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.

Physiological ß-amyloid clearance by the liver and its therapeutic potential for Alzheimer's disease.

Cerebral amyloid-ß (Aß) accumulation due to impaired Aß clearance is a pivotal event in the pathogenesis of Alzheimer's disease (AD). Considerable brain-derived Aß is cleared via transporting to the periphery. The liver is the largest organ responsible for the clearance of metabolites in the periphery. Whether the liver physiologically clears circulating Aß and its therapeutic potential for AD remains unclear. Here, we found that about 13.9% of Aß42 and 8.9% of Aß40 were removed from the blood when flowing through the liver, and this capacity was decreased with Aß receptor LRP-1 expression down-regulated in hepatocytes in the aged animals. Partial blockage of hepatic blood flow increased Aß levels in both blood and brain interstitial fluid. The chronic decline in hepatic Aß clearance via LRP-1 knockdown specific in hepatocytes aggravated cerebral Aß burden and cognitive deficits, while enhancing hepatic Aß clearance via LRP-1 overexpression attenuated cerebral Aß deposition and cognitive impairments in APP/PS1 mice. Our findings demonstrate that the liver physiologically clears blood Aß and regulates brain Aß levels, suggesting that a decline of hepatic Aß clearance during aging could be involved in AD development, and hepatic Aß clearance is a novel therapeutic approach for AD.

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.

Inhibiting α1-adrenergic receptor signaling pathway ameliorates AD-type pathologies and behavioral deficits in APPswe/PS1 mouse model.

The role of α1 adrenergic receptors (α1-ARs) signaling pathway in the pathogenesis of Alzheimer's disease (AD) has rarely been investigated. Clarifying the pathophysiological functions of α1-ARs in the AD brain is helpful for better understanding the pathogenesis and screening novel therapeutic targets of AD. This study included 2 arms of in vivo investigations: 1) 6-month-old female APPswe/PS1 mice were intravenously treated with AAV-PHP.eB-shRNA (α1-ARs)-GFP or AAV-PHP.eB-GFP for 3 months. 2) 3-month-old female APPswe/PS1 mice were daily treated with 0.5 mg/kg terazosin or an equal volume of saline for 6 months. SH-SY5Y cell lines bearing human amyloid precursor protein were treated with terazosin or saline for investigating possible mechanisms. α1-ARs knockdown mice exhibited improved behavioral performances in comparison with control mice. α1-ARs knockdown mice had significantly lower brain amyloid burden, as reflected by soluble Aß species, compact and total Aß plaques, than control mice. α1-ARs inhibitor terazosin substantially reduced Aß deposition, attenuated downstream pathologies including tau hyperphosphorylation, glial activation, neuronal loss, synaptic dysfunction et al., and rescued behavioral deficits in APPswe/PS1 mice. In vitro investigation demonstrated that α1-ARs inhibition down-regulated BACE1 expression, and promoted ser9 phosphorylation of GSK-3ß, thus reducing Aß production. This study indicates that inhibition of α1-ARs signaling pathway might represent a promising therapeutic strategy for AD.

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.

Blood cell-produced amyloid-ß induces cerebral Alzheimer-type pathologies and behavioral deficits.

It is traditionally believed that cerebral amyloid-beta (Aß) deposits are derived from the brain itself in Alzheimer's disease (AD). Peripheral cells such as blood cells also produce Aß. The role of peripherally produced Aß in the pathogenesis of AD remains unknown. In this study, we established a bone marrow transplantation model to investigate the contribution of blood cell-produced Aß to AD pathogenesis. We found that bone marrow cells (BMCs) transplanted from APPswe/PS1dE9 transgenic mice into wild-type (Wt) mice at 3 months of age continuously expressed human Aß in the blood, and caused AD phenotypes including Aß plaques, cerebral amyloid angiopathy (CAA), tau hyperphosphorylation, neuronal degeneration, neuroinflammation, and behavioral deficits in the Wt recipient mice at 12 months after transplantation. Bone marrow reconstitution in APPswe/PS1dE9 mice with Wt-BMCs at 3 months of age reduced blood Aß levels, and alleviated brain Aß burden, neuronal degeneration, neuroinflammation, and behavioral deficits in the AD model mice at 12 months after transplantation. Our study demonstrated that blood cell-produced Aß plays a significant role in AD pathogenesis, and the elimination of peripheral production of Aß can decrease brain Aß deposition and represents a novel therapeutic approach for AD.

Physiological clearance of amyloid-beta by the kidney and its therapeutic potential for Alzheimer's disease.

Amyloid-ß (Aß) accumulation in the brain is a pivotal event in the pathogenesis of Alzheimer's disease (AD), and its clearance from the brain is impaired in sporadic AD. Previous studies suggest that approximately half of the Aß produced in the brain is cleared by transport into the periphery. However, the mechanism and pathophysiological significance of peripheral Aß clearance remain largely unknown. The kidney is thought to be responsible for Aß clearance, but direct evidence is lacking. In this study, we investigated the impact of unilateral nephrectomy on the dynamic changes in Aß in the blood and brain in both humans and animals and on behavioural deficits and AD pathologies in animals. Furthermore, the therapeutic effects of the diuretic furosemide on Aß clearance via the kidney were assessed. We detected Aß in the kidneys and urine of both humans and animals and found that the Aß level in the blood of the renal artery was higher than that in the blood of the renal vein. Unilateral nephrectomy increased brain Aß deposition; aggravated AD pathologies, including Tau hyperphosphorylation, glial activation, neuroinflammation, and neuronal loss; and aggravated cognitive deficits in APP/PS1 mice. In addition, chronic furosemide treatment reduced blood and brain Aß levels and attenuated AD pathologies and cognitive deficits in APP/PS1 mice. Our findings demonstrate that the kidney physiologically clears Aß from the blood, suggesting that facilitation of Aß clearance via the kidney represents a novel potential therapeutic approach for AD.

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.

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.

Non-destructive detection of blueberry skin pigments and intrinsic fruit qualities based on deep learning.

BACKGROUND: This paper proposes a novel method to improve accuracy and efficiency in detecting the quality of blueberry fruit, taking advantage of deep learning in classification tasks. We first collected 'Tifblue' blueberries at seven different stages of maturity (10-70 days after full bloom) and measured the pigments of the blueberry skin and the total sugar and the total acid of the pulp. We then established a skin pigment contents prediction network (SPCPN), based on the correlation between the pigments and blueberry pictures, and also a fruit intrinsic qualities prediction network (FIQPN), based on the correlation between the pigments and fruit qualities. Finally, the SPCPN and FIQPN were consolidated into the blueberry quality parameters prediction network (BQPPN). RESULTS: The results showed that the anthocyanins in the blueberry skin were significantly correlated with the total sugar, total acid, and sugar / acid ratio of the fruit. After verification, the results also indicated that, for the prediction of anthocyanins, chlorophyll, and the anthocyanin / chlorophyll ratio, the SPCPN network model was found to achieve higher R2 (RMSE) values of 0.969 (0.139), 0.955 (0.005), 0.967 (15.4), respectively. The FIQPN network model was also able to evaluate the value of total sugar (R2 = 0.940, RMSE = 4.905), total acid (R2 = 0.930, RMSE = 2.034), and the sugar / acid ratio (R2 = 0.973, RMSE = 0.580). CONCLUSION: The above results indicated the potential for utilizing deep learning technology to predict the quality indicators of blueberry before harvesting. © 2020 Society of Chemical Industry.

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.

Emerging roles of class I PI3K inhibitors in modulating tumor microenvironment and immunity.

Immune system-mediated tumor killing has revolutionized anti-tumor therapies, providing long-term and durable responses in some patients. The phosphoinositide 3-kinase (PI3K) pathway controls multiple biological processes and is frequently dysregulated in malignancies. Enormous efforts have been made to develop inhibitors against class I PI3K. Notably, with the increasing understanding of PI3K, it has been widely accepted that PI3K inhibition not only restrains tumor progression, but also reshapes the immunosuppressive tumor microenvironment. In this review, we focus on the pivotal roles of class I PI3Ks in adaptive and innate immune cells, as well as other stromal components. We discuss the modulation by PI3K inhibitors of the tumor-supportive microenvironment, including eliminating the regulatory immune cells, restoring cytotoxic cells or regulating angiogenesis. The potential combinations of PI3K inhibitors with other therapies to enhance the anti-tumor immunity are also described.

Discovery and characterization of natural products as novel indoleamine 2,3-dioxygenase 1 inhibitors through high-throughput screening.

Indoleamine 2,3-dioxygenase 1 (IDO1) is emerging as a promising therapeutic target for the treatment of malignant tumors characterized by dysregulated tryptophan metabolism. However, the antitumor efficacy of existing small-molecule IDO1 inhibitors is still unsatisfactory, and the underlying mechanism remains largely undefined. To identify novel IDO1 inhibitors, an in-house natural product library of 2000 natural products was screened for inhibitory activity against recombinant human IDO1. High-throughput fluorescence-based screening identified 79 compounds with inhibitory activity > 30% at 20 µM. Nine natural products were further confirmed to inhibit IDO1 activity by > 30% using Ehrlich's reagent reaction. Compounds 2, 7, and 8 were demonstrated to inhibit IDO1 activity in a cellular context. Compounds 2 and 7 were more potent against IDO1 than TDO2 in the enzymatic assay. The kinetic studies showed that compound 2 exhibited noncompetitive inhibition, whereas compounds 7 and 8 were graphically well matched with uncompetitive inhibition. Compounds 7 and 8 were found to bind to the ferric-IDO1 enzyme. Docking stimulations showed that the naphthalene ring of compound 8 formed "T-shaped" π-π interactions with Phe-163 and that the 6-methyl-naphthalene group formed additional hydrophobic interactions with IDO1. Compound 8 was identified as a derivative of tanshinone, and preliminary SAR analysis indicated that tanshinone derivatives may be promising hits for the development of IDO1 inhibitors. This study provides new clues for the discovery of IDO1/TDO2 inhibitors with novel scaffolds.

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.