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

Inhibitory Effect of a Cytidine Analog on the Growth of Rabies Virus: Comparative Studies with Other Metabolic Inhibitors*

As a routine procedure in the study of the growth of rabies virus in tissue-culture systems (Fernandes et al., Virology 21:128, 1963; Wiktor et al., J. Immunol. 93:353, 1964), newborn mice were injected intracerebrally with various preparations of the tissue culture-passaged virus, and their brain tissues were removed for further study. On one occasion, adult mice injected intracerebrally with newborn mouse brain suspension of rabies virus showed signs of lymphocytic choriomeningitis (LCM) infection, as described by Armstrong and Lillie (Public Health Rept. U.S. 49:1019, 1934). The virus isolated from the mouse brain tissues was identified as LCMlike (LCMLC) in a neutralization test in mice injected with a mixture of the virus and serum obtained from rabbits immunized with guinea pig spleen infected with LCM (obtained through the kindness of Maurice Hilleman, Merck Sharp and Dohme, West Point, Pa.). As a result of this finding, all the tissue cultures maintained in the rabies virus laboratory were studied for the presence of LCM antigen by fluorescent antibodies, by use of either an indirect test against a nonlabeled human LCM convalescent serum (obtained through the kindness of J. Hotchin, New York State Department of Health, Albany, N.Y.) and labeled anti-human serum globulin, or by use of a direct test against serum obtained from guinea pigs immunized with LCM-infected guinea pig spleen (obtained through the kindness of Roger E. Wilsnak, Baltimore Biological Laboratories, Baltimore, Md.). The specificity of these tests was controlled by use of normal human sera that did not react with cell cultures infected with LCM virus. (Nonreactivity of these sera was verified as well by another laboratory.) The specificity of the guinea pig immune serum was established by Wilsnak and Rowe (J. Exptl. Med. 120:829, 1964); its lack of reactivity with uninfected cultures was confirmed in this laboratory. With the exception of rabies-infected dogkidney cultures and one series of rabbit-endothelium cultures, all of the other cell cultures chronically infected with six different strains of rabies virus eventually showed the presence of LCMLC in the fluorescent-antibody test (Table 1). In some cultures the LCMLC was detected soon after infection with rabies virus; in others, it was detected only several passages later. In strain LEP grown in human diploid cells, LCMLC was found only for a few passages. The origin of the six rabies strains was quite varied: LEP and HEP Flury were always maintained either in brains of newly hatched chicks or in chick embryo tissue (Koprowski, Bull. World Health Organ. 10:709, 1954) from the time of their isolation from a human source; the Pasteur and PM strains were always maintained in rabbit brain; and the CVS and Nishigahara strains were passaged in mouse brain. None of the cultures was exposed to other viruses than rabies, and none of the control cultures showed the presence of LCMLC even though these cultures were carried in the same laboratory and were handled by the same personnel. Properties of LCMLC. LCMLC could be isolated from some of the tissue cultures in an apparently \"pure\" form by passaging the infectious material in the presence of antirabies neutralizing serum (serum from a donkey immunized with phenol-treated sheep-brain vaccine, obtained through the kindness of K. Sikes, National Rabies Laboratories, Atlanta, Ga.). The character of the LCMLC preparations varied depending on the tissue-culture system used and the property of the rabies virus with which it was associated. For instance, the PM strain of rabies virus is pathogenic for adult mice injected intracerebrally, and the LCMLC isolated from the system also is lethal for adult mice. However, in contrast to the effect of the original PM strain, the LCMLC is noninfectious for adult guinea pigs, hamsters, and rabbits injected intracerebrally. The HEP Flury strain is pathogenic for newborn mice, but is nonlethal for adult mice in-

Detection of a lymphocytic choriomeningitis component in rabies virus preparations.

Rabies is a disease of major significance to human and veterinary medicine. Derivatives of the vaccine developed by Pasteur and associates 100 years ago (1885) are still in use in many parts of the world. During the past two decades considerable progress has been made in improving the efficiency and safety of rabies vaccines through the use of tissue-culture techniques. The Wistar Institute human diploid rabies vaccine, introduced in 1970, is now in general use in the United States and Western Europe. In spite of having a nearly perfect record of safety and efficacy, this vaccine is too costly for extensive use in many of the developing countries, particularly those where rabies vaccine is most needed. Furthermore, rabies is unique in that vaccine treatment is generally applied, in humans, after exposure to the virus: although postexposure vaccination is effective, the precise mechanism of its action is not yet clearly understood.

Tadeusz J. Wiktor

Papers

Inhibitory Effect of a Cytidine Analog on the Growth of Rabies Virus: Comparative Studies with Other Metabolic Inhibitors*

Detection of a lymphocytic choriomeningitis component in rabies virus preparations.

New Generation of Rabies Vaccine

Method of producing rabies vaccine

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. 1984.