Subject(s)
Cowpox virus/genetics , DNA, Viral/genetics , Genome, Viral , Gene Library , Restriction MappingABSTRACT
The nonstructural 36K protein of vaccinia virus (VV) mapped in HindIII-P and HindIII-J fragments of VV strain L-IVP has been expressed in E. coli as a fusion protein. The products of 36K gene preserved the antigen homology with the native protein 36K and the capacity to bind specific immunoglobulins of rabbit antiVV serum. The protective properties of 36K gene products and their joint effect with the immunodominant protein 35K were investigated. The non-structural 36K gene products showed no protective activity, but increased the production of specific antibodies in mice immunized with a mixture of both protein preparations, this increase being compatible with that observed after immunization with the inactivated virus preparations.
Subject(s)
Escherichia coli/genetics , Vaccinia virus/genetics , Viral Proteins/genetics , Animals , Cloning, Molecular , Mice , Mice, Inbred BALB C , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , Viral Vaccines/genetics , Viral Vaccines/immunologyABSTRACT
A virus, identified as cowpox virus by its biological properties and the results of the analysis of its DNA, was isolated from a sick 4-year-old child with a clinical picture of pox, though having had no contacts with known natural carriers of the causative agent of this infection. At the same time the isolated virus was found to differ from the reference strain, as well as from other isolates of vaccinia virus by some biological markers (and in particular by the structure of cytoplasmic inclusions of type A) and by the restriction profile of DNA. The Hind III maps indicating the location of restriction sites made it possible to localize the genome differences established in this study. The specific feature of this case was the previous close contact of the child with a mole which was probably the source of infection.
Subject(s)
Cowpox virus/classification , Cowpox/virology , Animals , Chick Embryo , Child, Preschool , Chlorocebus aethiops , Cowpox/pathology , Cowpox virus/genetics , Cowpox virus/isolation & purification , Cowpox virus/pathogenicity , DNA, Viral/genetics , Female , Humans , Mice , Microscopy, Electron , Rats , Restriction Mapping/methods , Vero CellsABSTRACT
The use of selected and nonselected markers for obtaining vaccinia virus recombinants is examined. The advantages of various selection systems are discussed.
Subject(s)
Genetic Markers , Recombination, Genetic , Vaccinia virus/geneticsABSTRACT
Comparative structural analysis of the 36K proteins of vaccinia, ectromelia, cowpox and variola viruses revealed that it is conservative among Orthopoxviruses. The possible role of this protein was suggested. A study of the synthesis kinetics of vaccinia and ectromelia virus 36K proteins established that they belong to late proteins. Electron microscopy of infected cells using protein A labelled with colloidal gold showed that the 36K protein is located in the viroplast and not coupled to virions or any cell organelles.
Subject(s)
Conserved Sequence , Genes, Viral , Vaccinia virus/genetics , Viral Nonstructural Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Chlorocebus aethiops , Microscopy, Electron , Molecular Sequence Data , Vaccinia virus/ultrastructureABSTRACT
This is a critical review of the modern methods of gene mapping in the vaccinia virus genome. Experimental data are given on the localization of the vaccinia virus genes. Major attention is paid to the functions of the proteins encoded by the mapped genes.
Subject(s)
Genes, Viral , Vaccinia virus/genetics , Restriction Mapping , Vaccinia virus/physiology , Viral Proteins/genetics , Viral Proteins/physiology , Virion , Virus ReplicationABSTRACT
We observed the expression of recombinant plasmids genes containing ectromelia virus DNA fragments in E. coli minicells. Using plasmids with vaccinia or ectromelia viruses DNA fragments inserted upstream of lacZ gene we showed that certain orthopoxvirus genome fragments carry out a promoter-like function in bacterial cells.
Subject(s)
DNA, Viral/genetics , Ectromelia virus/genetics , Escherichia coli/genetics , Vaccinia virus/genetics , Autoradiography , Base Sequence , Electrophoresis, Polyacrylamide Gel , Gene Expression , Genes, Viral , Molecular Sequence Data , Plasmids , Promoter Regions, GeneticABSTRACT
Two genetics markers: the herpes simplex virus thymidine kinase and Escherichia coli beta-galactosidase genes were inserted into the 36K protein gene of vaccinia virus located in a HindIII-P DNA fragment. An unstability of recombinant viruses with Lac(+)-phenotype were discovered. A mechanism of viruses unstable variants formation was proposed, it was confirmed by the results of hybridisation analyses of virus recombinant genomes. The importance of a late nonstructural 36K protein gene for virus reproduction was demonstrated.
Subject(s)
Genes, Viral , Vaccinia virus/genetics , Viral Proteins/genetics , Autoradiography , Cloning, Molecular , Escherichia coli/enzymology , Genetic Markers , Molecular Weight , Nucleic Acid Hybridization , Phenotype , Plasmids , Recombination, Genetic , Thymidine Kinase/genetics , beta-Galactosidase/geneticsABSTRACT
The HindIII DNA fragments of vaccinia virus strain L-IVP were cloned in pBR322 bacterial plasmid. A hybrid plasmids collection of pVHn series contains all fragments of virus genome except terminal HindIII-B and HindIII-G, and also a large HindIII-A. The latter was cloned in cosmid pHC79. The obtained collection of hybrid DNA molecules allows to carry out a wide range of molecular biological experiments on the vaccinia virus genome.
Subject(s)
Cloning, Molecular , DNA, Viral/genetics , Genes, Viral , Vaccinia virus/genetics , Blotting, Southern , DNA, Viral/chemistry , Deoxyribonuclease HindIII , Escherichia coli/genetics , Genetic Vectors , Plasmids , Restriction MappingABSTRACT
Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.
Subject(s)
DNA, Viral/genetics , Genes, Viral , Vaccinia virus/genetics , Viral Proteins/genetics , Amino Acid Sequence , Base Sequence , Blotting, Southern , Deoxyribonuclease HindIII , Molecular Sequence Data , Molecular Weight , RNA, Viral/genetics , Restriction Mapping , Viral Proteins/chemistryABSTRACT
Vaccinia virus gene encoding 36K protein was cloned in pUR290 bacterial expressing vector and resulted in the synthesis of a chimeric protein in E. coli. The chimeric protein consists of beta-galactosidase and virus protein in C-termini. It has virus antigen specificity. By monospecific antibody 36K protein of vaccinia virus was determined to be non-virion. It is localized in the cytoplasm of infected cells.
