High-throughput screening unveils nitazoxanide as a potent PRRSV inhibitor by targeting NMRAL1.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a major threat to the global swine industry, yet effective prevention and control measures remain elusive. This study unveils Nitazoxanide (NTZ) as a potent inhibitor of PRRSV both in vitro and in vivo. Through High-Throughput Screening techniques, 16 potential anti-PRRSV compounds are identified from a library comprising FDA-approved and pharmacopeial drugs. We show that NTZ displays strong efficacy in reducing PRRSV proliferation and transmission in a swine model, alleviating viremia and lung damage. Additionally, Tizoxanide (TIZ), the primary metabolite of NTZ, has been identified as a facilitator of NMRAL1 dimerization. This finding potentially sheds light on the underlying mechanism contributing to TIZ's role in augmenting the sensitivity of the IFN-ß pathway. These results indicate the promising potential of NTZ as a repurposed therapeutic agent for Porcine Reproductive and Respiratory Syndrome (PRRS). Additionally, they provide valuable insights into the antiviral mechanisms underlying NTZ's effectiveness.

Caffeic acid phenethyl ester: an effective antiviral agent against porcine reproductive and Respiratory Syndrome Virus.

Porcine Reproductive and Respiratory Syndrome (PRRS) presents a formidable viral challenge in swine husbandry. Confronting the constraints of existing veterinary pharmaceuticals and vaccines, this investigation centers on Caffeic Acid Phenethyl Ester (CAPE) as a prospective clinical suppressant for the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). The study adopts an integrated methodology to evaluate CAPE's antiviral attributes. This encompasses a dual-phase analysis of CAPE's interaction with PRRSV, both in vitro and in vivo, and an examination of its influence on viral replication. Varied dosages of CAPE were subjected to empirical testing in animal models to quantify its efficacy in combating PRRSV infections. The findings reveal a pronounced antiviral potency, notably in prophylactic scenarios. As a predominant component of propolis, CAPE stands out as a promising candidate for clinical suppression, showing exceptional effectiveness in pre-exposure prophylaxis regimes. This highlights the potential of CAPE in spearheading cutting-edge strategies for the management of future PRRSV outbreaks.

Enlarged tumour-draining lymph node with immune-activated profile predict favourable survival in non-metastatic colorectal cancer.

BACKGROUND: The tumour-draining lymph node (TDLN) plays a pivotal role in the suppression of malignant tumour, however, the immunological profile and prognostic differences between large TDLN (L-TDLN) and small TDLN (S-TDLN) in colorectal cancer (CRC) remain unclear. METHODS: We conducted a study using data from the Chinese National Cancer Center (CNCC) database, identifying 837 CRC patients with non-metastatic TDLN, and categorised them into L-TDLN and S-TDLN groups. The long-term survival outcomes and adjuvant therapy efficacy were compared between the two groups. Furthermore, we evaluated the differences in immune activation status and immune cell subsets between patients in L-TDLN and S-TDLN groups by RNA sequencing and immunohistochemical (IHC) staining. RESULTS: Patients with L-TDLN demonstrated better long-term outcomes compared to those with S-TDLN. Among patients with L-TDLN, there was no significant difference in long-term outcomes between those who received adjuvant chemotherapy and those who did not. The RNA sequencing data revealed a wealth of immune-activating pathways explored in L-TDLN. Furthermore, IHC analysis demonstrated higher numbers of CD3+ and CD8 + T cells in L-TDLN and the corresponding CRC lesions, as compared to patients with S-TDLN. CONCLUSION: Enlarged TDLN exhibited an activated anti-tumour immune profile and may serve as an indicator for favourable survival in non-metastatic CRC.

Conserved antigen structures and antibody-driven variations on foot-and-mouth disease virus serotype A revealed by bovine neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) serotype A is antigenically most variable within serotypes. The structures of conserved and variable antigenic sites were not well resolved. Here, a historical A/AF72 strain from A22 lineage and a latest A/GDMM/2013 strain from G2 genotype of Sea97 lineage were respectively used as bait antigen to screen single B cell antibodies from bovine sequentially vaccinated with A/WH/CHA/09 (G1 genotype of Sea97 lineage), A/GDMM/2013 and A/AF72 antigens. Total of 39 strain-specific and 5 broad neutralizing antibodies (bnAbs) were isolated and characterized. Two conserved antigenic sites were revealed by the Cryo-EM structures of FMDV serotype A with two bnAbs W2 and W125. The contact sites with both VH and VL of W125 were closely around icosahedral threefold axis and covered the B-C, E-F, and H-I loops on VP2 and the B-B knob and H-I loop on VP3; while contact sites with only VH of W2 concentrated on B-B knob, B-C and E-F loops on VP3 scattering around the three-fold axis of viral particle. Additional highly conserved epitopes also involved key residues of VP158, VP1147 and both VP272 / VP1147 as determined respectively by bnAb W153, W145 and W151-resistant mutants. Furthermore, the epitopes recognized by 20 strain-specific neutralization antibodies involved the key residues located on VP3 68 for A/AF72 (11/20) and VP3 175 position for A/GDMM/2013 (9/19), respectively, which revealed antigenic variation between different strains of serotype A. Analysis of antibody-driven variations on capsid of two virus strains showed a relatively stable VP2 and more variable VP3 and VP1. This study provided important information on conserve and variable antigen structures to design broad-spectrum molecular vaccine against FMDV serotype A.

MR Image Harmonization with Transformer.

Many clinical applications require medical image harmonization to combine and normalize images from different scanners or protocols. This paper introduces a Transformer-based MR image harmonization method. Our proposed method leverages the self-attention mechanism of the Transformer to learn the complex relationships between image patches and effectively transfer the imaging characteristics from a source image domain to a target image domain. We evaluate our approach to state-of-the-art methods using a publicly available dataset of brain MRI scans and show that it provides superior quantitative metrics and visual quality. Furthermore, we demonstrate that the proposed approach is highly resistant to fluctuations in image modality, resolution, and noise. Overall, the experiment results indicate that our approach is a promising method for medical image harmonization that can improve the accuracy and reliability of automated analysis and diagnosis in clinical settings.

Laparoscopic radical transverse colectomy with transrectal specimen extraction: A novel natural orifice specimen extraction procedure: A case report.

Transverse colon cancer accounts for about 10% of all colonic cancers. The resection of cancers in the transverse colon is technically more challenging, compared with other cancer locations in the colon because the variable anatomy of the middle colic vessels demands excellent surgical skills and the anatomical location of the transverse colon is related to major organs. We report a novel laparoscopic technique for the first time used in surgery of transverse colon cancer which combines a total intracorporeal anastomosis with natural orifice specimen extraction to solve the problems of traditional laparoscopic surgery. A 48-year-old male patient, whose diagnosis was transverse colon adenocarcinoma, was admitted to the hospital. The surgery was performed in accordance with the procedure of totally laparoscopic right hemicolectomy and the specimen was extracted by opening the rectum. Natural orifice specimen extraction surgery has many advantages, including less pain, better cosmesis and minimising risks of complications and also has comparable long-term outcomes compared to conventional laparoscopic surgery.

Comparative short-term and survival outcomes of three specimen extraction techniques in laparoscopic low rectal cancer surgery: does it affect ileostomy closure?

INTRODUCTION: This study aimed to compare the short-term and survival outcomes in laparoscopic low rectal cancer surgery with three different specimen extraction techniques, and whether it affects loop ileostomy closure. MATERIALS AND METHODS: A consecutive series of patients with low rectal cancer who underwent laparoscopic low anterior resection plus protective loop ileostomy (LAR-PLI) were enrolled. Three main techniques, namely specimen extraction through auxiliary incision (EXAI), specimen extraction through stoma incision (EXSI), and specimen eversion and extra-abdominal resection (EVER), were employed. The postoperative short-term and survival outcomes of the three techniques and the impact on loop ileostomy closure were compared. RESULTS: In all, 254 patients were enrolled in this study: 104 (40.9%) in the EXAI group, 104 (40.9%) in the EXSI group, and 46 (18.1%) in the EVER group. For primary surgery, EXAI group had significantly longer operative time (P < 0.001), more intraoperative bleeding (P < 0.001), longer length of abdominal incision (P<0.001), longer time to first flatus (P < 0.001), longer time to first defecation (P < 0.001), longer time to first eat (P < 0.001), and longer postoperative hospital stays (P = 0.005) than the EXSI and EVER groups. The primary postoperative complication rate in the EXAI and EVER group was significantly higher than in the EXSI group (P = 0.005). In loop ileostomy closure, EXAI group had significantly longer operative time (P = 0.001), more bleeding volume, and longer postoperative hospital stays (P < 0.001) than the EXSI and EVER groups. For survival outcomes, the 3-year local recurrence-free survival (LRFS) is 92.6% for all patients. The 3-year LRFS for patients in EXAI, EXSI, and EVER were 90.1%, 95.4%, and 92.7%, with P = 0.476. CONCLUSIONS: Our single-center results found that in LAR-PLI surgery for low rectal cancer, the short-term outcomes of specimen extraction through the stoma incision or anus were better than that through the auxiliary incision, but the 3-year LRFS was no statistically different.

Development and Evaluation of a Competitive Enzyme-Linked Immunosorbent Assay Based on Swine Monoclonal Antibodies for Detecting Neutralizing Antibodies against Senecavirus A.

Senecavirus A (SVA) is an emerging viral pathogen related to vesicular disease and neonatal mortality in swine, which results in enormous economic losses to the global swine industry. The clinical signs of SVA are indistinguishable from those of other vesicular diseases, such as foot-and-mouth disease, which is an economically devastating animal disease. Therefore, development of a rapid, sensitive, and specific diagnostic method for the detection of SVA infection is critical for the prevention and control of SVA and would help to rule out other exotic diseases. In this study, two whole-porcine anti-SVA antibodies (1M5 and 1M25) were produced using single B cell antibody technology. 1M5 and 1M25 possessed neutralizing activity against SVA but recognized different conformational epitopes that depended on the intact virion. Using 1M5 as the capture antibody and biotinylated 1M25 as the detection antibody, a reliable and rapid competitive enzyme-linked immunosorbent assay for detecting neutralizing antibodies (NAC-ELISA) against SVA was developed. Receiver-operating characteristic curve analysis showed that the sensitivity and specificity of the assay were 98.11% and 100%, respectively, with a cutoff percent inhibition value of 45%. The NAC-ELISA was specific for detecting SVA-specific antibodies, without cross-reactivity to other virus-infected sera. The results of the NAC-ELISA showed a strong agreement with the results of the virus neutralization test. Therefore, the NAC-ELISA developed in this study represents a sensitive, specific, and reliable tool for the detection of SVA-specific antibodies, which is applicable for serodiagnosis and serological surveillance of SVA and is conducive to the prevention and control of SVA. IMPORTANCE Senecavirus A (SVA) is an emerging picornavirus related to vesicular disease and neonatal mortality in swine, which results in enormous economic losses worldwide. Additionally, the clinical characteristics of the disease are indistinguishable from those of other vesicular diseases, such as foot-and-mouth disease. Therefore, developing tools for rapidly and accurately detecting SVA infection is critical and urgent. In this study, two porcine-derived monoclonal antibodies against SVA were generated, and a competitive ELISA for the detection of neutralizing antibodies (NAC-ELISA) against SVA was successfully developed using these two porcine monoclonal antibodies. The NAC-ELISA was SVA specific with no cross-reactivity to other related pathogens and had high sensitivity, specificity, and reproducibility for detecting SVA-specific antibody. Therefore, the NAC-ELISA developed in this study may be of great value as a simple and reliable tool for serodiagnosis or surveillance of SVA and may facilitate the prevention and control of SVA.

Lateral pelvic sentinel lymph node biopsy using indocyanine green fluorescence navigation: can it be a powerful supplement tool for predicting the status of lateral pelvic lymph nodes in advanced lower rectal cancer.

BACKGROUND: An innovative instrument for laparoscopy using indocyanine green (ICG) allows easy detection of sentinel lymph nodes (SLNs) in lateral pelvic lymph nodes (LPLNs). Here, we investigated the safety and efficacy of lateral pelvic SLN biopsy (SLNB) using ICG fluorescence navigation in advanced lower rectal cancer and evaluated the sensitivity and specificity of this technique to predict the status of LPLN. METHODS: From April 1, 2017 to December 1, 2020, we conducted lateral pelvic SLNB using ICG fluorescence navigation during laparoscopic total mesorectal excision and lateral pelvic lymph node dissection (LLND) in 23 patients with advanced low rectal cancer who presented with LPLN but without LPLN enlargement. Data regarding clinical characteristics, surgical and pathological outcomes, lymph node findings, and postoperative complications were collected and analyzed. RESULTS: We successfully performed the surgery using fluorescence navigation. One patient underwent bilateral LLND and 22 patients underwent unilateral LLND. The lateral pelvic SLN were clearly fluorescent before dissection in 21 patients. Lateral pelvic SLN metastasis was diagnosed in 3 patients and negative in 18 patients by frozen pathological examination. Among the 21 patients in whom lateral pelvic SLN was detected, the dissected lateral pelvic non-SLNs were all negative. All dissected LPLNs were negative in two patients without fluorescent lateral pelvic SLN. CONCLUSION: This study indicated that lateral pelvic SLNB using ICG fluorescence navigation shows promise as a safe and feasible procedure for advanced lower rectal cancer with good accuracy, and no false-negative cases were found. No metastasis in SLNB seemed to reflect all negative LPLN metastases, and this technique can replace preventive LLND for advanced lower rectal cancer.

Development of artificial blood loss and duration of excision score to evaluate surgical difficulty of total laparoscopic anterior resection in rectal cancer.

Purpose: Total laparoscopic anterior resection (tLAR) has been gradually applied in the treatment of rectal cancer (RC). This study aims to develop a scoring system to predict the surgical difficulty of tLAR. Methods: RC patients treated with tLAR were collected. The blood loss and duration of excision (BLADE) scoring system was built to assess the surgical difficulty by using restricted cubic spline regression. Multivariate logistic regression was used to evaluate the effect of the BLADE score on postoperative complications. The random forest (RF) algorithm was used to establish a preoperative predictive model for the BLADE score. Results: A total of 1,994 RC patients were randomly selected for the training set and the test set, and 325 RC patients were identified as the external validation set. The BLADE score, which was built based on the thresholds of blood loss (60 ml) and duration of surgical excision (165 min), was the most important risk factor for postoperative complications. The areas under the curve of the predictive RF model were 0.786 in the training set, 0.640 in the test set, and 0.665 in the external validation set. Conclusion: This preoperative predictive model for the BLADE score presents clinical feasibility and reliability in identifying the candidates to receive tLAR and in making surgical plans for RC patients.

Development of a double-antibody sandwich ELISA for rapidly quantitative detection of residual non-structural proteins in inactivated foot-and-mouth disease virus vaccines.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. Vaccination and surveillance against non-structure protein (NSP) are the most efficacious and cost-effective strategy to control this disease. Therefore, vaccine purity control is vital for successful prevention. Currently, vaccine purity is tested by an in-vivo test that recommended in the World Organization for Animal Health (WOAH), but it is time consuming and costly. Herein, we develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for quantitative detection of residual NSPs in inactivated FMD virus (FMDV) vaccines. In this assay, the monoclonal antibody 3A24 was selected as capture antibody and biotinylated 3B4B1 (Biotin-3B4B1) as detection antibody. A standard curve was developed using the NSP 3AB concentration versus OD value with the linear range of concentration of 2.5-160 ng/mL. The lowest limit of detection was 2.5 ng/mL. In addition, we determined 2.5 ng/mL of NSP as an acceptable threshold value of FMD vaccine purity using a dose-response experiment in cattle. The DAS-ELISA combined with the threshold value of FMD vaccine purity could provide a quick and simple tool for evaluation the antigenic purity of FMD vaccine during the manufacturing process.

Development of a machine learning algorithm to predict complications of total laparoscopic anterior resection and natural orifice specimen extraction surgery in rectal cancer.

BACKGROUND: Total laparoscopic anterior resection (tLAR) and natural orifice specimen extraction surgery (NOSES) has been widely adopted in the treatment of rectal cancer (RC). However, no study has been performed to predict the short-term outcomes of tLAR using machine learning algorithms to analyze a national cohort. METHODS: Data from consecutive RC patients who underwent tLAR were collected from the China NOSES Database (CNDB). The random forest (RF), extreme gradient boosting (XGBoost), support vector machine (SVM), deep neural network (DNN), logistic regression (LR) and K-nearest neighbor (KNN) algorithms were used to develop risk models to predict short-term complications of tLAR. The area under the receiver operating characteristic curve (AUROC), Gini coefficient, specificity and sensitivity were calculated to assess the performance of each risk model. The selected factors from the models were evaluated by relative importance. RESULTS: A total of 4313 RC patients were identified, and 667 patients (15.5%) developed postoperative complications. The machine learning model of XGBoost showed more promising results in the prediction of complication than other models (AUROC 0.90, P < 0.001). The performance was similar when internal and external validation was used. In the XGBoost model, the top four influential factors were the distance from the lower edge of the tumor to the anus, age at diagnosis, surgical time and comorbidities. In risk stratification analysis, the rate of postoperative complications in the high-risk group was significantly higher than in the medium- and low-risk groups (P < 0.001). CONCLUSION: The machine learning model shows potential benefits in predicting the risk of complications in RC patients after tLAR. This novel approach can provide reliable individual information for surgical treatment recommendations.

Creation of poxvirus expressing foot-and-mouth and peste des petits ruminant disease virus proteins.

OBJECTIVE: Foot-and-mouth disease (FMD) and Peste des petits ruminant disease (PPR) are acute and severe infectious diseases of sheep and are listed as animal diseases for compulsory immunization. However, there is no dual vaccine to prevent these two diseases. The Modified Vaccinia virus Ankara strain (MVA) has been widely used in the construction of recombinant live vector vaccine because of its large capacity of foreign gene, wide host range, high safety, and immunogenicity. In this study, MVA-GFP recombinant virus skeleton was used to construct dual live vector vaccines against FMD and PPR. METHODS: The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. RESULTS: The results showed that a recombinant virus without fluorescent labeling was obtained after multiple rounds of plaque screening. The recombinant virus successfully expressed the target proteins, and the empty capsid of FMDV could be observed by transmission electron microscope (TME), and the expression levels of foreign proteins (VP1 and VP3) detected by ELISA were like those detected in FMDV-infected cells. This study laid the foundation for the successful construction of a live vector vaccine against FMD and PPR. KEY POINTS: • A recombinant MVA expressing FMDVP12A3C and PRRV HF proteins • Both the FMDV and PRRV proteins inserted into the virus were expressed • The proteins expressed by the recombinant poxvirus were assembled into VLPs.

Downregulation of miR-122 by porcine reproductive and respiratory syndrome virus promotes viral replication by targeting SOCS3.

MicroRNAs are small non-coding RNA that regulate host anti-viral immune response. In this study, we used high-throughput sequencing to identify miRNAs that were differentially expressed upon PRRSV infection in porcine alveolar macrophages. We observed that the expression level of miR-122 was decreased upon PRRSV infection. Over-expression of miR-122 remarkably suppressed PRRSV replication, while blockage of endogenous miR-122 enhanced PRRSV replication. Moreover, over-expression of miR-122 reduced the protein level of porcine suppressor of cytokine signaling 3 (SOCS3), a negative regulator of JAK-STAT signaling, resulting in enhanced production of type Ⅰ IFN. Further analysis revealed that miR-122 decreased the expression of SOCS3 at the post-transcription level by targeting the 3' UTR region of SOCS3 mRNA. In conclusion, this study demonstrates that the expression of miR-122 was reduced during PRRSV infection. miR-122 impaired PRRSV replication by promoting the production of type I interferon. Our study may provide new insights into understanding PRRSV immune evasion mechanisms.

Prognostic and immunotherapeutic significance of mannose receptor C type II in 33 cancers: An integrated analysis.

Background: The type 2 mannose receptor C (MRC2) is involved in tumor biological processes and plays a new role in the remodeling of the extracellular matrix turnover. Previous studies have demonstrated MRC2 expression profiling and prognostic relevance in some tumor types. However, the clinical and immunotherapeutic value of MRC2 in pan-cancers remains controversial. Our study aimed to evaluate MRC2 expression pattern, clinical characteristics and prognostic significance in 33 cancers, explore the relationship between MRC2 and immune-related characteristics, and assess the prediction of MRC2 for the immunotherapeutic response. Methods: Transcriptional and clinical data of 33 cancers were downloaded from The Cancer Genome Atlas database (TCGA) database and two independent immunotherapeutic cohorts were obtained from GSE67501 and the IMvigor210 study. Next, patients stratified by MRC2 expression levels were displayed by Kaplan-Meier plot to compare prognosis-related indexes. Meanwhile, immune infiltrates of different cancers were estimated by tumor immune estimation resources (TIMER) and CIBERSORT. The ESTIMATE algorithm was used to estimate the immune and stromal scores in tumor tissues. MRC2 expression and immunological modulators, including immune inhibitors, immune stimulators, and MHC molecules, were screened through the TISIDB portal. Gene-set enrichment analysis analyses were performed to explore the underlying biological process of MRC2 across different cancers. The immunotherapeutic response prediction was performed in two independent cohorts (GSE78220: metastatic melanoma with pembrolizumab treatment and IMvigor210: advanced urothelial cancer with atezolizumab intervention). Results: MRC2 is expressed differently in many cancers and has been shown to have potential prognostic predicting significance. MRC2 was significantly associated with immune cell infiltration, immune modulators, and immunotherapeutic markers. Notably, the immunotherapeutic response group was associated with lower MRC2 expression in metastatic melanoma and advanced urothelial carcinoma cohort. Conclusion: This study demonstrated that MRC2 could be a prognostic indicator for certain cancer and is critical for tumor immune microenvironments. MRC2 expression level may influence and predict immune checkpoint blockade response as a potential indicator.

[A method for immortalizing swine monoclonal B cells secreting anti-PRRSV antibodies].

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.

Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses in pigs and cattle.

Foot-and-mouth disease (FMD) remains a very serious barrier to agricultural development and the international trade of animals and animal products. Recently, serotype O has been the most prevalent FMDV serotype in China, and it has evolved into four different lineages: O/SEA/Mya-98, O/ME-SA/PanAsia, O/ME-SA/Ind-2001 and O/Cathay. PanAsia-2, belonging to the O/ME-SA topotype, is prevalent in neighbouring countries and poses the risk of cross-border spread in China. This study aimed to develop a promising vaccine candidate strain that can not only provide the best protection against all serotype O FMDVs circulating in China but also be used as an emergency vaccine for the prevention and control of transboundary incursion of PanAsia-2. Here, two chimeric FMDVs (rHN/TURVP1 and rHN/NXVP1) featuring substitution of VP1 genes of the O/TUR/5/2009 vaccine strain (PanAsia-2) and O/NXYCh/CHA/2018 epidemic strain (Mya98) were constructed and evaluated. The biological properties of the two chimeric FMDVs were similar to those of the wild-type (wt) virus despite slight differences in plaque sizes observed in BHK-21 cells. The structural protein-specific antibody titres induced by the rHN/TURVP1 and wt virus vaccines in pigs and cows were higher than those induced by the rHN/NXVP1 vaccine at 28-56 dpv. The vaccines prepared from the two chimeric viruses and wt virus all induced the production of protective cross-neutralizing antibodies against the viruses of the Mya-98, PanAsia and Ind-2001 lineages in pigs and cattle at 28 dpv; however, only the animals vaccinated with the rHN/TURVP1 vaccine produced a protective immune response to the field isolate of the Cathay lineage at 28 dpv, whereas the animals receiving the wt virus and the rHN/NXVP1 vaccines did not, although the wt virus and O/GXCX/CHA/2018 both belong to the Cathay topotype. This study will provide very useful information to help develop a potential vaccine candidate for the prevention and control of serotype O FMD in China.

Development and Validation of a Competitive ELISA Based on Bovine Monoclonal Antibodies for the Detection of Neutralizing Antibodies against Foot-and-Mouth Disease Virus Serotype A.

The level of neutralizing antibodies in vaccinated animals is directly related to their level of protection against a virus challenge. The virus neutralization test (VNT) is a "gold standard" method for detecting neutralizing antibodies against foot-and-mouth disease virus (FMDV). However, VNT requires high-containment facilities that can handle live viruses and is not suitable for large-scale serological surveillance. In this study, a bovine broadly neutralizing monoclonal antibody (W145) against FMDV serotype A was successfully produced using fluorescence-based single-B-cell antibody technology. Using biotinylated W145 as a detector antibody and another bovine cross-reactive monoclonal antibody, E32, which was produced previously as a capture antibody, a competitive enzyme-linked immunosorbent assay for the detection of neutralizing antibodies (NAC-ELISA) against FMDV serotype A was developed. The specificity and sensitivity of the assay were evaluated to be 99.04% and 100%, respectively. A statistically significant correlation (r = 0.9334, P < 0.0001) was observed between the NAC-ELISA titers and the VNT titers, suggesting that the NAC-ELISA could detect neutralizing antibodies against FMDV serotype A and could be used to evaluate protective immunity.

[Expression and refolding of OLA â protein with peptides derived from sheeppox virus].

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-ß2m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a SuperdexTM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.