Coinfection with sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) causes sweet potato virus disease, with severe symptoms in sweet potato, decreasing sweet potato yield and root quality. However, the associated molecular mechanisms remain unknown. In this study, changes in mRNA and small RNA (sRNA) expression in storage roots and their 1‐ and 3‐week‐old sprouts infected with SPCSV, SPFMV, or SPCSV + SPFMV were analyzed via RNA sequencing and sRNA sequencing, respectively. The number of differentially expressed genes (DEGs) and differentially expressed microRNAs (DEMs) did not differ significantly between the roots and sprouts infected with only one virus. However, a higher number of DEGs or DEMs were observed in coinfected sprouts than in storage roots or sprouts infected with only one virus. The downregulated genes in coinfected samples were involved in phytohormone biosynthesis and signal transduction (storage roots) as well as photosynthesis, chloroplasts, and chloroplast thylakoid membranes (sprouts). Some defense‐related microRNAs of the miR156, miR164, miR165, and miR171 families were significantly upregulated in coinfected sprouts. Our results also showed that the coinfection increased the accumulation of SPFMV‐derived small interfering RNAs and upregulated the expression of several key genes in the RNA silencing pathway in sprouts. This study provides insights into the molecular basis of the synergistic effects of SPFMV and SPCSV on sweet potato storage roots and their sprouts.

The authors would like to make the following correction to this published paper [...]

Background & Aims: Immune targeting is likely required for functional cure of chronic hepatitis B (CHB). Tobevibart, a human monoclonal antibody against hepatitis B virus (HBV) surface antigen (HBsAg), neutralizes HBV and hepatitis delta virus (HDV). This study aimed to characterize effects of the engineered GAALIE Fc of tobevibart on HBV immune responses. Methods: We studied tobevibart and its equivalent HBC34*-GAALIE in vitro using electron microscopy, FcgR reporter cells, and primary human or mouse immune cells to assess HBsAg binding, dendritic cell (DC) activation, and T cell stimulation. Tobevibart-mediated binding of HBsAg to immune cells was evaluated also in a phase 1 clinical trial in patients with CHB. Results: The GAALIE Fc of tobevibart mediated gain of function in FcgR signaling in immune complexes (ICs) with HBsAg compared to wild-type (WT) Fc and increased binding of HBsAg to neutrophils and monocytes in vitro. Similarly, dosing of 300 mg tobevibart in patients with CHB mediated binding of HBsAg to these cells in vivo, concomitant with reducing HBsAg in circulation. In vitro, ICs of HBC34*-GAALIE and HBsAg activated human DCs significantly more than HBC34*-WT. These DCs presented antigen and stimulated HBsAg-specific human T cells. Similarly, ICs of HBC34*-GAALIE and HBsAg activated DCs from mice transgenic for human FcgRs and stimulated CD4+ T cells from immunized animals more than ICs with HBC34*- WT. Conclusions: We demonstrate that tobevibart combines the advantages of potent neutralization of HBV and HDV, FcgR-mediated reduction of HBsAg, and Fc-dependent enhancement of T cell responses. Tobevibart is currently under clinical investigation alone or in combination with other agents to treat patients with chronic hepatitis delta and to induce functional cure of patients with CHB. ### Competing Interest Statement LV, RW, RM, BG, ES, SVG, LER, YPC, JdI, AM, KET, SD, JME, LW, NC, JD, NS, AP, LS, DCloutier, GS, CHT, FAL, CHD, FB, AL, AA, DCorti, MAS are or were employees of Vir Biotechnology and may hold shares in Vir Biotechnology. LER, NC, GS, FAL, DC and MAS are listed as inventors on patent applications, which disclose the subject matter described in this manuscript. KA, MFY, HW, and EG served as advisors or received grant support from various industry partners. The remaining authors declare no conflict of interest. ### Clinical Trial NCT04423393 ### Funding Statement This clinical trial was designed, conducted and funded by Vir Biotechnology, Inc. as sponsor. David Belnap received funding from Vir Biotechnology through an agreement with University of Utah, related to the work described in this paper. The tobevibart phase 1 clinical study received editorial support by Lumanity Scientific Inc., which was funded by Vir Biotechnology. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: (A) For the phase 1 clinical trial (VIR-3434-1002, ClinicalTrials.gov, [NCT04423393][1]) Approval from the local Institutional Review Board or Independent Ethics Committee was obtained and informed consent was obtained from all participants prior to their participation in the study. These local review boards or committees were of the Medical Faculty of the University of Duisburg-Essen, Germany; London City & East Research Ethics Committee, Bristol, United Kingdom; University Hospital Birmingham, United Kingdom; Queen Mary Hospital, Hong Kong; University of Hong Kong, Hong Kong; Ethics Committee at Medical Center of Arensia Exploratory Medicine Limited Liability Company, Kyiv, Ukraine; Asan Medical Center, Seoul, South Korea; Gangnam Severance Hospital Yonsei University, South Korea; Korea University Anam Hospital, South Korea; Pusan National University Hospital, South Korea; Seoul National University Hospital, South Korea; Health and Disabilities Ethics Committee, Ministry of Health, Wellington, New Zealand; Romania Academy of Sciences, Bucharest, Romania; Singapore Health Services, Singapore. (B) For human samples obtained outside the above study for research in vitro, Whole blood, PBMCs, sera or plasma samples were obtained from human subjects under study protocols approved by the local Institutional Review Boards (Ethics Committees of the Canton Ticino, Switzerland or IRB of the University of California San Francisco or Advarra IRB for Quest Diagnostics, USA). All donors provided written informed consent for the use of blood and blood components. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04423393&atom=%2Fmedrxiv%2Fearly%2F2025%2F01%2F14%2F2025.01.13.25320453.atom

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin- neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 mu g of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 mu g completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

Pyroptosis, a newly discovered form of programmed cell death, has emerged as a promising antitumor approach by releasing pro-inflammatory cytokines and immunogenic substances upon cell rupture. However, non-specific pyroptosis potentially causes systemic damage to healthy tissues, and the heterogeneity of tumors limits the anti-tumor effect of pyroptosis. To address these challenges, we propose a simple yet powerful method for creating multifunctional hybrid oncolytic virus-mimicking nanovesicles (HVMNVs) that possess tumor-targeting capabilities, adjuvant activity, and the ability to evade immune clearance induced by virus modification. Based on this multifunctional nanovesicle, we developed a functional nanoparticle (HVMNVs@Fe-C) that significantly enhances the tumor targetability and cell uptake efficiency of the extremely small iron oxide nanoparticles (ESIONPs) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), leading to rapid tumor pyroptosis. More importantly, HVMNVs@Fe-C ingeniously achieves a synergistic effect by incorporating the adjuvantic and tumor-targeting properties of oncolytic viruses with the cell lytic and pro-inflammatory functions of pyroptosis in a single nanoparticle. This leads to a cascade of specific anti-tumor immune responses and reprogramming of the immunosuppressive tumor microenvironment, resulting in excellent tumor inhibition efficacy against primary melanoma growth and pulmonary metastasis. Overall, our novel strategy offers considerable promise for precisive targeted delivery and cancer immunotherapy.

Previous studies have shown that interferon gene-stimulating protein (STING) is essential for IFN-γ-inducible protein 16 (IFI16) as the DNA sensor and RNA sensor to induce transcription of type I interferon (IFN-I) and is essential for IFI16 to synergize with DNA sensor GMP-AMP (cGAMP) synthase (cGAS) in induction of IFN-I transcription. While other and our previous studies have shown that IFI16 enhanced retinoic acid-inducible gene I (RIG-I)-, which was an RNA sensor, and mitochondrial antiviral signaling (MAVS)-, which was the adaptor protein of RIG-I, induced production of IFN-I, so we wonder whether IFI16 regulates the signal pathway of RNA-RIG-I-MAVS-IFN-I in a STING-dependent manner. We used HEK 293T cells, which did not express endogenous STING and were unable to mount an innate immune response upon DNA transfection and found that IFI16 could enhance RIG-I- and MAVS-mediated induction of IFN-I in a STING-independent way. Furthermore, we found that upregulation of the expression of NF-kappa-B essential modulator (NEMO) by IFI16 was not the mechanism that IFI16 regulated the induction of IFN-I. In conclusion, we found that IFI16 regulated the signal pathway of RNA-RIG-I-MAVS-IFN-I in a STING-independent manner.

Critical bone defects complicate tissue graft-based surgeries, raising healthcare expenditures and underscoring scaffold-based tissue-engineering strategies to support bone reconstruction. Our study highlighted that the phase-compatible combination of inorganic nanorods, nanofibers, and hydrogels is promising for developing biomimetic and cell-instructive scaffolds since the bone matrix is a porous organic/inorganic composite. In brief, methacrylated gelatin (GelMA) was reacted with dopamine to form catechol-modified GeLMA (GelMA-C). The GelMA-C was nanocoated onto an iron-doped hydroxyapatite (FeHAp) nanorod via metal-catechol network coordination. The modified nanorod (FeHAp@GelMA-C) was loaded onto GelMA-based nanofibers. The nanorods loaded pre-fibers were electrospun onto GelMA solution and photochemically crosslinked to fabricate a fiber-reinforced hydrogel. The structural, mechanical, physicochemical, biocompatibility, swelling properties, osteogenic potential, and bone remodelling potential (using rat femoral defect model) of modified nanorods, simple hydrogel, and nanorod-loaded fiber-reinforced hydrogel were studied. The results supported that the interface interaction between GelMA-C/nanorods, nanorods/nanofibers, nanorods/hydrogels, and nanofiber/hydrogels significantly improved the microstructural and mechanical properties of the scaffold. Compared to pristine hydrogel, the nanorod-loaded fiber-reinforced scaffold better supported cellular responses, osteogenic differentiation, matrix mineralization, and accelerated bone regeneration. The nanorod-loaded fiber-reinforced hydrogel proved more biomimetic and cell-instructive for guided bone reconstruction.

Summary Pestiviruses, including classical swine fever virus, remain a concern for global animal health and are responsible for major economic losses of livestock worldwide. Despite high levels of vaccination, currently available commercial vaccines are limited by safety concerns, moderate efficacy, and required high doses. The development of new vaccines is therefore essential. Vaccine efforts should focus on optimizing antigen presentation to enhance immune responses. Here, we describe a simple herringbone‐dimer strategy for efficient vaccine design, using the classical swine fever virus E2 expressed in a rice endosperm as an example. The expression of rE2 protein was identified, with the rE2 antigen accumulating to 480 mg/kg. Immunological assays in mice, rabbits, and pigs showed high antigenicity of rE2. Two immunizations with 284 ng of the rE2 vaccine or one shot with 5.12 μg provided effective protection in pigs without interference from pre‐existing antibodies. Crystal structure and small‐angle X‐ray scattering results confirmed the stable herringbone dimeric conformation, which had two fully exposed duplex receptor binding domains. Our results demonstrated that rice endosperm is a promising platform for precise vaccine design, and this strategy can be universally applied to other Flaviviridae virus vaccines.

Newcastle disease (ND) is an acute septicemic infectious disease caused by Newcastle disease virus (NDV). Considering that vaccination is currently the main modality for the prevention of ND, it is essential to assess the effectiveness of clinical immunization. In this study, we have developed a blocking lateral flow assay (bLFA) strip for the rapid detection of NDV antibodies using the monoclonal antibody 9C1 against haemagglutinin-neuraminidase (HN), which allows for the determination of an NDV-specific antibody titer within 10 min at room temperature. In addition, the bLFA strip has no cross-reactivity with the positive serum of other avian pathogens including avian influenza subtypes H5, H7, and H9, MD, IBD, IB, EDS, and avian adenovirus. The ability of the bLFA strip for detecting a neutralizing antibody was also estimated. The results showed that the chicken NDV hyperimmunized serum had a complete blocking (100%) titer of 11 log 2, and half-blocking titer of 13 log 2, which are 4 times less than and the same as that of the HI test (13 log 2), and 8 and 2 times less than that of the VN test (14 log 2), respectively. A total of 510 clinical samples were tested for NDV antibodies. The coincidence rate between the results of the bLFA strip and HI test was 97.65%. Therefore, it is an ideal alternative method for assessing the clinical immunity of ND vaccines in the field in real-time.

Diabetes wounds take longer to heal due to extended inflammation, decreased angiogenesis, bacterial infection, and oxidative stress. These factors underscore the need for biocompatible and multifunctional dressings with appropriate physicochemical and swelling properties to accelerate wound healing. Herein, insulin (Ins)-loaded, and silver (Ag) coated mesoporous polydopamine (mPD) nanoparticles were synthesized (Ag@Ins-mPD). The nanoparticles were dispersed into polycaprolactone/methacrylated hyaluronate aldehyde dispersion, electrospun to form nanofibers, and then photochemically crosslinked to form a fibrous hydrogel. The nanoparticle, fibrous hydrogel, and nanoparticle-reinforced fibrous hydrogel were characterized for their morphological, mechanical, physicochemical, swelling, drug-release, antibacterial, antioxidant, and cytocompatibility properties. The diabetic wound reconstruction potential of nanoparticle-reinforced fibrous hydrogel was studied using BALB/c mice. The results indicated that Ins-mPD acted as a reductant to synthesize Ag nanoparticles on their surface, held antibacterial and antioxidant potential, and their mesoporous properties are crucial for insulin loading and sustained release. The nanoparticle-reinforced scaffolds were uniform in architecture, porous, mechanically stable, showed good swelling, and possessed superior antibacterial, and cell-responsive properties. Furthermore, the designed fibrous hydrogel scaffold demonstrated good angiogenic, anti-inflammatory, increased collagen deposition, and faster wound repair capabilities, therefore, it could be used as a potential candidate for diabetic wound treatment.

Porphyrin-based metal-organic frameworks (PMOFs) are a kind of crystal hybrid material with broad application prospects in energy, catalysis, biomedicine, and other fields. In this study, the La-TCPP PMOF nanocrystal was constructed using a porphyrin ligand and La ion. This material can produce a high loading rate on doxorubicin (DOX) owing to its special porous structure. The high loading rate of drug molecules and the reactive oxygen species (ROS) of the porphyrin ligand enable La-TCPP@DOX nanocrystal to produce a powerful killing effect on cancer cells under the synergistic attack of chemotherapy (CT) and photodynamic therapy (PDT). Finally, by modifying the targeted aptamer, the actual therapeutic effect of this special La-TCPP@DOX@Apt material on tumors was confirmed by applying the established mouse tumor model. The composite nanomaterial not only avoids the side effects caused by high concentrations of chemotherapeutic drugs, but also overcomes the limitation of PDT owing to insufficient light penetration and can inhibit and kill solid tumors under the condition of synergistic attack. This study is a complement to PMOF crystal materials, and its tumor-killing ability was achieved by loading drugs and introducing targeting molecules, which proves that the synergistic attack can more effectively inhibit and treat solid tumors. These studies have a reference and guiding significance for the treatment of cancer patients.

Sonodynamic therapy (SDT) has attracted intensive attention, but is still hindered by low sonosensitization and non-biodegradability of the traditional sonosensitizers. Herein, perovskite-type manganese vanadate (MnVO3 ) sonosensitizers integrating high reactive oxide species (ROS) production efficiency and appropriate bio-degradability are developed for enhanced SDT. Taking advantage of the intrinsic properties of perovskites such as narrow bandgap and substantial oxygen vacancies, MnVO3 shows a facile ultrasound (US)-triggered electrons-holes separation and restrained recombination, thus enhancing the ROS quantum yield in SDT. Furthermore, MnVO3 exhibits a considerable chemodynamic therapy (CDT) effect under the acidic condition probably owing to the presence of manganese and vanadium ions. Due to the presence of high-valent vanadium, MnVO3 can also eliminate glutathione (GSH) within the tumor microenvironment, which synergistically amplifies the efficacy of SDT and CDT. Importantly, the perovskite structure bestows MnVO3 with superior biodegradability, which alleviates the long-term presence of residues in metabolic organs after therapeutic actions. Based on these characteristics, US-assisted MnVO3 achieves an excellent antitumor outcome along with low systemic toxicity. Overall, perovskite-type MnVO3 may be promising sonosensitizers for highly efficient and safe treatment of cancer. The work attempts to explore the potential utility of perovskites in the design of degradable sonosensitizers.

Wound healing is a multifaceted process that involves hemostasis, inflammation, proliferation, and remodeling stages. Diabetic wounds affect the transition of the organized phases and result in delayed healing due to impaired angiogenesis, chronic inflammation, bacterial infection, and insufficient growth factors. Multifunctional heterostructural nanoparticles enriched minimally invasive hydrogels for on-demand procedural distribution to aid wound healing at various stages has become a promising strategy. Herein, silk fibroin-hyaluronic acid based injectable hydrogels incorporated with mace-like Au-CuS heterostructural nanoparticles (gAu-CuS HSs) were used to cure diabetic wounds. SF-HA and the rough surface of gAu-CuS HSs confer a synergistic hemostatic phase with a nano-bridge effect and rapidly close the wounds. During the inflammation stage, gAu-CuS HSs perform in-space resonance energy transfer under 808 nm laser irradiation which in return produces reactive oxygen species for bacterial destruction. The unusual mace-like rough structure of nanoparticles causes macrophage transfer to the M2 phenotype, regulates cytokine expression (interleukin 6, transforming factor-β1, interferon γ, and interleukin-10), promotes angiogenesis, and promotes cell multiplication and fibroblast emigration to the wound area during the proliferation and remodeling phase. Overall, the gAu-CuS HSs reinforced injectable hydrogel programmatically accelerates wound healing and could represent a versatile strategy for advanced diabetic wound healing.

Protein tyrosine phosphatase non-receptor type 14 (PTPN14) is a member of the protein tyrosine phosphatase (PTP) family which is a potential tumor suppressor. PTPs modulate the cellular level of tyrosine phosphorylation under normal and pathological conditions. Porcine epidemic diarrhea virus (PEDV) is one of the most important pathogens in the swine industry. Our previous membrane proteomics results showed that PTPN14 was markedly upregulated in PEDV-infected Vero cells. However, its biological roles in PEDV infection have not yet been investigated. In this study, we reported PTPN14 functions as a novel regulator of signal transducer and activator of transcription 3 (STAT3) phosphorylation during PEDV infection. Firstly, PTPN14 was markedly upregulated in PEDV-infected Vero cells with the decrease of STAT3 phosphorylation. Knockdown of PTPN14 or phosphatase inhibitor treatment promoted PEDV proliferation and increased the phosphorylation of STAT3 in Vero cells. On the contrary, overexpression of PTPN14 inhibits viral infection in Vero cells. Moreover, dephosphorylation of STAT3 by PTPN14 might occur in the cytoplasm but not in nucleus. Collectively, our results indicate that PTPN14 plays a negative role in regulating STAT3 activation in PEDV infected Vero cells and demonstrate another layer of regulation in PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. PEDV could remodel host membrane structures for their replication, assembly and escape from host cells. However, little is known about the host membrane proteins of PEDV infection. In this study, we analyzed differentially abundant proteins (DAPs) between PEDV infection group and control group and identified the polarity protein PARD3 as one of the most significantly DAPs. PARD3 is implicated in the formation of tight junctions at epithelial cell-cell contacts. Then, we found that PEDV infection promoted the degradation of PARD3 via the ubiquitin proteasome pathway. Moreover, knockdown of PARD3 promoted the proliferation of PEDV. Further study showed that the downregulation of PARD3 altered the normal morphology of the tight junction proteins and promoted apical and basolateral virus proliferation. Tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection and PEDV affected the barrier functions by causing degradation of PARD3. Taken together, this work is the first time to investigate the membrane protein profile of PEDV-infected cells using quantitative proteomics and suggests that PARD3 could be a potential novel antiviral protein against PEDV infection. SIGNIFICANCE: Membrane proteins are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are changed during in response to environmental stress. However, membrane proteins are difficult to study because of their hydrophobicity. Membrane proteomic methods using mass spectrometry analysis have been developed and applied for the characterization of the plasma membrane and subcellular organelles of various virus infected cells. Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen of importance to the swine industry, causing high mortality in neonatal piglets. Because PEDV infected Vero cells can lead to significant changes in cell membrane morphology and form syncytial lesions. Here, we isolated the membrane proteins of PEDV infected and control cells and applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially abundant proteins (DAPs) in PEDV-infected Vero cells and confirmed the DAPs by performing RT-qPCR and Western blot analysis. Among these differential proteins, we focused on a down-regulated protein PARD3 which is important for cell tight junction and cell polarity. Loss of PARD3 can destroy the tight junction of cells and promote the proliferation of PEDV in the apical and basolateral sides. These findings will provide valuable information to better understand the mechanisms underlying the host defense responses to PEDV infection.

Newcastle disease (ND) is an avian respiratory disease caused by the Newcastle disease virus (NDV) and is prevalent worldwide but lacks a reliable treatment to surmount it. The single-chain fragment variable (scFv) antibody is the smallest functional unit in an immunoglobulin retaining antigen-binding activities. Previous studies have demonstrated that by acting against the NDV phosphoprotein, scFv is capable of neutralizing the virus in infected cells. In this study, two anti-NDV scFv expressing cassettes driven by CAG promoter and EF1α promoter, respectively, were constructed using the piggyBac transposon plasmid backbone. The recombinant plasmids were transfected into DF-1 and 293T cells and four stable cell lines CAG-scFv-DF-1, EF1α-scFv-DF-1, CAG-scFv-293T and EF1α-scFv-293T were established after puromycin screening. RT-PCR results confirmed the presence of anti-NDV scFv mRNA in all four cell lines. Western blot results showed that scFv was detected in the culture supernatant of the CAG-scFv-DF-1 and EF1α-scFv-DF-1 lines, indicating that the scFv antibody was secretory after expression in the cells. Cytopathic effect assay showed that cells expressing scFv were more resistant to NDV F48E9 than those of control when the virus titer was not higher than 60×TCID50. Results in this study offer information to the generation of transgenic chickens resistant to NDV infection and for production of neutralizing antibodies against NDV.

Vaccination is an effective method to control the spread of classical swine fever virus (CSFV), which is a major cause of economic losses to the swine industry. Although serological detection assays are commonly used to assess immune status, current methods for monitoring of antibodies (Abs) are time-consuming, expensive, and require cell culture and virus manipulation. To address these problems, the E2 protein of CSFV was expressed in transgenic rice seeds as a labeled antigen for the development of an immunochromatographic test strip (ICTS) for rapid, precise, and cost-effective detection of Abs. The ICTS has a reasonable sensitivity of 1:128,000 for detection of serum Abs against CSFV and no cross-reactivity with Abs of other porcine viruses. The similarity of the results between the proposed ICTS and a commercial enzyme-linked immunosorbent assay was 94.1% (128/136) for detection of serum Abs from immunized animals and 92.3% (72/78) for detection of maternally derived Abs. The proposed assay was successfully used to monitor Abs against E2 of both pigs and rabbits immunized with a live attenuated vaccine or an E2 subunit vaccine. The results confirmed that the ICTS can be applied to detect Ab levels in animals with different immunological backgrounds. The ICTS based on plant-derived E2 is a relatively inexpensive, rapid, and accurate assay for detection of Abs against CSFV and avoids the risk of contamination by animal products. IMPORTANCE The E2 protein of classical swine fever virus (CSFV) was expressed in transgenic rice endosperms as a diagnostic antigen for use with a rapid colloidal gold assay for the detection of antibodies (Abs) against CSFV. This improved test was used to monitor Abs against the E2 protein in both pigs and rabbits immunized with a live attenuated vaccine or E2 subunit vaccine. The assay successfully detected Ab levels in serum samples from piglets with different immunological backgrounds. In contrast to current E2 protein-based diagnostic methods using Escherichia coli or insect cells as expression systems, plant-derived E2 avoids the limitations of low immunogenicity of eukaryotic expression systems and potential contamination of fetal bovine serum with bovine viral diarrhea virus in cell culture.

Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. As a notifiable terrestrial and aquatic animal disease listed by World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]), classical swine fever (CSF) has caused great economic losses to the swine industry worldwide during recent decades. Differentiation of infected and vaccinated animals (DIVA) is urgent for eradication of CSF. In this study, a diagnostic platform based on CRISPR/Cas13a was established with the ability to differentiate between classical swine fever virus (CSFV) virulent and vaccine strains. In combination with reverse transcription recombinase-aided amplification (RT-RAA), the detection limit for CSFV synthetic RNA templates reached 3.0 x 10(2) copies/mu L. In addition, with boiling and chemical reduction, heating unextracted diagnostic samples to obliterate nucleases (HUDSON) treatment was introduced to inactivate nucleases and release viral genome, achieving robust pretreatment of tested sample before CRISPR/Cas13a detection without the need to extract viral nucleic acids. HUDSON-RT-RAA-CRISPR/Cas13a can directly detect cell cultures of virulent Shimen strain and vaccine hog cholera lapinized virus (HCLV) strain, with the detection limit of 3.5 x 10(2) copies/mu L and 1.8 x 10(2) copies/mu L, respectively, which was equally sensitive to nested PCR (nPCR) and 100 times more sensitive than antigen enzyme-linked immunosorbent assay (ELISA). Meanwhile, HUDSON-RT-RAA-CRISPR/Cas13a showed no cross-reactivity with bovine viral diarrhea virus (BVDV), atypical porcine pestivirus (APPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2), exhibiting good specificity. At last, a total of 50 pig spleen samples with suspected clinical signs were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a, nPCR, and antigen ELISA in parallel. HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0% with nPCR and 82.0% coincident rate with antigen ELISA, respectively. IMPORTANCE Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. Methods with the ability to precisely differentiate classical swine fever virus (CSFV) virulent strains from vaccine strain hog cholera lapinized virus (HCLV) are urgently needed. Combining the high sensitivity of isothermal recombinase-aided amplification (RAA) with the accurate molecular sensing ability of Cas13a, we presented a novel method for CSFV detection without the need to extract viral nucleic acids, which showed great advantage to traditional detection methods for precise differentiation of CSFV virulent strains and vaccine strain, providing a novel powerful tool for CSF eradication.

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever.

SOCS3 belongs to the suppressor of cytokine signaling (SOCS) family, which function as negative factors in host immune responses. Prior studies have noted the importance of SOCS family proteins in immunosuppression induced by some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important swine-borne viruses and has threatened the global swine industry with huge economic losses since it was first described in the 1980s. PRRSV is the etiological agent of PRRS, which causes reproductive failure and respiratory disorders. PRRSV causes immunosuppression thus establishing persistent infection. In this study, it was observed that SOCS3 was upregulated in PRRSV-infected primary porcine alveolar macrophages (PAMs) and Marc-145 cells with dose-dependent effects, which depends on virus replication. Deletion of AP-1 binding motif located in SOCS3 promoter inhibited promoter activities, which indicates that AP-1 is essential for PRRSV-induced SOCS3. This result was confirmed by experiments using AP-1 inhibitor, whose pretreatment suppressed SOCS3 mRNA and protein expression. Further research showed that p38 was crucial for PRRSV-induced SOCS3 production. Importantly, SOCS3 enhanced PRRSV replication during infection. Taken together, this study indicates that PRRSV infection induced SOCS3 expression through p38/AP-1 signaling pathway. These results revealed the molecular basis of SOCS3 upregulation and would advance further understanding of the strategy for viral immune evasion.