Foot-and-mouth disease virus (FMDV) is a highly contagious picornavirus that affects cloven-hoofed animals and carries significant economic implications for the global livestock industry. FMDV features two Leader (L) protein isoforms, Lab and Lb, differing at their amino termini by 28 amino acids (La region). Currently, the activity of La protein sequences has not been investigated. To address this issue, the comparison study of biological and functional roles of Lab and Lb was performed as the La region alone did not independently perform protein function. We found that Lab and Lb significantly regulated FMDV replication and pathogenicity, and their coexistence afforded optimal FMDV properties. Subsequently, we observed that both L isoforms cleaved eukaryotic translation initiation factor 4G (eIF4G) I, suppressed type I and type III interferon (IFN) expression, and exhibited marked cytotoxicity, indicating that they were all key components in FMDV's antagonism of host antiviral defenses. Finally, the subcellular distribution of Lab and Lb was detected. Despite dual localization in cytoplasmic and nuclear compartments, both isoforms displayed different spatial distribution patterns, and Lb induced more pronounced morphological changes to host cells than Lab. Furthermore, bioinformatics predicted that the La region might contain a non-classical secretory signal peptide, potentially facilitating Lab distribution to the cell membrane or extracellular space. Collectively, the primary encoding role of La region was to control the intracellular distribution of L protein, as opposed to regulating its functional activity. This study may help to deepen our understanding of why FMDV encoded two isoforms of L protein.

Classical swine fever virus (CSFV) is a highly contagious pathogen that causes substantial economic losses in swine production, yet the mechanisms governing its cellular entry remain poorly defined. Here, through a combination of pharmacological inhibition, genetic knockout/overexpression, and co-immunoprecipitation, we identify annexin A2 (ANXA2) as a pivotal host determinant of CSFV entry into PK-15 cells. Confocal microscopy revealed that CSFV infection rapidly induced the translocation of ANXA2 to the cell surface, and functional assays confirmed that its plasma membrane localization was essential for efficient infection. Mechanistically, the viral envelope protein E2 specifically interacts with the heterotetrameric A2t complex (ANXA2/S100A10), establishing this complex as a functional cellular receptor for CSFV. ANXA2 also maintains clathrin at the plasma membrane and facilitates clathrin-dependent viral entry, as evidenced by inhibitor and siRNA knockdown studies. Intriguingly, ANXA2 depletion redirects viral entry to a caveolin-1-dependent pathway. We further demonstrate that ANXA2 competitively binds the C-terminal domain of caveolin-1, a site that overlaps with the binding region for the viral Erns protein, thereby suppressing the caveolin-1 route in wild-type cells. In summary, this study identifies the A2t complex as a CSFV receptor and reveals a crucial regulatory role for ANXA2 in viral entry pathway selection. These findings provide new insights into CSFV infection and a rationale for host-directed antiviral strategies.

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.

The whole life cycle of the highly pathogenic foot-and-mouth disease virus (FMDV) significantly depends on the host determinants to achieve its infection. ATG16L1 is well known to be required to form the autophagosomes membrane at the early steps of autophagy, while its non-autophagic roles in FMDV infection remain unclear. We found that following entry, FMDV O/Fujian/CHA/5/99 trafficked to early endosomes (EEs) and the trans-Golgi network (TGN), bypassing late endosomes (LEs) /lysosome and recycling endosomes (REs). This specific intracellular distribution mirrored the vesicular sorting pathway involving ATG16L1 that had been reported previously. Further analyses showed that ATG16L1 increased the internalization of FMDV and recruited EEs to facilitate the initial phase of FMDV infection. However, ATG16L1 degraded FMDV 2BC protein in the Golgi via its non-autophagic function to inhibit late stages of FMDV infection. To counteract this, membrane-associated 2BC interacted with ATG16L1 and mediated its reduction via the caspase pathway, thereby sustaining FMDV replication. In conclusion, our evidence suggested that ATG16L1 played dual roles in regulating the life cycle of FMDV.

Recently, serotype O foot-and-mouth disease viruses (FMDVs) belonging to four lineages (O/ME-SA/PanAsia, O/ME-SA/Ind2001, O/SEA/Mya-98, and O/Cathay) co-circulate in China, and the emergence of new variant viruses belonging to the Cathay lineage renders the existing vaccines less effective, which poses a serious threat to the livestock industries. The surface-exposed G-H loop of the VP1 structural protein of FMDV plays an important role in inducing neutralizing antibodies, generation of antigenic variants, and virus attachment. Here, we generated three recombinant FMDVs in which the G-H loops have been substituted with the corresponding sequences from the circulating strains of the PanAsia, Mya-98, and Cathay lineages based on an infectious cDNA clone of a chimeric FMDV, which carries amino acid substitutions in the leader protein and all surface proteins of FMDV O/XJ/CHA/2017. All mutant viruses exhibited a significantly improved replication capacity in BHK-21 cells and displayed relatively larger plaque morphologies compared with the parental virus. Chemically inactivated vaccines prepared from the parental virus and the mutant viruses all induced protective liquid-phase blocking ELISA (LPBE) antibodies against FMDVs in pigs after 28 days post vaccination (dpv), but only the vaccine with the substitution of the G-H loop of the Cathay strain induced protective neutralizing antibodies against the viruses of four lineages, while other vaccines exhibited excellent immunological cross-reactivity to the viruses of Ind2001, PanAsia, and Mya-98 lineages but did not for the Cathay virus at 28 dpv. Our studies indicated that the G-H loop of the Cathay virus plays a critical role in the antigenic drift of FMDV, which will provide key insights for designing porcinophilic FMDV vaccines in the future. • The swapping of the G-H loops of FMDVs notably improved replication capacity of FMDV in BHK-21 cells. • The swapping of the G-H loop of the Cathay virus obviously broadens antigenic coverage of FMDV vaccine. • The study showed that the G-H loop of the Cathay virus plays a critical role in antigenic drift of FMDV.

The E2 subunit vaccine has been considered a promising alternative to an attenuated classical swine fever (CSF) vaccine. However, it fails to induce a good cellular immune response. Given that immunogenic adjuvants can regulate the cellular immunity to achieve a maximum efficacy against antigens, immunostimulatory effects of porcine IL-28B on the CSF virus (CSFV) E2 subunit vaccine were evaluated in the present study. We expressed recombinant proteins E2-IL28B, E2, and IL-28B using CHO-S mammalian cells as an antigen expression platform, and three types of CSFV E2 subunit vaccines based on antigens E2-IL28B, E2 + IL-28B, and E2 were prepared, respectively. We found that both E2-IL28B and E2 + IL-28B antigens exhibited superior immunogenicity with dramatically induced antibody titers and neutralizing antibody levels than the E2 alone. Moreover, E2-IL28B or E2 + IL-28B, instead of E2, boosted cellular immune responses via obviously increasing the percentages of CD3+CD4+ T lymphocytes, promoting the lymphocyte proliferations, and enhancing the release of Th1-type cytokines. All results revealed that the inclusion of IL-28B, whether fused or mixed with E2, significantly elevated E2-induced immune potencies, suggesting that IL-28B could be used as a molecular adjuvant to optimize the design of E2 subunit vaccine for more effective controls of the CSF disease. • New CSF E2 subunit vaccine candidates were developed in which IL-28B was an immunoadjuvant • IL-28B significantly elevated the E2-induced immune potency whether it was fused or mixed with E2 • This study provided novel insights into the immunoregulatory properties of IL-28B used for the optimized subunit vaccine design

Classical swine fever virus (CSFV), a member of the Flaviviridae family, remains a major pathogen responsible for substantial economic losses in the global swine industry. Autophagy plays a critical role in the life cycle and virulence of CSFV, however, the mechanisms through which the virus regulates autophagy are still not fully understood. In this study, we identified ANXA2, a calcium-dependent phospholipid-binding protein, within autophagy-derived vesicles that facilitate CSFV transmission. We demonstrated that ANXA2 modulates CSFV release in a manner dependent on autophagy. Moreover, multiple lines of evidence, including Western blot, LC3 puncta formation, tandem fluorescence assay, and electron microscopy, consistently showed that ANXA2 promotes CSFV-induced autophagy. Mechanistically, ANXA2 overexpression reduced mTOR phosphorylation, while its knockout increased phosphorylation. Comprehensive binding assays revealed that both ANXA2 and the CSFV envelope protein E2 interact with mTOR with high affinity. Domain mapping further indicated that ANXA2 and E2 bind to distinct regions of mTOR, suggesting a synergistic mechanism for autophagy activation. Confocal microscopy showed that ANXA2 facilitates mTOR accumulation at the plasma membrane during infection. Importantly, relocalizing ANXA2 to mitochondria attenuated CSFV-induced autophagy. Collectively, these results indicate that ANXA2 modulates CSFV-triggered autophagy by controlling mTOR subcellular localization, thereby influencing viral production. This study unveils a novel strategy by which CSFV co-opts the ANXA2-mTOR axis to manipulate autophagic processes, highlighting potential targets for future antiviral interventions.

Camels, with the ability to survive under drought and chronic hunger, developed exceptional efficient lipid reserves and energy substance metabolic characteristics. Fibroblast growth factor (FGF) 21 is a hormone that regulates important metabolic pathways and energy homeostasis. However, the absence of a specific detection method for camel FGF21 impacts research on camels’ metabolic regulation. This study established a direct competition ELISA assay for detecting camel FGF21. Camel FGF21 antigen was expressed and purified through prokaryotic expression system. Polyclonal antibody was produced and purified via immunizing guinea pigs and affinity chromatography assay. Biotin-labeled FGF21 was synthesized artificially as the competitive antigen. After the determination of optimal conditions, including the working concentrations of the antibody and antigen, blocking solution, dilution buffer, and the competition reaction time, the standard curve with a typical “S” shape was generated using GraphPad Prism. The regression equation was Y = 0.1111 + (X−0.7894) × (2.162 − 0.1111)/(X−0.7894 + 15.76−0.7894), with the IC50 15.59 ng/mL, the limit of detection (LOD) 0.024 ng/mL, the limit of quantification (LOQ) 1.861 ng/mL, and the linear range IC20~IC80 2.0~119.22 ng/mL. The verification test showed that the recovery rate ranged from 91.34% to 98.9%, and the coefficients of variation for the intra- and inter-plate both were less than 10%, indicating that the ELISA method had high accuracy, good repeatability, and high stability. In addition, this ELISA method had the potential to detect FGF21 secretion levels in other species such as mouse, human, and pig. This study provided a rapid quantitative tool for conducting research on the FGF21 factor in camels.

ABSTRACT Neutralizing antibodies provide vital protection against foot-and-mouth disease virus (FMDV). The virus neutralization test (VNT) is a gold standard method for the detection of neutralizing antibodies. However, its application is limited due to the requirement for live virus and unsuitability for large-scale serological surveillance. In this study, a porcine broadly neutralizing monoclonal antibody (PO18-10) against FMDV was obtained from the heterologous sequentially vaccinated pig using single-B-cell antibody technology. A competitive enzyme-linked immunosorbent assay (C-ELISA) for detecting neutralizing antibodies against FMDV serotype O was developed using biotinylated PO18-10 as a detector antibody. The sensitivity and specificity of the assay were 100% and 99.55%, respectively, and the positive/negative coincidence rate with VNT was 94%, suggesting that C-ELISA based on natural host-derived monoclonal antibody (mAb) could be a promising tool to detect neutralizing antibodies against FMDV serotype O and evaluate the vaccine efficacy.IMPORTANCEFoot-and-mouth disease virus (FMDV) serotype O is one of the most prevalent serotypes in the world. The neutralizing antibody titers in primo-vaccinated animals are directly related to their level of protection against a virus challenge. The development of a safe, rapid, and accurate method for the detection of the neutralizing antibody is essential for the control and eradication of FMD. In this study, an inter-serotype broadly neutralizing monoclonal antibody PO18-10 was successfully produced using single-B-cell antibody technology from sequentially vaccinated pigs. A competitive ELISA based on this natural host-derived mAb for the detection of neutralizing antibodies against FMDV serotype O was developed and validated. The assay demonstrates high sensitivity, specificity, and coincidence rate with VNT, making it an alternative tool for confirming FMDV infection and evaluating the vaccine efficacy.

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.

Foot-and-mouth disease virus (FMDV) is highly infectious and lacks cross-protection among serotypes, with antibodies playing a key role in antiviral immunity. To map conserved epitopes on the FMDV surface that exhibit cross-serotype reactivity, we constructed a pig-specific B-cell receptor (BCR) library through single B-cell sorting and high-throughput sequencing. This led to the identification of 16 broadly reactive, non-neutralizing monoclonal antibodies (mAbs), with 10 targeting VP2 (pOTB-1, pOTB-10, pOTB-13, pOTB-33, pOTB-37, pONY-14, pONY-17, pONY-23, pONY-30, pONY-60) and 6 targeting VP3 (pOTB-6, pOTB-11, pOTB-22, pOTB-23, pONY-3, pONY-59). Among these, a novel free linear epitope was identified at the C-terminus of VP2, recognized by pOTB-1, with the minimal recognition motif "KE." Key residues, T53 and W101, within the complementarity-determining region (CDR) of the pOTB-1 heavy chain, interact with the carboxyl group of the C-terminal glutamate through hydrogen bonding, contributing to the free-form nature of the epitope. Competitive enzyme-linked immunosorbent assays (cELISA) showed that most non-neutralizing antibodies (nNAbs) interfered with the binding of neutralizing antibodies B82 (site 2) and C4 (site 4), confirming the overlap between non-neutralizing and neutralizing epitopes. It has been confirmed that nNAbs mediate antiviral activity in vivo through various mechanisms, such as the formation of immune complexes. These findings reveal new epitopes on VP2 and VP3 and their spatial overlap with neutralizing sites, enhancing our understanding of FMDV immunogenicity and providing novel targets for vaccine and therapeutic development.

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.

Non-structural protein 2 (NSP2) of PRRSV is highly variable and plays crucial roles in the virus’s life cycle. To elucidate the function of NSP2 during PRRSV infection, we identified SH3KBP1 as an NSP2-interacting host protein using mass spectrometry. Exogenous SH3KBP1 expression significantly inhibited PRRSV replication by enhancing IFN-I and related ISGs production. Conversely, SH3KBP1 knockdown promoted viral replication by downregulating IFN-I and ISGs levels. In vivo experiments revealed that Sh3kbp1-/- mice were more susceptible to VSV infection, exhibiting reduced serum IFN-β levels. Further investigation showed that SH3KBP1 enhances RIG-I signal transduction by increasing K63-linked polyubiquitination through interaction with the E3 ubiquitin ligase TRIM25. We also found that PRRSV infection and NSP2 overexpression induce the autophagic degradation of SH3KBP1, counteracting the host’s innate immune response. A critical interaction site was identified within the third polyproline-arginine motif in NSP2 (453PVPAPR458). Recombinant PRRSV lacking this motif displayed reduced virulence and decreased SH3KBP1 degradation. This study advances our understanding of how PRRSV interferes with the host immune response and offers valuable insights for developing novel attenuated vaccines against PRRSV.

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猪圆环病毒(PCV)是圆环病毒科圆环病毒属的单股环状闭合DNA病毒,是引起猪圆环病毒相关疾病(PCVAD)的主要病原,目前包括4个基因型:PCV1、PCV2、PCV3和PCV4.其中,PCV3是近年来新发的一种严重危害养猪业的基因型,已经在全球范围呈现广泛流行趋势,引起了国内外学者的广泛关注.本文主要从病原学、流行病学、临床症状、致病机理、免疫反应和诊断方法对PCV3近年来研究情况进行了综述,为PCV3后期的各方面研究提供参考.

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.

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.

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. The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. 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. • 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

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.