Disease Virus Proteins Research Articles

Quantitative Assessment of Newcastle Disease Virus Proteins Interactions with All Known Mucin Types of Chicken and Quail

Quantitative Assessment of Newcastle Disease Virus Proteins Interactions with All Known Mucin Types of Chicken and Quail

Study the Profile of Foot-and-mouth Disease Virus Protein by Electrophoresis and Identification of the Immunodominant Proteins.

Foot-and-mouth disease is an extremely infectious and occasionally fatal viral disease with a rapid onset and a short course that affects cloven-hoofed animals and results in considerable financial losses. Today, Foot-and-mouth disease is controlled by traditional inactivated vaccines. Due to the short duration of immunity, a study was conducted for proteins of the virus as well as obtaining immunodominant proteins to design more efficient vaccines against Foot-and-mouth disease virus. This research aims to study the profile of Foot-and-mouth disease virus protein by electrophoresis and identification of the immunodominant proteins. The purified Foot-and-mouth disease virus was purchased then the protein concentration of that solution was measured by Lowry method. SDS-PAGE was done to achieve the protein profiles of the virus and immunization of 5 guinea pigs was done, then blood samples were taken for obtaining serum. Finally, serology tests; double immunodiffusion, ELISA, and western blotting were used to evaluate antigen response to antibodies (antigenic immunization). The protein concentration was 3.5 mg/ml. In SDS-PAGE with 10% gel, the protein profile of the virus was observed. After immunization, by conducting double immunodiffusion tests, the sediment lines between the serum antibody and the antigen of the virus were formed. Also, The ELISA test showed that the antibodies were formed against the antigens. In the western blot test, two immunodominant proteins of the FMD virus were obtained. According to the results, the immunodominant proteins of the FMD virus were determined. These proteins can be used in immunological diagnostic methods and also novel vaccines.

Creation of poxvirus expressing foot-and-mouth and peste des petits ruminant disease virus proteins

Foot-and-mouth disease (FMD) and Peste des petits ruminant disease (PPR) are acute and severe infectious diseases of sheep and are listed as animal diseases for compulsory immunization. However, there is no dual vaccine to prevent these two diseases. The Modified Vaccinia virus Ankara strain (MVA) has been widely used in the construction of recombinant live vector vaccine because of its large capacity of foreign gene, wide hostrange, high safety, and immunogenicity. In this study, MVA-GFP recombinant virus skeleton was used to construct dual live vector vaccines against FMD and PPR. The recombinant plasmid pUC57-FMDV P1-2A3CPPRV FH was synthesized and transfected into MVA-GFP infected CEF cells for homologous recombination. The results showed that a recombinant virus without fluorescent labeling was obtained after multiple rounds of plaque screening. The recombinant virus successfully expressed the target proteins, and the empty capsid of FMDV could be observed by transmission electron microscope (TME), and the expression levels of foreign proteins (VP1 and VP3) detected by ELISA were like those detected in FMDV-infected cells. This study laidthe foundation for the successful construction of a live vector vaccine against FMD and PPR. • A recombinant MVA expressing FMDVP12A3C and PRRV HF proteins • Both the FMDV and PRRV proteins inserted into the virus were expressed • The proteins expressed by the recombinant poxvirus were assembled into VLPs.

Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability.

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

Comparison of novel recombinant nucleoprotein ELISA with commercial ELISA for Newcastle disease

In the present study, a novel ELISA method used recombinant nucleocapsid protein (rNP) as the coating agent. Recombinant Newcastle disease virus (NDV) protein was cloned and expressed in Escherichia coli. Though the rNP-ELISA results were consistent with commercial ELISA results for the NDV-negative sera samples, qualitatively and quantitatively variable (often reduced) results were obtained with NDV-positive sera. Although the rNP-ELISA results for NDV detection were inconclusive, further improvement and standardization of the rNP-ELISA approach, such as using multiple recombinant proteins as the ELISA coating agent and performing comprehensive statistical analyses of combined recombinant protein ELISA, should help counter Newcastle disease outbreaks by improving NDV detection.

Structural Perspectives of Beak and Feather Disease Virus and Porcine Circovirus Proteins.

Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel β-sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins.

Advances in Foot-and-Mouth Disease Virus Proteins Regulating Host Innate Immunity.

Foot-and-mouth disease (FMD) is a highly contagious disease that affects cloven-hoofed animals such as pigs, cattle, and sheep. The disease is caused by the foot-and-mouth disease virus (FMDV) which has a non-enveloped virion with icosahedral symmetry that encapsulates a positive-sense, single-stranded RNA genome of ∼8.4 kb. FMDV infection causes obvious immunosuppressive effects on the host. In recent years, studies on the immunosuppressive mechanism of FMDV have become a popular topic. In addition, studies have shown that many FMDV proteins are involved in the regulation of host innate immunity and have revealed mechanisms by which FMDV proteins mediate host innate immunity. In this review, advances in studies on the mechanisms of interaction between FMDV proteins and host innate immunity are summarized to provide a comprehensive understanding of FMDV pathogenesis and the theoretical basis for FMD prevention and control.

Prevalence of antibodies against Borna disease virus proteins in Japanese children with autism spectrum disorder.

Bornavirus infection is observed in both animals, including humans. However, bornavirus epidemiology in humans, especially in children, remains unclear. Here, we evaluated antibodies against bornaviruses in Japanese children with autism spectrum disorder (ASD) using immunofluorescence analysis, western blotting, and radio ligand assay. The prevalence of antibodies against bornavirus-specific speckles, N, and P proteins were 22%, 48%, and 33%, respectively, in the ASD children. According to our criteria, the prevalence of antibodies against bornaviruses was 7.4% in the ASD children. This is the first report of the serological prevalence of bornavirus in Japanese children. Our results provide valuable baseline-data regarding bornavirus epidemiology in children for future studies.

Infectious bursal disease virus protein VP4 suppresses type I interferon expression via inhibiting K48-linked ubiquitylation of glucocorticoid-induced leucine zipper (GILZ)

Viruses have developed a variety of methods to evade host immune response. Our previous study showed that infectious bursal disease virus (IBDV) inhibited type I interferon production via interaction of VP4 with cellular glucocorticoid-induced leucine zipper (GILZ) protein. However, the exact underlying molecular mechanism is still unclear. In this study, we found that IBDV VP4 suppressed GILZ degradation by inhibiting K48-linked ubiquitylation of GILZ. Furthermore, mutation of VP4 (R41G) abolished the inhibitory effect of VP4 on IFN-β expression and GILZ ubiquitylation, indicating that the amino acid 41R of VP4 was required for the suppression of IFN-β expression and GILZ ubiquitylation. Moreover, IBDV infection or VP4 expression markedly inhibited endogenous GILZ ubiquitylation. Thus, IBDV VP4 suppresses type I interferon expression by inhibiting K48-linked ubiquitylation of GILZ, revealing a new mechanism employed by IBDV to suppress host response.

A bivalent dendrimeric peptide bearing a T-cell epitope from foot-and-mouth disease virus protein 3A improves humoral response against classical swine fever virus

Three dendrimeric peptides were synthesized in order to evaluate their immunogenicity and their potential protection against classical swine fever virus (CSFV) in domestic pigs. Construct 1, an optimized version of a previously used dendrimer, had four copies of a B-cell epitope derived from CSFV E2 glycoprotein connected to an also CSFV-derived T-cell epitope through maleimide instead of thioether linkages. Construct 2 was similarly built but included only two copies of the B-cell epitope, and in also bivalent construct 3 the CSFV T-cell epitope was replaced by a previously described one from the 3A protein of foot-and-mouth disease virus (FMDV). Animals were inoculated twice with a 21-day interval and challenged 15days after the second immunization. Clinical signs were recorded daily and ELISA tests were performed to detect antibodies against specific peptide and E2. The neutralising antibody response was assessed 13days after challenge. Despite the change to maleimide connectivity, only partial protection against CSFV was again observed. The best clinical protection was observed in group 3. Animals inoculated with constructs 2 and 3 showed higher anti-peptide humoral response, suggesting that two copies of the B-cell epitope are sufficient or even better than four copies for swine immune recognition. In addition, for construct 3 higher neutralizing antibody titres against CSFV were detected. Our results support the immunogenicity of the CSFV B-cell epitope and the cooperative role of the FMDV 3A T-cell epitope in inducing a neutralising response against CSFV in domestic pigs. This is also the first time that the FMDV T-cell epitope shows effectivity in improving swine immune response against a different virus. Our findings highlight the relevance of dendrimeric peptides as a powerful tool for epitope characterization and antiviral strategies development.

Detection of Antibodies against Borna Disease Virus Proteins in an Autistic Child and Her Mother.

Volume 70, no 2, p.225-227, 2017. Page 226, Figure 1B and C should appear as shown below.

Visualization of Borna Disease Virus Protein Interactions with Host Proteins using in situ Proximity Ligation Assay

Borna disease virus type 1 (BDV) comprises highly conserved neurotropic non-segmented negative strand RNA-virus variants causing neurological and behavioral disorders in a wide range of mammalian a ...

The efficacy of chimeric vaccines constructed with PEP-1 and Ii-Key linking to a hybrid epitope from heterologous viruses

The heterologous epitope-peptide from different viruses may represent an attractive candidate vaccine. In order to evaluate the role of cell-permeable peptide (PEP-1) and Ii-Key moiety from the invariant chain (Ii) of MHC on the heterologous peptide chimeras, we linked the two vehicles to hybrid epitopes on the VP2 protein (aa197–209) of the infectious bursal disease virus and HN protein (aa345–353) of the Newcastle disease virus. The chimeric vaccines were prepared and injected into mice. The immune effects were measured by indirect ELISA. The results showed that the vehicle(s) could significantly boost immune effects against the heterologous epitope peptide. The Ii-Key-only carrier induced more effective immunological responses, compared with the PEP-1 and Ii-Key hybrid vehicle. The carrier–peptide hybrids all showed strong colocalization with major histocompatibility complex (MHC) class II molecules compared with the epitope-peptide (weakly-binding) after co-transfection into 293T cells. Together, our results lay the groundwork for designing new hybrid vaccines based on Ii-Key and/or PEP-1 peptides.

A critical role of interferon-induced protein IFP35 in the type I interferon response in cells induced by foot-and-mouth disease virus (FMDV) protein 2C.

Foot-and-mouth disease virus (FMDV) protein 2C is one of the most highly conserved viral proteins among the serotypes of FMDV. However, its effect on host cell response is not very clear. In our previous report, we showed that FMDV protein 2C interacts with cellular protein N-myc and STAT interactor (Nmi), inducing moderate apoptosis in cells. Here, we show that transfection of HEK293T cells with pEGFP-N1-2C or pEGFP-N1-Nmi induces activation of type I interferon promoters, leading to delayed vesicular stomatitis virus (VSV) growth. Using immunoprecipitation and confocal microscopy assays, we found that interferon-induced protein IFP35 interacts with Nmi. Knockdown of IFP35 expression by siRNA abolished pEGFP-N1-2C and pEGFP-N1-Nmi-induced activation of type I interferon promoters and restored VSV growth, suggesting that IFP35 plays a critical role in the type I interferon response induced by FMDV protein 2C. These findings may help to further understand cell responses to FMDV infection.

Amino acid esters substituted phosphorylated emtricitabine and didanosine derivatives as antiviral and anticancer agents.

Owing to the promising antiviral activity of amino acid ester-substituted phosphorylated nucleosides in the present study, a series of phosphorylated derivatives of emtricitabine and didanosine substituted with bioactive amino acid esters at P-atom were synthesized. Initially, molecular docking studies were screened to predict their molecular interactions with hemagglutinin-neuraminidase protein of Newcastle disease virus and E2 protein of human papillomavirus. The title compounds were screened for their antiviral ability against Newcastle disease virus (NDV) by their in ovo study in embryonated chicken eggs. Compounds 5g and 9c exposed well mode of interactions with HN protein and also exhibited potential growth of NDV inhibition. The remaining compounds exhibited better growth of NDV inhibition than their parent molecules, i.e., emtricitabine (FTC) and didanosine (ddI). In addition, the in vitro anticancer activity of all the title compounds were screenedagainst HeLa cell lines at 10 and 100μg/mL concentrations. The compounds 5g and 9c showed an effective anticancer activity than that of the remaining title compounds with IC50 values of 40 and 60μg/mL, respectively. The present in silico and in ovo antiviral and in vitro anticancer results of the title compounds are suggesting that the amino acid ester-substituted phosphorylated FTC and ddI derivatives, especially 5g and 9c, can be used as NDV inhibitors and anticancer agents for the control and management of viral diseases with cancerous condition.

Endopeptidase activity characterization of E. coli-derived infectious bursal disease virus protein 4 tubules

Viral protein 4 (VP4) is a serine protease that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 of infectious bursal disease virus. In this report, the recombinant VP4 with a His-tag and three mutants (VP4-S652A, VP4-K692A and VP4-S652A.K692A) were expressed in Escherichia coli. Soluble VP4 was purified using immobilized metal-ion affinity chromatography or sucrose density gradient following with gel-filtration chromatography. The purified VP4 has a tubular structure with 25-30 nm in width and ∼300 nm in length, as observed by transmission electron microscope. A similar tubular structure was also found for these three mutants. The endopeptidase activity of these VP4 tubules was characterized by fluorescence resonance energy transfer using a synthetic fluorogenic oligopeptide as a substrate. The results show that the tubule-like VP4 is a functional enzyme with K(m) of 43 ± 2 μM and k(cat) of 0.04 ± 0.01 min⁻¹; however, k(cat) of three mutants were significantly reduced. This is the first report to demonstrate that VP4 protein expressed in E. coli can self-assemble into functional tubule-like particles and its activity can be completely inhibited by 1 mM of Ni⁺² ions.

Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing theantibody response against synthetic foot-and-mouth disease virus peptide

To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, asynthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugatedto GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extracysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximalconjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultantpFMDV–GNP conjugates were then injected into BALB/c mice. Immunization withpFMDV–keyhole limpet hemocyanin (pFMDV–KLH) conjugate was also performed as acontrol. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titersagainst both pFMDV and the carriers were measured. For the pFMDV–GNPimmunization, specific antibodies against the synthetic peptide were detected in the sera ofmice injected with 2, 5, 8, 12, and 17 nm pFMDV–GNP conjugates. Maximalantibody binding was noted for GNPs of diameter 8–17 nm. The pFMDV–GNPsinduced a three-fold increase in the antibody response compared to the response topFMDV–KLH. However, sera from either immunized mouse group did not exhibitan antibody response to GNPs, while the sera from pFMDV–KLH-immunizedmice presented high levels of binding activity against KLH. Additionally, theuptake of pFMDV–GNP in the spleen was examined by inductively coupled plasmamass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). Thequantity of GNPs that accumulated in the spleen correlated to the magnitude of theimmune response induced by pFMDV–GNP. In conclusion, we demonstrated thesize-dependent immunogenic properties of pFMDV–GNP conjugates. Furthermore,we established that GNPs ranging from 8 to 17 nm in diameter may be idealfor eliciting a focused antibody response against a synthetic pFMDV peptide.

Vaccine development through terminal deletions of an infectious bursal disease virus protein 2 precursor variant

VP2 is the primary host-protective immunogen of infectious bursal disease virus (IBDV), the agent that causes the highly contiguous infectious bursal disease (IBD). Previous studies have shown that a C-terminal his-tagged 452 amino acid residue VP2 precursor variant (VP2-452H) can form an immunogenic subviral particle (SVP). A set of his-tagged N- and C-terminal VP2-452 deleting mutants (designated as N5-452H, N10-452H, N20-452H, N40-452H, VP2-441H, VP2-437H, VP2-411H and VP2-399H) was expressed in insect cells to discover the role of both N- and C-termini on the assembly of SVP and to develop an efficient SVP-based vaccine. Among these mutants, the expression level of N5-452H was the highest. Results of ultracentrifugation and electron microscopy also indicated that mutants of N-terminal deletion N10-452H, N20-452H and N40-452H or C-terminal deletion VP2-411H and VP2-399H lost the capability to self-assemble SVP. The other mutants, N5-452H, VP2-441H and VP2-437H, formed SVP. Additionally, SVP formed by N5-452H could not only be single-step purified by immobilized metal-ion affinity chromatography (IMAC), but it could also induce a high titer of neutralizing activity to protect chicks from the infection of IBDV at a low dosage (0.2 μg), suggesting that SVP formed by N5-452H can be an alternative vaccine candidate for the prevention of IBD.

English

Foot-and-mouth disease is caused by FMDV, an Aphthovirus of the viral family Picornaviridae. Peptide fragments of Foot-and-mouth disease virus protein can be used to select nonamers for use in rational vaccine design and to increase the understanding of roles of the immune system in infectious diseases. Analysis shows MHC class II binding peptides of protein from Foot- and-mouth disease virus are important determinant for protection of host form viral infection. In this assay we predicted the binding affinity of protein having 213 amino acids, which shows nonamers. These peptides are from a set of aligned peptides known to bind to a given MHC molecule as the predictor of MHC-peptide binding. MHCII molecules bind peptides in similar yet different modes and alignments of MHCII-ligands were obtained to be consistent with the binding mode of the peptides to their MHC class, this means the increase in affinity of MHC binding peptides may result in enhancement of immunogenicity of protein nonamers. Binding ability prediction of antigen peptides to major histocompatibility complex (MHC) class I & II molecules is important in vaccine development from Foot-and-mouth disease virus .

MDV-1 VP22: a transporter that can selectively deliver proteins into cells

The tegument protein VP22 of Marek's disease virus (MDV) was previously shown to be able to travel between cells. To further characterize the transport property of VP22 and assess whether it can be used for protein delivery, we investigated the subcellular localization of VP22 fused to five heterologous proteins, including green fluorescent protein, nucleoprotein of avian influenza virus, bovine IFN-gamma, F protein of Newcastle disease virus and VP2 protein of infectious bursa disease virus. The transport of these fusion proteins in monolayer cells was assayed by immunofluorescence assay. The results showed that all except VP2 could be delivered by VP22 of MDV serotype 1 (MDV-1), at different efficiencies. After being transported to surrounding cells, VP22 fused to avian influenza nucleoprotein, bovine IFN-gamma, or to F was localized in the nucleus. Our data suggest that VP22 of MDV-1 can be used as transport tool in protein delivery and that the original localization of cargo proteins may be changed after transport by MDV-1 VP22. Finally, infectious bursa disease VP2 protein could not be transported by MDV-1 VP22.