RNA viruses show high mutation frequencies partly because of a lack of the proofreading enzymes that assure fidelity of DNA replication. This high mutation frequency is coupled with high rates of replication reflected in rates of RNA genome evolution which can be more than a millionfold greater than the rates of the DNA chromosome evolution of their hosts. There are some disease implications for the DNA-based biosphere of this rapidly evolving RNA biosphere.

https://science.sciencemag.org/content/sci/215/4540/1577.full.pdf

Rapid evolution of RNA genomes

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

An analysis of the known cytotoxic and helper T cell epitopes has revealed similarity within their primary sequences These similar motifs, characteristic of the known determinants, have been incorporated into predictive templates that have been used successfully to define eight helper and three cytotoxic epitopes in four different proteins When the defined epitopes are segregated by restriction element, allele specific subpatterns emerge centering around the general pattern The presence of similarities argues that the binding of peptide antigens to class I and class II is similar in nature In addition, these motifs can be used to predict accurately areas within proteins capable of being recognized by individual MHC class I and class II molecules

A sequence pattern common to T cell epitopes.

Laboratory techniques in rabies , Laboratory techniques in rabies , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

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Laboratory Techniques in Rabies

Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

https://www.pnas.org/content/pnas/93/21/11341.full.pdf

Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety

Comparative nucleotide sequence analysis of the glycoprotein gene of antigenically altered rabies viruses1

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro

The rabies virus glycoprotein is the virion component responsible for induction of virus-neutralizing antibody and protection of animals against challenge. To determine its antigenic structure, a limited number of fragments were obtained by chemical cleavage and isolated by SDS-polyacrylamide gel electrophoresis. Eight large peptides, ranging in size from 32 to 77 amino acid residues, were mapped within the predicted amino acid sequence of the glycoprotein by determining the N-terminal sequence of each fragment. The antigenicity and immunogenicity of these peptides were then evaluated. Following inoculation of mice, all peptides induced significant levels of antibodies capable of binding to virus or purified viral glycoprotein in a solid-phase immunoassay. Fragments Cr1, Cr3, and Cr4, also induced significant levels of virus-neutralizing and complement-dependent lytic antibodies. Fragments Cr3 and Cr4 were immunoprecipitated by hyperimmune rabbit antirabies serum. To locate domains of the glycoprotein responsible for the stimulation of T helper cells, the peptides were tested for their capacity to stimulate proliferation of nylon wool-purified T cells obtained from spleens of rabies-immune mice. Peptides Cr1, Cr2, and Cr3 stimulated antigen-specific proliferation. Proliferation required the addition of syngeneic irradiated antigen-presenting cells and the proliferating cells were Lyt1-positive, Lyt2-negative. In addition, stimulation was obtained using a 36 amino acid synthetic peptide comprised of almost the entire sequence of Crl. Cytotoxic T cells are postulated to play a major role in protection against rabies infection. In vitro, glycoprotein and mixtures of glycoprotein fragments stimulated a secondary cytotoxic response (mediated by Lyt2-positive cells) comparable in magnitude with that stimulated by virus. In the presence of added T cell growth factors, fragment Cr4 also stimulated cytotoxic activity. The immunologic analysis of rabies virus glycoprotein fragments showed that specific immunologic functions are associated with different regions of the molecule. This finding should be a major consideration in the designing of a synthetic rabies vaccine.

Tadeusz J. Wiktor

Papers

Comparative nucleotide sequence analysis of the glycoprotein gene of antigenically altered rabies viruses1

Differences in cell-to-cell spread of pathogenic and apathogenic rabies virus in vivo and in vitro

The Antigenic Structure of the Rabies Virus Glycoprotein

Rage et génie dénétique: Développement de nouveaux vaccins

Recognition of antigenic variants of rabies virus by monoclonal antibodies