Otfried Marquardt

Identification and characterization of foot-and-mouth disease virus O1 Burgwedel/1987 as an intertypic recombinant

The foot-and-mouth disease virus field isolate Burgwedel/1987 subtype O1 was found to differ genetically from the antigenically related strain O1 Kaufbeuren within the region encoding the non-structural proteins. This genetic difference was indicated by the RNase mismatch cleavage method and confirmed by nucleotide sequencing. An alignment of sequences encoding proteinase 3C of the Burgwedel isolate and several other virus strains identified this isolate as an intertypic recombinant; the parent strains were O1 Kaufbeuren and a subtype C1 strain. Recombination occurred between nucleotide positions 5493 and 5521, within the region encoding peptide 3B1. Thus, the 5 three-quarters of the O1 genome were fused to the 3-terminal quarter of the C1 genome. Other contemporary isolates from the same district are not recombinants. Sequence alignment distinguished four patterns of proteinase 3C-coding sequences among the virus strains analysed: subtypes A12, C1 and O1 exhibit one pattern each, and another pattern is common to subtypes A5, A10 and O2.

Co-replication of several isotypes of foot-and-mouth disease virus

Genome segments of the foot-and-mouth disease virus isolates O1Lombardy and O3 Venezuela that encode, among other products, capsid protein VP1 were amplified using PCR, and the products were cloned and sequenced. The alignment of up to 11 O3-specific sequences revealed six silent nucleotide changes a well as six changes that cause amino acid substitutions in capsid protein VP1 at positions 45, 83, 141, 145, 170 and 178. The heterogeneity of three O1-specific sequences consisted of seven silent exchanges and amino acid changes at positions 85 and 134 on VP1. Amplification, subclonning and sequencing of cloned O3-specific cDNA was performed to examine the nature of the sequence heterogeneity. As no difference was found among five subcloned sequences, we conclude that the Taq polymerase copied the DNA correctly. The sequence heterogeneity observed with both virus isolates is, therefore, consistent with the quasispecies structure of foot-and-mouth disease virus. Furthermore, amino acid changes at a number of sites have been found to be involved in the formation or modulation of neutralizing epitopes. The novel aspect of this study is the ability to estimate, by cloning of PCR products, the number of virus isotypes, possibly varying in antigenicity, that are able to co-propagate. Seven isotypes of O3 Venezuela were identified. Some are of particular interest because they exhibit a change at VP1 codon 145 that causes the replacement of arginine, possibly essential for virus attachment to cells, by isoleucine.

Nucleotide sequence, genomic organization and cell-cycle-dependent expression of a Chlamydomonas 14-3-3 gene

Members of the 14-3-3 protein family have been identified as regulatory elements in intracellular signalling pathways and cell cycle control. Previously we reported the nucleotide sequence of a 14-3-3 cDNA cloned from the unicellular green alga Chlamydomonas reinhardtii. In this communication, we describe the nucleotide sequence, the genomic organization and the cell-cycle-dependent expression of the corresponding gene. The coding sequence of this gene was found to be interrupted by four introns of 124, 116, 81, and 659 bp, respectively. Introns 2-4 were found in conserved positions as compared to the Arabidopsis 14-3-3 genes. A counterpart to intron 1 absent in the Arabidopsis 14-3-3 genes was found in the human 14-3-3 epsilon gene.

Evidence for non-chromosomal hepatitis B virus surface (HBsAg)- and core antigen (HBcAg)-specific DNA sequences in a hepatoma cell line.

As demonstrated previously, a beta particle fraction isolated from the cytoplasm of PLC/PRF/5 cells contains hepatitis B virus (HBV)-specific DNA. Here, further evidence is provided that the specificity of the DNA for HBV is represented at least by sequences coding for the surface and core antigen (HBsAg and HBcAg). This was shown by two different hybridization techniques. One of them, the technique of Southern, distinguished these hybrid molecules formed from those containing HBV DNA integrated into chromosomes. The HBV-specific beta particle DNA forms two distinct bands separate from the high molecular weight cellular DNA.

Three strains of European foot-and-mouth disease virus are highly conserved in the 3'-termini and highly variable in the genes of two capsid proteins.

Restriction enzyme-generated subgenomic fragments of cloned cDNA prepared from RNA of the strain O1 Kaufbeuren (O1K) of foot-and-mouth disease virus (FMDV) were compared qualitatively and quantitatively for sequence complementarity with radioactive RNA from strains C Oberbayern (CObb) and A2 Spain (A2S) in hybridization experiments on nitrocellulose membranes. Quantitative comparison of nucleic acid sequences neighbouring (C Obb/O1K) or including (A2S/O1K) the 3 end of the virus genomes demonstrated more than 80% homology. In contrast, sequences coding for the capsid proteins VP1 (10%, CObb/O1K; 16 to 21%, A2S/O1K) and VP3 (12%, A2S/O1K) were remarkably heterologous. Sequences downstream from the gene for VP1, i.e. those coding for non-structural proteins, showed 23 to 51%f homology to both RNAs except for the area coding for protein P56a. Here, the observed homology was 82% to CObb and 39 to 46% to A2S.

Hepatitis B virus (HBV)-specific structures found in cytoplasmic extracts of cells producing HBV surface antigen (HBsAg) in vitro.

Two kinds of hepatitis B virus-specific particles are present in cytoplasmic extracts of hepatoma cells synthesizing hepatitis B virus (HBV) surface antigen. One class of particle contains the surface antigen of the virus, is 20S in size and has a buoyant density (in CsCl) of 1.2 g/ml. The second class of particle is a deoxyribonucleoprotein (CNP) with 1.3 g/ml buoyant density (in CsCl) and is 30S in size, the DNA of which contains HBV sequences thus proving virus specificity.