RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation.

As a RIG-I-like receptor, MDA5 plays a critical role in antiviral innate immunity by acting as a cytoplasmic double-stranded RNA sensor capable of initiating type I interferon pathways. Here, we show that RNF144B specifically interacts with MDA5 and promotes K27/K33-linked polyubiquitination of MDA5 at lysine 23 and lysine 43, which promotes autophagic degradation of MDA5 by p62. Rnf144b deficiency greatly promotes IFN production and inhibits EMCV replication in vivo. Importantly, Rnf144b-/- mice has a significantly higher overall survival rate than wild-type mice upon EMCV infection. Collectively, our results identify RNF144B as a negative regulator of innate antiviral response by targeting CARDs of MDA5 and mediating autophagic degradation of MDA5.

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.

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.

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.

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.

The Accessibility of Educational Public Service Facilities with Two-Step Mobile Search Taking Hetao District of Ganzhou City as an Example.

By using the two-step mobile search method of time and distance cost combined with Internet map data, taking the old urban district of Ganzhou city as research object, the spatial accessibility of primary and secondary schools from residential sites to residential areas was studied. The research shows that the reachability of primary and secondary schools in the old urban district of Ganzhou city is low, and most of the population fails to enjoy better education resources and the spatial distribution of education resources is not balanced. The reachability of primary education resources is high in geospatial area. The gathering area is the intersection of Ganjiang Street and Jiefang Street. The low-value areas are mainly the southwestern part of Nanwai Street and the northwestern area of Dongwai Street. The reachability of education resources shows a decreasing trend from the center to the surrounding areas. The education resources are concentrated in Ganjiang Street. Compared with traditional methods, the two-step mobile search method using Internet map data can more effectively and accurately reflect the accessibility of residential areas to primary and secondary schools in real time and can obtain the real conditions of traffic and roads more conveniently and also more accurat data acquisition.

[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.

The long non-coding RNA LNC_000397 negatively regulates PRRSV replication through induction of interferon-stimulated genes.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most significant threats to the global swine industry. It is of great importance to understand viral-host interactions to develop novel antiviral strategies. Long non-coding RNAs (lncRNAs) have emerged as critical factors regulating host antiviral immune responses. However, lncRNAs participating in virus-host interactions during PRRSV infection remain largely unexplored. METHOD: RNA transcripts of porcine alveolar macrophages (PAMs) infected with two different PRRSV strains, GSWW/2015 and VR2332, at 24 h post-infection were sequenced by high-throughput sequencing. Four programs namely, CNCI, CPC, PFAM, and phyloCSF, were utilized to predict the coding potential of transcripts. mRNAs co-localized or co-expressed with differentially expressed lncRNAs were considered as their targets. Fuction of lncRNAs was predicted by GO and KEGG analysis of their target mRNAs. The effect of LNC_000397 on PRRSV replication was validated by knockdown its expression using siRNA. Target genes of LNC_000397 were identified by RNA-Sequencing and validated by RT-qPCR. RESULT: In this study, we analyzed lncRNA and mRNA expression profiles of PRRSV GSWW/2015 and VR2332 infected porcine alveolar macrophages. A total of 1,147 novel lncRNAs were characterized, and 293 lncRNAs were differentially expressed. mRNAs co-localized and co-expressed with lncRNAs were enriched in pathogen-infection-related biological processes such as Influenza A and Herpes simplex infection. Functional analysis revealed the lncRNA, LNC_000397, which was up-regulated by PRRSV infection, negatively regulated PRRSV replication. Knockdown of LNC_000397 significantly impaired expression of antiviral ISGs such as MX dynamin-like GTPase 1 (MX1), ISG15 Ubiquitin-like modifier (ISG15), and radical S-adenosyl methionine domain containing 2 (RSAD2). CONCLUSIONS: LNC_000397 negatively regulated PRRSV replication by inducing interferon-stimulated genes (ISGs) expression. Our study is the first report unveiling the role of host lncRNA in regulating PRRSV replication, which might be beneficial for the development of novel antiviral therapeutics.

Two Cross-Protective Antigen Sites on Foot-and-Mouth Disease Virus Serotype O Structurally Revealed by Broadly Neutralizing Antibodies from Cattle.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that infects cloven-hoofed animals. Neutralizing antibodies play critical roles in antiviral infection. Although five known antigen sites that induce neutralizing antibodies have been defined, studies on cross-protective antigen sites are still scarce. We mapped two cross-protective antigen sites using 13 bovine-derived broadly neutralizing monoclonal antibodies (bnAbs) capable of neutralizing 4 lineages within 3 topotypes of FMDV serotype O. One antigen site was formed by a novel cluster of VP3-focused epitopes recognized by bnAb C4 and C4-like antibodies. The cryo-electron microscopy (cryo-EM) structure of the FMDV-OTi (O/Tibet/99)-C4 complex showed close contact with VP3 and a novel interprotomer antigen epitope around the icosahedral 3-fold axis of the FMDV particle, which is far beyond the known antigen site 4. The key determinants of the neutralizing function of C4 and C4-like antibodies on the capsid were ßB (T65), the B-C loop (T68), the E-F loop (E131 and K134), and the H-I loop (G196), revealing a novel antigen site on VP3. The other antigen site comprised two group epitopes on VP2 recognized by 9 bnAbs (B57, B73, B77, B82, F28, F145, F150, E46, and E54), which belong to the known antigen site 2 of FMDV serotype O. Notably, bnAb C4 potently promoted FMDV RNA release in response to damage to viral particles, suggesting that the targeted epitope contains a trigger mechanism for particle disassembly. This study revealed two cross-protective antigen sites that can elicit cross-reactive neutralizing antibodies in cattle and provided new structural information for the design of a broad-spectrum molecular vaccine against FMDV serotype O. IMPORTANCE FMDV is the causative agent of foot-and-mouth disease (FMD), which is one of the most contagious and economically devastating diseases of domestic animals. The antigenic structure of FMDV serotype O is rather complicated, especially for those sites that can elicit a cross-protective neutralizing antibody response. Monoclonal neutralization antibodies provide both crucial defense components against FMDV infection and valuable tools for fine analysis of the antigenic structure. In this study, we found a cluster of novel VP3-focused epitopes using 13 bnAbs against FMDV serotype O from natural host cattle, which revealed two cross-protective antigen sites on VP2 and VP3. Antibody C4 targeting this novel epitope potently promoted viral particle disassembly and RNA release before infection, which may indicate a vulnerable region of FMDV. This study reveals new structural information about cross-protective antigen sites of FMDV serotype O, providing valuable and strong support for future research on broad-spectrum vaccines against FMD.

Isolation and characterization of porcine monoclonal antibodies revealed two distinct serotype-independent epitopes on VP2 of foot-and-mouth disease virus.

Pigs are susceptible to foot-and-mouth disease virus (FMDV), and the humoral immune response plays an essential role in protection against FMDV infection. However, little information is available about FMDV-specific mAbs derived from single B cells of pigs. This study aimed to determine the antigenic features of FMDV that are recognized by antibodies from pigs. Therefore, a panel of pig-derived mAbs against FMDV were developed using fluorescence-based single B cell antibody technology. Western blotting revealed that three of the antibodies (1C6, P2-7E and P2-8G) recognized conserved antigen epitopes on capsid protein VP2, and exhibited broad reactivity against both FMDV serotypes A and O. An alanine-substitution scanning assay and sequence conservation analysis elucidated that these porcine mAbs recognized two conserved epitopes on VP2: a linear epitope (2KKTEETTLL10) in the N terminus and a conformational epitope involving residues K63, H65, L66, F67, D68 and L81 on two ß-sheets (B-sheet and C-sheet) that depended on the integrity of VP2. Random parings of heavy and light chains of the IgGs confirmed that the heavy chain is predominantly involved in binding to antigen. The light chain of porcine IgG contributes to the binding affinity toward an antigen and may function as a support platform for antibody stability. In summary, this study is the first to reveal the conserved antigenic profile of FMDV recognized by porcine B cells and provides a novel method for analysing the antibody response against FMDV in its natural hosts (i.e. pigs) at the clonal level.

The rescue and selection of thermally stable type O vaccine candidate strains of foot-and-mouth disease virus.

Inactivated foot-and-mouth disease virus (FMDV) vaccines have been used widely to control foot-and-mouth disease (FMD). However, the virions (146S) of this virus are easily dissociated into pentamer subunits (12S), which limits the immune protective efficacy of inactivated vaccines when the temperature is higher than 30 °C. A cold-chain system can maintain the quality of the vaccines, but such systems are usually not reliable in limited-resource settings. Thus, it is imperative to improve the thermostability of vaccine strains to guarantee the quality of the vaccines. In this study, four recombinant FMDV strains containing single or multiple amino acid substitutions in the structural proteins were rescued using a previously constructed FMDV type O full-length infectious clone (pO/DY-VP1). We found that single or multiple amino acid substitutions in the structural proteins affected viral replication to different degrees. Furthermore, the heat and acid stability of the recombinant viruses was significantly increased when compared with the parental virus. Three thermally stable recombinant viruses (rHN/DY-VP1Y2098F, rHN/DY-VP1V2090A-S2093H, and rHN/DY-VP1V2090A-S2093H-Y2098F) were prepared as inactivated vaccines to immunize pigs. Blood samples were collected every week to prepare sera, and a virus neutralization test showed that the substitutions S2093H and Y2098F, separately or in combination, did not affect the immunogenicity of the virus, but the Y2098F mutation increased the thermostability significantly (p < 0.05). Therefore, the rHN/DY-VP1Y2098F mutant should be considered for use in future vaccines.

Selection of Vaccine Candidate for Foot-and-Mouth Disease Virus Serotype O Using a Blocking Enzyme-Linked Immunosorbent Assay.

Foot-and-mouth disease (FMD) is a highly contagious disease and one of the most economically important diseases of livestock. Vaccination is an important measure to control FMD and selection of appropriate vaccine strains is crucial. The objective of this study was to select a vaccine candidate and to evaluate the potential of a blocking ELISA for detecting neutralizing antibodies (NA-ELISA) in vaccine strain selection. Binary ethylenimine inactivated vaccines, prepared from four representative circulating strains (FMDV O/Mya/98, SCGH/CHA/2016, O/Tibet/99, and O/XJ/CHA/2017) belonging to four lineages within three different topotypes of FMD virus (FMDV) serotype O in China, were used to vaccinate cattle (12-13 animals for each strain), sheep (12-13 animals for each strain), and pigs (10 animals for each strain). The results of immunogenicity comparison showed that O/XJ/CHA/2017 exhibited the highest immunogenicity among the four strains in pigs, cattle, and sheep both by NA-ELISA and virus neutralizing test (VNT). Cross-neutralization analysis indicated that O/XJ/CHA/2017 displayed broad antigen spectrum and was antigenically matched with other three representative strains both by NA-ELISA and VNT. In addition, A significant correlation (p < 0.0001) was observed between the NA-ELISA titers and the VNT titers for four representative strains. The results showed that O/XJ/CHA/2017 was a promising vaccine strain candidate and NA-ELISA was comparable to VNT in neutralizing antibodies detection and could be used as the reference test system for vaccine strain selection.

[Development of a sandwich ELISA for detecting 3AB non-structural protein of foot-and-mouth disease virus].

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.

Single Amino Acid Substitutions Surrounding the Icosahedral Fivefold Symmetry Axis Are Critical for Alternative Receptor Usage of Foot-and-Mouth Disease Virus.

The integrins function as the primary receptor molecules for the pathogenic infection of foot-and-mouth disease virus (FMDV) in vivo, while the acquisition of a high affinity for heparan sulfate (HS) of some FMDV variants could be privileged to facilitate viral infection and expanded cell tropism in vitro. Here, we noted that a BHK-adapted Cathay topotype derivative (O/HN/CHA/93tc) but not its genetically engineered virus (rHN), was able to infect HS-positive CHO-K1 cells and mutant pgsD-677 cells. There were one or three residue changes in the capsid proteins of O/HN/CHA/93tc and rHN, as compared with that of their tissue-originated isolate (O/HN/CHA/93wt). The phenotypic properties of a set of site-directed mutants of rHN revealed that E83K of VP1 surrounding the fivefold symmetry axis was necessary for the integrin-independent infection of O/HN/CHA/93tc. L80 in VP2 was essential for the occurrence of E83K in VP1 during the adaptation of O/HN/CHA/93wt to BHK-21 cells. L80M in VP2 and D138G in VP1 of rHN was deleterious, which could be compensated by K83R of VP1 for restoring an efficient infection of integrin-negative CHO cell lines. These might have important implications for understanding the molecular and evolutionary mechanisms of the recognition and binding of FMDV with alternative cellular receptors.

Identification of the largest non-essential regions of the C-terminal portion in 3A protein of foot-and-mouth disease virus for replication in cell culture.

BACKGROUND: Recent study has shown that the C-terminal portion of 3A (amino acids (aa) 81-153) is not essential for foot-and-mouth disease virus replication in cell culture, however, the complete C-terminal portion (aa 77-153) of 3A is highly variable and prone to occur deletions and mutations, therefore, we presume that this region plays a very limited role and probablely is completely nonessential for virus viability. METHODS: In this study, to identify the largest non-essential region of the C-terminal portion in 3A for FMDV viability, several deletions containing aa 80-153, 77-153 and 76-153 of 3A protein were introduced into an FMDV full-length infectious cDNA clone pOFS by the overlapping extension PCR. Additionally, to explore the importance of the highly conserved residue 76 L of 3A for the FMDV of Cathay topotype, two mutants containing 3A L76I and 3A L76V were generated based on the 3A deletion mutant by point mutation. We also introduced the enhanced green fluorescent protein (eGFP) into one of the 3A deletion mutants by the extension PCR to investigate the genetic flexibility of 3A to express foreign genes. All linearized full plasmids were transfected into BSR/T7 cells to rescue infectious foot-and-mouth disease viruses. The rescused viruses were analyzed by RT-PCR, nucleotide sequencing, immunofluorescence assay and western blot and were characterized by plaque assays and one-step growth kinetics. RESULTS: The results demonstrated that the deletion of aa 80-153 and aa 77-153 and the substitutions of 3A L76I and 3A L76V did not affect the production of infectious virus, while the fusion of the eGFP gene to the C-terminus of 3A resulted in nonviable FMDV. CONCLUSIONS: Our results firstly reported that the aa 77-153 rather than aa 81-153 of 3A protein was dispensable for FMDV replication in cell culture. This study is of great significance for development of FMD marker vaccine and foreign gene expression in the future.

Modification of the second translation initiation site restricts the replication of foot-and-mouth disease virus in PK-15 cells.

The translation initiation of foot-and-mouth disease virus (FMDV) occurs at two alternative initiation sites (Lab AUG and Lb AUG). Usually, the Lb AUG is more favorably used to initiate protein synthesis than the Lab AUG. To explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, this initiation codon was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA). Fortunately, the modifications did not prevent viral viability but influenced replication characteristics of some FMDV mutants in a cell-specific manner, as was shown by the similar replication in BHK-21 cells and delayed growth kinetics in PK-15 cells. This attenuated phenotype of FMDV mutants in PK-15 cells was found to be correlated with reduced abilities to cleave eIF4GI and suppress interference (IFN) expression. As leader (L) protein was reported to be responsible for eIF4GI cleavage and inhibition of IFN expression, the in vivo L protein synthesis was examined during the infection of FMDV mutants. Our results showed that not only the total yield of L proteins was severely influenced but also the individual yield of L protein was seen to be affected, which implied that both the relative usage of the two initiation sites and overall translation efficiency were changed by Lb AUG modifications. In addition, the in vitro translation activity was also negatively regulated by Lb AUG mutations. Collectively, these findings suggested that the restricted replications of Lb AUG-modified FMDVs were related to the delayed eIF4GI cleavage and decreased ability to block IFN expression but were mainly determined by the inefficient translation initiation. FMDVs precisely with modifications of Lb AUG initiation codon may represent safer seed viruses for vaccine production. KEY POINTS: • The polyprotein translation of FMDV initiates at two alternative initiation sites (Lab AUG and Lb AUG). In order to explore the effect of Lb AUG on FMDV replication and obtain FMDV with restricted replication, the Lb initiation AUG was mutated to a variety of non-AUG codons (UGG, AUC, CUG, and AAA), and four FMDV mutants with Lb AUG modification were generated. • We found that partial FMDV mutants grew almost as well as WT virus in BHK-21 cells, a typical cell line used for FMD vaccine production, but displayed impaired replication in IFN-competent PK-15 cells. • The attenuation of mutant FMDVs in PK-15 cells was found to be correlated with delayed eIF4GI cleavage and decreased ability to block IFN expression. • We proved that the attenuated phenotype of Lb AUG-modified FMDVs was mainly determined by the inefficient translation initiation, as demonstrated by the decrease of total yield of L proteins and individual production of L protein. • We successfully generated genetically engineered FMDV with attenuated phenotype. The approach of precise engineering of FMDV with the modification of initiation codon provides a safe platform to produce inactivated antigen vaccines.

Cellular Vimentin Interacts with Foot-and-Mouth Disease Virus Nonstructural Protein 3A and Negatively Modulates Viral Replication.

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.