R Stark

Genomic localization of hog cholera virus glycoproteins

A polyspecific antiserum has been used to identify four different glycoproteins in hog cholera virus (HCV)-infected cells termed gp55, gp48, gp44, and gp33 (Rümenapf et al, 1989, Virology 171, 18-27). Fusion proteins containing parts of the putative HCV-encoded glycoproteins were expressed in bacteria and served for the preparation of specific antibodies. These were used in radioimmunoprecipitation assays which revealed that gp48 and gp44 most likely share a common protein backbone. The order of the glycoproteins on the HCV genome was determined as follows: NH2-gp44/gp48-gp33-gp55-COOH.

Hog cholera virus--characterization of specific antiserum and identification of cDNA clones.

A specific antiserum was raised against the pestivirus inducing hog cholera (hog cholera virus, HCV). Using immunoprecipitation and SDS-PAGE, this antiserum served for comparison of HCV-induced proteins with those from a related and better characterized pestivirus, bovine viral diarrhea virus (BVDV). In addition to immunological relationships, the apparent molecular weights of some proteins induced by both viruses were quite similar. HCV genomic RNA was found to be about 12 kb in length, comparable to BVDV RNA. cDNA was synthesized starting from RNA isolated from partially purified virions and cloned in lambda gt11. Screening with the antiserum resulted in identification of several positive clones. Partial sequencing of one HCV-derived cDNA clone revealed a high degree of homology to a portion of the BVDV sequence.

Molecular characterization of positive-strand RNA viruses: pestiviruses and the porcine reproductive and respiratory syndrome virus (PRRSV)

Molecular characterization has become an important tool for the analysis of viruses including their classification. The manuscript focuses on the molecular analysis of two members of the genus pestivirus (hog cholera virus, HCV and bovine viral diarrhea virus, BVDV) and of the recently discovered porcine reproductive and respiratory syndrome virus (PRRSV). The first protein encoded within the single large pestivirus ORF is a nonstructural protein with autoproteolytic activity. The cleavage site between the protease and the capsid protein p14 has been predicted previously, but recent experimental data indicate that processing occurs at a different site. The capsid protein is followed by a putative internal signal sequence and three glycoproteins which are part of the virion envelope. According to a new proposal for the nomenclature of the structural proteins of pestiviruses they are termed C, E0, E1 and E2. The genomes of BVDV pairs isolated from animals which came down with mucosal disease were analyzed. The genomes from cytopathogenic (cp) BVD viruses may contain insertions highly homologous to cellular sequences. In addition, cp BVDV may differ from its non cytopathogenic (noncp) counterpart by mere rearrangement of viral sequences. The disease PRRS, which emerged a few years ago, is caused by a single strand RNA virus; the viral genome is of positive polarity and has a size of 15 kb. Data concerning morphology, morphogenesis and virion composition suggested already that PRRSV belongs to a group of so-called arteriviruses which comprises equine arteritis virus (EAV), lactate dehydrogenase elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). This conclusion has now been confirmed by analysis of genome organization, gene expression strategy and by comparison of deduced protein sequences.

Molecular characterization of hog cholera virus

An efficient tissue culture system was established which allowed to obtain substantial quantities of hog cholera virus (HCV) from the cell free tissue culture supernatant. After preparation of viral RNA and cDNA synthesis, the complete HCV genome was cloned and sequenced. Comparison with published BVDV sequences revealed a surprisingly high homology between HCV and BVDV at both the nucleotide and the amino acid level. In addition host cellular sequences were identified in BVDV genomes. The genomic localization of HCV glycoproteins was determined by the use of sequence specific antisera directed against bacterial fusion proteins. The order on the HCV genome was determined as follows: N-gp44/48-gp33-gp55-C. HCV gp33 and HCV gp55 were shown to be intracellularly linked by disulfide bridges. A cDNA fragment covering the genomic region that encodes the structural proteins of HCV was inserted into a vaccinia recombination vector. Expression studies with vaccinia/HCV recombinants led to identification of HCV specific glycoproteins which migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.

Molecular Biology of Pestiviruses and Comparison with HCV

Current knowledge on the molecular biology of pestiviruses, which represent a genus of the family Flaviviridae, is briefly summarized and compared with the data available for another genus of the virus family, the hepatitis C virus group. In particular, the genome organization of the two virus groups is compared with respect to the function of the different proteins. Furthermore, a special feature of the pestivirus bovine viral diarrhea virus is presented which is based on RNA recombination and concerns the development of a lethal disease during persistent infection. The recombination frequently involves cellular ubiquitin-coding mRNAs.