Inhibition of orf virus replication in goat skin fibroblast cells by the HSPA1B protein, as demonstrated by iTRAQ-based quantitative proteome analysis.

Orf virus (ORFV) infects sheep and goat tissues, resulting in severe proliferative lesions. To analyze cellular protein expression in ORFV-infected goat skin fibroblast (GSF) cells, we used two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ). The proteomics approach was used along with quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect differentially expressed proteins in ORFV-infected GSF cells and mock-infected GSF cells. A total of 282 differentially expressed proteins were identified. It was found that 222 host proteins were upregulated and 60 were downregulated following viral infection. We confirmed that these proteins were differentially expressed and found that heat shock 70-kDa protein 1B (HSPA1B) was differentially expressed and localized in the cytoplasm. It was also noted that HSPA1B caused inhibition of viral proliferation, in the middle and late stages of viral infection. The differentially expressed proteins were associated with the biological processes of viral binding, cell structure, signal transduction, cell adhesion, and cell proliferation.

Orf Virus VIR Antagonizes p53-Mediated Antiviral Effects to Facilitate Viral Replication.

As a zoonotic disease, ovine contagious pustular dermatitis (Orf) is a serious threat to sheep as well as humans. Orf virus (ORFV) interferon resistance protein (VIR) is the principal virulence protein that encodes a dsRNA-binding protein to inhibit host antiviral response. p53 is one of the key proteins of the host antiviral innate immunity. It not only enhances type I interferon secretion but also induces apoptosis in infected cells, and plays a crucial role in the immune response against various viral infections. However, it remains to be elucidated what role p53 plays in ORFV replication and whether ORFV's own protein VIR regulates p53 expression to promote self-replication. In this study, we showed that p53 has an antiviral effect on ORFV and can inhibit ORFV replication. In addition, ORFV nonstructural protein VIR interacts with p53 and degrades p53, which inhibits p53-mediated positive regulation of downstream antiviral genes. This study provides new insight into the immune evasion mediated by ORFV and identifies VIR as an antagonistic factor for ORFV to evade the antiviral response.

DDX56 cooperates with FMDV 3A to enhance FMDV replication by inhibiting the phosphorylation of IRF3.

The components of foot-and-mouth disease virus (FMDV) interact with host cellular proteins to promote self-replication and evade the host immune response. Previous studies have shown that FMDV 3A, 2C and 2B proteins interact with host cellular proteins involved in FMDV replication. However, whether the other host proteins have an impact on FMDV replication is less understood. In this study, we identified DDX56 as a positive regulator of FMDV replication. DDX56 overexpression increased FMDV replication, whereas DDX56 knockdown had the opposite effect. DDX56 interacted and cooperated with FMDV 3A to inhibit the type I interferon by reducing the phosphorylation of IRF3. Moreover, the D166 site of DDX56 played a role in increasing FMDV replication and cooperating with FMDV 3A to inhibit the phosphorylation of IRF3. Additionally, knockdown of DDX56 or FMDV 3A results also showed that DDX56 cooperated with FMDV 3A to inhibit the phosphorylation of IRF3. These results suggest that the interaction between FMDV 3A protein and the host protein DDX56 is critical for FMDV replication.

Advancement in TPL2-regulated innate immune response.

Tumor progression locus 2 (TPL2) is a serine/threonine kinase that belongs to the MAP3K family. The activated TPL2 regulates the innate immune-relevant signaling pathways, such as ERK, JNK, and NF-κB, and the differentiation of immune cells, for example, CD4+ T and NK cells. Therefore, TPL2 plays a critical role in regulating the innate immune response. The present review summarizes the recent advancements in the TPL2-regulated innate immune response.

Foot-and-mouth disease virus induces lysosomal degradation of NME1 to impair p53-regulated interferon-inducible antiviral genes expression.

Nucleoside diphosphate kinase 1 (NME1) is well-known as a tumor suppressor that regulates p53 function to prevent cancer metastasis and progression. However, the role of NME1 in virus-infected cells remains unknown. Here, we showed that NME1 suppresses viral replication in foot-and-mouth disease virus (FMDV)-infected cells. NME1-enhanced p53-mediated transcriptional activity and induction of interferon-inducible antiviral genes expression. FMDV infection decreased NME1 protein expression. The 2B and VP4 proteins were identified as the viral factors that induced reduction of NME1. FMDV 2B protein has a suppressive effect on host protein expression. We measured, for the first time, VP4-induced lysosomal degradation of host protein; VP4-induced degradation of NME1 through the macroautophagy pathway, and impaired p53-mediated signaling. p53 plays significant roles in antiviral innate immunity by inducing several interferon-inducible antiviral genes expression, such as, ISG20, IRF9, RIG-I, and ISG15. VP4 promoted interaction of p53 with murine double minute 2 (MDM2) through downregulation of NME1 resulting in destabilization of p53. Therefore, 5-flurouracil-induced upregulation of ISG20, IRF9, RIG-I, and ISG15 were suppressed by VP4. VP4-induced reduction of NME1 was not related to the well-characterized blocking effect of FMDV on cellular translation, and no direct interaction was detected between NME1 and VP4. The 15-30 and 75-85 regions of VP4 were determined to be crucial for VP4-induced reduction of NME1. Deletion of these VP4 regions also inhibited the suppressive effect of VP4 on NME1-enhanced p53 signaling. In conclusion, these data suggest an antiviral role of NME1 by regulation of p53-mediated antiviral innate immunity in virus-infected cells, and reveal an antagonistic mechanism of FMDV that is mediated by VP4 to block host innate immune antiviral response.

Development of a New RT-PCR with Multiple Primers for Detecting Southern African Territories Foot-and-mouth Disease Viruses.

INTRODUCTION: The extremely high genetic variation and the continuously emerging variants of foot-and-mouth disease virus (FMDV) of Southern African Territory (SAT) serotypes including SAT1, SAT2, and SAT3 make it necessary to develop a new RT-PCR for general use for monitoring viruses based on the updated genome information. MATERIAL AND METHODS: A FMDV SAT-D8 one-step RT-PCR was established based on the 1D2A2B genes of the SAT serotype viruses with a multiplex primer set. FMDV A, O, C, and Asia 1 serotypes, other vesicular disease viruses, inactivated SAT viruses, and 125 bovine, ovine, caprine and porcine tissue samples collected from the Chinese mainland were included for evaluating the assay. RESULTS: The new RT-PCR was proven to be specific without cross-reactions with Eurasian FMDV, swine vesicular disease virus (SVDV), Seneca valley virus (SVV), or other common viral pathogens of cattle, sheep, goat, and pig. An around 257 bp-sized amplicon clearly appeared when the inactivated SAT viruses were detected. However, all 125 samples collected from FMDV-susceptible animals from the Chinese mainland which has not known SAT epidemics showed negative results. CONCLUSIONS: A FMDV SAT-D8 one-step RT-PCR is a promising method for primary screening for FMDV SAT serotypes.

Transcriptome profiling indicating canine parvovirus type 2a as a potential immune activator.

Canine parvovirus type 2a (CPV-2a) is a variant of CPV-2, which is a highly contagious pathogen causing severe gastroenteritis and death in young dogs. However, how CPV-2 participates in cell regulation and immune response remains unknown. In this study, persistently infected MDCK cells were generated through culture passage of the CPV-2a-infected cells for ten generations. Our study showed that CPV-2a induces cell proliferation arrest and cell morphology alternation before the fourth generation, whereas, the cell morphology returns to normal after five times of passages. PCR detection of viral VP2 gene demonstrated that CPV-2a proliferate with cell passage. An immunofluorescence assay revealed that CPV-2a particles were mainly located in the cell nuclei of MDCK cell. Then transcriptome microarray revealed that gene expression pattern of MDCK with CPV-2a persistent infection is distinct compared with normal cells. Gene ontology annotation and Kyoto Encyclopedia of Genes and Genome pathway analysis demonstrated that CPV-2a infection induces a series of membrane-associated genes expression, including many MHC protein or MHC-related complexes. These genes are closely related to signaling pathways of virus-host interaction, including antigen processing and presentation pathway, intestinal immune network, graft-versus-host disease, and RIG-I-like helicases signaling pathway. In contrast, the suppressed genes mediated by CPV-2a showed low enrichment in any category, and were only involved in pathways linking to synthesis and metabolism of amino acids, which was confirmed by qPCR analysis. Our studies indicated that CPV-2a is a natural immune activator and has the capacity to activate host immune responses, which could be used for the development of antiviral strategy and biomaterial for medicine.

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.

Foot-and-mouth disease virus structural protein VP3 degrades Janus kinase 1 to inhibit IFN-γ signal transduction pathways.

Foot-and-mouth disease is a highly contagious viral disease of cloven-hoofed animals that is caused by foot-and-mouth disease virus (FMDV). To replicate efficiently in vivo, FMDV has evolved methods to circumvent host antiviral defense mechanisms, including those induced by interferons (IFNs). Previous research has focused on the effect of FMDV L(pro) and 3C(pro) on type I IFNs. In this study, FMDV VP3 was found to inhibit type II IFN signaling pathways. The overexpression of FMDV VP3 inhibited the IFN-γ-triggered phosphorylation of STAT1 at Tyr701 and the subsequent expression of downstream genes. Mechanistically, FMDV VP3 interacted with JAK1/2 and inhibited the tyrosine phosphorylation, dimerization and nuclear accumulation of STAT1. FMDV VP3 also disrupted the assembly of the JAK1 complex and degraded JAK1 but not JAK2 via a lysosomal pathway. Taken together, the results reveal a novel mechanism used by which FMDV VP3 counteracts the type II IFN signaling pathways.

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.

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.

Identification and analysis of differential miRNAs in PK-15 cells after foot-and-mouth disease virus infection.

The alterations of MicroRNAs(miRNAs) in host cell after foot-and-mouth disease virus (FMDV) infection is still obscure. To increase our understanding of the pathogenesis of FMDV at the post-transcriptional regulation level, Solexa high-throu MicroRNAs (miRNAs) play an important role both in the post-transcriptional regulation of gene expression and host-virus interactions. Despite investigations of miRNA expression ghput sequencing and bioinformatic tools were used to identify differentially expressed miRNAs and analyze their functions during FMDV infection of PK-15 cells. Results indicated that 9,165,674 and 9,230,378 clean reads were obtained, with 172 known and 72 novel miRNAs differently expressed in infected and uninfected groups respectively. Some of differently expressed miRNAs were validated using stem-loop real-time quantitative RT-PCR. The GO annotation and KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in immune response and cell death pathways.

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.

Detection of foot-and-mouth disease serotype O by ELISA using a monoclonal antibody.

An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, and A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Furthermore, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations.

Viroporin activity and membrane topology of classic swine fever virus p7 protein.

Viroporins are a group of viral proteins that participate in viral replication cycles, including modification of membrane permeability and promotion of viral release. Although biological data have been accumulated on viroporion-like proteins of other viruses belonging to family Flaviviridae, the viroporin activity and membrane topology of p7 protein from classical swine fever virus (CSFV), a member of the genus Pestivirus of the family Flaviviridae, are largely unknown. In this study, sequence analysis of the primary structure of p7 polypeptide demonstrates that p7 contains two putative transmembrane regions connected by a short hydrophilic segment. Expression of p7 protein in Escherichia coli leads to the permeabilization of bacterial cells to small molecules. The p7 protein also enhances the permeability of mammalian cells, increasing the intracellular Ca(2+) concentration and the permeability of cells to the translation inhibitor Hygromycin B. This protein is an integral membrane protein and can form homo-oligomers. It mainly localizes to the ER at the early stage of the expression and can be transferred to the plasma membrane at the late stage of the expression. Detergent permeabilization assays confirmed that the p7 protein is a 2-pass transmembrane protein and its N and C termini are exposed to the ER lumen. Deletion analysis showed that amino acid residues 41-63 may be essential for the viroporin activity of the protein. Our studies demonstrate that CSFV p7 possesses properties commonly associated with viroporins, which could be a potential target for the development of a therapeutic intervention for classic swine fever virus infection.

Detection of foot-and-mouth disease serotype O by ELISA using a monoclonal antibody.

An ELISA assay with monoclonal antibody (MELISA) was used to type serotype O of foot-and-mouth disease virus (FMDV). All FMDV serotype O reference strains were positive by MELISA, while other viruses such as FMDV serotypes Asia 1, C, A and classical swine fever virus, swine vesicular disease virus, and porcine reproductive and respiratory syndrome virus remained negative. Further, FMDV serotype O positive samples were able to be detected by MELISA. This assay may be particularly suitable for diagnosis of FMDV serotype O infection in field stations.

Immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 ORF2 protein.

BACKGROUND: Porcine circovirus type 2 (PCV2) is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS), which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs) have gained increasing interest for use in vaccines. METHODS: To study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol-gel/emulsion(oil-in-water/ethanol) method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry. RESULTS: HMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex. CONCLUSION: The findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.

Detection of foot-and-mouth disease virus RNA by reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for foot-and-mouth disease virus (FMDV) RNA. The amplification was able to finish in 45 min under isothermal condition at 64°C by employing a set of four primers targeting FMDV 2B. The assay showed higher sensitivity than RT-PCR. No cross reactivity was observed from other RNA viruses including classical swine fever virus, swine vesicular disease, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus. Furthermore, the assay correctly detected 84 FMDV positive samples but not 65 FMDV negative specimens. The result indicated the potential usefulness of the technique as a simple and rapid procedure for the detection of FMDV infection.