Detection of infectious bursal disease virus (IBDV) antibodies using chimeric plant virus-like particles.

The aim of the present study is to use Physalis mottle virus (PhMV) coat protein (CP) as a scaffold to display the neutralizing epitopes of Infectious bursal disease virus (IBDV) VP2. For this, three different chimeric constructs were synthesized by replacing the N-terminus of PhMV CP with tandem repeats of neutralizing epitopes of IBDV VP2 and expressed in Escherichia coli. Expression analysis revealed that all the three recombinant chimeric coat protein subunits are soluble in nature and self-assembled into virus-like particles (VLPs) as evidenced through sucrose density gradient ultracentrifugation. The chimeric VLPs were characterized by various biochemical and biophysical techniques and found that they are stable and structurally sound. When the chimeric VLPs were used as coating antigen, they were able to detect IBDV antibodies. These results indicated that the chimeric VLPs can be used as potential vaccine candidates for the control of IBDV, which needs to be further evaluated in animal models.

Tropical food legumes: virus diseases of economic importance and their control.

Diverse array of food legume crops (Fabaceae: Papilionoideae) have been adopted worldwide for their protein-rich seed. Choice of legumes and their importance vary in different parts of the world. The economically important legumes are severely affected by a range of virus diseases causing significant economic losses due to reduction in grain production, poor quality seed, and costs incurred in phytosanitation and disease control. The majority of the viruses infecting legumes are vectored by insects, and several of them are also seed transmitted, thus assuming importance in the quarantine and in the epidemiology. This review is focused on the economically important viruses of soybean, groundnut, common bean, cowpea, pigeonpea, mungbean, urdbean, chickpea, pea, faba bean, and lentil and begomovirus diseases of three minor tropical food legumes (hyacinth bean, horse gram, and lima bean). Aspects included are geographic distribution, impact on crop growth and yields, virus characteristics, diagnosis of causal viruses, disease epidemiology, and options for control. Effectiveness of selection and planting with virus-free seed, phytosanitation, manipulation of crop cultural and agronomic practices, control of virus vectors and host plant resistance, and potential of transgenic resistance for legume virus disease control are discussed.

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein.

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.

Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation.

Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs.

Chimeric tymovirus-like particles displaying foot-and-mouth disease virus non-structural protein epitopes and its use for detection of FMDV-NSP antibodies.

Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli (E. coli) was earlier shown to self-assemble into empty capsids that are nearly identical to the capsids formed in vivo. Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit (P>0.05).

Requirements for brome mosaic virus subgenomic RNA synthesis in vivo and replicase-core promoter interactions in vitro.

Based solely on in vitro results, two contrasting models have been proposed for the recognition of the brome mosaic virus (BMV) subgenomic core promoter by the replicase. The first posits that the replicase recognizes at least four key nucleotides in the core promoter, followed by an induced fit, wherein some of the nucleotides base pair prior to the initiation of RNA synthesis (S. Adkins and C. C. Kao, Virology 252:1-8, 1998). The second model posits that a short RNA hairpin in the core promoter serves as a landing pad for the replicase and that at least some of the key nucleotides help form a stable hairpin (P. C. J. Haasnoot, F. Brederode, R. C. L. Olsthoorn, and J. Bol, RNA 6:708-716, 2000; P. C. J. Haasnoot, R. C. L. Olsthoorn, and J. Bol, RNA 8:110-122, 2002). We used transfected barley protoplasts to examine the recognition of the subgenomic core promoter by the BMV replicase. Key nucleotides required for subgenomic initiation in vitro were found to be important for RNA4 levels in protoplasts. In addition, additional residues not required in vitro and the formation of an RNA hairpin within the core promoter were correlated with wild-type RNA4 levels in cells. Using a template competition assay, the core promoter of ca. 20 nucleotides was found to be sufficient for replicase binding. Mutations of the key residues in the core promoter reduced replicase binding, but deletions that disrupt the predicted base pairing in the proposed stem retained binding at wild-type levels. Together, these results indicate that key nucleotides in the BMV subgenomic core promoter direct replicase recognition but that the formation of a stem-loop is required at a step after binding. Additional functional characterization of the subgenomic core promoter was performed. A portion of the promoter for BMV minus-strand RNA synthesis could substitute for the subgenomic core promoter in transfected cells. The comparable sequence from Cowpea Chlorotic Mottle Virus (CCMV) could also substitute for the BMV subgenomic core promoter. However, nucleotides in the CCMV core required for RNA synthesis are not identical to those in BMV, suggesting that the subgenomic core promoter can induce the BMV replicase in interactions needed for subgenomic RNA transcription in vivo.