disease in 90–100-day-old pigs.

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Abstract Senecavirus A (SVA), previously called Seneca Valley virus, can cause vesicular

lesions in sows and a sharp decline in neonatal piglet production. In this study, a SVA

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strain was isolated from a pig herd in Shandong Province in China and identified as

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SVV-CH-SD. The full genome was 7286 nucleotides (nt) in length and contained a single open reading frame (ORF) of 6546 nt, encoding a 2182 amino acid (aa). A

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phylogenetic analysis showed that the isolate shares highest sequence homology (98.52%) with SVA strain USA-GBI26-2015. A genetic comparison of virulent and

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weakly virulent SVA strains showed that some amino acid residues may be associated with virulence. Animal challenge experiments showed that 90–100-day-old pigs

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inoculated with SVV-CH-SD intraorally and intranasally, intranasally, or

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intramuscularly developed low fever, blisters, and lameness. They had similar levels of neutralizing antibodies against SVA and viral loads in the serum and organs at 28 days post-challenge. However, 30–35- and 55–65-day-old pigs challenged with SVV-CH-SD showed no clinical signs, although anti-SVA neutralizing antibodies were detected. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.

Key words: Senecavirus A; SVA; pathogenesis; genomic sequence

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1. Introduction

Senecavirus A (SVA), a nonenveloped single-stranded RNA virus, belongs to the

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genus Senecavirus in the family Picornaviridae. The first SVA strain SVV-001 was

first identified in a culture of human retinal cells called PER.C6 (Hales et al., 2008). It

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had cytolytic activity and selectivity for tumor cell lines having neuroendocrine

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properties (Reddy et al., 2007). Actually, pigs were thought to be a natural host of SVA (Zhang et al., 2018). In addition, SVA virus was detected in mice, and

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houseflies, which indicated that mice and houseflies may also be the natural hosts (Joshi et al., 2016b).

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During the period 2007–2016, porcine idiopathic vesicular disease (PIVD)

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became prevalent among pigs in Canada (Pasma et al., 2008), the United States (Guo et al., 2016), Brazil (Leme et al., 2016; Leme et al., 2015; Vannucci et al., 2015), and China (Wu et al., 2017). Only SVA was identified as the causative agent of PIVD. Clinical and epidemiological characterization revealed that SVA is similar to foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine (Vannucci et al., 2015). Infected adult pigs showed various

clinical symptoms: fluid-filled and ruptured vesicles or ulcerative lesions on the snouts and coronary bands, lameness, anorexia, lethargy, cutaneous hyperemia, and fever (Leme et al., 2015; Wu et al., 2017; Zhu et al., 2017). Recently, SVA was found to be associated with epidemic transient neonatal losses, especially in herds containing SVA-affected sows (Canning et al., 2016; Gimenez-Lirola et al., 2016). However, whether SVA is a primary causative agent of acute neonatal mortality

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remains to be clarified, according to a previous report (Wang et al., 2018). Recent

research indicates that SVA was transmitted from carrier sows to contacted piglets

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(Maggioli et al., 2019).

There was an increase in the number of SVA outbreaks in swine in recent years,

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and it had resulted in significant economic losses ( Canning et al., 2016; Zhang et al.,

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2018). However, little has been reported on the pathogenesis of this virus. In this study, an SVA strain, SVV-CH-SD, was isolated and its molecular characteristics

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analyzed. Animal challenge experiments with piglets of different ages showed that it is only virulent in pigs aged 90–100 days. These data should be useful in studying the

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pathogenesis and controlling this disease in the future.

2. Materials and Methods 2.1 Cell reagents and clinical samples

Baby hamster kidney 21 (BHK-21) cells cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) were grown at 37 °C under 5% CO2. The epidermis of skin tissues samples were collected from unruptured and freshly ruptured vesicles on suspected SVA-infected pigs in Shandong province in China. The samples were stored in 0.01 M phosphate buffer containing 250 U/mL

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penicillin and 250 µg/mL streptomycin. The tissues were freeze–thawed three times, We generated tissue solutions by filtering the samples through 0.22 μm filters. The

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samples were stored at −80 °C until required.

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2.2 SVA detection with reverse transcription (RT)–PCR

Viral RNA was extracted from the field samples, and converted to cDNA with

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HiScript® Q RT SuperMix (+gDNA wiper) (Vazyme, Nanjing, China). 1 L of cDNA was used as the template for the subsequent PCR analysis with SVA-specific primers

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China).

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(Fan, H et al., 2019). The purified PCR products was sequenced (Genscript, Nanjing,

2.3 Virus isolation

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The virus was isolated by using BHK-21 cells as previous report (Qian et al.,

2016). The supernatants were harvested for further propagation until a cytopathic effect (CPE) was observed. The harvested CPE-positive cell cultures were examined for SVA with RT–PCR, as described above, and were also examined for foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV),

vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV) with previously described RT–PCRs (Fernandez et al., 2008; Lin et al., 1997; Lung et al., 2011; Nishi et al., 2019a). The only SVA-positive virus isolated was designated SVV-CH-SD. 2.4 Sequencing and phylogenetic analyses

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The whole genome of the isolate was sequenced. The complete genome of SVV-CH-SD was divided into three overlapping fragments. The three fragments were amplified with PCR using Prime STAR® HS (TaKaRa, Dalian, China). The primers

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are listed in Table 1. The PCR products were purified and cloned into the

pEASY®-Blunt Simple Cloning Vector (Transgene), and three positive clones of

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every fragment respectively were sequenced by Genscript (Nanjing, China). The

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overlapping sequences were assembled into the complete genomic sequence using the SeqMan II program in the DNASTAR software package (DNASTAR, Madison, WI,

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USA). The 5’- and 3’-UTR were determined using the 5’-full RACE core kit and 3’-full RACE core kit (TaKaRa, Dalian, China) with specific primers (Table 1). In

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addition, the sequences targeted by the primers designed based on existing SVA

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sequences were checked and confirmed by the PCR products sequences. A phylogenetic genomic analysis was performed with the MEGA version 7.0

program using the neighbor-joining method and bootstrap validation with 1,000 replications. 2.5 Indirect immunofluorescence assay (IFA)

An IFA was performed using a monoclonal antibody (mAb) 1G9 against SVA VP1, which made by using the purified recombinant VP1 protein expressed by E. coli system. BHK-21 cells were infected with SVV-CH-SD (MOI=0.01). At 18 h post-infection (h.p.i.), the cells were incubated with 100 L 1G9 mAb (diluted 1:200) for 1 h. After incubating with fluorescein isothiocyanate (FITC)-conjugated goat

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antibody against mouse IgG (H + L) (1:200) for 1 h, the cells were observed with an inverted fluorescence microscope (EVOS F1, AMG, USA).

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2.6 Western blotting analysis

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Cell lysates were separated with 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore). The membranes were incubated

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with 1G9 mAb (diluted 1:1000), and then incubated with

horseradish-peroxidase-labeled goat anti-mouse IgG antibody. The proteins were

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visualized with the Tanon Chemiluminescent Imaging System (Biotanon, China).

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2.7 Plaque assays

BHK-21 cells in 12-well plates were infected with SVV-CH-SD (MOI=0.01) for

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16 h in DMEM containing 1% low-melting-point agarose (Sigma-Aldrich) and 2% heat-inactivated FBS. For visualization, cells were overlain with 1% crystal violet in methanol for 3 h at 37 °C. 2.8 Viral growth curves

BHK-21 cells in 24-well plates, were incubated with the SVV-CH-SD ( MOI = 0.1 or 0.01). The infected cells were collected at 4, 8, 12, 16, 20, 24, and 32 h.p.i. for TCID50 determination. Finally, a one-step growth curve was constructed. 2.9 Transmission electron microscopy Negatively stained SVV-CH-SD was examined with transmission electron

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microscopy (TEM). Briefly, the SVA virus was purified with sucrose density gradient centrifugation. The virus was pelleted from the supernatant, resuspended in 500 μL of 0.01 M PBS (pH 7.2), and adsorbed onto Formvar-coated grids. The grids were then

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stained with 1% phosphotungstic acid (pH 7.0) and observed under TEM (Hitachi

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HT7700 TEM, Japan).

2.10 Analysis of recombination and virulence-related amino acids

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Recombination and mutation play key roles in the evolution of viruses. In this study, the RDP4 software was used to analyze the reorganization event(s) of

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SVV-CH-SD using different algorithms, including RDP, GENECONV, Bootscan,

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MaxChi, Chimaera, and SiScanand. some possible virulence-related amino acids in the VP1, 2C, 3A, and 3D proteins

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of SVV-CH-SD were aligned with the sequences of four virulent strains [CH-FJ-2017 (KY747510); KS15-01 (KX019804]; USA-SD15-26-2015 (KX778101); and USA-SD41901-2015 passage 1 (KT757281)] and one attenuated strain [SVV-001 (DQ641257)] using BioEdit V7.0.9.0. Three-dimensional models of these proteins were constructed online (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), and the

amino acids that might be associated with virulence were marked with different colors. 2.11 Pigs challenge experiments Pigs were challenged with the SVV-CH-SD strain. The animal experiment protocol was approved by the Animal Care and Ethics Committee of Nanjing

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Agricultural University, Nanjing, China. The procedures were conducted in accordance with the Guiding Principles for Biomedical Research Involving Animals.

The 30–35, 55–65, or 90–100 days old pigs (half male and half female) were selected

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and observed for 1 week to ensure none were symptomatic. They were free of SVA,

FMDV, SVDV, VSV, porcine reproductive and respiratory syndrome virus, classical

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swine fever virus, porcine circovirus type 2, and pseudorabies virus, detected with

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ELISA antibody kits and RT-PCR or PCR methods using the sera or oral swabs samples. Ten pigs aged 30–35 days were then randomly divided into two groups of

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five. Group 1-1 was inoculated intraorally and intranasally (half the inoculum was administered orally and the other half intranasally) with strain SVV-CH-SD (1 × 109

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TCID50/mL) and Group 1-2 was inoculated with DMEM and acted as the negative

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control (NC). Ten 55–65 days-old pigs were also randomly divided into two groups of five (Group 2-2 and Group 2-2). The inoculation routes and viral doses were the same as those in 30–35 days-old pigs experiment. Twenty pigs aged 90–100 days were then randomly divided into four groups of five. Group 3-1 was inoculated intraorally and intranasally, and Group 3-2 and Group 3-3 were inoculated intranasally or intramuscularly with the same dose. And Group 3-4 was inoculated with DMEM as

the negative control（NC）. All the pigs were given food and water and libitum. Their clinical signs were monitored daily for 28 days. At 0, 3, 7, 14, 21, or 28 days post-challenge (d.p.c.), the pig sera were collected, and the anti-SVA neutralizing antibody titers were determined as previous report (Joshi et al., 2016a). And the viral loads were determined with TaqMan real-time RT–PCR (AM et al., 2017). At 28 d.p.c., the pigs with clinical symptoms were euthanized for pathological examination.

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The organs, including the heart, liver, spleen, lung, kidneys, and inguinal lymph nodes

were collected for histopathological observation. The virus levels in these organs were

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determined with TaqMan real-time RT–PCR as described above. 2.13 Statistics

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Differences in antibody levels between the different groups were determined

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with one-way repeated-measures analysis of variance (ANOVA) and the least significance difference (LSD) test. Differences were considered statistically

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significant at p < 0.05.

3. Results and discussion

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3.1 A SVA strain isolated and identified from a pig with clinical signs Sudden outbreaks and spread of a swine infectious disease can lead to severe

economic losses in the pig industry. In 2015, the first case of an SVA-infected pig was reported in Guangzhou, China (Wu et al., 2017). And clinical cases of SVA infection have been increasing in many regions of China. In this study, we isolated

and identified a SVA strain SVV-CH-SD with BHK-21 cells. After the third blind passage, CPEs were observed at 36 h.p.i., including rounding, shrinkage, and the loss of adherence (Fig. 1A), which were consistent with a previous report (Qian et al., 2016). The IFA showed fluorescence reflecting the expression of the viral VP1 in cytoplasmic of the infected cells. Western blotting also confirmed the expression of SVA VP1 in the infected cells, and the molecular weight of VP1 protein was about 36

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kDa (Fig. 1B). The plaques were approximately 1.5 mm in diameter at 24 h.p.i. in the plaque assay (Fig. 1C). The maximum titer of the virus was 108.5 TCID50/mL at 20

h.p.i (Fig. 1D). The virions of the isolate had an icosahedral symmetry and a diameter

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of 27–30 nm (Fig. 1E), being similar to other SVA viruses (Hales et al., 2008).

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Science BHK-21 cells also support other picornaviruses, we used RT-PCR to detect

not shown here).

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FMDV and EMCV. The results showed that they did not exist in the cultures (Data

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3.3 Characterization of the complete-genome sequencing Molecular phylogenetic analyses are important in revealing the origins of

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viruses. Here, our results showed that the complete genomic sequence of

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SVV-CH-SD was 7286 nt in length (GenBank MH779611). It had the typical picornavirus L–4–3–4 genome layout. Phylogenetic analysis showed that SVA strains could be classified into four clades. The isolates from China mainly belonged to clade Ⅱand IV. And SVV-CH-SD strain belonged to clade IV (Fig. 2). SVV-CH-SD shared the highest homology (98.52%) with strain USA-GBI26-2015 (KT827250), and lower homology with the prototype strains SVV-001 (DQ641257) (93.56%) (Table 2). The

recombination analysis showed that SVV-CH-SD was probably the major parent of recombinant virus USA-IA40380-2015 (KT757280), and SVV-001 (DQ641257) might be the minor parent. The two most likely potential recombination breakpoints were both located in the 5-UTR (at nt 1 and nt 50). The approximate p value for the fragments corresponding to nucleotide positions 1–50 was 1.260 × 10−12 (Fig. 3). However, it was still not strong enough to conclude that this strain was a recombinant

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strain because the end sequences of RNA viruses have great similarities within species, and 50 nt might be too small.

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The VP1, 2C, 3A, and 3D proteins were related to virus virulence in the family Picornaviridae (Nishi et al., 2019b; Stenfeldt et al., 2018; Yuan et al., 2017; Zhu et

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al., 2011). In order to understand the putative virulence-related viral proteins and the

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key amino acid of SVV-CH-SD, four virulent SVA strains [CH-FJ-2017 (Yang et al., 2018); KS15-01 (Chen et al., 2016); USA-SD15-26-2015 (Fernandes et al., 2018;

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Joshi et al., 2016a); and USA-SD41901-2015 passage 1 (Buckley et al., 2019)] and one attenuated strain SVV-001 (Fernandes et al., 2018) were selected, and the

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sequence alignment results showed that several amino acid residues in the VP1, 2C,

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3A and 3D proteins may be related to its virulence (Table 3). In the three-dimensional models of these proteins, these amino acid residues were labeled in red (VP1), magenta (2C), orange (3A), and green (3D), with the PyMOL software (Fig 4). The locations of the amino acid residues on each protein might affect the proteins structure and the interaction between the viral protein and the cellular receptors. RNA viruses usually have constant mutations. The virulence determinants may also attribute to a

combination of various residues rather than certain individual ones. The roles of these amino acid residues in virulence of the SVA isolate should be studied with experiments in the future. 3.3 Pathogenicity of the isolate in pigs Challenge experiments were performed in pigs aged 30–35, 55–65, or 90–100

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days. All the pigs survived after viral infection and had similar anti-SVA neutralizing antibody (NA) levels at 14 and 28 d.p.c.. The body temperatures increased in all the challenged groups. In the 90–100-day-old pigs group, lameness was observed in all

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three infected rote groups at 4 d.p.c. and persisted until euthanasia: 3/5 pigs in group

1, 2/5 pigs in group 2, and 2/5 pigs in group 3 (Table 4). And those claudicatory pigs

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had cracked vesicles on the snout and ulcerative lesions on the coronary band (Fig.

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5A), which did not appear in 30–35-day-old and 55–65-day-old pigs groups. The incidence rate (40%) in the 90–100-day-old pigs challenged with SVA was lower in

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this study than the general incidence rate (66%) in experimental infections reported elsewhere (Joshi et al., 2016a). This may be attributable to the mild virulence of strain

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SVV-CH-SD and/or the nature of the pigs used in this study. Meanwhile, no oral or

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hoof lesions appeared throughout the experiment in pigs aged 30–35 or 55–65 days indicating that the pathogenesis of SVA infection is related to the age of the pigs. The viremia could be detected in the all infected pigs groups. The viral load levels in 90– 100-day-old pigs were significantly higher than those in 30–35, and 55–56-day-old pigs at 3 and 7 d.p.c.. In the 90–100-day-old pigs, the viral loads in the sera peaked at 3 d.p.c., with no significant differences among the three challenges groups. Then the

viremia levels declined rapidly and disappeared at 28 d.p.c. (Fig. 5B), demonstrating a strong causal relationship with the production of neutralizing antibodies. But obviously high viral loads were observed in the oral fluids at 3 and 7 d.p.c. (Fig. 5C), indicating that the oral fluid may play an important role in shedding of the virus. At the end of challenge experiment, the pigs with clinical symptoms were euthanized and the heart, liver, spleen, lung, kidneys, and inguinal lymph nodes

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tissues were collected for pathological examination and the viral load detection with

qPCR as previous reports (Leme et al., 2016; Dall et al., 2018; Fernandes et al., 2018).

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The results showed that no obvious micro lesion damages observed in those tissues

(Fig. 5D). And the viral DNA could be detected in those organ tissues, which being

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significantly lower than those in the oral fluids collected from challenged pigs tested

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at 3 and 7 d.p.c. (Fig. 5C). Here we could not make the correlation between the viral loads and the tissue damage. The micro lesion of the tongue and oral mucosa tissues

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should be detected in the future. In addition, it would be better to perform histopathology and immunohistochemistry on affected skin area, tonsil, bronchi,

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esophagus and intestines to understand well the pathogenesis of SVA infection.

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In summary, in this study, a virulent SVA strain was isolated and identified. It shares high sequence homology with US isolates. SVV-CH-SD infects pigs of different ages, but it is only virulent in 90–100-day-old pigs. It should be useful for studying the pathogenesis and transmission of SVA and controlling this disease in the future.

Competing interests The authors declare no competing interests.

Acknowledgements This work was supported by the National Natural Science Foundation (31502082), the China Agricultural Research System Foundation (CARS-36), the

Province (PAPD).

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Vannucci, F.A., Linhares, D.C., Barcellos, D.E., Lam, H.C., Collins, J., Marthaler, D., 2015. Identification and Complete Genome of Seneca Valley Virus in Vesicular Fluid and Sera 589-593.

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of Pigs Affected with Idiopathic Vesicular Disease, Brazil. Transbound Emerg Dis 62,

Wang, Z., Zhang, X., Yan, R., Yang, P., Wu, Y., Yang, D., Bian, C., Zhao, J., 2018. Emergence of

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a novel recombinant Seneca Valley virus in Central China, 2018. Emerging microbes & infections 7, 180.

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Wu, Q., Zhao, X., Bai, Y., Sun, B., Xie, Q., Ma, J., 2017. The First Identification and Complete Genome of Senecavirus A Affecting Pig with Idiopathic Vesicular Disease in China. Transbound Emerg Dis 64, 1633-1640.

Yang, F., Zhu, Z., Cao, W., Liu, H., Zhang, K., Tian, H., Liu, X., Zheng, H., 2018. Immunogenicity and protective efficacy of an inactivated cell culture-derived Seneca Valley virus vaccine in pigs. Vaccine 36, 841-846.

Yuan, T., Wang, H., Li, C., Yang, D., Zhou, G., Yu, L., 2017. T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication. Virus genes 53, 840-847. Zhang, X., Zhu, Z., Yang, F., Cao, W., Tian, H., Zhang, K., Zheng, H., Liu, X., 2018. Review of Seneca Valley Virus: A Call for Increased Surveillance and Research. Frontiers in microbiology 9, 940. Zhu, S., Ge, X., Gong, X., Guo, X., Chen, Y., Yang, H., 2011. Alteration of encephalomyocarditis virus pathogenicity due to a mutation at position 100 of VP1. Science China. Life sciences 54, 535-543. Zhu, Z., Yang, F., Chen, P., Liu, H., Cao, W., Zhang, K., Liu, X., Zheng, H., 2017. Emergence of

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novel Seneca Valley virus strains in China, 2017. Transbound Emerg Dis 64, 1024-1029.

ro of -p re lP na ur Jo Figure 1. Isolation and identification of SAV strain SVV-CH-SD. (A) Cytopathic effects (CPE) of the SVV-CH-SD isolates in infected BHK-21 cells. CPE effects,

such as rounding, shrinkage, and nonadherence, were observed in BHK-21 cells infected with SVV-CH-SD at 36 hpi. No CPE was observed in uninfected cells. BHK-21 cells were fixed and analyzed with immunofluorescence, using an anti-VP1 antibody. Fluorescent signals were detected in the SVA-infected BHK-21 cells, but only nonspecific fluorescence was observed in uninfected-BHK-21 cells. (B) Viral protein VP1 was detected with western blotting, using an anti-VP1 monoclonal

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antibody. (C) Viral plaque morphology in BHK-21 cells. At 24 hpi, plaques of 1.5 mm diameter were observed. (D) One-step growth curves for the viruses and viral

titers were determined with a TCID50 assay. Values are presented as the means ± SD

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of three independent experiments. (E) SVV-CH-SD particles were observed with

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TEM, with a diameter of approximately 27–30 nm. The SVA virus was purified with

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phosphotungstic acid.

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sucrose density gradient centrifugation and the particles were stained with 1%

ro of -p re lP na ur Jo Figure 2. Phylogenetic analysis based on the full-length genome of SVV-CH-SD and reference SVA strains. A phylogenetic tree was constructed using the

neighbor-joining method in the MEGA version 7.0 program, with bootstrap validation using 1,000 replications. Red circle indicates SVV-CH-SD and black triangles indicate other SVA strains isolated in China. The phylogenetic tree indicates that

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there are four clades of the virus, and that strain SVV-CH-SD belongs to clade IV.

ro of -p re lP na ur Jo Figure 3. Recombination analysis of strain SVV-CH-SD. The x-axis shows the position in the alignment, and the y-axis shows the bootstrap support (%). Major

parent plot (blue line) and minor parent plot (purple line) cross two areas (pink regions), which represent a weak recombinant signal (nt 1–50), and the p value was

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approximately 1.260 × 10−12.

ro of -p re lP na ur Jo Figure 4. Localization of amino acid residues that may be associated with viral virulence. We constructed three-dimensional structural models of the VP1, 2C, 3A,

and 3D proteins online (https://zhanglab.ccmb.med.umich.edu/I-TASSER/), using the PyMOL software. The potentially virulence-associated amino acids are labeled in red

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(VP1), magenta (2C), orange (3A), and green (3D).

ro of -p re lP na ur Jo Figure 5. Challenge of pigs with strain SVV-CH-SD. (A) Cracked vesicles on the snout of an affected pig and ulcerative lesions on the coronary band were observed.

(B) Monitoring the occurrence of viremia of pigs in different groups with TaqMan real-time PCR at 0, 3, 7, 14, 21, and 28 d.p.c., and the viral load in the sera up to the peak at 3 d.p.c.. The viral load in Group 3-1(90-100-day-old pigs, intraorally and intranasally), Group 3-2 (90-100-day-old pigs, Intramuscularly) and Group 3-3 (90-100-day-old pigs, intranasally) were significantly higher than those in Group 1-1 (30-35-day-old pigs, intraorally and intranasally) and Group 2-1 (55-56-day-old pigs,

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intraorally and intranasally) at 3 d.p.c. and 7d.p.c., *P<0.01. (C) The viral DNA levels in the tissues of the 90-100-day-old pigs groups were detected with qPCR. The virus was detected in almost all the organs tested, including the heart, liver, spleen, lung,

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kidney, and inguinal lymph nodes (LN). The highest viral load was in the oral fluids

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obtained at 3 and 7 d.p.c.. (D) Histopathological sections from pigs in the challenged groups. There were no apparent pathological changes in the heart, liver, spleen, lung,

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kidneys, or lymph nodes compared with the DMEM-treated control group.

Table 1. Primers for the amplification and identification of target genome. Primers

Sequences (5′–3′)

Positions

Amplicon size(bp)

5’RACE-R

TATGTGCTACCTATAGA

646-663

663

A-F

TTTGAAATGGGGGCTGGGCCCTC

1-24

3307

A-R

CCCCGCGTGGCAGAAGAAAGCTT

3285-3307

B-F

GCCACGCGGGGTCTGCCGGCTCATGCTGAC

3297-3326

B-R

TGTTTAAGAGCTTTGATCAGTCCTAATT

5770-5797

C-F

GCTCTTAAACACCTCGGTGAGC

5787-5808

C-R

TTTTCCCTTTTTTGTTCCGA

7267-7286

3’RACE-F

GTTTTAGCCCTGCGC

6613-6627

2501

1500

674

CH-DL-01-2016

CH-FJ-2017

CH-HN-2017

MH779611

China

KT321458

KX751944

KY747510

China

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SVA-HLJ-CHA-20

China

China

KY747511

KX377924

KY419132

China

China

Isolation/submissio

Siz

Percent

n year

e

identity

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origin

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HB-CH-2016

accession No.

Isolation source

pig

swine

re

CH-01-2015

geographic

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SVV-CH-SD

Gene

na

Virus designation

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Table 2. Senecavirus A isolates used in this study and the homology between SVV-CH-SD and those isolates

swine

2018/2019

2015/2017

SVA CH/FuJ/2017

6 728

96.28%

6 2016/2017

730

95.92%

3 porcine

2017/2017

728

97.86%

3 porcine

2017/2017

728

97.82%

3 swine

2016/2016

730

96.24%

0 China

pigs

2016/2017

16

SVA-GX-CH-2018

728

728

97.80%

3 MK039162.1

China

swine

2018/2019

726

97.09%

8 MH490944.1

China

swine

2017/2018

728

97.50%

6 KS15-01

KX019804

USA

porcine

2016/2016

728 1

98.45%

SVV-001

DQ641257

USA

cultures of

2002/2008

731

PER.C6 cells USA-GBI26-2015

KT827250

USA

93.56%

0

swine

2015/2016

711

98.52%

3 BRA-GO3-2015

KR063109

Brazil

pig

2015/2016

727

97.19%

0 Canadian

KC667560

Canada

swine

brain

2013/2013

735

11-55910-3

95.63%

6

Thailand-G103_SV

KY368743

Thailand

swine

2016/2017

729 2

3A

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_1-2016

95.24%

VP1

2C

6

6

9

9

1

1

2

2

h

2

3

3

7

6

7

2

3

5

7

1

3

3

6

8

8

8

3

9

1

3

6

2

3

4

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T

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Table 3. Some amino acids may be related to its virulence

3D

9

1

1

1

1

3

4

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e 1 2 1 9 9 6 S A T V D Y T I V T L T A D K E P A V S P si V te V Cs A T V D Y T I V T L T A D K E P A V S P H of C aK A T V D Y T I V T L T A D K E P A V S P H F m SJin 1 S U A T V D Y T I V T L T A D K E P A V S P 2o 5D S 0a 0 A 1 ci U A T V D Y T I V T L T A D K E P A V S P 1 7dS S A S G E A G F A V I K S S T E R G S V I N A D V 1 S V 5SVV-001 is an attenuated strain that has been reported, and other strains can cause blisters by D -02 artificially infecting pigs. 4 6-0 1 12 9 0 0 1 15 2 0 1 5 p

0 M

M

M

M

M

V

Table 4. Statistical analysis of the disease incidence and anti-SVA serum neutralizing antibodies of the pigs challenged with the SVV-CH-SD virus

Neutralizing Clinical symptoms

Days

antibody titers

Inoculation

(log2)

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Groups old of method pigs

Increased

Bad

Oral

Hoof

temperature

spirit

lesion

lesion

1/5

0/5

0/5

0/5

DMEM

0/5

0/5

Group

intraorally and

1/5

2-1

intranasally

14

intraorally and

1-1

intranasally

Group 1-2

0/5

1/5

1/5

1/5

3/5

9.0±0.71 9.4±0.55

1/5

1/5

1/5

2/5

9.4±0.55 9.6±0.55

intranasally

1/5

1/5

2/5

2/5

9.6±0.55 9.6±0.55

DMEM

0/5

0/5

0/5

0/5

0/5

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intranasally

ur

3-1

Intramuscularly

8.8±0.84 8.6±0.55

-

-

（hoof）

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3-2

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0/5

intraorally and

Group

-

0/5

Group

0/5

-

0/5

DMEM

2-2

0/5

0/5

55-65 Group

0/5

re

30-35

8.2±0.84 8.0±0.71

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Group

28 dpc

90-100

Group 3-3

Group 3-4

-

-

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