RESUMEN
Pseudorabies viruses (PRV) pose a major threat to the global pig industry and public health. Rapid, intuitive, affordable, and accurate diagnostic testing is critical for controlling and eradicating infectious diseases. In this study, a portable detection platform based on RPA-CRISPR/EsCas13d was developed. The platform exhibits high sensitivity (1 copy/µL), good specificity, and no cross-reactivity with common pathogens. The platform uses rapid preamplification technology to provide visualization results (lateral flow assays or visual fluorescence) within 1 h. Fifty pig samples (including tissues, oral fluids, and serum) were tested using this platform and real-time quantitative polymerase chain reaction (qPCR), showing 34.0 % (17 of 50) PRV positivity with the portable CRISPR/EsCas13d dual-readout platform, consistent with the qPCR results. These results highlight the stability, sensitivity, efficiency, and low equipment requirements of the portable platform. Additionally, a novel point-of-care test is being developed for clinical use in remote rural and resource-limited areas, which could be a prospective measure for monitoring the progression of pseudorabies and other infectious diseases worldwide.
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Sistemas CRISPR-Cas , Herpesvirus Suido 1 , Herpesvirus Suido 1/genética , Herpesvirus Suido 1/aislamiento & purificación , Animales , Porcinos , Sistemas CRISPR-Cas/genética , Seudorrabia/diagnóstico , Seudorrabia/virología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnósticoRESUMEN
Japanese encephalitis (JE), a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV), poses a serious threat to global public health. The low viremia levels typical in JEV infections make RNA detection challenging, necessitating early and rapid diagnostic methods for effective control and prevention. This study introduces a novel one-pot detection method that combines recombinant enzyme polymerase isothermal amplification (RPA) with CRISPR/EsCas13d targeting, providing visual fluorescence and lateral flow assay (LFA) results. Our portable one-pot RPA-EsCas13d platform can detect as few as two copies of JEV nucleic acid within 1â¯h, without cross-reactivity with other pathogens. Validation against clinical samples showed 100â¯% concordance with real-time PCR results, underscoring the method's simplicity, sensitivity, and specificity. This efficacy confirms the platform's suitability as a novel point-of-care testing (POCT) solution for detecting and monitoring the JE virus in clinical and vector samples, especially valuable in remote and resource-limited settings.
RESUMEN
Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.
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Infecciones por Alphavirus , Alphavirus , Embarazo , Femenino , Animales , Porcinos , Estudios Seroepidemiológicos , Infecciones por Alphavirus/diagnóstico , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina GRESUMEN
Seneca Valley virus (SVV), a non-enveloped positive single-stranded virus can cause vesicular disease in swine. However, the mechanisms by which SVV activates an innate immune response remain unknown. Mitofusin-2 (MFN2), a mitochondria-shaping protein regulating mitochondrial fusion and fission, plays a crucial role in innate immune responses. But, the roles of Mfn2 in SVV infection have not been elucidated. Here, we show that SVV inhibited Mfn2 expression and NLRP3 inflammasome, activating RIG-I/IRF7 signaling pathway to increase IFN-λ3 expression. Overexpression of Mfn2 inhibited RIG-I/IRF7 signaling pathway, thus decreasing IFN-λ3 expression and promoting SVV replication. Interestingly, overexpression of Mfn2 also activated NLRP3 inflammasome but did not inhibit SVV proliferation. That may mean the RIG-I/IRF7 signaling pathway plays a more important role in SVV proliferation in PK-15 cells. This study could provide important insights into the modulation of host metabolism during SVV infection and provide a strong theoretical basis for a better understanding of the pathogenic mechanism and immune activation mechanism of SVV.
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Inflamasomas , Picornaviridae , Animales , Proteína con Dominio Pirina 3 de la Familia NLR , Porcinos , Replicación ViralRESUMEN
Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.
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Abatacept/biosíntesis , Animales Modificados Genéticamente , Técnicas de Transferencia Nuclear , Trasplante de Piel , Abatacept/genética , Animales , Supervivencia de Injerto , Humanos , Queratinas/genética , Ratones , Regiones Promotoras Genéticas , Ratas , Porcinos/genética , Trasplante HeterólogoRESUMEN
BACKGROUND: We sought to establish a transgenic animal line skin-specifically overexpressing cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) as a reproducible source of xenogeneic skin grafts with extended survival for wound coverage. We tested this strategy in mice based on a previously established transgenic mouse line that stably and skin-specifically expresses CTLA4Ig for lifetimes and generations. METHODS: CTLA4Ig expression was examined by immunohistochemical assay, and its bio-activity was tested by mixed lymphocyte reaction. The survival of transgenic mouse skin grafted onto rat burn wounds was observed. The impact of transgenic skin grafting on recipient immunity was evaluated by inspecting the survival of the wild-type skin grafted along with transgenic skin onto a separate wound on the same rat. The circulatory CTLA4Ig protein in recipient was detected by sandwich enzyme-linked immunosorbent assay, and its impact on recipient lymphocyte response against donor antigen was tested by mixed lymphocyte reaction. RESULTS: The transgenic CTLA4Ig protein suppressed lymphocyte proliferation in vitro, and the transgenic skin graft survival was remarkably prolonged compared with the wild-type skin derived from the same mouse strain. The survival of the wild-type skin grafted along with transgenic skin exhibited no significant difference from that grafted alone. Circulatory CTLA4Ig protein was detected in recipients, however, no significantly reduced recipient lymphocyte response against donor antigen was observed. CONCLUSION: transgenic expression of CTLA4Ig may be a potential and safe method to prolong xenogenic skin graft survival in burn wounds, and transgenic animal lines can be established as a reproducible source of skin grafts with extended survival for wound coverage.
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Antígenos CD/fisiología , Quemaduras/cirugía , Supervivencia de Injerto/fisiología , Trasplante de Piel/métodos , Trasplante Heterólogo , Animales , Quemaduras/inmunología , Quemaduras/metabolismo , Antígeno CTLA-4 , Terapia de Inmunosupresión , Ratones , Ratones Transgénicos , Ratas , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To select the optimal pregnancy time window of embryonic pig skin precursor tissue for xenotransplantation and study its ability in wound repair. METHODS: Skin precursor tissues were obtained from pig fetus of fetal age of 35, 42, 56, 70 days, and were minced into microskin and transplanted to dorsal wounds of BALB/c nude mice, then they were covered with residual skin after plastic surgery of patients or adult pig skin (white). The characteristics of growth and development were observed after transplantation. Pathological examination was performed on 6 and 12 post operation weeks respectively to observe the tissue structure and tumorigenicity. RESULTS: Skin precursor tissues from fetal pig survived and developed after transplantation, and the microskin fused. New tissue area from skin precursor tissues with fetal age of 42 days was (47 +/- 6) mm2, which was higher than that of 35 days (18 +/- 8 mm2), 56 days (31 +/- 12 mm2), 70 days (20 +/- 8 mm2, P < 0.05). The skin precursor developed into "intact skin" with hair, sebaceous glands and sweat glands, and melanocytes were also detected in epidermis. The newly-grown skin tissue included epidermal and dermal layer, and obvious dermal papillae. Teratoma was not found after transplantation in skin precursor tissue with fetal age of 56, 70 days. CONCLUSION: Fetal pig skin precursor tissue with fetal age of 56 days can be used to repair wound as xenotransplantation.
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Trasplante de Tejido Fetal , Trasplante de Piel , Trasplante Heterólogo , Animales , Feto , Edad Gestacional , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Porcinos , Cicatrización de HeridasRESUMEN
Skin graft rejection is a typical cellular immune response, mainly mediated by T cells. Cytotoxic T lymphocyte associated antigen 4-immunoglobin (CTLA4-Ig) extends graft survival by blocking the T cell co-stimulation pathway and inhibiting T cell activation. To investigate the efficacy of CTLA4-Ig in prolonging skin graft survival, human CTLA4-Ig (hCTLA4-Ig) was engineered to overexpress in mouse skin by transgenesis using the K14 promoter. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay indicated that the expression of CTLA4-Ig remained skin-specific and relatively constant compared to the internal control protein, AKT, through seven generations. The presence and concentration of the hCTLA4-Ig protein in transgenic mouse sera was determined by enzyme-linked immunosorbent assay (ELISA), and the results indicated that the serum CTLA4-Ig concentration also remained constant through generations. Survival of transgenic mouse skins grafted onto rat wounds was remarkably prolonged compared to that of wild-type skins from the same mouse strain, and remained comparable among all seven generations. This suggested that the bioactive hCTLA4-Ig protein was stably expressed in transgenical mice through at least seven generations, which was consistent with the stable skin-specific CTLA4-Ig expression. The results demonstrated that the transgenic expression of hCTLA4-Ig in skin driven by the K14 promoter remained constant through generations, and a transgenic line can be established to provide transgenic skin with extended survival reproducibly.