RESUMEN
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.
Asunto(s)
Infecciones por Picornaviridae , Picornaviridae , ARN Viral , Enfermedades de los Porcinos , Animales , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Porcinos , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/veterinaria , Infecciones por Picornaviridae/virología , ARN Viral/genética , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Sensibilidad y Especificidad , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedad Vesicular Porcina/diagnóstico , Enfermedad Vesicular Porcina/virología , Fiebre Aftosa/diagnóstico , Fiebre Aftosa/virología , Brasil , Reproducibilidad de los ResultadosRESUMEN
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.
Asunto(s)
Proteasas Virales 3C , Picornaviridae , Proteolisis , ARN Helicasas , Proteasas Virales 3C/metabolismo , Animales , Cromatografía Liquida , Inmunoprecipitación , Picornaviridae/enzimología , Picornaviridae/genética , Picornaviridae/crecimiento & desarrollo , Picornaviridae/fisiología , Unión Proteica , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , Porcinos/virología , Enfermedad Vesicular Porcina/virología , Espectrometría de Masas en Tándem , Replicación ViralRESUMEN
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.
Asunto(s)
Benzotiazoles/química , Proteínas de la Cápside/genética , Diaminas/química , Picornaviridae/aislamiento & purificación , Quinolinas/química , Enfermedad Vesicular Porcina/diagnóstico , Animales , Límite de Detección , Picornaviridae/genética , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/virologíaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática/veterinaria , Enfermedad Vesicular Porcina/diagnóstico , Vacunas de Partículas Similares a Virus/inmunología , Animales , Femenino , Ratones Endogámicos BALB C , Mutación , Pruebas de Neutralización/veterinaria , Sensibilidad y Especificidad , Porcinos , Enfermedad Vesicular Porcina/virologíaRESUMEN
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.
Asunto(s)
Infecciones por Picornaviridae/veterinaria , Picornaviridae/patogenicidad , Enfermedad Vesicular Porcina/patología , Enfermedad Vesicular Porcina/virología , Animales , China , Picornaviridae/aislamiento & purificación , Infecciones por Picornaviridae/patología , Infecciones por Picornaviridae/virología , Porcinos , VirulenciaRESUMEN
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.
Asunto(s)
Genoma Viral , Picornaviridae/genética , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/virología , Animales , China/epidemiología , Enfermedades Transmisibles Emergentes/veterinaria , Enfermedades Transmisibles Emergentes/virología , Filogenia , Picornaviridae/aislamiento & purificación , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedad Vesicular Porcina/epidemiologíaRESUMEN
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.
Asunto(s)
Enfermedades Transmisibles Emergentes/veterinaria , Brotes de Enfermedades/veterinaria , Infecciones por Picornaviridae/veterinaria , Picornaviridae/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Enfermedades de los Porcinos/epidemiología , Animales , Brasil/epidemiología , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Enterovirus Humano B/genética , Enterovirus Humano B/aislamiento & purificación , Picornaviridae/genética , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/virología , ARN Viral/análisis , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/epidemiología , Enfermedad Vesicular Porcina/virologíaRESUMEN
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 7285â¯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.
Asunto(s)
Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Infecciones por Picornaviridae/veterinaria , Picornaviridae/clasificación , Picornaviridae/genética , Enfermedad Vesicular Porcina/epidemiología , Enfermedad Vesicular Porcina/virología , Animales , Línea Celular , China/epidemiología , Enfermedades Transmisibles Emergentes/historia , Efecto Citopatogénico Viral , Genes Virales , Genoma Viral , Historia del Siglo XXI , Filogenia , Picornaviridae/aislamiento & purificación , Picornaviridae/fisiología , Porcinos , Enfermedad Vesicular Porcina/historiaRESUMEN
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.
Asunto(s)
Dexametasona/farmacología , Inmunosupresores/farmacología , Infecciones por Picornaviridae/veterinaria , Picornaviridae , Enfermedades de los Porcinos/virología , Animales , Animales Recién Nacidos , Anticuerpos Antivirales/sangre , Porcinos , Enfermedad Vesicular Porcina/virología , Esparcimiento de Virus/efectos de los fármacosRESUMEN
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.
Asunto(s)
Filogenia , Picornaviridae/genética , Enfermedad Vesicular Porcina/epidemiología , Enfermedad Vesicular Porcina/virología , Animales , China/epidemiología , Genoma Viral , Picornaviridae/aislamiento & purificación , PorcinosRESUMEN
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.
Asunto(s)
Enfermedades Transmisibles Emergentes/veterinaria , Genoma Viral/genética , Picornaviridae/genética , Enfermedad Vesicular Porcina/epidemiología , Proteínas Virales/genética , Animales , China/epidemiología , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Brotes de Enfermedades/veterinaria , Femenino , Genómica , Masculino , Filogenia , Picornaviridae/aislamiento & purificación , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Porcinos , Enfermedad Vesicular Porcina/virología , Cultivo de VirusRESUMEN
Senecavirus A (SVA), previously known as Seneca Valley virus, was first isolated in the United States in 2002. SVA was associated with porcine idiopathic vesicular disease in Canada and the USA in 2007 and 2012, respectively. Recent increase in SVA outbreaks resulting in neonatal mortality of piglets and/or vesicular lesions in sows in Brazil, the USA and Canada point to the necessity to study the pathogenicity and molecular epidemiology of the virus. Here, we report the analysis of the complete coding sequences of SVA from 2 clinical cases and 9 assembly yard environmental samples collected in 2015 in Canada, along with 22 previously released complete genomes in the GenBank. With this combined data set, the evolution of the SVA over a 12-month period in 2015/2016 was evaluated. These SVA isolates were characterized by a rapid accumulation of genetic variations driven mainly by a high nucleotide substitution rate and purifying selection. The SVA sequences clustered in clearly defined geographical areas with reported cases of SVA infection. No transmission links were identified between assembly yards, suggesting that point source introductions may have occurred. In addition, 25 fixed non-synonymous mutations were identified across all analyzed strains when compared to the prototype SVA strain (SVV-001). This study highlights the importance of monitoring SVA mutations for their role in increased virulence and impact on SVA diagnostics.
Asunto(s)
Genoma Viral , Picornaviridae/genética , Porcinos/virología , Animales , Canadá/epidemiología , Brotes de Enfermedades , Filogenia , Picornaviridae/clasificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Enfermedad Vesicular Porcina/epidemiología , Enfermedad Vesicular Porcina/virología , Estados Unidos/epidemiologíaRESUMEN
Swine vesicular disease (SVD) is a highly contagious viral disease that causes vesicular disease in pigs. The importance of the disease is due to its indistinguishable clinical signs from those of foot-and-mouth disease, which prevents international trade of swine and related products. SVD-specific antibody detection via an enzyme-linked immunosorbent assay (ELISA) is the most versatile and commonly used method for SVD surveillance and export certification. Inactivated SVD virus is the commonly used antigen in SVD-related ELISA. A recombinant SVD virus-like particle (VLP) was generated by using a Bac-to-Bac baculovirus expression system. Results of SVD-VLP analyses from electron microscopy, western blotting, immunofluorescent assay, and mass spectrometry showed that the recombinant SVD-VLP morphologically resemble authentic SVD viruses. The SVD-VLP was evaluated as a replacement for inactivated whole SVD virus in competitive and isotype-specific ELISAs for the detection of antibodies against SVD virus. The recombinant SVD-VLP assay produced results similar to those from inactivated whole virus antigen ELISA. The VLP-based ELISA results were comparable to those from the virus neutralization test for antibody detection in pigs experimentally inoculated with SVD virus. Use of the recombinant SVD-VLP is a safe and valuable alternative to using SVD virus antigen in diagnostic assays.
Asunto(s)
Enterovirus Humano B , Enfermedad Vesicular Porcina/diagnóstico , Animales , Western Blotting/veterinaria , Ensayo de Inmunoadsorción Enzimática/veterinaria , Técnica del Anticuerpo Fluorescente/veterinaria , Microscopía Electrónica/veterinaria , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Porcinos , Enfermedad Vesicular Porcina/virologíaRESUMEN
Senecavirus A (SVA) is the only member of genus Senecavirus that causes vesicular lesions in pigs. We have characterized seven SVA isolates from different swine farms in Guangdong, China. The most variable isolate, CH-DL-01-2016, contained a single amino acid insertion at position 219-220 and a 16 amino acid insertion at position 250-251. The VP1 protein also had four nucleotide changes when compared to 31 other SVA VP1 sequences obtained from GenBank. These mutations have not been identified before. Phylogenetic trees demonstrated that the new SVA isolates were clustered into two different clades and shared 96.3%-97.1% similarity with US strains and 97.9%-98.3% similarity with Brazilian stains on nucleotide level, respectively. Prediction of antigenic epitope indicated that SVA VP1 protein contained both potential B-cell and potential T-cell epitopes. This report provides information about variation analysis in SVA in southern China.
Asunto(s)
Picornaviridae/genética , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/virología , Animales , China/epidemiología , Granjas , Femenino , Filogenia , Picornaviridae/inmunología , Picornaviridae/aislamiento & purificación , Alineación de Secuencia/veterinaria , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedad Vesicular Porcina/epidemiologíaRESUMEN
The use of swine oral fluid (OF) for the detection of nucleic acids and antibodies is gaining significant popularity. Assays have been developed for this purpose for endemic and foreign animal diseases of swine. Here, we report the use of OF for the detection of virus and antibodies in pigs experimentally infected with swine vesicular disease virus (SVDV), a virus that causes a disease clinically indistinguishable from the economically devastating foot-and-mouth disease. Viral genome was detected in OF by real-time reverse transcription polymerase chain reaction (RRT-PCR) from 1 day post-infection (DPI) to 21 DPI. Virus isolation from OF was also successful at 1-5 DPI. An adapted competitive ELISA based on the monoclonal antibodies 5B7 detected antibodies to SVDV in OF starting at DPI 6. Additionally, using isotype-specific indirect ELISAs, SVDV-specific IgM and IgA were evaluated in OF. IgM response started at DPI 6, peaking at DPI 7 or 14 and declining sharply at DPI 21, while IgA response started at DPI 7, peaked at DPI 14 and remained high until the end of the experiment. These results confirm the potential use of OF for SVD surveillance using both established and partially validated assays in this study.
Asunto(s)
Anticuerpos Antivirales/sangre , Enterovirus Humano B/inmunología , Fiebre Aftosa/virología , Genoma Viral/genética , Enfermedad Vesicular Porcina/virología , Animales , Anticuerpos Monoclonales , Enterovirus Humano B/genética , Enterovirus Humano B/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/veterinaria , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Saliva/virología , PorcinosRESUMEN
Senecavirus A (SVA) infection was recently confirmed in pigs in Brazil. In March, 2015, an outbreak of vesicular disease occurred in Guangdong, China, characterized by vesicular lesions in sows and acute death of neonatal piglets. Cumulative incidence of porcine idiopathic vesicular disease in farm A was 258, which had a total number of 5500 sows. Sows in farm B displayed typical vesicular symptoms by May, 2015, which also had 5500 sows. A total of 278 and 142 of 5500 sows in farm B demonstrated lame and presented vesicles, respectively, associated with a total of 186 mortality in piglets. Routine differential diagnoses for swine vesicular disease were carried out to exclude infection with foot-and-mouth disease virus, swine vesicular disease virus, vesicular exanthema of swine virus and vesicular stomatitis virus. In this study, seven pairs of primer were designed to amplify the complete genome of SVA in RT-PCR assays. Sequence alignment showed that this Chinese strain shares 94.4-97.1% sequence identity to other eight strains of SVA. This is the first report of SVA in China and provides information about the association between SVA infection and vesicular disease.
Asunto(s)
Infecciones por Picornaviridae/veterinaria , Picornaviridae/genética , Enfermedad Vesicular Porcina/virología , Animales , China/epidemiología , Brotes de Enfermedades/veterinaria , Picornaviridae/aislamiento & purificación , Infecciones por Picornaviridae/virología , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedad Vesicular Porcina/epidemiologíaRESUMEN
Senecavirus A (SV-A) may cause vesicular disease and neonatal mortality in pigs, and was first detected in Brazil in 2015. Samples including tissues and serum from pigs with suspected vesicular diseases were collected from January to August in 2015 from farms in the states of Minas Gerais, Santa Catarina, Goiás and Rio Grande do Sul, Brazil, and tested for the presence of SV-A by reverse transcriptase PCR. All samples were negative for foot and mouth disease virus, as well as 13 other infectious agents associated with vesicular diseases in pigs. SV-A was detected by PCR in 65/265 (24.5%) specimens. A 530 base pair fragment sequenced from the VP1 protein coding region indicated a high genetic distance from SV-A in other countries, but a common origin among the Brazilian isolates.
Asunto(s)
Infecciones por Picornaviridae/veterinaria , Picornaviridae/fisiología , Enfermedades de los Porcinos/epidemiología , Proteínas Virales de Fusión/genética , Secuencia de Aminoácidos , Animales , Brasil/epidemiología , Filogenia , Picornaviridae/genética , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Análisis de Secuencia de ARN/veterinaria , Porcinos , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/virologíaRESUMEN
Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.
Asunto(s)
Brotes de Enfermedades , Microbiología Ambiental , Moscas Domésticas/virología , Ratones/virología , Picornaviridae/aislamiento & purificación , Enfermedad Vesicular Porcina/epidemiología , Enfermedad Vesicular Porcina/virología , Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America, where sporadic outbreaks in cattle and pigs can cause clinical signs that are similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific differential diagnostic assays that are suitable for decision making in the field. RT-LAMP assays have been developed for vesicular diseases such as FMD and swine vesicular disease (SVD) but there is currently no RT-LAMP assay that can detect VS virus (VSV), nor are there any multiplex RT-LAMP assays which permit rapid discrimination between these 'look-a-like' diseases in situ. This study describes the development of a novel RT-LAMP assay for the detection of VSV focusing on the New Jersey (VSNJ) serotype, which has caused most of the recent VS cases in the Americas. This RT-LAMP assay was combined in a multiplex format combining molecular lateral-flow devices for the discrimination between FMD and VS. This assay was able to detect representative VSNJV's and the limit of detection of the singleplex and multiplex VSNJV RT-LAMP assays were equivalent to laboratory based real-time RT-PCR assays. A similar multiplex RT-LAMP assay was developed to discriminate between FMDV and SVDV, showing that FMDV, SVDV and VSNJV could be reliably detected within epithelial suspensions without the need for prior RNA extraction, providing an approach that could be used as the basis for a rapid and low cost assay for differentiation of FMD from other vesicular diseases in the field.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Estomatitis Vesicular/diagnóstico , Virus de la Estomatitis Vesicular New Jersey/aislamiento & purificación , Animales , Bovinos/virología , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/virología , América Central , Fiebre Aftosa/virología , ARN Viral/genética , Sensibilidad y Especificidad , América del Sur , Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/virología , Enfermedad Vesicular Porcina/virología , Temperatura , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular New Jersey/clasificación , Virus de la Estomatitis Vesicular New Jersey/genéticaRESUMEN
Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates.