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.

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.

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.

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.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

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.

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.

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.

[Secreted expression of nonstructural protein gene 3ABC of foot-and-mouth disease virus in Sf9 cells and activity analysis].

Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.

The effect of bovine IFN-alpha on the immune response in guinea pigs vaccinated with DNA vaccine of foot-and-mouth disease virus.

In this study, we constructed recombinant plasmid pcDNA3.1/P12X3C3D including P1, 2A, 3C, 3D and part of 2B gene of FMDV and pcDNA3.1/IFN containing the gene encoding bovine IFN-alpha. We inoculated the DNA vaccine pcDNA3.1/P12X3C3D with or without pcDNA3.1/IFN to evaluate the efficiency of this DNA vaccine and the immunogenicity of DNA vaccine enhanced by the co-delivery with pcDNA3.1/IFN. After two times of vaccination with DNA vaccine, all of guinea pigs were challenged with 103 ID50 FMDV type O. Anti-FMDV antibody levels were detected by ELISA and T lymphocyte proliferation response was tested by MTT assay. The result shows that guinea pigs inoculated by pcDNA3.1/P12X3C3D alone or with pcDNA3.1/IFN generated specific antibodies and induced an FMDV-specific T lymphocyte proliferation response. FMDV challenge tests showed that one in four guinea pigs immunized by pcDNA3.1/P12X3C3D with pcDNA3.1/IFN was protected from the FMDV serotype O infection. This result indicated that the efficiency of the DNA vaccine was enhanced by co-delivery with pcDNA3.1/IFN. However, the protection rate was considerably lower than that immunized with conventional FMD vaccine.

[Expression of recombinant plasmid pcDNA3.1/P12X3C with multi-genes of foot-and-mouth disease virus in BHK-21 cells].

In order to obtain the gene P12X3C of Foot-and-Mouth Disease Virus (FMDV) that includes full length P1, 2A, 3C and a part of 2B, the site mutation strategy was used. After being digested by Kpn I and Xba I respectively, the gene P12X3C was cloned into the pcDNA3.1 (+) expression vector. The recombinant plasmid was checked by restriction enzyme analysis and nucleic acid sequencing, and then named pcDNA3.1/P12X3C. Further, BHK-21 cells was transfected with pcDNA3.1/P12X3C by using lipoid. The proteins of Foot-and-Mouth Disease Virus, which were expressed in BHK-21 cells, were confirmed by sandwich-ELISA and fluoroscopy. The result shows the gene P12X3C is cloned into eukaryotic expression plasmid, and the recombinant eukaryotic expression plasmid pcDNA3.1/P12X3C could express proteins of Foot-and-Mouth Disease Virus in BHK-21 cells, which have immunocompetence. This study demonstrates that delivery of a recombinant eukaryotic expression plasmid containing P12X3C coding regions results in the assembly of FMDV capsid structures, which will offer experimental base to DNA vaccine of FMDV.