Of the seven serotypes of foot-and-mouth disease virus (FMDV) strains circulating globally, serotype Asia1 has been effectively eradicated in China through systematic vaccination in livestock. The structural characteristics of serotype Asia1 may enhance its immunogenicity compared to other serotypes. Herein, we present a preliminary exploration of Asia1-binding B-cell receptor repertoire, containing 3571 clones, and identified 17 porcine-derived neutralizing monoclonal antibodies (pnAbs) from the top 33 high-frequency clonotypes. The majority of pnAbs (14/17) recognized the epitopes on VP2, with a common determinant at residue 72 (D) on the B-C loop; two pnAbs (2/17) recognized a novel epitope spanning VP2 and VP3; and the remaining one (1/17) bound to the C-terminus of VP1. Furthermore, the antigenic structures on VP2 and spanning VP2 and VP3 were respectively elucidated by determining the cryo-EM structures of FMDV serotype Asia1 in complexes with two pnAbs, PAS5 and PAS12. The light chain of PAS5, forming the majority of contact sites with the viral particle, focuses on the βB, B-C loop, βC and H-I loop of VP2, with key determinants at residues 68, 72 and 77 around the three-fold axis, corresponding to antigenic site 2. The contact sites of both VH and VL of PAS12 uncover a novel antigenic structure comprising the B-C, and H-I loops on VP2, and the B-B knob and βB on VP3, with key determinants at residue 73 on VP2 and 59 on VP3. Subsequently, site-directed competitive ELISA analysis of sera from primary and booster vaccinated pigs revealed a balanced antibody response profile, suggesting a potentially even immunodominance among antigenic site 2, VP1 G-H loop, and the novel antigenic structure spanning VP2 and VP3 on FMDV serotype Asia1. Compared to the focused immunodominance observed in other serotypes, this balanced antigenic recognition across VP1, VP2, and VP3 of FMDV serotype Asia1 reflects a diversified antibody response that may contribute to effective neutralization and protection.

Vaccination with inactivated whole-virus vaccines remains the most effective measure for controlling foot-and-mouth disease virus (FMDV) transmission and disease outbreaks. However, the existing type O FMDV vaccines show suboptimal efficacy and antigenic mismatch to the circulating Cathay viruses in China, thus requiring the development of a new vaccine. The VP1 G-H loop is a hypervariable region and plays a pivotal role in the protective immunity induced by FMDV vaccines. Here, we engineered four recombinant FMDVs with insertions of a 20-amino acid (aa) or a 24-aa G-H loop epitope of a prevalent Cathay strain upstream or downstream of the RGD (Arg-Gly-Asp) motif. The recombinant viruses with insertions upstream of the RGD motif retained parental virus-like plaque morphology and replication kinetics and maintained genetic stability even after 20 serial passages. In contrast, the downstream insertion variants exhibited small plaque morphology, reduced growth capacity, and acquired 1 or 2 aa mutations in the capsid proteins by passage 20. The parental virus vaccine induced high titer protective mean neutralizing antibodies (> 1:128) against viruses of the Mya98, PanAsia, and Ind-2001 lineages but failed to elicit protective mean neutralizing antibodies (< 1:22) to the Cathay virus after 28 days vaccination (dpv) in pigs. In contrast, vaccines containing upstream insertions both exhibited protective immune response to viruses of four lineages. Especially, pigs vaccinated with vaccine containing a 24-aa insertion produced significantly higher mean neutralizing antibody against the Cathay virus (p < 0.01), compared to those vaccinated with vaccine having a 20-aa insertion, indicating that the recombinant virus with 24-aa insertion has great potential as a vaccine candidate for serotype O FMD control. This study provides crucial insights for designing FMDV vaccines in the future. • This study firstly reported that FMDV can tolerate a 24-aa insertion in the VP1 G-H loop • The G-H loop insertions at different sites of FMDV VP1 have different impacts on viral replication capacity • Vaccines containing the G-H loop insertions can induce markedly high neutralizing antibodies to the Cathay virus

This study delves into the antiviral efficacy of Formononetin (FMN) and Mizoribine (MZR) against the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a virus with a considerable economic impact and a current void in effective treatments. FMN and MZR were found to inhibit various PRRSV strains in vitro, predominantly in the early stages of viral infection. Noteworthy was the observation of their synergistic effects when combined with Ribavirin. The study underscores the antiviral potential of FMN and MZR, particularly emphasizing their low cytotoxicity at specific concentrations. These results position FMN and MZR as promising antiviral agents against PRRSV, underscoring their low cytotoxicity and efficacy in early-stage viral inhibition. Such findings pave the way for their potential inclusion in future PRRSV management strategies.

The foot-and-mouth disease virus (FMDV) serotype O contains at least five neutralizing antigenic sites, yet the structural relationship and antibody abundance remain poorly characterized. This study identifies six distinct neutralizing antigenic sites by evaluating 27 host-derived neutralizing antibodies (NAbs) using competitive enzyme-linked immunosorbent assay (cELISA). These sites include the VP1 G-H loop, VP1 C-terminus, site 2, site 4, site 6, and site 7. Notably, classical sites 1 and 5 were reclassified into the VP1 G-H loop and VP1 C-terminus classes. Sites 2 and 4 align with classical classifications, targeting independent epitopes on VP2 and VP3, respectively. We identified two novel sites: site 6, which involves extensive interactions with the G-H loop, C-terminus of VP1 and VP3, and site 7, which interacts with both VP2 and VP3. Sera from cattle, sheep, and pigs immunized with four serotype O lineages (O/SCGH/2016, O/Mya/98, O/Tibet/99, and O/XJ/2017) were used to evaluate the immunodominance of these sites. NAb responses favored site 4 for O/SCGH/2016 and the VP1 G-H loop for O/XJ/2017. Immunization effectiveness varied by strains and host species: O/XJ/2017 and O/Tibet/99 were effective in sheep, while O/Mya/98 showed reduced efficacy; O/Tibet/99 showed good immunogenicity in pigs. No significant differences were observed in cattle. There is a strong correlation (r = 0.8693) between NAb levels at site 6 and virus neutralization tests, suggesting its potential for use in alternative testing methods. This study describes the spatial distribution of neutralizing sites and highlights strain-specific immunodominant epitopes and differential antibody responses across species, providing valuable insights for FMD prevention and control. IMPORTANCE:The antigenic structure of the foot-and-mouth disease virus (FMDV) serotype O is complex, and the immunodominant epitopes among different lineages remain poorly understood. This study classified the capsid surface epitopes into six distinct antigenic sites utilizing 27 neutralizing antibodies (NAbs) by paired competitive ELISAs (cELISAs). High-affinity NAbs were selected for site-directed cELISAs to assess antibody abundance in serum from cattle, sheep, and pigs vaccinated with various inactivated FMDV serotype O vaccines. Additionally, liquid-phase blocking ELISA (LPBE) and virus neutralization test (VNT) were employed to measure total antibody and NAb titers. Results indicated that immunodominant sites vary among different strains, with each strain exhibiting different immunogenicity across the three animal species. Notably, antibody titers from NAb pO18-10, targeting site 6 on VP1 and VP3, correlated strongly with VNT results. This study provides comprehensive insights into the antigenic structure of FMDV serotype O and lays the groundwork for developing new methods to detect NAbs.

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-β 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.

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.

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.

为了探究猪源IFN-λ3蛋白能否在CHO-S悬浮细胞中表达以及表达蛋白的抗口蹄疫病毒(FMDV)活性,根据NCBI上的猪源IFN-λ3序列,构建真核表达载体pcDNA3.4-IFNλ3-His,将其转染至CHO-K1贴壁细胞中,用间接免疫荧光试验和Western-blot验证重组质粒是否表达IFN-λ3蛋白.将pcDNA3.4-IFNλ3-His转染至CHO-S悬浮细胞中大量制备IFN-λ3蛋白,用AKTA蛋白纯化系统纯化蛋白,用SDS-PAGE和Western-blot方法鉴定纯化出的蛋白,用细胞毒性试验分析纯化蛋白是否具有细胞毒性,用实时荧光定量PCR、Western-blot和空斑试验三种方法研究纯化蛋白的抗FMDV活性.结果显示,重组质粒pcDNA3.4-IFNλ3-His在CHO-K1细胞中瞬时表达IFN-λ3,在CHO-S细胞中以分泌形式表达IFN-λ3;用AKTA系统能够纯化出纯度较高的IFN-λ3蛋白;纯化出的IFN-λ3对PK-15细胞无明显毒性,能够抑制FMDVRNA的复制、减弱病毒蛋白翻译活动以及降低子代病毒的生成.本研究为发展新型FMDV防控策略提供了新方向.

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.

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.

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune and systemic inflammatory diseases with both licensed and off-label indications. Recent studies indicated that IVIg-mediated immunomodulation and anti-inflammation are closely associated with the IgG sialylation, especially with IgG crystallizable fragment (Fc) sialylation. The sialic acid levels of the IgG molecules and Fc fragments in 12 IVIg preparations from six Chinese manufacturers were evaluated. The Fc fragments were derived from the papain digestion of IVIg, followed by affinity and size exclusion chromatography. The sialic acid levels in Fc fragments and IVIg preparations were determined by high-performance liquid chromatography with fluorescence detection, after the sialic acid residues were released from the proteins. The results showed that the sialic acid levels in Chinese IVIg preparations ranged from 0.875 (mol/mol IgG) to 1.085 (mol/mol IgG), and the sialic acid levels in Fc fragments were from 0.321 (mol/mol Fc) to 0.361 (mol/mol Fc). Furthermore, the sialic acid levels of IVIg preparations and Fc fragments from different Chinese manufactures were significantly different. These findings will contribute to an increased understanding of Chinese IVIg preparations and the relationship between the sialic acid levels in IVIg preparations and their clinical efficacy in future clinical studies.

UAV networks often partition into separated clusters due to the high node and link dynamic. As a result, network connectivity recovery is an important issue in this area. Existing solutions always need excessive movement of nodes and thus lead to low recovery efficiency in terms of the time and energy consumption. In this paper, we for the first time study the issue of how to utilize cooperative communication technology to improve the connectivity recovery efficiency in UAV networks. We propose a Cooperative Communication based Connectivity Recovery algorithm for UAV Networks, named C 3 RUN. The key novelty in C 3 RUN is nodes can proactively find better locations to establish more efficient cooperative communication links, than the ones from passively leveraging on existing opportunities. We conduct extensive simulations to evaluate the performance of C 3 RUN. The simulation results reveal that C 3 RUN can not only achieve connectivity recovery with less nodes and shorter distance to move, but also always finish recovery with less time, when comparing with existing work. Furthermore, C 3 RUN can achieve 100% success ratio for connectivity recovery.

The IgG activities of antigen recognition, Fc-mediated complement activation and cellular Fcγ-receptors (FcγRs) binding are critical for intravenous immunoglobulin (IVIg) immunotherapy in a variety of immune deficiency diseases. Further, these activities could be influenced by different plasma sources and the IVIg manufacturing processes of different manufacturers. This study evaluated and compared the biological activities of IgG in 7 IVIg preparations produced by different Chinese manufacturers. By using ELISA and two-dimensional immunoblotting, the binding capacity and antibody repertoire of IVIg against typical pathogenic antigens were investigated. Further, Fc-mediated complement activation and receptor binding activities were measured by the haemolysis method and flow-cytometric assay respectively. The results showed that all of the preparations tested have a broad spectrum of antibodies against the E. coli O157:H7 proteome, and each IVIg has its own distinct antibody repertoire. Compared to the European Pharmacopoeia IgG standard, the mean indices of the pathogenic antigen binding capacity, complement activation activity and FcγRs binding activity in Chinese preparations were 152%, 143% and 87%. The biological activities varied widely among the 7 IVIg preparations, and no significant differences were observed between the different batches of most IVIgs from the same manufacturer. This study will contribute to the improvement of the IVIg product quality evaluation system and an increased understanding of the variety of IgG biological activities in Chinese IVIg preparations.

In this paper, 3 classic suffix type algorithms: QS, Tuned BM and BMHq were improved by reducing the average cost of basic operations. Firstly, the multi-windows method was used to let the calculations of the jump distance run in parallel and pipelining. Secondly, the comparison unit was increased to integer to reduce the total number and the average cost of comparisons. Especially for BMHq, the jump distance was increased by good prefix rule and the operations to get the jump distance were simplified by unaligned integer read. Thus, 3 algorithms named QSMI, TBMMI and BMHqMI were presented. These algorithms are faster than other known algorithms in many cases.

We designed a series of epitope proteins containing the G–H loops of three topotypes of foot-and-mouth disease virus (FMDV) serotype O and promiscuous artificial Th sites and selected one epitope protein (designated as B4) with optimal immunogenicity and cross-reactivity. Three out of five pigs immunized intramuscularly with this B4 were protected against virulent FMDV challenge after a single inoculation, while all pigs co-immunized with B4 and polyinosinic–cytidylic acid [poly(I:C)] conferred complete protection following FMDV challenge. Additionally, we demonstrated that all pigs co-immunized with B4 and poly(I:C) elicited FMDV-specific neutralizing antibodies, total IgG antibodies, type I interferon (IFN-α/β) and cytokines IFN-γ. In contrast, some pigs immunized with B4 alone produced parameters mentioned above, while some not, suggesting that poly(I:C) reduced animal-to-animal variations in both cellular and humoral responses often observed in association with epitope-based vaccines and up-regulated T-cell immunity often poorly observed in protein-based vaccines. We propose that poly(I:C) is an effective adjuvant for this epitope-based vaccine of FMDV. This combination could yield an effective and safe candidate vaccine for the control and eradication of FMD in pigs.

We investigated the enhanced immune response of a recombinant T cell immunogen as an effective cellular immune adjuvant. The T cell immunogen named TI contained several T cell epitopes from the VP1, VP4, 3A and 3D proteins of foot-and-mouth disease virus (FMDV) and two pan-T helper (T(H)) cell sites to broaden the immunogenicity of the protein. Meanwhile, another fusion protein named OA-VP1 was expressed in bacteria, which contained two VP1 proteins of O and Asia1 type FMDV. Mice were vaccinated with commercially inactivated vaccine or OA-VP1 protein with or without the TI immunogen. The results show that mice inoculated with inactivated vaccine or OA-VP1 protein supplemented with TI immunogen produced significantly higher level of neutralizing antibodies (P < 0.01 or P < 0.05) than the mice only inoculated with inactivated vaccine or OA-VP1 protein by microneutralization assay. An obvious increase in T cell number by flow cytometric analysis and significantly higher concentration of IFN-gamma secreted in culture media of spleen lymphocytes were observed in groups supplemented with TI immunogen (P < 0.01). TI immunogen was an effective stimulator for humoral and cellular immunity and could help improve the immunogenicity of inactivated vaccine or protein subunit vaccine.

Three different routes of Foot-and-mouth disease virus (FMDV) infection of piglets, namely intranasal (i.n.) through drops, intradermal (i.d.) into the foot, and intramuscular (i.m.) were compared regarding the onset and severity of the disease. The results showed that the i.d. injection of the virus resulted in the fastest onset of the disease. The i.m. injection led to a delayed onset, but the final effect was identical with i.d. injection. Moreover, the i.m. injection was simpler to perform and easier to evaluate. Therefore, the i.m. injection of piglets is recommended as the optimal infection route for evaluation of the FMDV vaccine potency.

In the study, a method called "hardening in liquid phase" for preparing chitosan granules with glutaraldehyde as crosslinker and Tween 80 as surfactant and paraffin liquid as dispersant was established. The chitosan granules were light yellow and insoluble in water or oil, but they swelled in acid solution and narrowed in neutral or alkaline solution. Furthermore, some of characteristics of the chitosan granules were revealed. (a) Stability: Their shapes were stable at pH 7.0 and pH 8.0 and -30°C~120°C. The shelf life is at least one year in vitro at room temperature. (b) Safety: Some experiments of their lethal effect to suckling mice and pathogenicity to mature mice proved the chitosan granules were harmless. (c) Antiviral activity: Some suckling mice injected with chitosan granules were still alive or delayed death compared with control group when they challenged with foot-and-mouth disease virus (FMDV). Such anti-FMDV capacity could maintain 1 week and was the strongest on the third day.

OBJECTIVE:The purpose of this research is to establish a simple rapid amplification of cDNA ends (RACE) strategy for direct mapping of the 3' end and 5' end of the genomic RNA of Newcastle disease virus (NDV), and to analyze the leader and trailer sequence of NDV strains belonging to different genotypes.METHODS:Classic RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) was specifically modified for mapping both ends of the NDV genome. 3'-RACE was carried out by genomic RNA ligation with 5' end phosphated adaptor CL+, and the 5' end was obtained by first strand cDNA with adaptor CL+.RESULTS:A modified RLM-RACE strategy was established in this paper, which proved simple, low-cost, repetitive and could be specifically used to map genome ends of NDV. By using this method, the leader and trailer sequence of 5 NDV strains, termed JS/5/05/Go, JS/07/04/Pi, JS/07/16/Pi, JS/7/05/Ch and JS/9/05/Go, belonging to genotype III, VI and VII was determined, respectively.CONCLUSION:The initial 8nt at the 3' and 5' ends of the genome of genotype I-VI NDV strains were complementary, whereas, the complementary sequences of strain JS/5/05/Go were up to 9 nt due to a mutation from T to C at the 9th nt in the 5' end. The 3' end of NDV genomic and anti-genomic RNA was predicted to form a potential hairpin structure. The U-->C(T-->C) mutation was located in the circle part of the hairpin in the 5' end of anti-genomic RNA, and had no visible influence on the formation of RNA secondary structure. However, the sequence of the circle part of the hairpin was changed from 3'-UUUC-5' to 3'-UCUC-5', more similar to the 3'-UCUUA-5' in the hairpin of genomic RNA.

The test for alloy combustibility was carried out by using CO2 laser. A 430 watt and 6 mm diameter laser spot impinges directly on the sample within normal atmospheric pressure and temperature. The results show that the ignition time of the non-burning titanium alloy is the longest in the eight examined titanium alloys, and is 3.5 times the length of TC4 alloy. The ignition temperature of the non-burning alloy is 2991 °C, and is higher than that of TC4 alloy by 1976 °C. On the condition of high temperature and rich oxygen, the surface of alloy forms a melting layer which plays roles of oxygen insulation, heat insulation and burning products insulation. This is the mechanism of combustion resistance.