Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Histomorphic analysis and expression of mRNA and miRNA in embryonic gonadal differentiation in Chinese soft-shelled turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is extensively cultured in Asia for its nutritional and medical value. Gonadal differentiation is fantastic in turtles, whereas morphologic, mRNA, and miRNA expressions were insufficient in the turtle. In this study, ovaries and testes histomorphology analysis of 14-23 stage embryos were performed, and mRNA and miRNA expression profiles were analyzed. Histomorphology analysis revealed that gonads were undifferentiated at embryonic stage 14. Ovarian morphological differentiation became evident from stage 15, which was characterized by the development of the cortical region and degeneration of the medullary region. Concurrently, testicular morphological differentiation was apparent from stage 15, marked by the development of the medullary region and degeneration of the cortical region. qRT-PCR results showed that Cyp19a1 and Foxl2 exhibited female-specific expression at stage 15 and the expression increased throughout most of the embryonic development. Dmrt1, Amh, and Sox9 displayed male-specific expression at stage 15 and tended to increase substantially at later developmental stages. The expression of miR-8356 and miR-3299 in ZZ gonads were significantly higher than that in ZW gonads at stage 15, 17 and 19, and they had the highest expression at stage 15. While the expression of miR-8085 and miR-7982 had the highest expression at stage 19. Furthermore, chromatin remodeler genes showed differential expression in female and male P. sinensis gonads. These results of master sex-differentiation genes and morphological characteristics would provide a reference for the research of sex differentiation and sex reversal in turtles. Additionally, the expression of chromatin remodeler genes indicated they might be involved in gonadal differentiation of P. sinensis.

Molecular characterization and potential function of Rxrγ in gonadal differentiation of Chinese soft-shelled turtle (Pelodiscus sinensis).

Retinoid X receptor (RXR) is a member of the ligand-dependent nuclear receptor family. Previous studies revealed that RXRs are involved in reproduction in vertebrates. However, information on the function of RXRs in turtles is scarce. In this study, the Rxrγ cDNA sequence of Pelodiscus sinensis was cloned and analyzed, and a polyclonal antibody was constructed. RXRγ protein showed a positive signal in both mature and differentiated gonads of the turtle. Subsequently, the function of the Rxrγ gene in gonadal differentiation was confirmed using short interfering RNA (RNAi). The full-length cDNA sequence of the Rxrγ gene in P. sinensis was 2152 bp, encoding 407 amino acids and containing typical nuclear receptor family domains, including the DNA-binding domain (DBD), ligand-binding domain (LBD), and activation function 1 (AF1). Moreover, gonadal Ps-Rxrγ showed sexual dimorphism expression patterns in differentiated gonads. Real-time quantitative PCR results revealed that the Rxrγ gene was highly expressed in the turtle ovary. RNAi treatment increased the number of Sertoli cells in ZZ embryonic gonads. Furthermore, RNA interference upregulated Dmrt1 and Sox9 in ZZ and ZW embryonic gonads. However, Foxl2, Cyp19a1, Stra8, and Cyp26b1 were downregulated in embryonic gonads. The results indicated that Rxrγ participated in gonadal differentiation and development in P. sinensis.

Prokaryotic expression of the V protein of the peste des petits ruminants virus and development of an indirect ELISA.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

Effect of Different Phosphates on Pyrolysis Temperature-Dependent Carbon Sequestration and Phosphorus Release Performance in Biochar.

Carbon sequestration is the primary function of biochar. Hence, it is necessary to design biochar with high carbon (C) retention and low C loss. In this study, three P compounds, including KH2PO4, Ca(H2PO4)2, and NH4H2PO4, were premixed with corn stalk (1:4, w/w), aiming to produce biochars (CSB+K, CSB+Ca, and CSB+N) with high C sequestration and slow release of P at three temperatures (300, 500, and 700 °C). The addition of all P sources obviously increased C retention, with the order of NH4H2PO4 (65.6-83.5%) > Ca(H2PO4)2 (60.4-78.2%) > KH2PO4 (50.1-76.1%), compared with the pristine biochar (47.8-73.6%). The addition of Ca(H2PO4)2 and KH2PO4 led to an increase in aromaticity and graphitization, as evidenced by H/C, FTIR, Raman and XPS analysis, whereas an opposite result occurred on CSB+N. Furthermore, all three phosphates reduced C loss of biochars with H2O2 oxidation, and CSB+Ca showed the best effect. Ca(H2PO4)2 and KH2PO4 pretreated biochars had higher resistance to K2Cr2O7 oxidation and thermal treatment. In contrast, the C loss of NH4H2PO4-added biochar at 500 and 700 °C with K2Cr2O7 oxidation was increased by 54% and 36%, respectively. During the pyrolysis process, Ca(H2PO4)2 was transformed into insoluble Ca2P2O7, leading to the lowest P release rate of CSB+Ca. This study indicates that co-pyrolysis of corn stalk and Ca(H2PO4)2 is optimal for increasing C retention, enhancing C stability and improving slow-release performance of P regardless of pyrolysis temperature.

Molecular cloning and characterization of Sirt1 and its role in the follicle of juvenile Chinese soft-shelled turtle (Pelodiscus sinensis).

Sirt1 is a member of the Sirtuins family that regulates ovarian senescence, follicular development, and oocyte maturation in vertebrates. To understand its role in the ovary of Pelodiscus sinensis, we cloned the full-length cDNA of Ps-Sirt1 and characterized its potential function by intraperitoneally injecting agonist (resveratrol) and antagonist (EX527) in the female juvenile turtle. The full-length cDNA of Ps-Sirt1 was 2106 bp, comprising 203 bp 5'UTR, a 226 bp 3'UTR, and a 1677 bp ORF encoding 558 amino acids. The calculated molecular weight of predicted protein was 63 kDa, and the isoelectric point was 4.65. The predicted protein comprised a conserved Sir2 domain. Amino acid sequence alignment and phylogenetic analyses showed that Ps-Sirt1 was most closely related to turtles, and distantly related to fish. Expression pattern analysis showed Ps-Sirt1 was highest expressed in ovary, followed by testis, liver, heart, and brain. In the ovarian differentiation processes, Sirt1 showed significantly higher expression at embryonic stage 15 and 21. In the testis differentiation process, Sirt1 expression was downregulated at embryonic stages 15-19. Activated and inactivated Sirt1 decreased the number of primordial follicles in juvenile turtles. Bcl2, Bax, mTOR, and rpS6 expressions were up-regulated, whereas GnRH, Fshb, p50, and p65 were down-regulated after agonist treatment. The inaction of Sirt1 with antagonist up-regulated GnRH, Fshb, p65, p53, Foxo3a, Bcl2, Bax, mTOR, and rpS6, but down-regulated p50. In summary, Sirt1 might be involved in the ovarian follicle development of P. sinensis.

Molecular characterization and functional analysis of Esr1 and Esr2 in gonads of Chinese soft-shelled turtle (Pelodiscus sinensis).

Estrogens and their receptors play crucial roles in regulating the gonadal development of vertebrates. To clarify the roles of estrogen receptors in the gonadal development of turtles, estrogen receptors (Esr1 and Esr2) in Chinese soft-shelled turtle (Pelodiscus sinensis) were identified and characterized, and their function in gonads was investigated by intraperitoneal injection of agonist propylpyrazoletriol (PPT) and diarylpropionitrile (DPN), and antagonist ICI 182,780 (ICI). Ps-Esr1 encoded a 588 amino acid protein and Ps-Esr2 encoded a 556 amino acid protein. The two receptors contained classic domains, including the DNA-binding domain and ligand-binding domain, and amino acid sequences showed high homology with other turtles. Ps-Esr1 showed the highest expression in the testis, followed by the ovary and liver. However, Ps-Esr2 showed the highest expression in the ovary, followed by the brain and testis. Ps-Esr1 expression showed an up-regulation trend in gonadal differentiation. Histomorphometric analysis showed that the number of follicles increased in female juvenile turtles treated with DPN or PPT. In addition, Tsc2, GnRH, and Fshß were up-regulated in ovaries of turtles treated with agonists, while Sycp3 and Picalm were up-regulated in testes of turtles treated with agonists. Treatment with the antagonist decreased the number of sperm in matured turtles. Stra8, Scyp3, Dmc1, Picalm, Evl, Boule, and Cdk1 were up-regulated in testis after antagonist treatment. The results indicated that Esr1 might play an important role in gonadal differentiation, and the two estrogen receptors might be involved in the spermatogenesis of the turtle. These results could provide a reference for further research on the function of the estrogen signal in male vertebrates.

Retrospective genomic analysis of the first Lumpy skin disease virus outbreak in China (2019).

Lumpy skin disease caused by Lumpy skin disease virus (LSDV) is a severe systemic disease affecting cattle and other ruminants. Lumpy skin disease was first reported in northwest China in August 2019 and has severely threatened the cattle breeding industry in China. However, there have been limited genomic studies of LSDV from the first outbreak and its subsequent epidemics. This study aims to characterize the comparative genomic evolution of the LSDV strain from the first outbreak in China. The etiological agent was isolated in a Madin-Darby bovine kidney cell culture and subsequently identified by PCR and Sanger sequencing of six selected genes. The genome sequence was determined using Illumina sequencing and analyzed through genome alignment and phylogenetic tree. The results showed that all six genes were successfully amplified and genetically clustered into LSDV. The virus presented the highest homology to strain China/GD01/2020, which shared 100% identities among 150 open reading frames (ORFs), and 97.1-99.7% identities among additional 6 ORFs. Bayesian inference tree analysis revealed that the virus shared a common ancestor with LSDV strains from China and Vietnam. The study provides an additional genomic data for LSDV tracking and control in China and neighboring countries.

Induction of Robust and Specific Humoral and Cellular Immune Responses by Bovine Viral Diarrhea Virus Virus-Like Particles (BVDV-VLPs) Engineered with Baculovirus Expression Vector System.

Bovine viral diarrhea virus (BVDV) is an important animal pathogen that affects cattle. Infections caused by the virus have resulted in substantial economic losses and outbreaks of BVDV are reported globally. Virus-like particles (VLPs) are promising vaccine technology largely due to their safety and strong ability to elicit robust immune responses. In this study, we developed a strategy to generate BVDV-VLPs using a baculovirus expression vector system (BEVS). We were able to assemble BVDV-VLPs composed of dimerized viral proteins E2 and Erns, and the VLPs were spherical particles with the diameters of about 50 nm. Mice immunized with 15 µg of VLPs adjuvanted with ISA201 elicited higher levels of E2-specific IgG, IgG1, and IgG2a antibodies as well as higher BVDV-neutralizing activity in comparison with controls. Re-stimulation of the splenocytes collected from mice immunized with VLPs led to significantly increased levels of CD3+CD4+T cells and CD3+CD8+T cells. In addition, the splenocytes showed dramatically enhanced proliferation and the secretion of Th1-associated IFN-γ and Th2-associated IL-4 compared to that of the unstimulated control group. Taken together, our data indicate that BVDV-VLPs efficiently induced BVDV-specific humoral and cellular immune responses in mice, showing a promising potential of developing BVDV-VLP-based vaccines for the prevention of BVDV infections.

Fabrication of a double-layer membrane cathode based on modified carbon nanotubes for the sequential electro-Fenton oxidation of p-nitrophenol.

To improve the electrocatalytic efficiency of the cathode and provide a wider pH range in the electro-Fenton process, N-doped multi-walled carbon nanotubes (NCNTs) and ferrous ion complexed with carboxylated carbon nanotubes (CNT-COOFe2+) were used to fabricate the diffusion layer and catalyst layer of a membrane cathode, respectively. The morphology, structure, and composition of CNT-COOFe2+ were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). The oxygen reduction performance of NCNT was evaluated using cyclic voltammetry (CV) and the rotating disk electrode technique (RDE). In addition, a potential application of the cathode in sequential electro-Fenton degradation of p-nitrophenol (p-NP) was investigated. The results revealed that iron was successfully doped on the carboxylated carbon nanotubes in ionic complexation form and the content of iron atoms in CNT-COOFe2+ was 2.65%. Furthermore, the defects on the tube walls provided more reactive sites for the electro-Fenton process. A combination of CV and RDE data indicated that NCNT had better electrocatalytic H2O2 generation activity with a more positive onset potential and higher cathodic peak current response than CNT. A p-NP removal rate of 96.04% was achieved within 120 min, and a mineralization efficiency of 80.26% was obtained at 180 min in the sequential electro-Fenton process at a cathodic potential of - 0.7 V vs SCE and neutral pH. The activity of the used cathode was restored simply through electro-reduction at - 1.0 V vs SCE, and a p-NP removal rate of more than 70% was obtained at 60 min after six regeneration cycles.

Interaction between Translocation-associated membrane protein 1 and σC protein of novel duck reovirus controls virus infectivity.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is associated with high mortality in Pekin ducklings. σC is an outer capsid protein encoded by the S1 genome segment of NDRV which mediates attachment to host cells. Our previous studies using immunoprecipitation and mass spectrometry found that σC coprecipitated with some host proteins including Translocation-associated membrane protein 1 (TRAM1). However, the interaction between σC and TRAM1 has not been further confirmed experimentally. In this study, we utilized coimmunoprecipitation assays, glutathione S-transferase pull-down, and confocal microscopy to confirm the interaction between σC and TRAM1. In addition, knockdown of TRAM1 using siRNA and overexpression of TRAM1 gene were conducted to explore its effect on virus replication. The result showed that TRAM1 silencing benefits while overexpression inhibits viral replication. This study confirms the important role TRAM1 during NDRV infection which can help develop new approaches for NDRV disease prevention and control.

Novel H5N6 avian influenza virus reassortants with European H5N8 isolated in migratory birds, China.

Five novel H5N6 influenza viruses, including four highly pathogenic avian influenza viruses and one low pathogenic avian influenza virus, were isolated from migratory birds in Ningxia, China, in November 2017. To understand the genetic origination of the novel H5N6 virus, and the infectivity and pathogenicity of the four highly pathogenic avian influenza viruses in mammals, phylogeographic analyses and infection studies in mice were performed. The phylogenetic and phylogeographic analyses showed that the H5N6 isolates, which are closely related to the viruses from Korea, Japan and the Netherlands, originated from reassortant virus between H5N8 and HxN6 viruses from western Russia. The animal study revealed that the SBD-87 isolate presented moderate virulence in mice, suggesting a potential public risk to humans and a potential threat to public health.

Identification of a conserved linear epitope using monoclonal antibody against non-structural protein 3A of foot-and-mouth disease virus with potential for differentiation between infected and vaccinated animals.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral disease of cloven-hoofed animals. Vaccination is a key element in the control of FMD among countries where the disease is enzootic. Differentiating infected from vaccinated animals in herds after immunization is an important component of effective eradication strategies. Non-structural protein (NSP) 3A of FMDV is as part of a larger detected antigen that is used for this differential diagnosis. Here, we generated a specific monoclonal antibody (MAb) against FMDV non-structural protein called 3A10, and further defined the linear epitopes recognized by the MAb 3A10 using a series of peptides that expressed GST-fused protein. Using Western blot, it was showed that the 5-aa peptide 126ERTLP130 of 3A was the minimal epitope reactive to MAb 3A10. Alanine-scanning mutagenesis analysis revealed that Arg127 and Leu129 were crucial for MAb 3A10 binding to 126ERTLP130. Furthermore, sequence alignment analysis, indicated that the epitope 126ERTLP130 recognized by 3A10 was shown to be conserved among seven serotypes of FMDV strains. The synthetic peptide Elisa demonstrated that this epitope peptide could be recognized by sera from FMDV-infected pigs and cattle, but negative reactivity to unvaccinated and vaccinated healthy animal sera. Thus, the MAb reagents and the linear epitopes defined herein provide theoretical and technical support for the development of diagnostic tools for infection differentiating FMDV infected from vaccinated animals.

Ultrasensitive electrochemiluminescence detection of Staphylococcus aureus via enzyme-free branched DNA signal amplification probe.

As a main cause of foodborne diseases, pathogenic bacteria have threatened the health and well-being of human communities. There is a need of fastness, accuracy and sensitivity in the method of detecting pathogenic bacteria. Classical signal amplification assays usually employ enzymes as biocatalysts to generate amplified signals, but the strict experimental conditions and complicated instruments restrict their application. In this work, we demonstrated an enzyme-free branched DNA (bDNA)-based signal amplification electrochemiluminescence (ECL) assay for ultrasensitive detection of pathogenic bacteria. Firstly, the capture probes and the amplification probes group were carefully designed by our research group. The detecting ECL signal of Staphylococcus aureus (S. aureus) was amplified by bDNA technique through the layer-by-layer signal amplification. The sensitivity was greatly improved by the use of multiple Ru(bpy)32+ (TBR)-labeled ECL probes. Secondly, the whole process of the detection was carried out in the absence of enzyme, without the need to control the reaction conditions strictly. Thirdly, the designed amplification probes group could be used for the analysis of other pathogenic bacteria, virus, tumor markers, biomarkers, etc. For the detection of S. aureus, the limit of detection (LOD) of the method was 2 pM for standard DNA, with the linear range from 20 pM to 100 nM. Last but not least, the LOD of the S. aureus asymmetric PCR products was 5 pM, with the linear range from 10 pM to 50 nM. The sensitivity was 1-2 orders in magnitude higher than that of the common detection assays.

An miR-200 Cluster on Chromosome 23 Regulates Sperm Motility in Zebrafish.

Besides its well-documented roles in cell proliferation, apoptosis, and carcinogenesis, the function of the p53-microRNA axis has been recently revealed in the reproductive system. Recent studies indicated that miR-200 family members are dysregulated in nonobstructive azoospermia patients, whereas their functions remain poorly documented. The aim of this study was to investigate the function of the miR-200 family on zebrafish testis development and sperm activity. There was no substantial difference in testis morphology and histology between wild-type (WT) and knockout zebrafish with deletion of miR-200 cluster on chromosome 6 (chr6-miR-200-KO) or on chromosome 23 (chr23-miR-200-KO). Interestingly, compared with WT zebrafish, the chr6-miR-200-KO zebrafish had no difference on sperm motility, whereas chr23-miR-200-KO zebrafish showed significantly improved sperm motility. Consistently, ectopic expression of miR-429a, miR-200a, and miR-200b, which are located in the miR-200 cluster on chromosome 23, significantly reduced motility traits of sperm. Several sperm motility-related genes, such as amh, wt1a, and srd5a2b have been confirmed as direct targets of miR-200s on chr23. 17α-ethynylestradiol (EE2) exposure resulted in upregulated expression of p53 and miR-429a in testis and impairment of sperm motility. Strikingly, in p53 mutant zebrafish testis, the expression levels of miR-200s on chr23 were significantly reduced and accompanied by a stimulation of sperm motility. Moreover, the upregulation of miR-429a associated with EE2 treatment was abolished in testis with p53 mutation. And the impairment of sperm activity by EE2 treatment was also eliminated when p53 was mutated. Together, our results reveal that miR-200 cluster on chromosome 23 controls sperm motility in a p53-dependent manner.

Potential Contributions of miR-200a/-200b and Their Target Gene-Leptin to the Sexual Size Dimorphism in Yellow Catfish.

Sexual size dimorphism is the consequence of differential expression of sex-biased genes related to feeding and growth. Leptin is known to regulate energy balance by regulating food intake. In order to investigate the molecular mechanism of sexual size dimorphism in yellow catfish (Pelteobagrus fulvidraco), the expression of leptin (lep) and its functional receptor (lepr) were detected during larval development. Both lep and lepr have lower expression in males than in females during 1-4 weeks post hatching. 17a-Methyltestosterone (MT) treatment resulted in decreased expression of lep and lepr in both male and female larval fish. Interestingly, the mRNA levels of lep and lepr in juvenile male were significantly decreased compared with juvenile female during short-term fasting periods. Lep was predicted to be a potential target of miR-200a and miR-200b that had an opposite expression pattern to lep in male and female larvas. The results of luciferase reporter assay suggested that lep is a target of miR-200a/-200b. Subsequently, male hormone and fasting treatment have opposite effects on the expression of miR-200a/-200b and lep between males and females. In summary, our results suggest that sexual size dimorphism in fish species is probably caused by the sexually dimorphic expression of leptin, which could be negatively regulated by miR-200a/-200b.

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8TLLEDRILT16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr8 and Asp12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

Identification of a conformational neutralizing epitope on the VP1 protein of type A foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype A and protect against future pandemics. In this study, we generated a serotype A FMDV-specific potent neutralizing monoclonal antibody (MAb), 6C9, which recognizes a conformation-dependent epitope. MAb 6C9 potently neutralized FMDV A/XJBC/CHA/2010 with a 50% neutralization titer (NT50) of 4096. Screening of a phage-displayed random 12-mer peptide library revealed that MAb 6C9 bound to phages displaying the consensus motif YxxPxGDLG, which is highly homologous to the 135YxxPxxxxxGDLG147 motif found in the serotype A FMDV virus-encoded structural protein VP1. To further verify the authentic epitope recognized by MAb 6C9, two FMDV A/XJBC/CHA/2010 mutant viruses, P138A and G144A, were generated using a reverse genetic system. Subsequent micro-neutralization assays and double-antibody sandwich (DAS) ELISA analyses revealed that the Pro138 and Gly144 residues of the conformational epitope that are recognized by 6C9 are important for MAb 6C9 binding. Importantly, the epitope 135YxxPxxxxxGDLG147 was highly conserved among different topotypes of serotype A FMDV strains in a sequence alignment analysis. Thus, the results of this study could have potential applications in the development of novel epitope-based vaccines and suitable a MAb-based diagnostic method for the detection of serotype A FMDV and the quantitation of antibodies against this serotype.

Identification of a conserved conformational epitope in the VP2 protein of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, a potent FMDV serotype-independent monoclonal antibody (MAb) 3D9 was generated. Screening of a phage-displayed random 12-peptide library revealed that MAb 3D9 bound to phages displaying a consensus motif GVYxxAYxW that is highly homologous to the 89GVYxxxxxxxAYxxxxW105 motif in the FMDV VP2 protein. Importantly, this conformational epitope was highly conserved among all seven serotypes of FMDV analyzed in sequence alignments. To further verify the authentic epitope recognized by MAb 3D9, a FMDV O/YS/CHA/05 mutant virus V90A was generated using a reverse genetics system. The results revealed that Val90 was an important residue for MAb 3D9 binding within this conformational epitope. Thus, we finely mapped a conserved conformational epitope onto the FMDV VP2 protein. These results could be applied in the development of epitope-based vaccines and suitable MAb-based diagnostic methods for various FMDV serotype-independent tests.