IMpRH server: an RH mapping server available on the Web.

SUMMARY: The INRA-Minnesota Porcine Radiation Hybrid (IMpRH) Server provides both a mapping tool (IMpRH mapping tool) and a database (IMpRH database) of officially submitted results. The mapping tool permits the mapping of a new marker relatively to markers previously mapped on the IMpRH panel. The IMpRH database is the official database for submission of new results and queries. The database not only permits the sharing of public data but also semi-private and private data.

Topology of bovine rotavirus (RF strain) VP6 epitopes by real-time biospecific interaction analysis.

An automated biosensor system designed for measuring molecular interactions in real-time (BIAcore) was used to characterize monoclonal antibodies (Mabs) raised against the inner capsid protein (VP6) of the bovine rotavirus (RF strain). Six Mabs, all reactive in Western blot and in indirect immunofluorescence assays, were mapped, using purified recombinant VP6. These Mabs were delineated into several groups of antibodies. Interactions of selected monoclonal antibodies with purified viral particles were studied by the BIAcore methodology. We showed that some Mabs did not react with single-shelled particles. Conversely, several Mabs reacted with single-shelled particles and one antibody reacted with both single-shelled and double-shelled particles. The latter Mab seemed to interact with VP6 through the holes of the outer capsid and its interaction with the double-shelled particles induced a significant decapsidation. These results allowed a better characterization of the epitopes of VP6. The localization of the epitopes in the viral particle is discussed in comparison with a pepscan study that determined the reactivity of Mabs with nested heptapeptides derived from the whole VP6 molecule.

Identification of cytotoxic T cell epitopes on the VP3 and VP6 rotavirus proteins.

It has been shown previously that the viral glycoprotein VP7 is a major target of cytotoxic T lymphocytes (CTLs) induced by bovine rotavirus RF in C57BL/6 mice. Here we show that these RF-specific CTLs recognize target cells infected with a recombinant vaccinia virus (rVV) expressing the SA11 (simian rotavirus) VP6 but not those infected with rVVs that express RF VP1, VP2 or VP3 core proteins. After immunization of mice with insect cells infected with recombinant baculoviruses that express the corresponding RF proteins a strong specific CTL response was generated against target cells infected with VP6 rVV and VP3 rVV, a weak one against the VP2 rVV but none against the VP1 rVV. Kb-restricted CTL epitopes were identified on VP6 and VP3 using allele-specific motifs. When peptides corresponding to these epitopes were used to restimulate in vitro the spleen cells from RF-immunized mice, CTLs specific for each peptide and its corresponding rVV were induced.

Inhibition of in vitro reconstitution of rotavirus transcriptionally active particles by anti-VP6 monoclonal antibodies.

Six monoclonal antibodies specific for the major capsid protein of rotavirus, VP6, previously characterized, were tested in a biological assay for their capacity to block the transcriptase activity associated with the single-shelled particles. The results showed that two MAbs (RV-50 and RV-133), specific for distinct antigenic sites, were able to block the transcription when they were incubated with a purified baculovirus-expressed group A VP6, prior to the reconstitution of the single-shelled particles from the cores, suggesting that at least two domains are involved in active single-shelled particle reconstitution. The results obtained previously from immunochemistry of synthetic peptides did not allow us to attribute this biological activity to a particular linear sequence of the protein, the domain involved being probably complex and dependent on the folding of the protein. However, the C-terminal end, which is necessary for binding into single-shelled particles could be necessary but not sufficient to restore the transcription, since neither of these two MAbs reacted significantly with peptides of this region. These two MAbs will be useful reagents to study the interactions between VP6 and the cores.

In vitro reconstitution of rotavirus transcriptional activity using viral cores and recombinant baculovirus expressed VP6.

Purified baculovirus-expressed group A rotavirus VP6 polypeptide was shown to be active in the recovery of the transcriptase activity associated with the reconstitution of the single-shelled rotavirus particle. Recombinant VP6 polypeptide was able to restore the transcriptional activity in purified viral cores from both SA-11 and RF rotavirus strains. Recombinant group C VP 6 (Cowden strain) is capable of binding as a trimer to group A viral core particles but unable to restore the transcriptase activity, suggesting that the binding of the polypeptide to cores is not the only requirement to restore the transcriptase activity. The VP 6 group A polypeptide was shown to bind as a monomer to viral cores, indicating that trimerization of VP 6 may be not required for reconstitution of the polymerase activity.

Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses.

VP6 of group C (Cowden strain) rotavirus was expressed in the baculovirus system. The recombinant protein, expressed to a high level in insect cells, was purified by ion-exchange chromatography. The purified protein was proven to be trimeric. The effect of pH on the trimer's stability was investigated. Coexpression of VP6 from group A (bovine strain RF) and VP6 from group C in the baculovirus system did not result in the formation of chimeric trimers. Coexpression of VP2 from group A rotavirus (bovine strain RF) and VP6 from group C in the baculovirus system led to the formation of chimeric, empty, single-shelled particles. These results demonstrate conservation in the domains necessary for binding to VP2 in different serogroups of VP6. The locations of the domains involved in trimerization and in the interaction with VP2 are discussed.