Interfering factors in the diagnosis of Senecavirus A.

BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.

Seneca Valley Virus Enters PK-15 Cells via Caveolae-Mediated Endocytosis and Macropinocytosis Dependent on Low-pH, Dynamin, Rab5, and Rab7.

Seneca Valley virus (SVV), a new pathogen resulting in porcine vesicular disease, is prevalent in pig herds worldwide. Although an understanding of SVV biology pathogenesis is crucial for preventing and controlling this disease, the molecular mechanisms for the entry and post-internalization of SVV, which represent crucial steps in viral infection, are not well characterized. In this study, specific inhibitors, Western blotting, and immunofluorescence detection revealed that SVV entry into PK-15 cells depends on low-pH conditions and dynamin. Furthermore, results showed that caveolae-mediated endocytosis (CavME) contributes crucially to the internalization of SVV, as evidenced by cholesterol depletion, downregulation of caveolin-1 expression by small interfering RNA knockdown, and overexpression of a caveolin-1 dominant negative (caveolin-1-DN) in SVV-infected PK-15 cells. However, SVV entry into PK-15 cells did not depend on clathrin-mediated endocytosis (CME). Furthermore, treatment with specific inhibitors demonstrated that SVV entry into PK-15 cells via macropinocytosis depended on the Na+/H+ exchanger (NHE), p21-activated kinase 1 (Pak1), and actin rearrangement, but not phosphatidylinositol 3-kinase (PI3K). Electron microscopy showed that SVV particles or proteins were localized in CavME and macropinocytosis. Finally, knockdown of GTPase Rab5 and Rab7 by siRNA significantly inhibited SVV replication, as determined by measuring viral genome copy numbers, viral protein expression, and viral titers. In this study, our results demonstrated that SVV utilizes caveolae-mediated endocytosis and macropinocytosis to enter PK-15 cells, dependent on low pH, dynamin, Rab5, and Rab7. IMPORTANCE Entry of virus into cells represents the initiation of a successful infection. As an emerging pathogen of porcine vesicular disease, clarification of the process of SVV entry into cells enables us to better understand the viral life cycle and pathogenesis. In this study, patterns of SVV internalization and key factors required were explored. We demonstrated for the first time that SVV entry into PK-15 cells via caveolae-mediated endocytosis and macropinocytosis requires Rab5 and Rab7 and is independent of clathrin-mediated endocytosis, and that low-pH conditions and dynamin are involved in the process of SVV internalization. This information increases our understanding of the patterns in which all members of the family Picornaviridae enter host cells, and provides new insights for preventing and controlling SVV infection.

Seneca Valley Virus Induces DHX30 Cleavage to Antagonize Its Antiviral Effects.

Seneca Valley virus (SVV) is a new pathogen associated with porcine idiopathic vesicular disease (PIVD) in recent years. However, SVV-host interaction is still unclear. In this study, through LC-MS/MS analysis and coimmunoprecipitation analysis, DHX30 was identified as a 3Cpro-interacting protein. 3Cpro mediated the cleavage of DHX30 at a specific site, which depends on its protease activity. Further study showed that DHX30 was an intrinsic antiviral factor against SVV that was dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of viral infection. RIP-seq showed comparatively higher coverage depth at SVV 5'UTR, but the distribution across SVV RNA suggested that the interaction had low specificity. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. Interestingly, DHX30 was determined to interact with 3D in an SVV RNA-dependent manner. Thus, DHX30 negatively regulated SVV propagation by blocking viral RNA synthesis, presumably by participating in the viral replication complex. IMPORTANCE DHX30, an RNA helicase, is identified as a 3Cpro-interacting protein regulating Seneca Valley virus (SVV) replication dependent on its helicase activity. DHX30 functioned as a viral-RNA binding protein that inhibited SVV replication at the early stage of virus infection. DHX30 expression strongly inhibited double-stranded RNA (dsRNA) production. In addition, 3Cpro abolished DHX30 antiviral effects by inducing DHX30 cleavage. Thus, DHX30 is an intrinsic antiviral factor that inhibits SVV replication.

Validation of a competitive enzyme-linked immunosorbent assay to improve the serological diagnosis of swine vesicular disease.

Swine vesicular disease (SVD) is an infectious viral disease of pigs. The clinical symptoms of SVD are indistinguishable from other vesicular diseases. In countries free of vesicular diseases, rapid SVD diagnosis and differentiation from other vesicular diseases are essential. In this report, a competitive enzyme-linked immunosorbent assay (cELISA) was developed and validated to improve the current SVD serological diagnosis. In this cELISA, an anti-SVD monoclonal antibody (mAb) captures the recombinant SVD virus-like particle (SVD-VLP) antigen, and 5B7 mAb is used as a competitor to compete with polyclonal antibodies in SVD-positive sera. The cut-off value of the SVD-VLP based cELISA (SVD-VLP cELISA) is ≥ 65% inhibition (%). The determined diagnostic specificity was 99.2%. SVD-VLP cELISA successfully detected SVD antibodies in the sera of SVD-infected animals and produced a diagnostic sensitivity of 100%. A panel of SVD positive sere including outbreak samples (n = 11) and samples (n = 5) from experimentally inoculated pigs, were correctly identified as positive by the SVD-VLP cELISA. In terms of reducing false positives detected by the currently used cELISA (5B7 cELISA), the performance of SVD-VLP cELISA is comparable to the gold standard virus neutralization test.

La maladie vésiculeuse du porc (SVD) est une maladie virale infectieuse des porcs. Les symptômes cliniques de la SVD sont indiscernables des autres maladies vésiculaires. Dans les pays exempts de maladies vésiculaires, un diagnostic rapide de la SVD et une différenciation avec les autres maladies vésiculaires sont essentiels. Dans ce rapport, un test immuno-enzymatique compétitif (cELISA) a été développé et validé pour améliorer le diagnostic sérologique actuel de la SVD. Dans ce cELISA, un anticorps monoclonal anti-SVD (mAb) capture l'antigène recombinant de particules de type virus SVD (SVD-VLP), et le mAb 5B7 est utilisé comme compétiteur pour concurrencer les anticorps polyclonaux dans les sérums positifs pour la SVD. La valeur seuil du cELISA basé sur SVD-VLP (cELISA SVD-VLP) est ≥ 65 % d'inhibition (%). La spécificité diagnostique déterminée était de 99,2 %. SVD-VLP cELISA a détecté avec succès des anticorps SVD dans les sérums d'animaux infectés par SVD et a produit une sensibilité diagnostique de 100 %. Un panel de sérums positifs pour la SVD, comprenant des échantillons d'épidémie (n = 11) et des échantillons (n = 5) de porcs inoculés expérimentalement, a été correctement identifié comme positif par le cELISA SVD-VLP. En termes de réduction des faux positifs détectés par le cELISA actuellement utilisé (5B7 cELISA), les performances du cELISA SVD-VLP sont comparables au test de neutralisation du virus de référence.(Traduit par Docteur Serge Messier).

Retrospective Characterization of the 2006-2007 Swine Vesicular Disease Epidemic in Northern Italy by Whole Genome Sequence Analysis.

Advances in the epidemiological tracing of pathogen transmission have been largely driven by the increasing characterisation of whole-genome sequence data obtained at a finer resolution from infectious disease outbreaks. Dynamic models that integrate genomic and epidemiological data further enhance inference on the evolutionary history and transmission dynamics of epidemic outbreaks by reconstructing the network of 'who-infected-whom'. Swine Vesicular Disease (SVD) was present in Italy from 1966 until 2015, and since the mid-1990s, it has mainly been circulating within Italy's central-southern regions with sporadic incursions to the north of the country. However, a recrudescence of SVD in northern Italy was recorded between November 2006 and October 2007, leading to a large-scale epidemic that significantly affected the intensive pig industry of the Lombardy region. In this study, by using whole-genome sequence data in combination with epidemiological information on disease occurrences, we report a retrospective epidemiological investigation of the 2006-2007 SVD epidemic, providing new insights into the transmission dynamics and evolutionary mode of the two phases that characterised the epidemic event. Our analyses support evidence of undetected premises likely missed in the chain of observed infections, of which the role as the link between the two phases is reinforced by the tempo of SVD virus evolution. These silent transmissions, likely resulting from the gradual loss of a clear SVD clinical manifestation linked to sub-clinical infections, may pose a risk of failure in the early detection of new cases. This study emphasises the power of joint inference schemes based on genomic and epidemiological data integration to inform the transmission dynamics of disease epidemics, ultimately aimed at better disease control.

Eradication of Swine Vesicular Disease in Italy.

Swine vesicular disease (SVD) is a contagious viral disease of pigs clinically indistinguishable from other vesicular diseases, such as foot and mouth disease, vesicular stomatitis, vesicular exanthema of swine, and idiopathic vesicular disease. In Italy, where SVD was first reported in 1966, an eradication program started in 1995. The program, updated in 2008, was based on regionalization, complete control on pig movements, improvement of pig farms biosecurity, appropriate cleansing and disinfection procedures of vehicles approved for pig transportation, and a testing program using both serological and virological assays. In cases of confirmed SVD virus infection a stamping-out policy was applied. In the period 2009 to 2019, between 300,000 and 400,000 pigs were serologically tested each year. The last SVD outbreak was notified in 2015, and the last seropositive pig was detected in 2017. SVD surveillance is still ongoing and no proof of virus activity has been detected so far. All available data support the complete SVD virus eradication from the Italian pig industry.

Development of an improved dual-promoter-based reverse genetics system for emerging Senecavirus A.

Senecavirus A (SVA), a recently emerging picornavirus, poses a great threat to the swine industry because it causes swine idiopathic vesicular disease and epidemic transient neonatal losses. Thus far, the progress in SVA viral pathogenesis studies and vaccine development remains sluggish, and an available and convenient reverse genetics system would undoubtedly promote relevant research. Herein, we established an improved universal dual-promoter reverse genetics system with an SVA-specific hammerhead ribozyme and hepatitis delta virus ribozyme at both terminals of the viral genome; this system could be applied to rescue all SVA strains by both eukaryotic and prokaryotic RNA polymerase systems. The genome of the clone-derived Chinese field strain CH/HeN-2018 was assembled into the universal vector pcDNA-rSVAuni through the Gibson assembly technique. Moreover, two silent mutations, G6848C and C7163 G, were separately engineered into the full-length cDNA clone with one step site-directed mutagenesis to create a KpnI restriction enzyme site, which served as a unique genetic marker. The viruses, designated rCH/HeN-2018-T7, rCH/HeN-2018-CMV, rCH/HeN-2018-6484 m and rCH/HeN-2018-7163 m, were successfully rescued through both CMV- and T7-dependent pathways, and their biological properties were further evaluated. The results showed that all four viruses grew rapidly in PK-15 cells and exhibited viral titers and growth kinetics similar to those of parental wtCH/HeN-2018. The established reverse genetics system is easily operated and can be applied to rescue all SVA strains in a short time, which will be helpful for studying SVA biology, including viral pathogenesis, antiviral therapies and vaccine development.

Development of a novel SYBR green I-based quantitative RT-PCR assay for Senecavirus A detection in clinical samples of pigs.

Porcine vesicular disease caused by Senecavirus A (SVA) is a newly emerging disease in many countries. Based on clinical signs only, it is very challenging to distinguish SVA infection from other similar diseases, such as foot and mouth disease, swine vesicular disease, and vesicular stomatitis. Therefore, it is crucial to establish a detection assay for the clinical diagnosis of SVA infection. In this study, a pair of specific primers were designed based on the highly conserved L/VP4 gene sequence of SVA. The established SYBR green I-based quantitative reverse transcription polymerase chain reaction (qRT-PCR) method was used to detect SVA nucleic acids in clinical samples. The limit of detection SVA nucleic acids by qRT-PCR was 6.4 × 101 copies/µL, which was significantly more sensitive than that by gel electrophoresis of 6.4 × 103 copes/µL. This assay was specific and had no cross-reaction with other seven swine viruses. Using SYBR green I-based qRT-PCR, the SVA positive rates in experimental animal samples and field samples were 67.60% (96/142) and 80% (24/30) respectively. The results demonstrate that SYBR green I-based qRT-PCR is a rapid and specific method for the clinical diagnosis and epidemiological investigation of related vesicular diseases caused by SVA.

Development and evaluation of swine vesicular disease isotype-specific antibody ELISAs based on recombinant virus-like particles.

Swine vesicular disease (SVD) is a contagious viral disease of pigs. The clinical signs of SVD are indistinguishable from other vesicular diseases, such as senecavirus A infection (SVA) and foot-and-mouth disease (FMD). Rapid and accurate diagnostic tests of SVD are considered essential in countries free of vesicular diseases. Competitive ELISA (cELISA) is the serological test used routinely. However, although cELISA is the standard test for SVD antibody testing, this test produces a small number of false-positive results, which caused problems in international trade. The current project developed a SVD isotype antibody ELISA using recombinant SVD virus-like particles (VLP) and an SVD-specific monoclonal antibody (mAb) to reduce the percentage of false positives. The diagnostic specificities of SVD-VLP isotype ELISAs were 98.7% and 99.6% for IgM and IgG. The SVD isotype ELISAs were SVD-specific, without cross-reactivity to other vesicular diseases. A panel of 16 SVD-positive reference sera was evaluated using the SVD-VLP isotype ELISAs. All sera were correctly identified as positive by the two combined SVD-VLP isotype ELISAs. Comparison of the test results showed a high level of correlation between the SVDV antigen isotype ELISAs and SVD-VLP isotype ELISAs. 303 sera from animals lacking clinical signs and history of SVDV exposure were identified positive using SVD cELISA. These samples were examined using SVD-VLP isotype ELISAs. Of the 303 serum samples, five were positive for IgM, and five of 303 were positive for IgG. Comparable to virus neutralization test results, SVD isotype ELISAs significantly reduced the false-positive samples. Based on above test results, the combined use of cELISA and isotype ELISAs can reduce the number of false-positive samples and the use of time-consuming virus neutralization tests, with benefit for international trade in swine and related products.

Pathogenicity of two Chinese Seneca Valley virus (SVV) strains in pigs.

Seneca Valley virus (SVV) has been identified as the causative agent of SVV-associated vesicular disease (SAVD). To investigate the pathogenicity of two newly isolated SVV strains (GD-S5/2018 and GD04/2017) in China, experimental infections of pigs were performed. In pig experiments, both SVV strains successfully infected all animals, evidenced by presence of virus shedding and robust protective antibody responses. SVV GD-S5/2018 infection resulted in characteristic clinical signs, and ulcerative lesions on the tongue and gums. However, SVV GD04/2017 did not cause any clinical symptoms except depression in pigs during the experiment. Taken together, these results demonstrate that SVV GD-S5/2018 is a virulent strain for pigs, whereas SVV GD04/2017 is nearly avirulent. The established animal models for SVV infection will be utilized to dissect the immunity and pathogenesis, and develop vaccines and antivirals.

The risk of introduction of swine vesicular disease virus into Kenya via natural sausage casings imported from Italy.

Pig production in Kenya is hampered by seasonal markets. As an alternative outlet for the finished pigs, several value-added meat-processing firms have been established. Sausage, which is produced using casings derived from intestines of pigs, is one form of processed meats. Kenya imports several kgs of natural casings every year; and a recent concern is Swine vesicular disease virus (SVDV), which has never been reported in Kenya, might be introduced via natural casings imported from Italy. To determine conditions (with associated probabilities) that could lead to the introduction of SVDV, a quantitative risk assessment model was developed. Using Monte Carlo simulations at 10,000 iterations, the probability of introducing SVDV was estimated to be 1.9x10-8. Based on the suggested volume of import and mitigations used in the analysis, contaminated casings derived from an estimated 0.003 (Range = 8.1x10-8 - 0.08) infected pigs will be included in the consignment each year. The critical pathway analysis revealed that rigorous surveillance programs in Italy have a potential to dramatically reduce the risk of introducing SVDV into Kenya by this route.

Emergence and complete genome of Senecavirus A in pigs of Henan Province in China, 2017.

Senecavirus A (SVA) the only member of the Senecavirus genus within the Picornaviridae family, is an emerging pathogen causing swine idiopathic vesicular disease and epidemic transient neonatal losses. Here, SVA strain (CH-HNKZ-2017) was isolated from a swine farm exhibiting vesicular disease in Henan Province of Central China. A phylogenetic analysis based on complete genome sequence indicated that CH-HNKZ-2017 was closely related to US-15-40381IA, indica- ting that a new SVA isolate had emerged in China.

Reverse transcriptase droplet digital PCR to identify the emerging vesicular virus Senecavirus A in biological samples.

Senecavirus A (SVA) belonging to the family Picornaviridae, genus Senecavirus was incidentally isolated in 2002 from the PER.C6 (transformed foetal retinoblast) cell line. However, currently, this virus is associated with vesicular disease in swine and it has been reported in countries such as the United States of America, Canada, China, Thailand and Colombia. In Brazil, the SVA was firstly reported in 2015 in outbreaks of vesicular disease in swine, clinically indistinguishable of Foot-and-mouth disease, a contagious viral disease that generates substantial economic losses. In the present work, it was standardized a diagnostic tool for SVA based on RNA reverse transcriptase droplet digital PCR (RT-ddPCR) using one-step and two-step approaches. Analytical sensitivity and specificity were done in parallel with real-time PCR, RT-qPCR (one-step and two-step) for comparison of sensitivity and specificity of both methods. In the standardization of RT-ddPCR, the double-quenched probe and the temperature gradient were crucial to reduce background and improve amplitude between positive and negative droplets. The limit of detection and analytical specificity of techniques of one-step techniques showed superior performance than two-step methods described here. Additionally, the results showed 94.2% concordance (p < 0.001) for RT-ddPCR and RT-qPCR using the one-step assay approach and biological samples from Brazilian outbreaks of Senecavirus A. However, ddRT-PCR had a better performance than RT-PCR when swine serum pools were tested. According to the results, the one-step RT-ddPCR and RT-qPCR is highlighted to be used as an auxiliary diagnostic tool for Senecavirus A and for viral RNA absolute quantification in biological samples (RT-ddPCR), being a useful tool for vesicular diseases control programs.

Fully automated and integrated multiplex detection of high consequence livestock viral genomes on a microfluidic platform.

Differential diagnosis of diseases that share common clinical signs typically requires the performance of multiple independent diagnostic tests to confirm diagnosis. Diagnostic tests that can detect and discriminate between multiple differential pathogens in a single reaction may expedite, reduce costs, and streamline the diagnostic testing workflow. Livestock haemorrhagic diseases like classical swine fever (CSF), African swine fever (ASF), and vesicular diseases, such as foot-and-mouth disease (FMD), vesicular stomatitis (VS), and swine vesicular disease (SVD) can have an enormous impact on the livestock industry and economy of countries that were previously free of the diseases. Thus, rapid diagnosis of these diseases is critical for disease control. Here, we describe the development and initial laboratory validation of a novel fully automated user-developed assay for simultaneous detection and differentiation of multiple viruses of veterinary importance in a single reaction with minimal user-intervention. The user only performs sample loading, placement of consumables and reagents, selection and initiation of assay while all other processes (i.e., nucleic acid extraction, multiplex RT-PCR, reverse dot blot detection and result reporting) are performed fully automated. The current assay has a turn-around time of approximately 6 hr and can simultaneously process up to 24 samples. The automated assay accurately and specifically detected 37 laboratory amplified strains of the five target viruses, including all seven serotypes of FMD virus, three genotypes of CSF virus, and two serotypes of VS virus. The assay also detected targeted viruses in a variety of clinical samples collected from infected animals, such as oral fluid, oral swab, nasal swab, whole blood, serum, as well as tonsil, spleen, kidney, and ileum. No cross-reactivity was observed with 15 nontarget viruses that affect livestock and samples from clinically healthy animals. To our knowledge, this is the first fully automated and integrated assay for simultaneous detection of multiple high consequence veterinary pathogens.

Isolation and phylogenetic analysis of an emerging Senecavirus A in China, 2017.

Senecavirus A (SVA), which is associated with porcine vesicular disease and high mortality in neonatal piglets, is a small non-enveloped RNA virus and a member of Picornaviridae family. An emerging SVA strain, named SVA CH/FuJ/2017, was isolated from vesicular liquid and vesicular lesion tissue from piglets with vesicular disease in Fujian province, China. In our study, the complete genome sequence of SVA CH/FuJ/2017 strain has been determined. The viral genome was 7285 nt in length. The homology analysis indicated that the gene sequences of polyprotein and VP1 in SVA CH/FuJ/2017 shared highest nucleotide identities with American SVA isolates; and polyprotein showed the highest similarity with American SVA isolates. The phylogenetic analysis based on polyprotein and VP1 nucleotide sequences indicated that SVA CH/FuJ/2017 was closely related to American SVA isolates. The results revealed that the novel SVA strain was closely related to those SVA strains that were isolated in America. Hence, the retrospective study is important for tracing the probable origin of China SVA strains.

Dexamethasone treatment did not exacerbate Seneca Valley virus infection in nursery-age pigs.

BACKGROUND: Senecavirus A, commonly known as Seneca Valley virus (SVV), is a picornavirus that has been infrequently associated with porcine idiopathic vesicular disease (PIVD). In late 2014 there were multiple PIVD outbreaks in several states in Brazil and samples from those cases tested positive for SVV. Beginning in July of 2015, multiple cases of PIVD were reported in the United States in which a genetically similar SVV was also detected. These events suggested SVV could induce vesicular disease, which was recently demonstrated with contemporary US isolates that produced mild disease in pigs. It was hypothesized that stressful conditions may exacerbate the expression of clinical disease and the following experiment was performed. Two groups of 9-week-old pigs were given an intranasal SVV challenge with one group receiving an immunosuppressive dose of dexamethasone prior to challenge. After challenge animals were observed for the development of clinical signs and serum and swabs were collected to study viral shedding and antibody production. In addition, pigs were euthanized 2, 4, 6, 8, and 12 days post inoculation (dpi) to demonstrate tissue distribution of virus during acute infection. RESULTS: Vesicular disease was experimentally induced in both groups with the duration and magnitude of clinical signs similar between groups. During acute infection [0-14 days post infection (dpi)], SVV was detected by PCR in serum, nasal swabs, rectal swabs, various tissues, and in swabs from ruptured vesicles. From 15 to 30 dpi, virus was less consistently detected in nasal and rectal swabs, and absent from most serum samples. Virus neutralizing antibody was detected by 5 dpi and lasted until the end of the study. CONCLUSION: Treatment with an immunosuppressive dose of dexamethasone did not drastically alter the clinical disease course of SVV in experimentally infected nursery aged swine. A greater understanding of SVV pathogenesis and factors that could exacerbate disease can help the swine industry with control and prevention strategies directed against this virus.

The Distribution of Different Clades of Seneca Valley Viruses in Guangdong Province, China.

Seneca Valley virus (SVV), a newly determined etiological agent of vesicular disease in swine, causes porcine idiopathic disease and occasional acute death in piglets. Recently, an increased number of SVV infection cases have been reported in the United States (US) and China, resulting in significant economic losses to the swine industry. The first identification of SVV in China was reported in Guangdong Province, a major swine producing province. The cases of SVV were continuously reported in Guangdong in 2015 and 2016. However, the spread of SVV in Guangdong in 2017 remains unknown. In this study, we determined two new SVV strains, CH-GD-2017-1 and CH-GD-2017-2, from Guangdong. The genetic analysis suggested that the two Guangdong strains showed different characteristics to previous Guangdong strains. They showed lower nucleotide similarity with strains isolated in 2015 and 2016, and were more similar to the US strains. Phylogenetic analyses indicated that the new strains were clustered in a different clade with previous Guangdong strains. We found 28 mutated amino acids in the new strains, compared with the first Guangdong strain, SVV CH-01-2015. In the geographic analysis, we found that the US and China reported more SVV cases than other countries, and most of the SVV cases were reported in east and central China-of which, Guangdong Province is one of the major epidemic regions. In conclusion, our findings indicate that SVV continued to spread in Guangdong Province in 2017, and two different clades of SVVs have emerged in this region.

Identification and genomic characterization of the emerging Senecavirus A in southeast China, 2017.

Senecavirus A (SVA) is an emerging non-enveloped virus with a single-stranded, positive-sense RNA genome that belongs to the Senecavirus genus in the Picornaviridae family. Senecavirus A-associated swine idiopathic vesicular disease and epidemic transient neonatal losses have caused substantial economic losses for the swine industry. Here, we describe a case of re-emerging vesicular disease among sows and finishing pigs on a swine farm in Fujian Province of southeast China. Other causative pathogens, including FMDV, SVDV and VSV, were excluded, and a novel SVA strain, CH-FJZZ-2017, was isolated. Sequencing and phylogenetic analysis of the complete genome and individual viral proteins revealed that CH-FJZZ-2017 is closely related to the US strains in 2015. The results further showed that Chinese SVAs have formed two distinct subclades with 2016 as the turning point. Viruses causing outbreaks after late 2016 shared higher nucleotide identities with the US strains in 2015. There is still some evolutionary distance between CH-FJZZ-2017 and other strains isolated in late 2016, suggesting that Chinese SVA isolates have been evolving in different directions. This study provides a basis for the development of effective prevention and control strategies.

Adaptive Immune Responses following Senecavirus A Infection in Pigs.

Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of αß T cells, especially CD4+ T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8+ and double-positive CD4+ CD8+ T cells (effector/memory T cells) expressing interferon gamma (IFN-γ) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host.IMPORTANCE Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4+ T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.