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

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

Structures of Foot-and-mouth Disease Virus with neutralizing antibodies derived from recovered natural host reveal a mechanism for cross-serotype neutralization.

The development of a universal vaccine against foot-and-mouth disease virus (FMDV) is hindered by cross-serotype antigenic diversity and by a lack of knowledge regarding neutralization of the virus in natural hosts. In this study, we isolated serotype O-specific neutralizing antibodies (NAbs) (F145 and B77) from recovered natural bovine hosts by using the single B cell antibody isolation technique. We also identified a serotype O/A cross-reacting NAb (R50) and determined virus-NAb complex structures by cryo-electron microscopy at near-atomic resolution. F145 and B77 were shown to engage the capsid of FMDV-O near the icosahedral threefold axis, binding to the BC/HI-loop of VP2. In contrast, R50 engages the capsids of both FMDV-O and FMDV-A between the 2- and 5-fold axes and binds to the BC/EF/GH-loop of VP1 and to the GH-loop of VP3 from two adjacent protomers, revealing a previously unknown antigenic site. The cross-serotype neutralizing epitope recognized by R50 is highly conserved among serotype O/A. These findings help to elucidate FMDV neutralization by natural hosts and provide epitope information for the development of a universal vaccine for cross-serotype protection against FMDV.

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.

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.

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization.

Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the ßB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3E70 forms a key component of neutralizing epitopes, and the determination of the VP3E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

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.

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.

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.

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.

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.

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.

Engineering Responses to Amino Acid Substitutions in the VP0- and VP3-Coding Regions of PanAsia-1 Strains of Foot-and-Mouth Disease Virus Serotype O.

The presence of sequence divergence through adaptive mutations in the major capsid protein VP1, and also in VP0 (VP4 and VP2) and VP3, of foot-and-mouth disease virus (FMDV) is relevant to a broad range of viral characteristics. To explore the potential role of isolate-specific residues in the VP0 and VP3 coding regions of PanAsia-1 strains in genetic and phenotypic properties of FMDV, a series of recombinant full-length genomic clones were constructed using Cathay topotype infectious cDNA as the original backbone. The deleterious and compensatory effects of individual amino acid substitutions at positions 4008 and 3060 and in several different domains of VP2 illustrated that the chain-based spatial interaction patterns of VP1, VP2, and VP3 (VP1-3), as well as between the internal VP4 and the three external capsid proteins of FMDV, might contribute to the assembly of eventually viable viruses. The Y2079H site-directed mutants dramatically induced a decrease in plaque size on BHK-21 cells and viral pathogenicity in suckling mice. Remarkably, the 2079H-encoding viruses displayed a moderate increase in acid sensitivity correlated with NH4Cl resistance compared to the Y2079-encoding viruses. Interestingly, none of all the 16 rescued viruses were able to infect heparan sulfate-expressing CHO-K1 cells. However, viral infection in BHK-21 cells was facilitated by utilizing non-integrin-dependent, heparin-sensitive receptor(s) and replacements of four uncharged amino acids at position 3174 in VP3 of FMDV had no apparent influence on heparin affinity. These results provide particular insights into the correlation of evolutionary biology with genetic diversity in adapting populations of FMDV.IMPORTANCE The sequence variation within the capsid proteins occurs frequently in the infection of susceptible tissue cultures, reflecting the high levels of genetic diversity of FMDV. A systematic study for the functional significance of isolate-specific residues in VP0 and VP3 of FMDV PanAsia-1 strains suggested that the interaction of amino acid side chains between the N terminus of VP4 and several potential domains of VP1-3 had cascading effects on the viability and developmental characteristics of progeny viruses. Y2079H in VP0 of the indicated FMDVs could affect plaque size and pathogenicity, as well as acid sensitivity correlated with NH4Cl resistance, whereas there was no inevitable correlation in viral plaque and acid-sensitive phenotypes. The high affinity of non-integrin-dependent FMDVs for heparin might be explained by the differences in structures of heparan sulfate proteoglycans on the surfaces of different cell lines. These results may contribute to our understanding of the distinct phenotypic properties of FMDV in vitro and in vivo.

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.

Engineering viable foot-and-mouth disease viruses with increased acid stability facilitate the development of improved vaccines.

Foot-and-mouth disease virus (FMDV), the most acid-unstable virus among picornaviruses, tends to disassemble into pentamers at pH values slightly below neutrality. However, the structural integrity of intact virion is one of the most important factors that influence the induction of a protective antibody response. Thus, improving the acid stability of FMDV is required for the efficacy of vaccine preparations. According to the previous studies, a single substitution or double amino acid substitutions (VP1 N17D, VP2 H145Y, VP2 D86H, VP3 H142D, VP3 H142G, and VP1 N17D + VP2 H145Y) in the capsid were introduced into the full-length infectious clone of type O FMDV vaccine strain O/HN/CHN/93 to develop seed FMDV with improved acid stability. After the transfection into BSR/T7 cells of constructed plasmids, substitution VP1 N17D or VP2 D86H resulted in viable and genetically stable FMDVs, respectively. However, substitution VP2 H145Y or VP1 N17D + VP2 H145Y showed reverse mutation and additional mutations, and substitution VP3 H141G or VP3 H141D prevented viral viability. We found that substitution VP1 N17D or VP2 D86H could confer increased acid resistance, alkali stability, and thermostability on FMDV O/HN/CHN/93, whereas substitution VP1 N17D was observed to lead to a decreased replication ability in BHK-21 cells and mildly impaired virulence in suckling mice. In contrast, substitution VP2 D86H had no negative effect on viral infectivity. These results indicated that the mutant rD86H carrying substitution VP2 D86H firstly reported by us could be more adequate for the development of inactivated FMD vaccines with enhanced acid stability.

Knockout of mitochondrial voltage-dependent anion channel type 3 increases reactive oxygen species (ROS) levels and alters renal sodium transport.

It has been suggested that voltage-dependent anion channels (VDACs) control the release of superoxide from mitochondria. We have previously shown that reactive oxygen species (ROS) such as superoxide (O2̇̄) and hydrogen peroxide (H2O2) stimulate epithelial sodium channels (ENaCs) in sodium-transporting epithelial tissue, including cortical collecting duct (CCD) principal cells. Therefore, we hypothesized that VDACs could regulate ENaC by modulating cytosolic ROS levels. Herein, we find that VDAC3-knockout(KO) mice can maintain normal salt and water balance on low-salt and high-salt diets. However, on a high-salt diet for 2 weeks, VDAC3-KO mice had significantly higher systolic blood pressure than wildtype mice. Consistent with this observation, after a high-salt diet for 2 weeks, ENaC activity in VDAC3-KO mice was significantly higher than wildtype mice. EM analysis disclosed a significant morphological change of mitochondria in the CCD cells of VDAC3-KO mice compared with wildtype mice, which may have been caused by mitochondrial superoxide overload. Of note, compared with wildtype animals, ROS levels in VDAC3-KO animals fed a normal or high-salt diet were consistently and significantly increased in renal tubules. Both the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL) and the mitochondrial ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mito-TEMPO) could reverse the effect of high-salt on ENaC activity and systolic blood pressure in the VDAC3-KO mice. Mito-TEMPO partially correct the morphological changes in VDAC3-KO mice. Our results suggest that knocking out mitochondrial VDAC3 increases ROS, alters renal sodium transport, and leads to hypertension.

Lack of urea transporters, UT-A1 and UT-A3, increases nitric oxide accumulation to dampen medullary sodium reabsorption through ENaC.

Although the role of urea in urine concentration is known, the effect of urea handling by the urea transporters (UTs), UT-A1 and UT-A3, on sodium balance remains elusive. Serum and urinary sodium concentration is similar between wild-type mice (WT) and UT-A3 null (UT-A3 KO) mice; however, mice lacking both UT-A1 and UT-A3 (UT-A1/A3 KO) have significantly lower serum sodium and higher urinary sodium. Protein expression of renal sodium transporters is unchanged among all three genotypes. WT, UT-A3 KO, and UT-A1/A3 KO acutely respond to hydrochlorothiazide and furosemide; however, UT-A1/A3 KO fail to show a diuretic or natriuretic response following amiloride administration, indicating that baseline epithelial Na+ channel (ENaC) activity is impaired. UT-A1/A3 KO have more ENaC at the apical membrane than WT mice, and single-channel analysis of ENaC in split-open inner medullary collecting duct (IMCD) isolated in saline shows that ENaC channel density and open probability is higher in UT-A1/A3 KO than WT. UT-A1/A3 KO excrete more urinary nitric oxide (NO), a paracrine inhibitor of ENaC, and inner medullary nitric oxide synthase 1 mRNA expression is ~40-fold higher than WT. Because endogenous NO is unstable, ENaC activity was reassessed in split-open IMCD with the NO donor PAPA NONOate [1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine)], and ENaC activity was almost abolished in UT-A1/A3 KO. In summary, loss of both UT-A1 and UT-A3 (but not UT-A3 alone) causes elevated medullary NO production and salt wasting. NO inhibition of ENaC, despite elevated apical accumulation of ENaC in UT-A1/A3 KO IMCD, appears to be the main contributor to natriuresis in UT-A1/A3 KO mice.

Implication of Broadly Neutralizing Bovine Monoclonal Antibodies in the Development of an Enzyme-Linked Immunosorbent Assay for Detecting Neutralizing Antibodies against Foot-and-Mouth Disease Virus Serotype O.

Vaccination with inactivated vaccines is still the main measure to control foot-and-mouth disease (FMD) in areas where the disease is endemic, and the level of neutralizing antibody in vaccinated animals is directly related to their protection against virus challenge. Currently, neutralizing antibody is mainly detected using the virus neutralization test (VNT) based on cell culture, which is laborious and time-consuming and requires restrictive biocontainment facilities. In this study, two broadly neutralizing antibodies (bnAbs), E46 and F128, were successfully produced using techniques for the isolation of single B cells from peripheral blood mononuclear cells (PBMCs) from bovines sequentially immunized with three topotypes of foot-and-mouth disease virus (FMDV) serotype O. Based on these bnAbs, a blocking enzyme-linked immunosorbent assay (ELISA) for detecting neutralizing antibodies (NA-ELISA) against FMDV serotype O was developed. The specificity and sensitivity of the test were estimated to be 99.21% and 100%, respectively. A significant correlation (P < 0.01) was observed between the NA-ELISA titers and the VNT titers for all sera from vaccinated animals and for all tested strains, suggesting that the NA-ELISA could detect neutralizing antibodies against FMDV serotype O strains of wide antigenic and molecular diversity and could be used for the evaluation of protective immunity.

Recombination of host cell mRNA with the Asia 1 foot-and-mouth disease virus genome in cell suspension culture.

Foot-and-mouth disease virus (FMDV) exhibits high mutation rates during replication. In this study, an isolate of FMDV serotype Asia-1 was serially passaged in a BHK-21 cell monolayer and then adapted to serum-free BHK-21 cell suspension culture to produce a seed virus for production of an inactivated vaccine. Analysis of the sequence encoding the structural proteins of the virus at various passages showed the presence of overlapping peaks in sequencing electropherograms after nucleotide 619 of VP1 in viruses recovered from the fourth passage in suspension culture, suggesting the possible introduction of an insertion or deletion into this portion of the viral genome of our seed virus stock. To evaluate this phenomenon, a virus designated "Vac-Asia1-VDLV", was isolated by plaque purification from the tenth passage in suspension culture. Sequencing results showed that a 12-nt-long exogenous sequence was inserted into the 3' end of the VP1 coding region at the position where the original overlapping peaks were identified. Analysis of the host cell transcriptome showed that the 12-nt sequence was identical to a highly expressed sequence in BHK-21 cells, strongly suggesting that recombination between the FMDV genome and host cell mRNA produced the recombinant virus. A growth curve showed that the virus with the 12-nt insertion reached a peak earlier than the parental strain and that this virus had acquired the ability to bind to the cell surface by a mechanism that was not dependent on integrin or the heparan sulfate receptor. This novel pathogen-host cell recombination event is discussed in terms of the mechanism of viral RNA replication and the phenotypic constraints of FMDV biology and evolution.

The molecular characteristic analysis of PRRSV GSWW/2015 strain and its pathogenicity to pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a severe disease, causing great economic losses to the pig industry. The causative agent, porcine reproductive and respiratory syndrome virus (PRRSV) is highly variable. Since the emergence of highly pathogenic PRRSV (HP-PRRSV) in China in 2006, this virus strain has undergone extensive variation. To investigate the genetic variation and pathogenicity of currently isolated PRRSV GSWW/2015 strain, its whole genome was sequenced and analyzed for the specific variation in NSP2, GP3 and GP5 regions. Pigs were challenged with the isolated virus to investigate its pathogenicity. METHODS: The PRRSV GSWW/2015 strain was isolated by seeding the viral material in Marc-145 cells. The virus specific cytopathic effect (CPE) was confirmed by indirect immunofluorescent assay (IFA) and PCR to detect the virus protein and RNA. Nine pairs of primers were designed to obtain the complete genome by PCR. All PCR fragments were cloned into T-vector for sequencing. The genetic variation of GSWW/2015 strain was analyzed by multiple sequence alignments. Nineteen PRRSV-free piglets were intranasally challenged with 108 copies of GSWW virus, while seven piglets were housed together as contact-infected control. Clinical signs were recorded daily after challenge. Blood samples were obtained every week and the viral titer was detected by quantitative real-time PCR (qRT-PCR). The PRRSV specific antibody was detected by LSI ELISA kit. RESULTS: The complete genome of PRRSV GSWW/2015 strain (GenBank accession number KX767091) was obtained. The whole genome of this strain shares 88.5 and 60.6% identity with VR-2332 and LV respectively, indicating that it belongs to the North American type (NA-type). Sequence alignments revealed that GSWW/2015 strain has a discontinuous deletion of 30 amino acids in NSP2, which is similar with HP-PRRSV. Some amino acids mutations can be observed in antigenic epitope regions of GP3 and GP5 compared with earlier strains of HP-PRRSV. Some piglets showed typical clinical signs of PRRSV after challenge. Only four pigs showed viremia within 3 days after challenge, most pigs showed peaked viremia after 21-28 days including 7 contact-infected pigs. Two pigs were detected to be positive for antibody to PRRSV at 14 days post infection (DPI), and 11 pigs (11/26) show seroconversion for PRRSV at 49 DPI. Twelve piglets died of PRRSV infection within two months. CONCLUSIONS: The genome of PRRSV GSWW/2015 strain shows the features of HP-PRRSV with 30 discontinuous amino acids deletion in NSP2 and some new amino acid mutations in epitope regions of GP5 and GP3, which might alter the antigenicity of the virus. Furthermore, the virus showed high virulence to piglets as reported in HP-PRRSV, and induced long-lasting viremia and low level of antibody responses. This work further enriched our knowledge on PRRSV evolution and pathogenicity.