- Volume 68, Issue 11, 1987

The number and size of proteins associated with hepatitis delta virus (HDV) from serum and liver (human, chimpanzee and woodchuck) in the acute and chronic stages of HDV infection were analysed by immunoblotting. HDV particles in serum were separated from serum proteins by gel filtration and peak fractions of HDV antigens were subjected to PAGE. Immunoblotting with human anti-HDV-positive sera and 125I-labelled Protein A revealed two bands of 27K and 29K. It was not possible to identify any core-like structure from liver homogenates by CsCl gradient centrifugation. HDV proteins from such gradients were degraded to a size of 14K as determined by immunoblotting. HDV RNA was found in fractions at a density of 1·5 g/ml. However, direct homogenization of liver tissue in gel electrophoresis sample buffer, followed by PAGE and immunoblotting resulted in identification of HDV-associated proteins of 27K and 29K, indicating that HDV proteins in liver tissue are the same size as those in serum, but that they degrade rapidly. There was no difference in size of HDV proteins in liver samples from humans, chimpanzees or woodchucks.

Human rotaviruses containing rearranged genomes were found to package up to 1800 additional base pairs in virus particles. The viruses compared were indistinguishable in respect of their particle diameters and their apparent S values. Particles containing rearranged genomes were found to be of a higher density than rotavirus particles containing a standard genome as determined by equilibrium density ultracentrifugation. The increase in density was directly proportional to the number of additionally packaged base pairs.

To determine the extent and nature of the antigenic variation of four U.S.A. serotypes of bluetongue virus (BTV), the complete nucleotide sequence was determined for cDNA clones representing the L2 dsRNA of BTV serotype 13, the gene that codes for the outer capsid neutralization antigen (VP2). The predicted amino acid sequence of the protein was compared with the VP2 sequences of the U.S.A. serotypes of BTV-10, BTV-11 and BTV-17. Diagon comparisons, hydropathic plots and analyses of potential secondary structure of the four proteins indicated that all four VP2 proteins were structurally similar. However, the VP2 protein of BTV-13 was found to exhibit only 40% homology with the VP2 species of the other three viruses. The comparative sequence data indicated that there were regions of the proteins with greater variability than other regions, as expected for proteins that vary antigenically but are structurally similar.

A 646 bp fragment derived from a full length cDNA clone of genomic segment 9 of bovine rotavirus (NCDV strain) was inserted into Escherichia coli expression plasmid pEXl. The fragment encodes amino acids 50 to 265 of the major vital neutralization antigen VP7, a 326 amino acid long outer shell glycoprotein. Several transformed bacterial clones were isolated in which the recombinant plasmid directed the synthesis of a cro-β-galactosidase -VP7 fusion protein that was recognized by rabbit polyclonal antibodies against NCDV rotavirus. Sera from rabbits immunized with the fusion protein specifically reacted with VP7 among NCDV virion polypeptides. The chimeric polypeptide was also specifically recognized by two monoclonal antibodies against UK strain rotavirus VP7 that exhibited virus-neutralizing activity. However, immune sera to the chimeric polypeptide showed no neutralizing activity against bovine rotavirus. These results are discussed in view of a recent report that a fusion VP7-β-galactosidase polypeptide comprising 35 more amino acids at the carboxy terminus was able to induce neutralizing antibodies in mice to simian rotavirus SA11

The structures at the 5′ termini of the two genomic RNA species of cherry leaf roll virus (CLRV) were found to be neither 7-methylguanosine caps, nor 5′-hydroxyl groups or 5′-phosphate groups. CLRV RNA could be iodinated using Na125I and Iodogen; the labelled moiety appeared to be covalently linked to the RNA and could be removed by treatment with Pronase or proteinase K without altering the electrophoretic mobility of the RNA. The infectivity of RNA was greatly diminished by these enzymes, but such treatment impaired neither the fidelity nor the efficiency of translation of the RNA in vitro. Electrophoresis of the acetone-precipitable material released from the RNA by ribonuclease A treatment yielded a single radioactive component corresponding in mobility to a protein with a mol. wt. of about 3500. This molecule (VPg) presumably corresponds to the protease-sensitive structure needed for infectivity. An antiserum prepared against RNA of the birch isolate of CLRV precipitated homologous VPg as well as those of the rhubarb, dogwood, walnut and Sambucus racemosa isolates of CLRV, but not the VPgs of arabis mosaic nepovirus or tomato black ring nepovirus.