Protective effects of the secondary metabolites from Quercus salicina Blume against gentamicin-induced nephrotoxicity in zebrafish (Danio rerio) model.

To reveal the protective effect on the nephrotoxicity of Quercus salicina Blume(QS), a traditional medicine for the treatment of urolithiasis, the 50 % ethanol extract from the branches and leaves of QS was chemically studied by systematic solvent extraction and HPLC chromatography. Two phenolic acids and three flavonoids were identified by nuclear magnetic resonance spectroscopy, namely Ferulic acid (1), p-Hydroxycinnamic acid (2), Hesperidin (3), Formononetin (4), and Quercetin (5). At the same time, the gentamicin-induced nephrotoxicity of zebrafish was used as a model for the first time. The antioxidant activity of these derivatives with good antioxidant activity screened from free radical scavenging experiments in vitro (DPPH and ABTS) was evaluated in vivo, including protein levels (LPO, NO, GSH, and SOD), kidney injury factor (KIM-1), zebrafish kidney pathology and real-time PCR. The results showed that metabolites 1, 3, and 5 had strong antioxidant activity, and oxidative stress in renal tissue was significantly reduced; KIM-1, TNF-α, and IL-6 mRNA expression in a dose-dependent manner, which preliminarily revealed the protective effect of the secondary metabolites of QS on nephrotoxicity, and preliminarily discussed the structure-activity relationship. This study provides an experimental basis for further exploring the mechanism of QS in the kidney.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs.

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

Improvement of functional dyspepsia with Suaeda salsa (L.) Pall via regulating brain-gut peptide and gut microbiota structure.

PURPOSE: The traditional Chinese herbal medicine Suaeda salsa (L.) Pall (S. salsa) with a digesting food effect was taken as the research object, and its chemical composition and action mechanism were explored. METHODS: The chemical constituents of S. salsa were isolated and purified by column chromatography, and their structures were characterized by nuclear magnetic resonance. The food accumulation model in mice was established, and the changes of the aqueous extract of S. salsa in gastric emptying and intestinal propulsion rate, colonic tissue lesions, serum brain-gut peptide hormone, colonic tissue protein expression, and gut microbiota structure were compared. RESULTS: Ten compounds were isolated from S. salsa named as naringenin (1), hesperetin (2), baicalein (3), luteolin (4), isorhamnetin (5), taxifolin (6), isorhamnetin-3-O-ß-D-glucoside (7), luteolin-3'-D-glucuronide (8), luteolin-7-O-ß-D-glucuronide (9), and quercetin-3-O-ß-D-glucuronide (10), respectively. The aqueous extract of S. salsa can improve the pathological changes of the mice colon and intestinal peristalsis by increasing the rate of gastric emptying and intestinal propulsion. By adjusting the levels of 5-HT, CCK, NT, SS, VIP, GT-17, CHE, MTL, and ghrelin, it can upregulate the levels of c-kit, SCF, and GHRL protein, and restore the imbalanced structure of gut microbiota, further achieve the purpose of treating the syndrome of indigestion. The effect is better with the increase of dose. CONCLUSION: S. salsa has a certain therapeutic effect on mice with the syndrome of indigestion. From the perspective of "brain-gut-gut microbiota", the mechanism of digestion and accumulation of S. salsa was discussed for the first time, which provided an experimental basis for further exploring the material basis of S. salsa.

Spike 1 trimer, a nanoparticle vaccine against porcine epidemic diarrhea virus induces protective immunity challenge in piglets.

Porcine epidemic diarrhea virus (PEDV) is considered the cause for porcine epidemic diarrhea (PED) outbreaks and hefty losses in pig farming. However, no effective commercial vaccines against PEDV mutant strains are available nowadays. Here, we constructed three native-like trimeric candidate nanovaccines, i.e., spike 1 trimer (S1-Trimer), collagenase equivalent domain trimer (COE-Trimer), and receptor-binding domain trimer (RBD-Trimer) for PEDV based on Trimer-Tag technology. And evaluated its physical properties and immune efficacy. The result showed that the candidate nanovaccines were safe for mice and pregnant sows, and no animal death or miscarriage occurred in our study. S1-Trimer showed stable physical properties, high cell uptake rate and receptor affinity. In the mouse, sow and piglet models, immunization of S1-Trimer induced high-level of humoral immunity containing PEDV-specific IgG and IgA. S1-Trimer-driven mucosal IgA responses and systemic IgG responses exhibited high titers of virus neutralizing antibodies (NAbs) in vitro. S1-Trimer induced Th1-biased cellular immune responses in mice. Moreover, the piglets from the S1-Trimer and inactivated vaccine groups displayed significantly fewer microscopic lesions in the intestinal tissue, with only one and two piglets showing mild diarrhea. The viral load in feces and intestines from the S1-Trimer and inactivated vaccine groups were significantly lower than those of the PBS group. For the first time, our data demonstrated the protective efficacy of Trimer-Tag-based nanovaccines used for PEDV. The S1-Trimer developed in this study was a competitive vaccine candidate, and Trimer-Tag may be an important platform for the rapid production of safe and effective subunit vaccines in the future.

Enhanced antigen-specific CD8 T cells contribute to early protection against FMDV through swine DC vaccination.

Foot-and-mouth disease virus (FMDV) remains a challenge for cloven-hooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines.IMPORTANCEAlthough the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines.

A novel recombination porcine epidemic diarrhea virus isolated from Gansu, China: Genetic characterization and pathogenicity.

Porcine epidemic diarrhea virus (PEDV) is an acute and highly contagious porcine enteric coronavirus. It has caused serious economic losses of pig industry in China. Here we insolated a current PEDV field strain named GS2022, analyzed the characters of genetic variation and pathogenicity. The results demonstrated that the GS2022 strain was belong to a newly defined subgroup G2 d, forming an independent branch which mainly contains strains isolated in China from 2017 to 2023. Notably, there are multiple mutations and extensive N-glycosylation compared to CV777 strain and PT-P5 strain, therefore the structure of GS2022 strain is different from 6U7K and 7W6M. Animal pathogenicity test showed that GS2022 strain could cause severe clinical signs and the high level of virus shedding in 7-day-old piglets. But recovery of diarrhea after 5 days, and no pathological damage to important organs. Further study on 3-day-old piglets also indicated GS2022 strain have pathogenicity. In this study no piglets died, which make it possible for that GS2022 strain become a candidate vaccine. These results are helpful to understand the epidemiology, molecular characteristics, evolution, and antigenicity of PEDV circulating in China. It also provides reference for designing effective vaccines against PEDV.

Self-Assembling E2-Based Nanoparticles Improve Vaccine Thermostability and Protective Immunity against CSFV.

Classical swine fever virus (CSFV) is a highly contagious pathogen causing significant economic losses in the swine industry. Conventional inactivated or attenuated live vaccines for classical swine fever (CSF) are effective but face biosafety concerns and cannot distinguish vaccinated animals from those infected with the field virus, complicating CSF eradication efforts. It is noteworthy that nanoparticle (NP)-based vaccines resemble natural viruses in size and antigen structure, and offer an alternative tool to circumvent these limitations. In this study, we developed an innovative vaccine delivery scaffold utilizing self-assembled mi3 NPs, which form stable structures carrying the CSFV E2 glycoprotein. The expressed yeast E2-fused protein (E2-mi3 NPs) exhibited robust thermostability (25 to 70 °C) and long-term storage stability at room temperature (25 °C). Interestingly, E2-mi3 NPs made with this technology elicited enhanced antigen uptake by RAW264.7 cells. In a rabbit model, the E2-mi3 NP vaccine against CSFV markedly increased CSFV-specific neutralizing antibody titers. Importantly, it conferred complete protection in rabbits challenged with the C-strain of CSFV. Furthermore, we also found that the E2-mi3 NP vaccines triggered stronger cellular (T-lymphocyte proliferation, CD8+ T-lymphocytes, IFN-γ, IL-2, and IL-12p70) and humoral (CSFV-specific neutralizing antibodies, CD4+ T-lymphocytes, and IL-4) immune responses in pigs than the E2 vaccines. To sum up, these structure-based, self-assembled mi3 NPs provide valuable insights for novel antiviral strategies against the constantly infectious agents.

Self-Assembling Nanovaccine Fused with Flagellin Enhances Protective Effect against Foot-and-Mouth Disease Virus.

Nanovaccines based on self-assembling nanoparticles (NPs) can show conformational epitopes of antigens and they have high immunogenicity. In addition, flagellin, as a biological immune enhancer, can be fused with an antigen to considerably enhance the immune effect of antigens. In improving the immunogenicity and stability of a foot-and-mouth disease virus (FMDV) antigen, novel FMDV NP antigens were prepared by covalently coupling the VP1 protein and truncated flagellin containing only N-terminus D0 and D1 (N-terminal aa 1-99, nFLiC) with self-assembling NPs (i301). The results showed that the fusion proteins VP1-i301 and VP1-i301-nFLiC can assemble into NPs with high thermal tolerance and stability, obtain high cell uptake efficiency, and upregulate marker molecules and immune-stimulating cytokines in vitro. In addition, compared with monomeric VP1 antigen, high-level cytokines were stimulated with VP1-i301 and VP1-i301-nFLiC nanovaccines in guinea pigs, to provide clinical protection against viral infection comparable to an inactivated vaccine. This study provides new insight for the development of a novel FMD vaccine.

Local and systemic immune responses induced by intranasal immunization with biomineralized foot-and-mouth disease virus-like particles.

Introduction: Foot-and-mouth disease virus (FMDV) infects the host by invading mucosal epithelial cells of the respiratory or digestive tract. Therefore, establishing a specific antiviral mucosal immune barrier can effectively block viral invasion. Methods: We evaluated local mucosal and systemic immune responses elicited by intranasal immunization of mice with foot-and-mouth disease (FMD) calcium phosphate mineralized virus-like particles (CaP-VLPs) and tested whether three commercial mucosal adjuvants enhanced the immunogenicity of the antigen. The biosafety of the vaccine was verified through gross observation and pathological analysis of the lungs. Results: CaP-VLPs effectively induced secretion of IgA (sIgA) from multiple sites in mouse mucosa and produced anti-FMD-specific IgG in the serum. Splenic lymphocytes specifically proliferated and secreted IFN-γ following antigen stimulation, indicating the vaccine can induce a certain level of cellular immune response. Finally, the pathological examination confirmed that CaP-VLPs did not cause substantial damage to the lungs of animals after immunization via mucosal administration. Notably, the vaccine mixed with S adjuvant increased the content of sIgA and serum IgG, and the high level of IgG in serum was maintained at least 7 weeks. Discussion: Overall, this study reveals that FMD CaP-VLPs can induce good local mucosal immune and systemic immune response through intranasal immunization, and the immune response was specifically enhanced by S adjuvant. These data support that CaP-VLPs-S as a candidate mucosal vaccine for the prevention of FMD vaccine infection.

Establishment and application of a solid-phase blocking ELISA method for detection of antibodies against classical swine fever virus.

BACKGROUND: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. OBJECTIVES: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. METHODS: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. RESULTS: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. CONCLUSIONS: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

In Vitro Analysis of TGF-ß Signaling Modulation of Porcine Alveolar Macrophages in Porcine Circovirus Type 2b Infection.

Porcine circovirus 2 (PCV2) has been recognized as an immunosuppressive pathogen. However, the crosstalk between this virus and its host cells in related signaling pathways remains poorly understood. In this study, the expression profiles of 84 genes involved in transforming growth factor-beta (TGF-ß) signaling pathway were probed in PCV2b-infected primary porcine alveolar macrophages (PAMs) by using an RT2 profiler PCR array system. The protein expression levels of cytokines involved in the TGF-ß signaling pathway were determined with a RayBiotech fluorescent Quantibody® porcine cytokine array system. Results showed that 48, 30, and 42 genes were differentially expressed at 1, 24, and 48 h after infection, respectively. A large number of genes analyzed by a co-expression network and implicated in transcriptional regulation and apoptosis were differentially expressed in PCV2b-infected PAMs. Among these genes, TGF-ß, interleukin-10, CCAAT/enhancer-binding protein beta (C/EBPB), growth arrest, and DNA-damage-inducible 45 beta (GADD45B), and BCL2 were upregulated. By contrast, SMAD family member 1 (smad1) and smad3 were downregulated. These results suggested that the TGF-ß signaling pathway was repressed in PAMs at the early onset of PCV2 infection. The inhibited apoptosis was indicated by the upregulated C/EBPB, GADD45B, and BCL2, and by the downregulated smad1 and smad3, which possibly increased the duration of PCV2 replication-permissive conditions and caused a persistent infection. Our study may provide insights into the underlying antiviral functional changes in the immune system of PCV2-infected pigs.

Quercus salicina Blume: Research Progress in Chemistry and Pharmacodynamics (1959-2021).

The crude extracts of different parts (leaves and shoots) of Quercus salicina Blume (QS) have shown considerable effect in urolithiasis. QS has been widely used in clinical practice and has attracted great research interest The relevant published literature, however, reveals only partial education of its chemical components and bio-active mechanisms, and only two review articles have summarized the QS research progress. In this review, a comprehensive and systematic review of chemistry and pharmacodynamics of QS was carried out using the international authoritative databases (1959-2021), focusing on phenols and flavonoids, and their effect such as urinary stone dissolution, anti-inflammatory, anti-diabetes, anti-bacterial, antioxidant, and anti-allergy activities as well as toxic effects. The aim of review is to provide the most recent and effective literature support for further basic research and application development.

Identification of a new cell-penetrating peptide derived from the african swine fever virus CD2v protein.

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP]3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery.

Potent Protective Immune Responses to Senecavirus Induced by Virus-Like Particle Vaccine in Pigs.

Senecavirus A (SVA) is the pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical symptoms of PIVD are similar to those of acute foot-and-mouth disease and also can result in the death of newborn piglets, thus entailing economic losses. Vaccine immunization is the most effective way to prevent and control SVA. Among all SVA vaccines reported, only the SVA inactivated vaccine has been successfully developed. However, to ensure the elimination of this pathogen, safer and more effective vaccines are urgently required. A virus-like particles (VLPs)-based vaccine is probably the best alternative to inactivated vaccine. To develop an SVA VLPs vaccine and evaluate its immune effect, a prokaryotic expression system was used to produce SVA capsid protein and assemble VLPs. The VLPs were characterized by affinity chromatography, sucrose density gradient centrifugation, ZetaSizer and transmission electron microscopy. Meanwhile, the SVA CH-HB-2017 strain was used to infect pigs and to determine infection routes and dose. Experimental pigs were then immunized with the SVA VLPs vaccine emulsified in an ISA 201 adjuvant. The results showed that the VLPs vaccine induced neutralizing and specific antibodies at similar levels as an inactivated SVA vaccine after immunization. The level of INF-γ induced by the VLPs vaccine gradually decreased-similar to that of inactivated vaccine. These results indicated that VLPs vaccine may simultaneously cause both cellular and humoral immune responses. Importantly, after the challenge, the VLPs vaccine provided similar levels of protection as the inactivated SVA vaccine. In this study, we successfully obtained novel SVA VLPs and confirmed their highly immunogenicity, thus providing a superior candidate vaccine for defense and elimination of SVA, compared to the inactivated vaccine.

Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots.

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10-7 and 1.35 × 10-8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

Porcine Circovirus Type 2 Induces Single Immunoglobulin Interleukin-1 Related Receptor (SIGIRR) Downregulation to Promote Interleukin-1ß Upregulation in Porcine Alveolar Macrophage.

Multisystemic inflammation in pigs affected by porcine circovirus type 2 (PCV2) indicates the disordered expression of inflammatory cytokines. However, the PCV2-induced expression profile of inflammation cytokines and its regulating mechanism remain poorly understood. In this study, inflammatory cytokines and receptors in porcine alveolar macrophages (PAMs) after PCV2 infection were profiled in vitro by an RT2 ProfilerTM PCR array assay. The regulatory mechanism of interleukin-1ß (IL-1ß) expression was investigated. Results showed that 49 of 84 inflammation cytokines and receptors were differentially expressed (p < 0.05, absolute fold change ≥2) in PAMs at different stages post-PCV2 infection. Moreover, the overexpression of single-immunoglobulin interleukin-1 related receptor (SIGIRR) or the blocking of NF-κB activation by its inhibitor markedly decreased IL-1ß secretion. This finding suggested that PCV2-induced overexpression of IL-1ß was associated with the downregulation of SIGIRR and the activation of NF-κB. Furthermore, the excessive activity of NF-κB in SIGIRR-knockout PAMs cell line, indicating that SIGIRR negatively regulated IL-1ß production by inhibiting the activation of NF-κB. Overall, PCV2-induced downregulation of SIGIRR induction of NF-κB activation is a critical process in enhancing IL-1ß production in PAMs. This study may provide insights into the underlying inflammatory response that occurs in pigs following PCV2 infection.

Proteomic analysis of murine bone marrow derived dendritic cells in response to peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV) poses a great threat to livestock husbandry, especially goat farming due to its high mortality and morbidity. Dendritic cells (DCs), as the principal stimulators of naive Th cells were widely used in antigen processing and presenting. In the previous study, we tested the effects of PPRV on murine bone marrow derived dendritic cells (BMDCs) including surface markers and cytokines. While the aim of this study is to detect the proteomic profile of BMDCs stimulated with PPRV towards key proteins involved in. Following PPRV stimulation, 110 differentially expressed proteins (DEPs) were identified through iTRAQ labelling with LC-MS/MS approach, of which 94 DEPs were up-regulated and 16 DEPs were down-regulated, respectively. Among them 15 out of 110 DGPs were related to innate immune system, three were involved in cell apoptosis, RPS15a and Smox were related to translation of viral mRNA. Additionally, western blot analysis showed identical results to iTRAQ analysis. There will be profound significance for understanding antigen-presenting of BMDCs after stimulation with PPRV.

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus.

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.

Specific detection of foot-and-mouth disease serotype Asia 1 virus by carboxyl-magnetic beads conjugated with single-domain antibody.

BACKGROUND: Immunomagnetic nanobead (IMNB) labeled with specific antibody, has been demonstrated to be useful for the capturing and detection of viruses. RESULTS: In this study, we developed an imunomagnetic bead based on carboxyl-magnetic beads (MNB) labeled with a single-domain antibody (sdAb) for capturing foot-and-mouth disease (FMD) Asia 1 virus. After magnetic separation, complexes of MNB-sdAb-virus were detected with either a sandwich ELISA or QDs-C5 probe under a fluorescence microscope, and the complexes were used as templates for extraction of total RNA for amplification of the VP1 or 3D gene fragments using RT-PCR and real-time RT-PCR. The Asia 1 VLPs were efficiently captured through IMNB with a high binding rate of 5.09 µg of antigen/µl of bead suspension. Moreover, this method has been successfully used to capture Asia 1 antigen in synthetic samples. CONCLUSION: Ultimately, a specific and highly sensitive capture FMDV Asia 1 tool has been established that has the potential to enhance the sensitivity and reliability when diagnosing FMDV Asia 1.

Diagnosis of porcine circovirus type 2 infection with a combination of immunomagnetic beads, single-domain antibody, and fluorescent quantum dot probes.

The use of a specific antibody conjugated with nanobeads, forming immunomagnetic nanobeads (IMNBs), has been demonstrated to be useful for the capture and detection of viruses. In this study, IMNBs functionalized with a single-domain antibody against the capsid protein (Cap) of porcine circovirus type 2 (PCV2), hereafter denoted as psdAb, were evaluated and used to capture PCV2. Quantum dots (QDs) conjugated with psdAb were used as a fluorescence probe to visualize PCV2 captured by IMNBs. The specificity and sensitivity of this method were further evaluated using common pathogens of pig viral disease and PCV2. To assess its practicality, clinical samples were tested in this study. The results showed that 2.57 ± 0.13 mg Cap or 0.97 ± 0.064 × 10(6) copies of PCV2 particles could be captured by 1 mg of IMNBs in 30 min. This suggests that the IMNBs have the ability to efficiently capture PCV2 with good specificity, as there was no cross-reaction with other pathogens, and with strong sensitivity, with a detection limit as low as 10(3) copies/ml of PCV2 particles. Moreover, PCV2 in inguinal lymph node, lung, spleen, serum, and fecal samples was successfully detected by IMNBs. The results demonstrate that this method is promising for the rapid and effective detection of PCV2 in complex clinical samples.