A universal design of restructured dimer antigens: Development of a superior vaccine against the paramyxovirus in transgenic rice.

The development of vaccines, which induce effective immune responses while ensuring safety and affordability, remains a substantial challenge. In this study, we proposed a vaccine model of a restructured "head-to-tail" dimer to efficiently stimulate B cell response. We also demonstrate the feasibility of using this model to develop a paramyxovirus vaccine through a low-cost rice endosperm expression system. Crystal structure and small-angle X-ray scattering data showed that the restructured hemagglutinin-neuraminidase (HN) formed tetramers with fully exposed quadruple receptor binding domains and neutralizing epitopes. In comparison with the original HN antigen and three traditional commercial whole virus vaccines, the restructured HN facilitated critical epitope exposure and initiated a faster and more potent immune response. Two-dose immunization with 0.5 µg of the restructured antigen (equivalent to one-127th of a rice grain) and one-dose with 5 µg completely protected chickens against a lethal challenge of the virus. These results demonstrate that the restructured HN from transgenic rice seeds is safe, effective, low-dose useful, and inexpensive. We provide a plant platform and a simple restructured model for highly effective vaccine development.

PRRSV non-structural protein 5 inhibits antiviral innate immunity by degrading multiple proteins of RLR signaling pathway through FAM134B-mediated ER-phagy.

Viruses employ various evasion strategies to establish prolonged infection, with evasion of innate immunity being particularly crucial. Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant pathogen in swine industry, characterized by reproductive failures in sows and respiratory distress in pigs of all ages, leading to substantial economic losses globally. In this study, we found that the non-structural protein 5 (Nsp5) of PRRSV antagonizes innate immune responses via inhibiting the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs), which is achieved by degrading multiple proteins of RIG-I-like receptor (RLR) signaling pathway (RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7). Furthermore, we showed that PRRSV Nsp5 is located in endoplasmic reticulum (ER), where it promotes accumulation of RLR signaling pathway proteins. Further data demonstrated that Nsp5 activates reticulophagy (ER-phagy), which is responsible for the degradation of RLR signaling pathway proteins and IFN-I production. Mechanistically, Nsp5 interacts with one of the ER-phagy receptor family with sequence similarity 134 member B (FAM134B), promoting the oligomerization of FAM134B. These findings elucidate a novel mechanism by which PRRSV utilizes FAM134B-mediated ER-phagy to elude host antiviral immunity.IMPORTANCEInnate immunity is the first line of host defense against viral infections. Therefore, viruses developed numerous mechanisms to evade the host innate immune responses for their own benefit. PRRSV, one of the most important endemic swine viruses, poses a significant threat to the swine industry worldwide. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to degrade multiple proteins of RLR signaling pathways, which play important roles in IFN-I production. Moreover, FAM134B-mediated ER-phagy was further proved to be responsible for the protein's degradation. Our study highlights the critical role of ER-phagy in immune evasion of PRRSV to favor replication and provides new insights into the prevention and control of PRRSV.

Pseudorabies Virus Regulates the Extracellular Translocation of Annexin A2 To Promote Its Proliferation.

Pseudorabies virus (PRV) infection causes enormous economic losses to the pork industry and severe health consequences in many hosts. Annexin A2 (ANXA2) is a membrane-associated protein with various intracellular functions associated with many viral infections. However, the role of ANXA2 in alphaherpesvirus replication is still not explored. In the present study, we identified the interaction between ANXA2 and PRV US3. The deficiency of ANXA2 significantly restricted PRV proliferation. PRV infection or US3 overexpression led to ANXA2 extracellular translocation. Furthermore, we confirmed that PRV or US3 could lead to the phosphorylation of the Tyr23 ANXA2 and Tyr419 Src kinase, which was associated with the ANXA2 cell surface transposition. US3 can also bind to Src in an ANXA2-independent manner and enhance the interaction between Src and ANXA2. Additionally, inhibitors targeting ANXA2 (A2ti-1) or Src (PP2) could remarkably inhibit PRV propagation in vitro and protect mice from PRV infection in vivo. Collectively, our findings broaden our understanding of the molecular mechanisms of ANXA2 in alphaherpesvirus pathogenicity and suggest that ANXA2 is a potential therapeutic target for treating alphaherpesvirus-induced infectious diseases. IMPORTANCE PRV belongs to the alphaherpesvirus and has recently re-emerged in China, causing severe economic losses. Recent studies also indicate that PRV may pose a potential public health challenge. ANXA2 is a multifunctional calcium- and lipid-binding protein implicated in immune function, multiple human diseases, and viral infection. Herein, we found that ANXA2 was essential to PRV efficient proliferation. PRV infection resulted in the extracellular translocation of ANXA2 through phosphorylation of ANXA2 and Src. ANXA2 and Src formed a complex with PRV US3. Importantly, inhibitors targeting ANXA2 or Src prevented PRV infection in vitro and in vivo. Therefore, our studies reveal a novel strategy by which alphaherpesvirus modifies ANXA2 to promote its replication and highlight ANXA2 as a target in developing novel promising antivirus agents in viral therapy.

Potential mechanisms mediating PM2.5-induced alterations of H3N2 influenza virus infection and cytokine production in human bronchial epithelial cells.

Exposure to particulate matter (PM) has been associated with increased hospital admissions for influenza. Airway epithelial cells are a primary target for inhaled environmental insults including fine PM (PM2.5) and influenza viruses. The potentiation of PM2.5 exposure on the effects of influenza virus on airway epithelial cells has not been adequately elucidated. In this study, the effects of PM2.5 exposure on influenza virus (H3N2) infection and downstream modulation of inflammation and antiviral immune response were investigated using a human bronchial epithelial cell line, BEAS-2B. The results showed that PM2.5 exposure alone increased the production of pro-inflammatory cytokines including interleukin-6 (IL-6) and IL-8 but decreased the production of the antiviral cytokine interferon-ß (IFN-ß) in BEAS-2B cells while H3N2 exposure alone increased the production of IL-6, IL-8, and IFN-ß. Importantly, prior exposure to PM2.5 enhanced subsequent H3N2 infectivity, expression of viral hemagglutinin protein, as well as upregulation of IL-6 and IL-8, but reduced H3N2-induced IFN-ß production. Pre-treatment with a pharmacological inhibitor of nuclear factor-κB (NF-κB) suppressed pro-inflammatory cytokine production induced by PM2.5, H3N2, as well as PM2.5-primed H3N2 infection. Moreover, antibody-mediated neutralization of Toll-like receptor 4 (TLR4) blocked cytokine production triggered by PM2.5 or PM2.5-primed H3N2 infection, but not H3N2 alone. Taken together, exposure to PM2.5 alters H3N2-induced cytokine production and markers of replication in BEAS-2B cells, which in turn are regulated by NF-κB and TLR4.

Development of an immunochromatographic strip for rapid detection of H7 subtype avian influenza viruses.

BACKGROUND: H7N9 avian influenza virus (AIV) including highly and low pathogenic viruses have been detected in China since 2013. H7N9 AIV has a high mortality rate after infection in humans, and most human cases have close contacted with poultry in the live poultry market. Therefore, it is necessary to develop a rapid point-of-care testing (POCT) technique for H7N9 AIV detection. METHODS: The H7N9 AIV was inactivated and purified, and was used as the antigen to immunize BALB/c. Twelve H7-HA specific monoclonal antibodies (McAbs) were produced through the hybridoma technique. The McAb 10A8 was conjugated with colloid gold as detecting antibody; McAb 9B6 was dispensed on the nitrocellulose membran as the capture test line and the Goat-anti mouse IgG antibody was dispensed as control line respectively. The immunochromatographic strip was prepared. RESULTS: The analysis of ELISA and virus neutralization test showed that the obtained McAbs specifically recognized H7 HA. Based on the prepared strip, the detection of H7 AIV was achieved within 10 min. No cross-reaction occurred between H7 AIVs and other tested viruses. The detection limit of the strip for H7 was 2.4 log10EID50/0.1 mL for chicken swab samples. CONCLUSION: The McAbs were specific for H7 and the immunochromatographic strip developed in this study was convenient, rapid and reliable for the detection of H7 AIV. The strip could provide an effective method for the rapid and early detection of H7 AIV.

Study on the Dynamic Proliferation of JEV in BHK-21 Cells.

INTRODUCTION: Epidemic Japanese encephalitis is one of the most important zoonotic diseases that cause central nervous system damage. The vaccination has become the most effective and economical measure for its control. Hence, real-time monitoring of Japanese encephalitis virus (JEV) proliferation is crucial to optimize virus inoculation, culturing conditions, and virus harvest time. METHODS: The proliferation dynamics of JEV in BHK-21 cells was studied by combining the established quantitative PCR method with the conventional TCID50 assay in this study. RESULTS: The proliferation curve determined by the 2 methods has a definite parallel relationship, but the quantitative real-time PCR method (4 h) is faster and more sensitive than the TCID50 method (3-4 days). The determination results of TCID50 showed that the highest viral titer was 105.44 TCID50/0.1 mL and 104.86 TCID50/0.1 mL in cell suspension and culture supernate, respectively, while the virus RNA copies reached the peak at 1.0 × 107.5 copies/µL and 1.0 × 105.6 copies/µL in cell suspension and culture supernate, respectively. CONCLUSION: The comprehensive analysis showed that the best time for JEV proliferation in BHK-21 cell was 60 h post infection.

Purification of cell-derived Japanese encephalitis virus by dual-mode chromatography.

Purification of the enveloped virus poses a challenge as one must retain viral infectivity to preserve immunogenicity. The traditional process of virus purification is time-consuming, laborious and hard to scale up. Here, a rapid, simple and extensible laboratory program for the purification of Japanese encephalitis virus (JEV) was developed by using differential centrifugation, ultrafiltration, Sepharose 4 fast flow gel chromatography, and CaptoTM Core 700 chromatography. The entire process recovered 61.64% of the original virus, and the purified virus particles maintained good activity and immunogenicity. The purification process described has potential application in large-scale production of high-purity JEV.

Parasitic Ferromagnetism in Few-Layered Transition-Metal Chalcogenophosphate.

Since the rise of two-dimensional (2D) semiconductors, it seems that electronic devices will soon be upgraded with spintronics, in which the manipulation of spin degree of freedom endows it obvious advantages over conventional charge-based electronics. However, as the most crucial prerequisite for the above-mentioned expectation, 2D semiconductors with adjustable magnetic interaction are still rare, which has greatly hampered the promotion of spintronics. Recently, transition metal phosphates have attracted tremendous interest due to their intrinsic antiferromagnetism and potential applications in spintronics. In the work described herein, parasitic ferromagnetism is achieved for the first time by exfoliating an antiferromagnetic chalcogenophosphate to a few layers. Taking the transition metal chalcogenophosphate Mn2P2S6 as an example, the antiferromagnetic transition at the Néel temperature is completely suppressed, and the magnetic behaviors of the as-obtained few-layered Mn2P2S6 are dominated by parasitic ferromagnetism. We experimentally verify an electron redistribution by which part of the Mn 3d electrons migrate and redistribute on P atoms in few-layered Mn2P2S6 due to the introduced Mn vacancies. The results demonstrated here broaden the tunability of the material's magnetic properties and open up a new strategy to rationally design the magnetic behaviors of 2D semiconductors, which could accelerate the applications of spintronics.

Fine mapping of linear B cell epitopes on capsid protein of porcine circovirus 3.

Porcine circovirus type 3 (PCV3) is an emerging swine pathogen associated with acute porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and multisystemic inflammation. Current evidence shows that PCV3 is spread worldwide, and its high incidence may pose a threat to the global pig industry. Capsid (Cap) protein is the sole structural protein which plays an important role in inducing protective immunity against PCV3 infection. In this study, monoclonal antibodies (mAbs) against Cap protein of PCV3 were produced by the hybridoma technique. Subsequently, 12 serial overlapping peptides (P1 to P12) spanning the entire region of Cap were synthesized to determine the B cell epitope regions using the mAbs. Results from dot-blot and peptide ELISA identified that P3, P9, and P10 were the major B cell antigenic regions. Fine mapping by shorter N- and C-terminal truncated peptides confirmed that the motifs 57NKPWH61, 140KHSRYFT146, and 161QSLFFF166 were linear B cell epitopes, which were highly conserved among different PCV3 strains. Interestingly, we found that the motif 140KHSRYFT146 was highly conserved in all reported types of PCVs (i.e., PCV1, PCV2, PCV3, and PCV4), except for the substitution (Y → K → R) of the first residue. This is the first research to identify B cell epitopes of PCV3 Cap, and these findings may lead to a better understanding of the antibody-antigen interaction and provide some guidance for PCV3 vaccine design.Key points• The recombinant Cap protein of PCV3 was expressed and purified in soluble form. • PCV3 Cap-specific mAbs prepared in this study had no cross-reactivity with PCV1/PCV2 Cap. • This is the first report of three conserved linear B cell epitopes on PCV3 Cap. • The minimal residues of the epitopes were 57-61 aa, 140-146 aa, and 161-166 aa.

A strip test for the optical determination of influenza virus H3 subtype using gold nanoparticle coated polystyrene latex microspheres.

A strip test is described for the optical determination of influenza virus H3 subtype. It utilizes gold nanoparticle (AuNP) coated polystyrene latex microspheres (PS) as the label and a sandwich format. The AuNP and PS particles were linked using monoclonal antibodies against influenza virus as the bridge. Under the optimal conditions, the visual detection limit of the AuNP-PS-based strip test was as low as 1/16 hemagglutination unit (HAU). It was 64 times higher than that of 10 nm (4 HAU) AuNP-based strip tests. Quantitative analysis showed that the detection limit of the AuNP-PS-based strip is 0.016 HAU. The AuNP-PS-based strip test showed no cross-reactivity to the other subtypes (H1, H5, H7, or H9) of influenza viruses. Graphical abstract .

The bovine herpesvirus 1 regulatory proteins, bICP4 and bICP22, are expressed during the escape from latency.

Following acute infection of mucosal surfaces by bovine herpesvirus 1 (BoHV-1), sensory neurons are a primary site for lifelong latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Two viral regulatory proteins (VP16 and bICP0) are expressed within 1 h after calves latently infected with BoHV-1 are treated with dexamethasone. Since the immediate early transcription unit 1 (IEtu1) promoter regulates both BoHV-1 infected cell protein 0 (bICP0) and bICP4 expressions, we hypothesized that the bICP4 protein is also expressed during early stages of reactivation from latency. In this study, we tested whether bICP4 and bICP22, the only other BoHV-1 protein known to be encoded by an immediate early gene, were expressed during reactivation from latency by generating peptide-specific antiserum to each protein. bICP4 and bICP22 protein expression were detected in trigeminal ganglionic (TG) neurons during early phases of dexamethasone-induced reactivation from latency, operationally defined as the escape from latency. Conversely, bICP4 and bICP22 were not readily detected in TG neurons of latently infected calves. In summary, it seems clear that all proteins encoded by known BoHV-1 IE genes (bICP4, bICP22, and bICP0) were expressed during early stages of dexamethasone-induced reactivation from latency.

The evaluation of an immunoperoxidase assay applicable in antiviral drug screening.

Bovine viral diarrhea virus (BVDV) fall into cytopathic (CP) and noncytopathic (NCP) biotypes, based on their ability to kill cultured cells. NCP-BVDV can not be titrated by conventional means as used for CP-BVDV, which has impeded the identification of antiviral drugs targeting NCP-BVDV virus strains. In this study, the application of an immunoperoxidase assay in the screening of antiviral drugs was tested using two known BVDV inhibitors, ribavirin and ammonium chloride (NH4Cl). Phospholipase C inhibitor U73122 was identified to affect BVDV infection by using this immunoperoxidase assay. In addition, the results of immunoperoxidase assay were validated by real-time PCR. Taken together, the immunoperoxidase assay is a useful and versatile method suitable for antiviral drug screening targeting NCP-BVDV.

Comparison of two docking methods for peptide-protein interactions.

BACKGROUND: The importance of peptides in regulatory interactions has caused peptide-protein docking to attract the attention of many researchers. A variety of methods for molecular modeling of peptide-protein docking, such as local search and global search, are currently used. RESULTS: The interactions of 11 peptides and CSFV E2 protein were evaluated by the GalaxyPepDock and FlexX/ SYBYL programs, respectively. The assessment scores of all the peptides were correlated with their KD values. The final results showed that a moderate correlation coefficient was represented between KD values and CScores of predicted models by FlexX/ SYBYL. CONCLUSION: Our results demonstrate that considering the flexibility of the peptide is better than searching for more potential binding sites on the target protein surface while performing peptide-protein molecular docking. These data provide reasonable evidence for the molecular design of peptides and guidance for the functional assignment of target proteins. © 2018 Society of Chemical Industry.

Long non-coding RNA NEAT1 regulates permeability of the blood-tumor barrier via miR-181d-5p-mediated expression changes in ZO-1, occludin, and claudin-5.

The blood-tumor barrier (BTB) constitutes an efficient organization of tight junctions that limits the delivery of chemotherapeutic drugs to brain tumor tissues and impacts the treatment of glioma. Long non-coding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression, some lncRNAs play a crucial role in BTB permeability. However, the function of lncRNAs in BTB permeability is still largely unclear. Here, we have identified lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1), was remarkably up-regulated in glioma endothelial cells (GECs) obtained from an in vitro BTB model. Knockdown of NEAT1 impaired the integrity and increased the permeability of the BTB, accompanied by downregulation of expression of the tight junction proteins ZO-1, occludin and claudin-5 in GECs. Both bioinformatics data and results of luciferase reporter assays demonstrated that NEAT1 influenced BTB permeability by binding to miR-181d-5p. Knockdown of NEAT1 also down-regulated the expression of sex determining region Y-box protein 5 (SOX5), which was defined as a direct and functional downstream target of miR-181d-5p. SOX5 interacts with the promoter region of ZO-1, occludin and claudin-5 in GECs. In conclusion, knockdown of NEAT1 increased BTB permeability by binding to miR-181d-5p and then reducing tight junction protein expression by targeting SOX5. These results suggest an important role for NEAT1 in regulating BTB permeability and provide an additional strategy for treating glioma.

Discovery of highly potent, selective, covalent inhibitors of JAK3.

A useful and novel set of tool molecules have been identified which bind irreversibly to the JAK3 active site cysteine residue. The design was based on crystal structure information and a comparative study of several electrophilic warheads.

Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1 (S1P1).

The synthesis and structure-activity relationship (SAR) of a series of pyridyl-isoxazole based agonists of S1P1 are discussed. Compound 5b provided potent in vitro activity with selectivity, had an acceptable pharmacokinetic profile, and demonstrated efficacy in a dose dependent manner when administered orally in a rodent model of arthritis.

Genome sequencing and analysis of a novel recombinant porcine epidemic diarrhea virus strain from Henan, China.

Porcine epidemic diarrhea virus (PEDV) has caused devastating impact on pig-rearing industry in China and current vaccine is not effective against the circulating PEDV variants. In the present study, the full-length genome sequence from a PEDV isolate (CH/HNQX-3/14) was determined. The complete genome sequence analysis showed that the CH/HNQX-3/14 possessed unique deletion regions in the S and ORF3 genes. It was identified as a recombinant strain using phylogenetic analysis and recombination detection program. Further analyses of the full-length sequence suggest that CH/HNQX-3/14 is a natural recombinant between the attenuated vaccine strains (CV777 and DR13) and circulating wild-type strain (CH/ZMDZY/11). The recombination occurred not only in structural protein-coding region (S1 and N genes) but also in non-structural protein-coding region (replicases 1a and ORF3 genes). These results provided new evidence that PEDV strains circulating in China underwent recombination between vaccine and field strains, suggesting that recombination contributes to the genetic diversity of PEDV. Our findings provide valuable information on PEDV evolution and underscore the need for ongoing surveillance of this economically important swine disease.

Molecular epidemiology of outbreak-associated pseudorabies virus (PRV) strains in central China.

In several parts of China, there have been a large number of pseudorabies (PR) outbreaks which have devastated many swine farms even though the herds had been previously immunized with gE-deleted vaccines (Bartha-K61). The emergence of these outbreak-associated PRV strains might indicate that Bartha-K61 vaccine could not provide effective protection and poses challenges for current serologic diagnostics of anti-PRV antibodies. Here, we performed phylogenetic analyses based on partial gE, gB, and gC genes to provide information about the molecular epidemiology, diagnostics, and immune protection in these outbreak-associated PRV strains. Our results indicated that the maximal nucleotide sequence divergence for gE, gB, and gC genes are 1.7, 0.4, and 2.7 % within the cluster where outbreak-associated PRV strains were located, and are 2.3, 2.7, and 7.6 % with other clusters in the phylogenetic trees, respectively. Phylogenetic analyses revealed that gE, gB, and gC genes of the twelve outbreak-associated PRV strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor with strains Ea-China-1999, Fa-China-2001, and BJ-China-2008. The genetic relationship between these outbreak-associated PRV strains and strain Bartha is not close which may genetically explain the emergence of PR outbreaks in Bartha-K61-vaccinated swine farms. We suggest that these outbreak-associated PRV strains originate from earlier strains in local regions in China.

A 550 Mbit/s real-time visible light communication system based on phosphorescent white light LED for practical high-speed low-complexity application.

In this paper, we first experimentally demonstrate a 550 Mbit/s real-time visible light communication (VLC) system based on nonreturn-to-zero on-off keying (NRZ-OOK) modulation of a commercial phosphorescent white light LED. The 3-dB modulation bandwidth of such devices is only a few megahertz. We proposed an analog pre-emphasis circuit based on NPN transistors and an active post-equalization circuit based on an amplifier to enhance the 3-dB bandwidth of VLC link. Utilizing our proposed pre-emphasis and post-equalization circuits, the 3-dB bandwidth of VLC link could be extended from 3 to 233 MHz with blue-filter, to the best of our knowledge, which is the highest ever achieved in VLC systems reported. The achieved data rate was 550 Mbit/s at the distance of 60 cm and the resultant bit-error-ratio (BER) was 2.6 × 10(-9). When the VLC link operated at 160 cm, the data rate was 480 Mbit/s with 2.3 × 10(-7) of BER. Our proposed VLC system is a good solution for high-speed low-complexity application.

Proteomic analysis reveals the antiviral effects of baicalin on pseudorabies virus.

Pseudorabies virus (PRV) poses a significant threat to livestock and even humans. Baicalin, a bioactive flavonoid glycoside with medicinal potential, has been reported to have various biological activities. However, its inhibitory effect on PRV remains poorly understood. In this study, we proved that baicalin effectively inhibits PRV infection. Proteomic analysis revealed that baicalin reduces the expression of 14 viral proteins, which are associated with virus replication, release and immune evasion. Furthermore, the abundance of 116 host proteins was altered by PRV infection, but restored to normal levels after treatment with baicalin. Pathway analysis indicated that baicalin mitigates reactive oxygen species (ROS) and suppresses abnormal mitochondrion by reducing the expression of NFU1 iron­sulfur cluster scaffold homolog (NFU1) protein induced by PRV. Notably, baicalin also activates the complete coagulation cascade by increasing the expression of coagulation factor III (F3) protein and enhances nucleoplasm by upregulating the expression of solute carrier family 3 member 2 (SLC3A2) and CCAAT enhancer binding protein beta (CEBPB) proteins, contributing to its inhibitory effects on PRV. Our findings implied that baicalin has the potential to be developed as an anti-PRV drug and provide insights into the underlying molecular basis.