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

Mechanism of the cytopathic effect of rabies virus in tissue culture.

The study of rabies virus and its components with hybridoma antibodies is of particular importance for several reasons. First, rabies, a rhabdovirus, is unique in its ability to infect all warm-blooded animals, including man. It is a strictly neurotropic virus, replicating in neurons and spreading through axons and nerve cells from the site of infection to the central nervous system; the virus is never detected in the blood of infected animals. Transmission occurs in most cases by bites or scratches and only on very rare occasions by inhalation.

Monoclonal Antibodies against Rabies Virus

Clinical and antibody responses of human volunteers to four different serial production lots of human diploid cell vaccine (HDCV) each with a different antigenic value are described. Three to four doses of HDCV administered over a period of 14 days produced high levels of virus neutralizing antibodies with an average titer up to 20 times higher than the titer elicited by four doses of duck embryo vaccine. Antibodies were still present one year after completion of vaccination. Only minimal differences in antibody response could be observed between groups receiving vaccines of different antigenic values. Untoward reactions to the vaccine were few. The possibility of using the vaccine as part of postexposure human prophylaxis against rabies is discussed.

Immunization schedules for the new human diploid cell vaccine against rabies

Spleen lymphocytes from rabbits immunized with live or inactivated rabies virus were exposed in vitro to rabies antigens. Blastogenesis of sensitized spleen cells was measured by incorporation of 3H thymidine into the DNA of actively proliferating cells. The maximum stimulation ratio by rabies antigens was observed 8 days after immunization and animals remained sensitized for at least 175 days. No differences were noted when either live or inactivated viruses were used for immunization or for stimulation of spleen lymphocytes. Purified rabies virions, “soluble antigens,” and glycoprotein fraction showed a stimulating effect but not the nucleocapsid fraction of rabies virions.

In Vitro Stimulation of Rabbit Lymphocytes after Immunization with Live and Inactivated Rabies Vaccines

Summary
The phosphorylation of structural proteins of ten rhabdoviruses has been studied. Each virus preparation has been found to possess at least one phosphoprotein which, in the case of Chandipura, Cocal, Piry viruses or vesicular stomatitis virus (VSV), Indiana and New Jersey serotypes, is the minor NS protein. In rabies virus the sole phosphoprotein appears to be the N protein of the nucleocapsid. For Mokola and Lagos bat viruses, two or three phosphoproteins have been observed, one of which is the N protein. Spring viraemia of carp virus (SVCV) preparations contain two phosphoproteins one of which is, or has a mobility close to, that of the N protein. Kern Canyon virus preparations also possess two phosphoproteins, one with the same electrophoretic mobility as (and possible identity to), the virus glycoprotein while the other has a mobility slightly slower than the N protein. Antigenically the core proteins of rabies, Mokola and Lagos bat viruses have been shown to be related to each other but distinct from those of SVCV or VSV.

Tadeusz J. Wiktor

Papers

Mechanism of the cytopathic effect of rabies virus in tissue culture.

Monoclonal Antibodies against Rabies Virus

Immunization schedules for the new human diploid cell vaccine against rabies

In Vitro Stimulation of Rabbit Lymphocytes after Immunization with Live and Inactivated Rabies Vaccines

Structural phosphoproteins associated with ten rhabdoviruses.