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Relationship between commercial kits and the VP3-based ELISA. OD measurements using the IDEXX kit were obtained as for Fig. 4 and compared with those obtained from serum dilutions of 1/100 (A) and 1/500 (B) in the VP3 ELISA. When the KPL kit was used, chicken sera were diluted 1/100 and OD values were compared with those obtained from serum dilutions of 1/100 (C) and 1/500 (D) in the VP3 ELISA. The linear regression formula and correlation coefficient are shown at the top of each plot.
Source publication
The routine technique for detecting antibodies specific to infectious bursal disease virus (IBDV) is a serological evaluation by enzyme-linked immunosorbent assay (ELISA) with preparations of whole virions as the antigens. To avoid using complete virus in the standard technique, we have developed two new antigens through the expression of the VPX a...
Contexts in source publication
Context 1
... With the KPL ELISA, the correlation coefficient was lower at both dilutions (R 2 between 0.54 and 0.64) and values moved in a broader range, but the results were still in good agreement (Fig. 4C and D). For VP3 there was an overall lower correlation with both kits. The best correlation (R 2 0.643) was obtained with the KPL kit at a 1:100 dilution (Fig. 5C). In this case, the correlation of the results with those from the IDEXX kit was lower (R 2 0.5) (Fig. 5A and B). On the basis of these correlations, it is clear that the recombinant VPX-based ELISA is a good alternative to currently available kits, yielding similar if not superior results. Correlation between seroneutralization and ...
Context 2
... values moved in a broader range, but the results were still in good agreement (Fig. 4C and D). For VP3 there was an overall lower correlation with both kits. The best correlation (R 2 0.643) was obtained with the KPL kit at a 1:100 dilution (Fig. 5C). In this case, the correlation of the results with those from the IDEXX kit was lower (R 2 0.5) (Fig. 5A and B). On the basis of these correlations, it is clear that the recombinant VPX-based ELISA is a good alternative to currently available kits, yielding similar if not superior results. Correlation between seroneutralization and ELISA titers. Seroneutralization is the reference technique for diagnosing IBDV. Therefore, establishing a ...
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The prevalence of infectious bursal disease virus (IBDV) was studied in chickens, which had not been vaccinated against IBD. Fifty sera and forty-six bursae of Fabricius from chickens showing impaired growth, collected from 7 IBD vaccination-free farms in Japan were used for virus neutralization (VN) tests and RT-PCR for detection of IBDV genome co...
Infectious bursal disease virus (IBDV) causes infectious bursal disease (IBD), an immunosuppressive disease of poultry. The current classification scheme of IBDV is confusing because it is based on antigenic types (variant and classical) as well as pathotypes. Many of the amino acid changes differentiating these various classifications are found in...
The immunochromatographic assay is an alternative method for simple and rapid detection of Infectious bursal disease virus (IBDV) in chickens using colloidal gold-antibody conjugate. The whole-virus antigen of IBDV (UPM04190 isolate) and the high-affinity polyclonal antibodies directed against IBDV were blotted onto nitrocellulose membranes for tes...
Cytokines are important mediators and regulators of host responses against foreign antigen, with their main function to orchestrate the functional activities of the cells of the immune system. However little is known about the role of cytokines in pathogenesis and immune responses caused by infectious bursa disease virus (IBDV). The aim of this stu...
The full-length RNA genomes of a chicken embryonic fibroblast (CEF)-nonpermissive, very virulent infectious bursal disease virus (IBDV) (strain HK46) were amplified into cDNAs by reverse transcription-PCR. The full-length cDNAs were sequenced and subcloned into a eukaryotic expression vector, from which point mutations were introduced into the VP2...
Citations
... Previous publications reported the development of diagnostic assays based on the production of the VP3 protein using E.coli (Wang et al., 2008) or insect cells (Martínez-Torrecuadrada et al., 2000) as expression systems. The use of a plant-based expression platform may represent an alternative method for the low-cost production of large amounts of recombinant proteins not only for vaccinal but also for diagnostic use (Rage et al., 2020). ...
Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7–100.0) and 94.17% specificity (95% CI: 88.4–97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.
... There are several commercial ELISA kits for the detection of antibodies against IBDV, and most of them use the whole virion as the antigen. The substitution of the whole virion by recombinant proteins produced in heterologous systems or by synthetic peptides is a trend in the diagnostic field due to the biological safety, ease of production and cost reduction [17][18][19][20][21][22]. ...
... There are very few reports evaluating the performance of diagnostic devices based on recombinant proteins of IBDV [17,18,[20][21][22]. We developed an ELISA based on a recombinant VP3 to assess the immunological status of our cohort of vaccinated and nonvaccinated chickens from Paraná State, Brazil. ...
... Despite the agreement between both ELISA results (our rVP3 and the commercial ELISA), the correlation of O.D. values between them was low (R 2 = 0.24). Martínez-Torrecuadrada and coworkers [18] also compared their results with commercial tests and showed a high correlation when a recombinant VP2 precursor was used in the ELISA, contrasting the lower correlation with a recombinant VP3 test (R 2 < 0.4). This low concordance can be reflection of the antigen used to sensitize the plates and the balance of anti-VP2 and anti-VP3 antibodies in the antiserum of each animal [17]. ...
Background
Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria.
Results
We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera.
Conclusions
The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.
... For the above reasons, VP3 seems to be a good candidate for use in enzyme-linked immunosorbent assay (ELISA) diagnostic kits for evaluation of the immune status of birds. A number of papers have been published describing the use of Escherichia coli- [19,20] or baculovirus-expression-system-derived [21] recombinant VP3 as an antigen in ELISA. When choosing a system for the heterologous expression of a recombinant protein, many factors should be taken into account, since each system possesses its own advantages with respect to post-translational modifications, correct folding, and quantitative yield of the target polypeptide. ...
... Nevertheless, the need remains to make such ELISA systems cheaper and easier to manufacture. Most commercially available ELISA systems for anti-IBDV antibody detection are based on whole virion preparations, which are expensive and time-consuming to obtain [21]. The use of recombinant antigens as diagnostic kit components could make the manufacturing of the kits cheaper and, ultimately, reduce expenses for poultry veterinary services. ...
Infectious bursal disease virus (IBDV), which infects young chickens, is one of the most important pathogens that harm the poultry industry. Evaluation of the immune status of birds before and after vaccination is of great importance for controlling the disease caused by this virus. Therefore, the development of low-cost and easy-to-manufacture test systems for IBDV antibody detection remains an urgent issue. In this study, three expression systems (bacteria, yeast, and human cells) were used to produce recombinant VP3 protein of IBDV. VP3 is a group-specific antigen and hence may be a good candidate for use in diagnostic tests. Comparison of the antigenic properties of the obtained polypeptides showed that the titres of antibodies raised in chickens against bacteria- or human-cell-derived recombinant VP3 were high, whereas the antibody level against yeast-derived recombinant VP3 was low. The results of an enzyme-linked immunosorbent assay (ELISA) of sera from IBDV-infected chickens demonstrated that the recombinant VP3 produced in E. coli would be the best choice for use in test systems.
... To date, several ELISA kits for IBD are on the market and the most popular kits use the whole virus. However, the precursor of VP2 and even the neutralizing epitopes expressed in heterologous systems also perform adequately to detect anti IBDV antibodies (Martinez-Torrecuadrada et al. 2000b;Sahithi et al. 2019). ...
... The characterization included antigenic and immunological assays and the subsequent development of an indirect ELISA. Although SVP have been produced in bacteria, yeast and insect cells (Martinez-Torrecuadrada et al. 2000b;Rogel et al. 2003;Dey et al. 2009;Wang et al. 2016), the production of IBDV-SVP in plants has remained, to the best of our knowledge, unexplored. ...
... In a previous study, the researchers of two different groups reported optimal SVP concentrations of 0.5-1.5 µg/well or 1 µg/mL to be used in an in-house ELISA (Martinez-Torrecuadrada et al. 2000b;Dey et al. 2009). Similarly, in this study, the optimal concentration was 95 ng/well, which corresponds to 1.9 µg/mL, of the plantderived antigen. ...
Infectious bursal disease virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds, thus causing important economic losses in the poultry industry. Multimeric particles with different architectures based on the capsid protein VP2 have been widely produced for different purposes. We hereby show the production and easy recovery of IBDV subviral particles (SVP) from transiently transformed Nicotiana benthamiana. The SVP, which were observed by electronic microscopy, proved to be antigenically and immunogenically similar to the virion. Indeed, anti-IBDV antibodies from samples of infected birds recognized these SVP and, when injected intramuscularly, these subviral particles also evoked a humoral immune response in chickens. We developed an in-house ELISA using SVP as coating reagent that demonstrated to be highly accurate and in good agreement with a commercial ELISA. This study demonstrates that the recombinant antigen generated and the technology used to produce it are suitable for developing a diagnostic tool against Infectious bursal disease.
... The trimmed VP2 and VP3 genes of IBDV generated a VLP in baculovirus expression system. 58 Attenuated pathogens are commonly excellent inducers of T cell as well as B cell responses, but as discussed earlier have chances of reversion to a more virulent phenotype. Non-infectious subunits of pathogens such as recombinant proteins, peptides or sugars are poorly immunogenic and have to be formulated with immune-stimulating adjuvants. ...
Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of young chickens. Although first observed about 60 years ago, to date, the disease is responsible for major economic losses in the poultry industry worldwide. IBD virus (IBDV), a double-stranded RNA virus, exists as two serotypes with only serotype 1 causing the disease in young chickens. The virus infects the bursa of Fabricius of particularly the actively dividing and differentiating lymphocytes of the B-cells lineage of immature chickens, resulting in morbidity, mortality, and immunosuppression. Immunosuppression enhances the susceptibility of chickens to other infections and interferes with vaccination against other diseases. Immunization is the most important measure to control IBD; however, rampant usage of live vaccines has resulted in the evolution of new strains. Although the immunosuppression caused by IBDV is more directed toward the B lymphocytes, the protective immunity in birds depends on inducement of both humoral and cell-mediated immune responses. The interference with the inactivated vaccine induced maternally derived antibodies in young chicks has become a hurdle in controlling the disease, thus necessitating the development of newer vaccines with improved efficacy. The present review illustrates the overall dynamics of the virus and the disease, and the recent developments in the field of virus diagnosis and vaccine research.
... To overcome these problems, development of IBD recombinant vaccine as a novel strategy has been introduced using different expression systems. Studies showed that the expressed VP2 proteins in E. coli, yeast, viral vectors, and even plants protect specific pathogen-free (SPF) chickens against IBDV infection (Wu et al. 2004;Taghavian et al. 2013;Huang et al. 2004;Li et al. 2004;Martínez-Torrecuadrada et al. 2000;Gomez et al. 2013;Zanetti et al. 2014). The VP2 protein contains major epitopes that stimulate neutralizing antibodies when folding in the correct manner (Pradhan et al. 2012;Rong et al. 2005;Pitcovski et al. 2003). ...
... The protection against IBDV is achieved by vaccination with inactivated IBDV vaccines; however, administration of the vaccines is associated with limitations due to a poor efficiency (Müller et al. 2012;Mahgoub 2012;Berg 2000;Müller et al. 2012). Several studies have been shown that vaccination of chickens with rVP2 protein of IBDV strains could stimulate neutralizing antibodies required for protection of chickens against IBD (Wu et al. 2004;Taghavian et al. 2013;Huang et al. 2004;Li et al. 2004;Martínez-Torrecuadrada et al. 2000;Gomez et al. 2013;Zanetti et al. 2014). Besides the induction of these antibodies, use of potent molecules that augment immunogenicity without the side effects is a novel idea to increase the immunogenicity and protective efficacy of vaccines. ...
Currently, a variety of immunostimulatory molecules are used as adjuvants in combination with poor immunogenic recombinant infectious bursal disease virus (IBDV) vaccine antigen. TIR domain of Toll-like receptors (TLRs) triggers the interleukin-1 (IL-1) signaling cascade which plays a critical role in immune responses against infectious agents. For this study, the bacmid DNA pBac-VP2 of a field isolate was generated and transfected to Sf9 insect cells to produce recombinant VP2 (rVP2). In addition, based on in silico analysis, the cDNA encoding the conserved TIR domain of TLR7 (TIR-TLR7) contain the IL-1 receptor was synthesized and used as an adjuvant. We further evaluated the ability of rVP2/TIR-TLR7 vaccine candidate to induce specific immune responses in specific pathogen-free (SPF) chickens and compared the efficacy by challenging with the virulent IBDV. The results indicate that rVP2/TIR-TLR7-adjuvanted vaccine elicited humoral immune responses and protected chickens against IBDV infection. Higher levels of neutralizing IBDV antibody titers were observed in chickens intramuscularly immunized with rVP2/TIR-TLR7 than those injected with rVP2 alone. The antibody titers were higher in chickens which received booster injection. Furthermore, chickens immunized with rVP2/TIR-TLR7 in the prime and boost groups had significant protection against a virus challenge. The survival rate was 70 and 90% after the primary and booster rVP2/TIR-TLR7 vaccinations, respectively, and indicated that they were protected from virus challenge. Despite the unvaccinated control group, the immunized chickens with rVP2/TIR-TLR7 did not show any clinical signs and histopathological changes of the bursa. Our results demonstrate that TIR-TLR7 is a potential vaccine adjuvant and can induce antigen-specific immune responses induced by rVP2 for protection against IBDV infection.
... The inactivated and attenuated vaccines may associated with poor efficiency and emergence of new IBDV variants (4). Because of these limitations, developing new VP2 recombinant subunit vaccines using various prokaryotic and eukaryotic expression vectors (14)(15)(16)(17)(18)(19)(20)(21)(22) TLR7 motifs can be potentiated for developing a vaccine capable of stimulating effective immune response against IBDV. As shown in table 2 the eight chimera constructs were slightly differing in physicochemical properties. ...
Infectious bursal disease virus (IBDV) causes highly contagious and immunosuppressive disease in young chickens worldwide. The control of infectious bursal disease (IBD) depends mainly on vaccination and strict hygiene management of poultry farms, but the disease continues to pose an important threat to the commercial poultry industry. Recently, second-generation vaccines based on expression of VP2 in various vectors have been developed as new strategies for vaccination against IBD. A series of the vaccines were made using different adjuvant to examine their immunogenicity. In this study we explore the idea of using TLR7 as bio adjuvant to stimulate immune responses against IBDV. Eight conserved TLR7 motifs were found among Homo sapiens, Mus musculus, and Gallus gallus following alignment of the related sequences. Each of the TLR7 motif was fused to VP2 fragment and VP2/TLR7-1 to-8 constructs were designed. By using in silico analysis include physicochemical properties determination, protein structures prediction, antigenic site determination, and evaluation of model quality, one of the chimeric proteins was subjected to introduce as vaccine candidate. The results indicate that some TLR7 motifs can be potentially used as bio adjuvant for induction of immune responses against IBDV. It is necessary to determine the potential role of the peptide in induction of immunity against IBDV infection in chicken.
... In contrast, baculovirus based systems have been exploited extensively to generate large quantities of recombinant proteins that are antigenically similar to their native counterparts. In recent years, baculoviral system has been used widely to produce recombinant proteins that have been used in several diagnostic assays (Wan et al., 1995;Pastey and Samal, 1998;Martinez-Torrecuadrada et al., 2000;Lopez et al., 2007). In addition, several rapid protocols have been developed for optimum level of recombinant protein production using wave bioreactors resulting in generation of several liters of recombinant baculoviruses from infected Sf9 cells within a few days (Weber et al., 2002;Elias et al., 2007;Kadwell and Hardwicke, 2007). ...
... However, whole virus purification required the propagation of large quantities of virus in embryonic system and depends on difficult and expansive processes. Recombinant protein-based serological assays have been developed for the diagnosis of many animal viral infections, including chicken infectious bursal disease virus (Torrecuadrada et al., 2000), infectious bronchitis virus (Ndifuna et al., 1998), avian influenza virus (Tumpey et al., 2005), porcine circovirus type 2 (Nawagitgul et al., 2002), porcine reproductive and respiratory syndrome virus (Witte et al., 2000), and many others. These assays based on recombinant proteins provide a simple, highly sensitive, noninfectious , and inexpensive tool for antibody detection. ...
... Epitope mapping studies of VP2 have shown at least two conformational epitopes on hypervariable region of the VP2 protein (hvVP2) between residues 206 to 350, which elicits virus QHXWUDOL]LQJ DQWLERGLHV ,VPDLO HW DO Vakharia et al., 1994). Hence, the VP2 protein is the most important candidate to produce vaccines and diagnostic tests (Martinez et al., 2000). The sequence of hvVP2 gene is used for most molecular phylogeny studies and serves to uniquely identify variant, classic, and very virulent IBDV strains %UDQGW HW DO -DFNZRRG HW DO 6LQFH the hvVP2 protein may be used for protective immunity against IBDV or diagnostic tests, the aim of this study was to increase expression of the hvVP2 protein in E.coli system. ...
Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease, an infectious disease of global economic importance in poultry. Structural protein VP2 of IBDV is the most frequently studied protein due to its significant roles in virus attachment, protective immunity, and serotype specificity. The objective of the present study was to improve the expression of hypervariable region of VP2 protein (hvVP2) in Escherichia coli (E.coli). The results showed that the hvVP2 was expressed in very low amount in E.coli. But, codon optimized hvVP2 protein showed significantly enhanced protein expression level. The coding sequence of hvVP2 was amplified and then identified by polymerase chain reaction (PCR) and sequencing. To achieve high-level expression of hvVP2 protein, we optimized hvVP2 gene base on E. coli preferred codons and synthesized the optimized gene. The synthetical gene was cloned into expression vector pET-26b and expressed in E.coli BL21 (DE3). After induction with Isopropyl-D-1-Thiogalactopyranoside (IPTG) and optimization the conditions of expression, the hvVP2 protein was relatively increased and identified by SDS-PAGE and Western blotting. Productive conformation can now be used for structure-based design purposes as well as structure-function relation of VP1 protein. It is suggested that the codon optimized hvVP2-His protein may be a useful option (but it is not enough) for developing diagnostic tests and immunization proposes.
