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SVA VP1, VP2, and VP3 purified proteins. Purity of the proteins eluted from cobalt affinity columns were visualized by SDS-PAGE. Molecular weights are determined by comparison to kaleidoscope pre-stained standards (Bio-rad, Hercules, CA) run on the same gel
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Background
Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathi...
Citations
... Current diagnostic approaches for SVA include serum neutralization assay [15], indirect enzyme-linked immunosorbent assay [16], competitive enzyme-linked immunosorbent assay [17], reverse transcription nested polymerase chain reaction [17], TaqMan-based quantitative reverse transcription PCR (qRT-PCR) [18], reverse transcription loop-mediated isothermal amplification [19], reverse transcription droplet digital PCR (RT-ddPCR) [20], and SYBR green I-based quantitative RT-PCR [21], each with inherent advantages and limitations. For instance, serum neutralization assay requires extended experimental periods; nested PCR is prone to cross-contamination; and techniques like qRT-PCR demand costly equipment. ...
Background
Senecavirus A (SVA), identified in 2002, is known to cause porcine idiopathic vesicular disease (PIVD), which presents with symptoms resembling other vesicular diseases. This similarity complicates field diagnosis. Conventional molecular diagnostic techniques are limited by their cost, sensitivity, and requirement for complicated instrumentation. Therefore, developing an effective and accurate diagnostic method is crucial for timely identification and isolation of affected pigs, thereby preventing further disease spread.
Methods
In this study, we developed a highly-specific and ultra-sensitive SVA detection method powered by CRISPR/Cas12a. To enhance the availability in laboratories with varied equipment conditions, microplate reader and ultraviolet light transilluminator were introduced. Moreover, PCR amplification has also been incorporated into this method to improve sensitivity. The specificity and sensitivity of this method were determined following the preparation of the recombinant Cas12a protein and optimization of the CRISPR/Cas12a-based trans-cleavage system.
Results
The method demonstrated no cross-reactivity with ten kinds of viruses of swine. The minimum template concentration required to activate substantial trans-cleavage activity was determined to be 10⁶ copies/µL of SVA templates. However, when PCR amplification was incorporated, the method achieved a detection limit of one copy of SVA templates per reaction. It also exhibited 100% accuracy in simulated sample testing. The complete testing process does not exceed three hours.
Conclusions
Importantly, this method utilizes standard laboratory equipment, making it accessible for use in resource-limited settings and facilitating widespread and ultra-sensitive screening during epidemics. Overall, the development of this method not only broadens the array of tools available for detecting SVA but also holds significant promise for controlling the spread of PIVD.
... The virus solution purified by sucrose density gradient centrifugation after low-speed centrifugation at 40,000 rpm for 4 h at 4°C, was examined with transmission electron microscopy (TEM) as described previously (12,18). In addition, the viral titer was determined from BHK-21 cells using the median tissue culture infective dose (TCID 50 ) and plaque assay as described previously (8,12,19). For TCID 50 assay, CPE in BHK-21 cells were monitored for 72 h, and the viral titers were calculated according to the Spearman-Karber method (12). ...
Porcine idiopathic vesicular disease (PIVD), one of several clinically indistinguishable vesicular diseases of pigs, is caused by the emerging pathogen Senecavirus A (SVA). Despite the widespread prevalence of porcine SVA infection, no effective commercial vaccines for PIVD prevention and control are available, due to high costs associated with vaccine testing in pigs, considerable SVA diversity, and SVA rapid evolution. In this study, SVA CH/JL/2022 (OP562896), a novel mutant SVA strain derived from an isolate obtained from a pig farm in Jilin Province, China, was inactivated then combined with four adjuvants, MONTANIDETM GEL02 PR (GEL 02), MONTANIDETM ISA 201 VG (ISA 201), MONTANIDETM IMG 1313 VG N (IMS1313), or Rehydragel LV (LV). The resulting inactivated SVA CH/JL/2022 vaccines were assessed for efficacy in mice and found to induce robust in vivo lymphocyte proliferation responses and strong IgG1, IgG2a, and neutralizing antibody responses with IgG2a/IgG1 ratios of <1. Furthermore, all vaccinated groups exhibited significantly higher levels of serum cytokines IL-2, IL-4, IL-6, and IFN as compared to unvaccinated mice. These results indicate that all vaccines elicited both Th1 and Th2 responses, with Th2 responses predominating. Moreover, vaccinated mice exhibited enhanced resistance to SVA infection, as evidenced by reduced viral RNA levels and SVA infection-induced histopathological changes. Collectively, our results demonstrate that the SVA-GEL vaccine induced more robust immunological responses in mice than did the other three vaccines, thus highlighting the potential of SVA-GEL to serve an effective tool for preventing and controlling SVA infection.
... In addition, VP2 is crucial for determining the cell tropism of SVA (Wang et al., 2023) and mediating virus binding to the cell receptor anthrax toxin receptor 1 (ANTXR1) (Miles et al., 2017). Although VP2 is the major structural protein that induces neutralizing antibodies (Dvorak et al., 2016;Maggioli et al., 2018;Wen et al., 2022), few neutralizing epitopes have been identified. Thus, identifying the epitopes of the SVA capsid protein VP2 has potential application value for analyzing the function and antigenicity of the VP2 protein. ...
Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, ¹⁷⁷SLGTYYR¹⁸³ and ²⁶⁶SPYFNGL²⁷², were identified by peptide scanning. The epitope ¹⁷⁷SLGTYYR¹⁸³ was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that ¹⁷⁷SLGTYYR¹⁸³ locates on “the puff” region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.
... The antigenic sites are located in VP's external viral capsid proteins (VP1, VP2 and VP3), which are involved in the neutralization of picornaviruses (Maggioli et al., 2018). Among these VP proteins, VP2 showed to be an ideal target for the specific detection of SVA antibodies as it is more conserved and immunogenic than VP1 and VP3 (Dvorak et al., 2017). This study aimed to develop and standardize an indirect ELISA based on a recombinant VP2 (rVP2) of SVA to detect antibodies against the virus in Brazilian pig herds. ...
... however, only 14 sequences (GenBank accession numbers: KR063107, KR0631008, KR063109, MF615501, MF615502, MF615503, MF615504, MF615505, MF615506, MF615507, MF615508, MF615509, MF615510, and MZ456812), which presented the full length genome, were used in the study. As VP2 has been shown to be the viral protein with a higher antibody response (Dvorak et al., 2017) and has more epitopes for antibody recognition (Yang et al., 2007), it was selected to be the target in this study. From the viral polyprotein, the full-length VP2 gene, composed of 852 nucleotides, was identified based on its respective cleavage sites (Hales et al., 2008). ...
... Currently, several SVA antibody detection methods are available, such as indirect immunofluorescence (IF), virus neutralization assays and enzyme-linked immunosorbent assays, targeting different SVA structural proteins, including VP1, VP2, and VP3 (Gimenez-Lirola et al., 2016;Dvorak et al., 2017;Goolia et al., 2017;Bai et al., 2021). Among the three SVA structural proteins, VP2 showed higher antibody responses than VP1 and VP3 and showed higher affinity binding on an avidity ELISA (Dvorak et al., 2017). ...
Senecavirus A (SVA) is a nonenveloped, single-stranded RNA virusThe icosahedral viral particle is composed of four structural proteins: VP1, VP2, VP3 and VP4, among which VP2 is strongly involved in the antibody immune response. The virus causes vesicles on the snout and feet in pigs, which are clinically indistinguishable from other vesicular diseases such as foot-and-mouth disease. Outbreaks of SVA have been reported worldwide since 2014; however, its prevalence in Brazil remains unknown. In this study, the VP2 structural protein was produced and purified from E. coli, and recombinant VP2 (rVP2), based on the most recent Brazilian strain, was used to develop an indirect ELISA to identify antibodies against SVA in Brazilian swine herds. Sensitivity and specificity values of the rVP2 ELISA were determined using receiver operating characteristic analysis performed on 43 SVA positive and 219 negative serum samples. In addition, serum samples from pigs immunized with eight distinct Brazilian SVA inactivated strains were tested with the rVP2 ELISA. For the specificity of the assay, ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 22 (1): gmr19118 J.S.G. Rieger et al. 2 17 serum samples from vesicular stomatitis virus (VSV) from naturally infected pigs were tested. The rVP2 ELISA was found to have 100% specificity and 74.4% sensitivity. The performance of the assay using samples collected during the SVA outbreak, had a sensitivity of 100%, and with those collected nine months after the outbreak it had a sensitivity of 73.4%. The rVP2 ELISA developed here was able to detect specific SVA antibodies in acute disease and recovered pigs, and no cross-reactivity with VSV was observed. This assay has potential as a useful tool for monitoring SVA infection and could help to improve disease diagnosis.
... In the early stage of the disease, NAs are predominantly composed of IgM antibodies, and SVA-specific IgG antibodies appear later and are detected in the serum on day 7 post-infection [12,13]. Importantly, VP1 and VP3 IgG antibodies are undetectable following resolution of the disease, while VP2 IgG antibodies can persist for up to 35 days after SVA infection [14]. Thus, VP2 is an ideal diagnostic target for specific detection of antibodies against SVA. ...
... At present, three diagnostic methods are used to detect antibodies against SVA. One method is based on virus neutralization tests (VNT), and the other two methods are blocking enzyme-linked immunosorbent assay (bELISA) using monoclonal antibodies and indirect ELISA (iELISA) using VP2 protein [14][15][16]. However, diagnostic methods based on epitopes to detect antibodies directed against SVA are lacking. ...
... Previous studies have shown that antibody responses to VP2 were higher than those to VP1 and VP3, and VP2 would be a reliable target to detect antibodies directed against SVA [14]. The structural protein of SVA is composed of four structural proteins, VP4, VP2, VP3, and VP1, which contain the major epitope region [20,21]. ...
Background
Senecavirus A (SVA) is a pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical signs are similar to those of foot-and-mouth disease, porcine vesicular disease, and vesicular stomatitis. Therefore, identification of SVA as a cause of PIVD is important to eliminate this emerging pathogen.
Methods
In this study, an indirect ELISA based on the VP2 epitope (VP2-epitp-ELISA) was developed to detect antibodies directed against SVA.
Results
A novel linear epitope ( ²⁷¹ GLRNRFTTGTDEEQ ²⁸⁴ ) was first identified at the C-terminus of the VP2 protein by epitope mapping. The diagnostic performance of VP2-epitp-ELISA was estimated by testing a panel of known background sera from swine. Under the optimum test conditions, when the cutoff value was 37%, the diagnostic sensitivity (Dn) and diagnostic specificity (Dp) of the assay were 91.13% and 91.17%, respectively. The accuracy of VP2-epitp-ELISA was validated and further compared with that of commercial diagnostic kits. The diagnostic results showed that VP2-epitp-ELISA did not cross-react with serum positive for other idiopathic vesicular diseases and had a concordance rate of 90.41% with the Swinecheck ® SVA bELISA.
Conclusions
These results indicate that VP2-epitp-ELISA is suitable for specific detection of antibodies against SVA in swine.
... As reported in recent work by Houston et al. (2020) [48], the diagnosis can be achieved by PCR and qRT-PCR virus isolation from vesicular material [42,[49][50][51][52][53][54][55][56][57], in situ hybridization (ISH) [39,42,58], and immunohistochemistry (IHC) [44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61]. The detection of the nucleic acid of SVA by conventional and quantitative RT-PCR is regarded as the gold-standard test for etiological diagnosis and is considered a fast, sensitive, and specific method with numerous different viral targets reported [48]. ...
... Antibody detection methods available consist of indirect immunofluorescence (IF), virus neutralization assays, competitive enzyme-linked immunosorbent assays (cELISAs), and indirect ELISAs targeting different structural proteins, including VP1, VP2, and VP3 [39,41,42,47,59,60]. As reported by Gimenez-Lirola and colleagues in 2016 [47], VP1 indirect ELISA provided a diagnostic sensitivity and specificity of 93% and 99%, VP2 ELISA yield 94.2% sensitivity and 89.7% specificity while VP3 protein showed minimal immunoreactivity, based on ROC analysis [59]. ...
... Antibody detection methods available consist of indirect immunofluorescence (IF), virus neutralization assays, competitive enzyme-linked immunosorbent assays (cELISAs), and indirect ELISAs targeting different structural proteins, including VP1, VP2, and VP3 [39,41,42,47,59,60]. As reported by Gimenez-Lirola and colleagues in 2016 [47], VP1 indirect ELISA provided a diagnostic sensitivity and specificity of 93% and 99%, VP2 ELISA yield 94.2% sensitivity and 89.7% specificity while VP3 protein showed minimal immunoreactivity, based on ROC analysis [59]. Moreover, the authors demonstrated that viral shedding is present also in animals without clinical disease with SVA-specific IgG response, suggesting that the virus may be circulating subclinically [47]. ...
Swine production represents a significant component in agricultural economies as it
occupies over 30% of global meat demand. Infectious diseases could constrain the swine health
and productivity of the global swine industry. In particular, emerging swine viral diseases are
omnipresent in swine populations, but the limited knowledge of the pathogenesis and the scarce
information related to associated lesions restrict the development of data-based control strategies
aimed to reduce the potentially great impact on the swine industry. In this paper, we reviewed and
summarized the main pathological findings related to emerging viruses, such as Senecavirus A, Torque
teno sus virus, and Linda virus, suggesting a call for further multidisciplinary studies aimed to fill this
lack of knowledge and better clarify the potential role of those viral diseases in swine pathology.
... Up to now, outbreaks of PIVD caused by SVV have been reported in most provinces of China. In recent years, some laboratory test methods have been developed that can detect SVV infection, such as iELISA, cELISA, virus neutralization test assay, and reverse transcription polymerase chain reaction (RT-PCR) [14][15][16][17][18]. However, there are no commercial vaccines that can inoculate against SVV infection. ...
... Whole blood was collected at 0, 14,21,28,35,42, and 49 days to detect neutralizing antibody titers, from the first immunization to the end of the immunization period (49 days). During the protection challenge period, whole blood was collected every 2 days. ...
Seneca Valley virus (SVV), also known as Senecavirus A (SVA), is a non-enveloped and single-strand positive-sense RNA virus, which belongs to the genus of Senecavirus within the family Picornaviridae. Porcine idiopathic vesicular disease (PIVD) caused by SVV has frequently been prevalent in America and Southeast Asia (especially in China) since the end of 2014, and has caused continuing issues. In this study, an SVV strain isolated in China, named SVV LNSY01-2017 (MH064435), was used as the stock virus for the preparation of an SVV-inactivated vaccine. The SVV culture was directly inactivated using binary ethyleneimine (BEI) and β-propiolactone (BPL). BPL showed a better effect as an SVV inactivator, according to the results of pH variation, inactivation kinetics, and the detection of VP1 content during inactivation. Then, SVV inactivated by BPL was subsequently emulsified using different adjuvants, including MONTANIDETM ISA 201 VG (ISA 201) and MONTANIDETM IMG 1313 VG N (IMS 1313). The immunoreactivity and protection efficacy of the inactivated vaccines were then evaluated in finishing pigs. SVV-BPL-1313 showed a better humoral response post-immunization and further challenge tests post-immunization showed that both the SVV-BPL-201 and SVV-BPL-1313 combinations could resist challenge from a virulent SVV strain. The SVV LNSY01-2017-inactivated vaccine candidate developed here represents a promising alternative to prevent and control SVV infection in swine.
... These monoclonal antibodies have a high diagnostic capacity for vesicular lesions that are clinically uncertain, and they effectively distinguish suspected SVV infections from other vesicular disease viruses (Yang et al., 2012). A simple ELISA based on the detection of antibodies to SVA VP2 can aid in the diagnosis of vesicular disease caused by SVA (Dvorak et al., 2017). RT-PCR is the most widely used method for detecting SVV RNA. ...
... Previous studies have shown that VP1, VP2, and VP3 proteins can induce the production of neutralizing antibodies. The antigenicity is relatively strong and conservative, and the proteins can be considered the main diagnostic target antigen of SVV [16][17][18][19]. ...
... The capsid protein of SVV is composed of four structural proteins: VP1, VP2, VP3, and VP4, which contain the main epitope region. Dvorak used a modified prokaryotic expression vector pET-24b to express and then purified VP1, VP2, and VP3 proteins and coated the three recombinant proteins with ELISA plates, respectively [18]. Through the verification of a large number of clinical samples, it was found that the sensitivity and specificity of the ELISA plate coated with the VP2 protein alone were better than that of the ELISA plate coated with the VP1 and VP3 proteins. ...
Background
Seneca Valley virus (SVV) is a picornavirus that causes vesicular disease in swine. Clinical characteristics of the disease are similar to common viral diseases such as foot-and-mouth disease virus, porcine vesicular disease virus, and vesicular stomatitis virus, which can cause vesicles in the nose or hoof of pigs. Therefore, developing tools for detecting SVV infection is critical and urgent.
Methods
The neutralizing antibodies were produced to detect the neutralizing epitope.
Results
Five SVV neutralizing monoclonal antibodies (mAb), named 2C8, 3E4, 4C3, 6D7, and 7C11, were generated by immunizing mouses with ultra-purified SVV-LNSY01-2017. All five monoclonal antibodies exhibited high neutralizing titers to SVV. The epitopes targeted by these mAbs were further identified by peptide scanning using GST fusion peptides. The peptide ¹⁵³ QELNEE ¹⁵⁸ is defined as the smallest linear neutralizing epitope. The antibodies showed no reactivity to VP2 single mutants E157A. Furthermore, the antibodies showed no neutralizing activity with the recombinant virus (SVV-E157A).
Conclusions
The five monoclonal antibodies and identified epitopes may contribute to further research on the structure and function of VP2 and the development of diagnostic methods for detecting different SVV strains. Additionally, the epitope recognized by monoclonal antibodies against VP2 protein may provide insights for novel SVV vaccines and oncolytic viruses development.
... Moreover, the recombination among SVA strains has been reported recent years [19], suggesting a continuous evolution of SVA. To limit the spread of SVA, a series of diagnostic methods have been established and used for surveillance of SVA in pigs [20][21][22][23][24], and our lab has developed an inactivated vaccine previously that can protect pigs against SVA infection [25]. Appropriate immunization schedules are critical for control of diseases. ...
Senecavirus A (SVA) is a newly porcine virus that has been detected in many countries since its first detection in pigs in Canada in 2007, and it remains endemic in many countries in Asia and America, which has become a substantial problem for the pig industry. Vaccination is a potentially effective strategy for the prevention and control of SVA infection. Our lab has developed a SVA vaccine candidate previously. In this study, the antibody response to the prepared vaccine in sows and their offspring was evaluated. Vaccination of sows with inactivated SVA vaccines during pregnancy elicited SVA-specific virus-neutralizing antibodies. Vaccination with a high dose of SVA vaccine followed a booster immunization contributed to a long-term duration of the persistence of maternally derived neutralizing antibodies (MDAs) in the milk of the sows (>14 days). In contrast, vaccination with a single low dose of SVA vaccine resulted in a short-term persistence of MDAs in the milk (2–7 days). The MDAs could be efficiently transferred from the sows to their offspring through the colostrum/milk but not the umbilical cord blood. The antibody titers and the duration of the persistence of MDAs in the offspring are highly associated with the antibody levels in the milk from the sows. Vaccination of sows with a booster dose of SVA vaccine resulted in a longer-lasting MDAs in their offspring (persisted for at least 90 days). However, vaccination with the single low dose of vaccine only brought about 42 days of MDAs persistence in their offspring. The effect of MDAs on active immunization with SVA vaccine in offspring was further evaluated, which showed that vaccination of the SVA vaccine in the presence of MDAs at the titer of ≈1:64 or less could overcome the MDAs’ interference and give rise to effective antibody response. This will help for establishing the optimal times and schedules for SVA vaccination in pigs.
