Double synergic chitosan-coated poly (lactic-co-glycolic) acid nanospheres loaded with nucleic acids as an intranasally administered vaccine delivery system to control the infection of foot-and-mouth disease virus.

BACKGROUND & AIMS: The spread of foot-and-mouth disease virus (FMDV) through aerosol droplets among cloven-hoofed ungulates in close contact is a major obstacle for successful animal husbandry. Therefore, the development of suitable mucosal vaccines, especially nasal vaccines, to block the virus at the initial site of infection is crucial. PATIENTS AND METHODS: Here, we constructed eukaryotic expression plasmids containing the T and B-cell epitopes (pTB) of FMDV in tandem with the molecular mucosal adjuvant Fms-like tyrosine kinase receptor 3 ligand (Flt3 ligand, FL) (pTB-FL). Then, the constructed plasmid was electrostatically attached to mannose-modified chitosan-coated poly(lactic-co-glycolic) acid (PLGA) nanospheres (MCS-PLGA-NPs) to obtain an active nasal vaccine targeting the mannose-receptor on the surface of antigen-presenting cells (APCs). RESULTS: The MCS-PLGA-NPs loaded with pTB-FL not only induced a local mucosal immune response, but also induced a systemic immune response in mice. More importantly, the nasal vaccine afforded an 80% protection rate against a highly virulent FMDV strain (AF72) when it was subcutaneously injected into the soles of the feet of guinea pigs. CONCLUSIONS: The nasal vaccine prepared in this study can effectively induce a cross-protective immune response against the challenge with FMDV of same serotype in animals and is promising as a potential FMDV vaccine.

Cinnamyl Chalcone Based AIE Fluorescent Probes for Sensitive Detection of Hydrazine and its Application in Living Cells.

Widely utilized in the chemical industry and agriculture, hydrazine is easily absorbed by living things and can cause physical harm when in touch for an extended period of time. As a result, a novel cinnamaldehyde chalcone C5 was produced by Friedel Crafts process and aldol condensation reaction. Triphenylamine was used as the raw material for hydrazine determination in both reactions. Chalcone C5 exhibits significant AIE behavior in a mixed mixture of ethanol and water in addition to having great selectivity and a low detection limit (0.119 nm) for hydrazine. The solvent effect test revealed a linear relationship between the Stokes shift of C5 in the solvent and the rise in solvent orientation polarization. It is important to note that C5 is not harmful to MCF-7 cells, mouse kidney cells, or pig kidney cells. Furthermore, research on cell imaging has demonstrated that probe C5 may be utilized to image the fluorescence of hydrazine in active MCF-7 cells.

ptsI gene in the phosphotransfer system is a potential target for developing a live attenuated Salmonella vaccine.

Salmonella enterica serovar Typhimurium causes invasive non­typhoidal Salmonella diseases in animals and humans, resulting in a high mortality rate and huge economic losses globally. As the prevalence of antibiotic­resistant Salmonella has been increasing, vaccination is thought to be the most effective and economical strategy to manage salmonellosis. The present study aimed to investigate whether dysfunction in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), which is critical for carbon uptake and survival in macrophages, may be adequate to generate Salmonella­attenuated vaccine strains. A Salmonella strain (KST0555) was generated by deleting the ptsI gene from the PTS and it was revealed that this auxotrophic mutant was unable to efficiently utilize predominant carbon sources during infection (glucose and glycerol), reduced its invasion and replication capacity in macrophages, and significantly (P=0.0065) lowered its virulence in the setting of a mouse colitis model, along with a substantially decreased intestinal colonization and invasiveness compared with its parent strain. The reverse transcription­quantitative PCR results demonstrated that the virulence genes in Salmonella pathogenicity island-1 (SPI-1) and -2 (SPI-2) and the motility of KST0555 were all downregulated compared with its parent strain. Finally, it was revealed that when mice were immunized orally with live KST0555, Salmonella­specific humoral and cellular immune responses were effectively elicited, providing protection against Salmonella infection. Thus, the present promising data provides a strong rationale for the advancement of KST0555 as a live Salmonella vaccine candidate and ptsI as a potential target for developing a live attenuated bacterial vaccine strain.

Synthesis of nitrogen-doped graphene quantum dots (N-GQDs) from marigold for detection of Fe3+ ion and bioimaging.

Graphene quantum dots (GQDs) are synthesized by the method of high-temperature pyrolysis from marigold granules and subsequently nitrogen-doped graphene quantum dots (N-GQDs) are synthesized from ethylenediamine by hydrothermal treatment, which shows a strong blue emission with 7.84% quantum yield (QY). This will be used in detection of Fe3+ in water environments and the field of bioimaging.

Effective mucosal live attenuated Salmonella vaccine by deleting phosphotransferase system component genes ptsI and crr.

Salmonella enterica is a major human pathogen that causes invasive non-typhoidal Salmonellosis (iNTS), resulting in significant morbidity and mortality. Although a number of pre-clinical and clinical studies have reported on the feasibility of developing a safe and effective vaccine against iNTS, there have been no licensed Salmonella vaccines available to protect against NTS strains. Vaccine formulations of highest priority for NTS are live attenuated vaccines, which can elicit effective induction of intestinal mucosal and intracellular bacteria-specific cell mediated immune responses. Since glucose is crucial for intracellular survival and replication in host cells, we constructed strains with mutations in components of the glucose uptake system, called the phosphotransferase system (PTS), and compared the relative virulence and immune responses in mice. In this study, we found that the strain with mutations in both ptsI and crr (KST0556) was the most attenuated strain among the tested strains, and proved to be highly effective in inducing a mucosal immune response that can protect against NTS infections in mice. Thus, we suggest here that KST0556 (ΔptsIΔcrr) is a potential live vaccine candidate for NTS, and may also be a candidate for a live delivery vector for heterologous antigens. Moreover, since PTS is a well-conserved glucose transporter system in both Gramnegative and Gram-positive bacteria, the ptsI and crr genes may be potential targets for creating live bacterial vectors or vaccine strains.

A novel biphenyl-derived salicylhydrazone Schiff base fluorescent probes for identification of Cu2+ and application in living cells.

A novel biphenyl-derived salicylhydrazone Schiff base (BSS) fluorescent probes for highly sensitive and selective identification of Cu2+ has been synthesized. In addition, the recognition has been proved experimentally. The results indicated that the complex forms a 1:1 complex with Cu2+ shows fluorescent quenching. Furthermore, the detection limit of 1.54×10-8M. More interesting, the probe BSS not only have a good biocompatibility in living cells, but also the sense behavior of Cu2+ in the cell nucleus.

A new pyrazoline-based probe of quenched fluorescent reversible recognition for Cu2+ and its application in cells.

A new pyrazoline-based probe D was designed and synthesized, which can be used as a highly sensitive, selective and reversible recognizing fluorescent to detect Cu2+. The recognition properties of this compound was investigated by UV-vis absorption and fluorescence spectrophotometry. The results showed that the probe D forms a 1:1 complex with Cu2+ and displayed a linear fluorescence response to Cu2+ with a detection limit of 1.94×10-7M. In addition, the probe have a good biocompatibility in living cells.

Biological function of Foot-and-mouth disease virus non-structural proteins and non-coding elements.

Foot-and-mouth disease virus (FMDV) represses host translation machinery, blocks protein secretion, and cleaves cellular proteins associated with signal transduction and the innate immune response to infection. Non-structural proteins (NSPs) and non-coding elements (NCEs) of FMDV play a critical role in these biological processes. The FMDV virion consists of capsid and nucleic acid. The virus genome is a positive single stranded RNA and encodes a single long open reading frame (ORF) flanked by a long structured 5'-untranslated region (5'-UTR) and a short 3'-UTR. The ORF is translated into a polypeptide chain and processed into four structural proteins (VP1, VP2, VP3, and VP4), 10 NSPs (L(pro), 2A, 2B, 2C, 3A, 3B1-3, 3C(pro), and 3D(pol)), and some cleavage intermediates. In the past decade, an increasing number of studies have begun to focus on the molecular pathogenesis of FMDV NSPs and NCEs. This review collected recent research progress on the biological functions of these NSPs and NCEs on the replication and host cellular regulation of FMDV to understand the molecular mechanism of host-FMDV interactions and provide perspectives for antiviral strategy and development of novel vaccines.

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 µg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.

Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase.

Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na(+)/H(+) exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses.

The application of virus-like particles as vaccines and biological vehicles.

Virus-like particles (VLPs) can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding the genetic material and potential replication probability. In addition, VLPs possess several features including can be rapidly produced in large quantities through existing expression systems, highly resembling native viruses in terms of conformation and appearance, and displaying repeated cluster of epitopes. Their capsids can be modified via genetic insertion or chemical conjugation which facilitating the multivalent display of a homologous or heterogeneous epitope antigen. Therefore, VLPs are considered as a safe and effective candidate of prophylactic and therapeutic vaccines. VLPs, with a diameter of approximately 20 to 150 nm, also have the characteristics of nanometer materials, such as large surface area, surface-accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Therefore, assembled VLPs have great potential as a delivery system for specifically carrying a variety of materials. This review summarized recent researches on VLP development as vaccines and biological vehicles, which demonstrated the advantages and potential of VLPs in disease control and prevention and diagnosis. Then, the prospect of VLP biology application in the future is discussed as well.

A long-lasting protective immunity against chronic toxoplasmosis in mice induced by recombinant rhoptry proteins encapsulated in poly (lactide-co-glycolide) microparticles.

Toxoplasma gondii infection in humans and animals is a worldwide zoonosis. Prevention and control of toxoplasmosis based on vaccination is one of the promising strategies. In the present study, recombinant T. gondii rhoptry proteins 38 and 18 (TgROP38 and TgROP18) were encapsulated into poly (lactide-co-glycolide) (PLG) (1:1), respectively, to obtain the stable water-in-oil-in-water double emulsion. Female Kunming mice were then immunized with the protein vaccines twice at a 2-week interval. Eight weeks after the second immunization, 10 mice from each group were challenged with T. gondii PRU strain (genotype II). The entrapment rates of PLG-rROP38 and PLG-rROP18 ranged from 65.5 to 77.7% and 58.1 to 72.3%, respectively. Immunization of mice with rROP38 and rROP18 proteins encapsulated into PLG microparticles elicited strongly humoral and cell-mediated responses against T. gondii, associated with relatively high levels of total IgG, IgG2a isotype, and IFN-γ, as well as the mixed Th1/Th2 immunity responses. Immunization with various protein vaccines induced significant reduction of the brain cysts after chronic infection with the T. gondii PRU strain, and the most effective protection was achieved in the PLG-rROP38-rROP18-immunized mice, with a cyst reduction of 81.3%. The findings of the present study indicated that recombinant rhoptry antigens encapsulated in PLG could maintain the protein immunogenicity in an extended period and elicit effective protection against chronic T. gondii infection, which has implications for the development of long-lasting vaccines against chronic toxoplasmosis in animals.

Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1.

Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantitatively study the host cell gene expression profile, in order to achieve an unbiased overview of the protein expression changes in BHK-21 cells infected with FMDV serotype Asia 1. The SILAC-based approach identified overall 2,141 proteins, 153 of which showed significant alteration in the expression level 6 h post FMDV infection (57 up-regulated and 96 down-regulated). Among these proteins, six cellular proteins, including three down-regulated (VPS28, PKR, EVI5) and three up-regulated (LYPLA1, SEC62 and DARs), were selected according to the significance of the changes and/or the relationship with PKR. The expression level and pattern of the selected proteins were validated by immunoblotting and confocal microscopy. Furthermore, the functions of these cellular proteins were assessed by small interfering RNA-mediated depletion, and their functional importance in the replication of FMDV was demonstrated by western blot, reverse transcript PCR (RT-PCR) and 50% Tissue Culture Infective Dose (TCID50). The results suggest that FMDV infection may have effects on the expression of specific cellular proteins to create more favorable conditions for FMDV infection. This study provides novel data that can be utilized to understand the interactions between FMDV and the host cell.

NLRP3 Inflammasome Activation by Viroporins of Animal Viruses.

Viroporins are a group of low-molecular-weight proteins containing about 50-120 amino acid residues, which are encoded by animal viruses. Viroporins are involved in several stages of the viral life cycle, including viral gene replication and assembly, as well as viral particle entry and release. Viroporins also play an important role in the regulation of antiviral innate immune responses, especially in inflammasome formation and activation, to ensure the completion of the viral life cycle. By reviewing the research progress made in recent years on the regulation of the NLRP3 inflammasome by viroporins of animal viruses, we aim to understand the importance of viroporins in viral infection and to provide a reference for further research and development of novel antiviral drugs.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+) concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region.

Three-dimensional structure of foot-and-mouth disease virus and its biological functions.

Foot-and-mouth disease (FMD), an acute, violent, infectious disease of cloven-hoofed animals, remains widespread in most parts of the world. It can lead to a major plague of livestock and an economical catastrophe. Structural studies of FMD virus (FMDV) have greatly contributed to our understanding of the virus life cycle and provided new horizons for the control and eradication of FMDV. To examine host-FMDV interactions and viral pathogenesis from a structural perspective, the structures of viral structural and non-structural proteins are reviewed in the context of their relevance for virus assembly and dissociation, formation of capsid-like particles and virus-receptor complexes, and viral penetration and uncoating. Moreover, possibilities for devising novel antiviral treatments are discussed.

Topology and biological function of enterovirus non-structural protein 2B as a member of the viroporin family.

Viroporins are a group of transmembrane proteins with low molecular weight that are encoded by many animal viruses. Generally, viroporins are composed of 50-120 amino acid residues and possess a minimum of one hydrophobic region that interacts with the lipid bilayer and leads to dispersion. Viroporins are involved in destroying the morphology of host cells and disturbing their biological functions to complete the life cycle of the virus. The 2B proteins encoded by enteroviruses, which belong to the family Picornaviridae, can form transmembrane pores by oligomerization, increase the permeability of plasma membranes, disturb the homeostasis of calcium in cells, induce apoptosis, and cause autophagy; these abilities are shared among viroporins. The present paper introduces the structure and biological characteristics of various 2B proteins encoded by enteroviruses of the family Picornaviridae and may provide a novel idea for developing antiviral drugs.

Virus-like particles in picornavirus vaccine development.

Virus-like particles (VLP), which are similar to natural virus particles but do not contain viral genes, have brought about significant breakthroughs in many research fields because of their unique advantages. The ordered repeating epitopes of VLP can induce immunity responses similar to those prompted by natural viral infection; thus, VLP vaccines are regarded as candidate alternatives to whole-virus vaccines. As picornavirus has serious impacts on human and animal health, the development of efficient and safe vaccines is a key endeavor in preventing virus infections. The characteristics of picornavirus capsid proteins allow the development of VLP vaccines. This paper investigates research scenarios and progress on picornavirus VLP vaccines with the aim of providing a reference for researchers focusing on virology and vaccinology.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.