VP7 Protein Of Bluetongue Virus Research Articles

A blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for detecting ASFV antibodies

African swine fever virus (ASFV) is a highly lethal pathogen of domestic and wild pigs. Due to no vaccines or drugs available, early accurate diagnosis and eradication of infected animals are the most important measures for ASFV prevention and control. Bluetongue virus (BTV) core-like particles (CLPs) are non-infectious hollow nanoparticles assembled from the BTV VP3 and VP7 proteins, which could be used as a platform for presenting foreign epitopes. In this study, the secondary structure of BTV VP7 protein was analyzed and predicted using the IEDB Analysis resource. Based on the prediction results of the VP7 protein, the chimeric CLPs with an ASFV P54 epitope were successfully prepared through the BAC-to-BAC baculovirus expression system and sucrose gradient centrifugation. Based on the chimeric CLPs and mAb 2E4 against AFSV P54 epitope, a blocking ELISA for detecting AFSV antibodies was established, and its reaction conditions were optimized. Through comprehensive evaluation of the method, the results showed the chimeric CLPs-based blocking ELISA displayed the best detection performance, with an AUC of 0.9961, a sensitivity of 97.65%, and a specificity of 95.24% in ROC analysis. Compared with western blot and a commercial c-ELISA for detecting anti-ASFV antibodies, this method had an excellent agreement of 96.35% (kappa value = 0.911) and 97.76% (kappa value = 0.946) with the other tests, respectively. This ELISA also had high repeatability, with CV < 10%, and no cross-reaction with the serum antibodies against other swine viruses or Orbivirus. In brief, this was the first report on developing a blocking ELISA based on virus-like nanoparticles chimerized with an antigenic epitope of ASFV P54 for serological diagnosis of ASFV.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6–97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8–100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

In silico, in ovo and in vitro antiviral efficacy of phosphorylated derivatives of abacavir: an experimental approach

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A–K). Among the synthesized title compounds (5A–K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

A multi-species indirect ELISA for detection group-specific antibodies against VP7 protein of bluetongue virus

Abstract In the present study, a multispecies indirect ELISA for the detection of serogroup-specific antibodies against the VP7 protein of bluetongue virus (BTV) has been developed. The assay was optimized using a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Serum samples (n = 515) from small and large domestic ruminants were used to optimize the assay. The percent positivity (PP) values were used for the estimation of cut-off values. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >20.24% PP value was accepted as the cut-off at which DSn was 96.05% with 92.9–98.1 (95%) confidence interval and DSp was 98.47 with 96.1–99.6 (95%) confidence interval. The above results indicate that the multispecies i-ELISA assay is sensitive, specific and minimize the dependence of species-specific conjugated antibodies for serological investigation of bluetongue in small and large ruminants.

Synthesis, Spectral Characterization, Docking Studies and Antiviral Activity of Phosphorylated Derivatives of Lopinavir Intermediate

AbstractLopinavir (LPV) intermediate, a core unit of the HIV‐protease inhibitor is phosphorylated and screened for their binding efficacy with the Hemagglutinin‐Neuraminidase (HN) protein of Newcastle disease virus (NDV) and of VP7 protein of Bluetongue virus (BTV). The docking simulations were encouraged for their synthesis and screened their antiviral activity against NDV in the Chicken Embryonated eggs. The compounds which have exhibited higher binding energy were selected for antiviral evaluation on target pathogens such as NDV and BTV using in in ovo and in vitro methods. The results revealed that the compounds, 5a, 5b, 5i and 5j showed good activity against NDV and 5a, 5b, 5g and 5j exhibited noble antiviral activity against BTV in both in ovo and in vitro than the remaining title compounds.

A competitive ELISA for detection of group specific antibody to bluetongue virus using anti-core antibody

Bluetongue virus (BTV) is transmitted by biting midges, which infects domestic and wild ruminants. In present study, a competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of serogroup-specific antibodies against VP7 protein of BTV has been developed. The assay measures the competition between a group specific antibody against core protein of BTV and a test serum to an optimized concentration of BTV recombinant-VP7 (r-VP7) antigen. Serum samples (n = 895) collected from small and large ruminants were used to optimize the C-ELISA. Percent inhibition (PI) values were used for estimation of the cut-off value for the C-ELISA. On receiver operator characteristic (ROC) analysis, different cut-off values along with their diagnostic sensitivity (DSn) and diagnostic specificity (DSp) were obtained. Among these, >50% PI value was accepted as cut-off at which DSn and Dsp was achieved as 97.6% and 98.0% respectively, at >95% confidence interval. Results show the present C-ELISA assay described to be sensitive, specific and reliable and could be adopted for serological investigation of small and large ruminants.

Preparation and Characterization of a Monoclonal Antibody Against the Core Protein VP7 of the 25thSerotype of Bluetongue Virus

Bluetongue virus (BTV) is a member of the genus Orbivirus, within the family Reoviridae. The VP7 protein of BTV is used for developing group-specific serological assays. To prepare monoclonal antibody (MAb) against VP7 of the 25th serotype BTV, the RNA S7 encoding VP7 was cloned into prokaryotic expression vectors pET-28a (+) and pGEX-6P-1 to generate recombinant plasmids. The recombinant protein VP7 was expressed in Escherichia coli BL21 (DE3), respectively. The results of SDS-PAGE revealed that the VP7 was expressed and the molecular mass of recombinant fusion protein pET-28a (+)/VP7 and pGEX-6P-1/VP7 was approximately 44 kDa and 64 kDa, respectively. The Western blot analysis indicated that the recombinant VP7 possessed good immunoreactivity. After purification, pET-28a (+)/VP7 was used to immunize BALB/c mice, while pGEX-6P-1/VP7 was used to screen for well-to-well MAb-secreting hybridomas. The hybridoma cell line 3H7 against recombinant VP7 that secreted MAbs was obtained. The isotype of 3H7 was identified as IgG1. The purification of recombinant VP7 protein and the monoclonal antibody will have potential applications on competitive ELISA format for BT-specific serum detection method.

Isolation of a Bluetongue virus group-specific monoclonal antibody and application to a diagnostic competitive ELISA.

The Bluetongue virus (BTV) VP7 protein represents an important group-specific antigen that can serve as a basis for diagnostic tests. Here, we report the generation of a novel BTV group-specific monoclonal antibody (Mab; herein named 4H7) that recognizes a conformational epitope in the VP7 protein. We used a phage-displayed peptide screen and site-directed mutagenesis to define the VP7 amino acid residues that most strongly contribute to the conformational epitope recognized by Mab 4H7. Amino acid residues at positions 175, 185, 186, and 278 of the BTV VP7 protein strongly contributed to Mab 4H7 binding. These key amino acid residues are conserved among all BTV serotypes, whereas related Orbiviruses possess at least one amino acid substitution at these positions. We developed a competitive enzyme-linked immunosorbent assay (c-ELISA) using Mab 4H7 and recombinant BTV VP7 protein to detect serum antibodies against this BTV group-specific VP7 epitope. The c-ELISA was used to screen 833 clinical samples collected from animals in three provinces of China. BTV seroprevalence in the three provinces ranged from 25.42 to 47.45 %. This work provides the foundation for a new diagnostic c-ELISA that can be further applied to BTV surveillance activities and informs our understanding of the structure of the BTV VP7 protein.

Serological and molecular evidence of bluetongue in sheep and goats in Uttar Pradesh, India

Cross-sectional experimental study was conducted with the objective to estimate the seroprevalence on the basis of antibodies to VP7 protein of bluetongue virus by competitive enzyme linked immunosorbent assay (c-ELISA), to test the neutralizing ability of the antibody to reference strains of 4 BTV serotypes (BTV-1, 2, 10 and 23) by micro serum neutralization assay (m-SNT), to check the presence of BTV dsRNA and to isolate and characterize bluetongue virus (BTV). A total of 91 serum and 26 whole blood samples were obtained from sheep and goat. The study was conducted between September and November, 2012 when the culicoides midges’ activity is maximal. The animals were observed for clinical signs of BTV infection and serum samples were obtained from all animals for c-ELISA and m-SNT. Further, blood samples were collected from the c-ELISA positive animals and subjected to virus isolation and nested RT-PCR. Out of 91 animals tested, 26 (28.6%) were found to be seropositive by c-ELISA and one sheep showed neutralizing antibody against BTV-1 serotype at a titer of 1.2 log10. Multivalent logistic regression analysis of risk factors like age, sex, body condition and species of animals were considered during the serological study and found that species of animal had significant influence (χ2 = 17.111, P 0.05) on seropositivity. It was worth enough to conclude that higher seroprevalence among goats indicated that goats would be the most important animals in the epidemiology of BTV with less clinical manifestation due to development of acquired immunity as the result of continuous exposure. Keywords : Bluetongue virus (BTV), competitive enzyme linked immunosorbent assay (c-ELISA), goat, seroprevalence, sheep, micro serum neutralization assay (m-SNT), nested reverse transcription polymerase chain reaction (RT-PCR) African Journal of Biotechnology Vol. 12(19), pp. 2699-2705

Evidence for BTV-4 circulation in free-ranging red deer (Cervus elaphus) in Cabañeros National Park, Spain

Bluetongue (BT) is an infectious disease of wild and domestic ruminants caused by bluetongue virus (BTV). BTV-4 spread through southern Spain from 2004 to 2006, whereas a BTV-1 outbreak that started in southern Spain in 2007 is still ongoing. Vaccination and movement restriction regulations are applied to domestic ruminants to control BT, but the potential reservoir role of wild European ungulates has not been clarified so far. The aim of this study was to describe the epidemiology of BTV in the wild free-ranging red deer (Cervus elaphus) population of Cabañeros National Park (CNP) in central Spain during the BTV-4 and BTV-1 epizootics, assessing the potential role of this deer population as a BTV reservoir. Blood samples from 2885 (2542 adults, 208 calves and 135 undetermined) wild red deer were collected from 2005 to 2010 in CNP and surrounding hunting estates. All sera were tested for antibodies against the BTV VP7 protein by ELISA. Ninety-four of the ELISA-positive samples were analysed by serum neutralization to detect BTV-4 and BTV-1 specific antibodies, and 142 blood samples were analysed by RT-PCR for BTV RNA. A total of 371 (12.9%) out of the 2,885 deer (35/208 calves, 307/2,542 adults, and 29/135 undetermined) were positive for antibodies against BTV. Prevalence increased in adult deer from 2005–2006 to 2008–2009, declining thereafter. No positive samples for BTV-1 were found by serum neutralization, whereas 43 deer (38 adults, four calves and one undetermined) were positive for BTV-4 specific antibodies. No BTV RNA positive deer were found by RT-PCR. Antibody detection throughout the study period suggests a maintained circulation of BTV in red deer. However, the lack of BTV RNA detection suggests a minor transmission risk to livestock.

Production, Characterization and Assay Application of a Purified, Baculovirus-Expressed, Serogroup Specific Bluetongue Virus Antigen

The predominant serodiagnostic assay used in many countries to detect bluetongue virus (BTV) infections is a competitive enzyme-linked immunosorbent assay (c-ELISA) which employs two critical reagents: a cell culture-derived BTV antigen and group-specific monoclonal antibody (Mab). Ongoing difficulties have been reported by laboratories in the production and quality control of the native antigen reagent which relies on the presence of adequate molar quantities and appropriate presentation of the major BTV core protein VP7. To address this important issue, a recombinant baculovirus was constructed containing a cDNA copy of genome segment 7 of BTV serotype 11 and used to infect insect cells which, in turn, expressed high levels of theVP7 protein with an estimated molecular mass of 39 kDa. In its purified form, this recombinant protein could be detected by group-specific Mabs designated 3.17.A3 and 8A3B.6 produced against BTV serotypes 1 and 17, respectively, as well as by polyclonal bovine antibodies raised against North American and South African BTV serotypes. No reactivity was observed by Western blot analysis with these two Mabs suggesting that the common antigenic determinants, on the BTV VP7 protein, were mainly conformational. It was interesting to note that the purified recombinant VP7 protein demonstrated a greater degree of reactivity with Mab 8A3B.6 compared to that exhibited with Mab 3.17.A3 when evaluated in an ELISA. Due to its antigenic similarity to the native antigen, the recombinant protein was found to be a suitable replacement for use in a c-ELISA to detect BTV-specific antibodies with the added advantage that it could be consistently produced and was, therefore, amenable to quality control testing for purity, stability and other standards.