Vaccinia

Vaccinia virus Vaccinology Vacodur 16 Vacoflux Z Vacoperm 100... 

Live vectors (131,133) are another appHcation of genetic engineering. In this case, the genes from a pathogen are inserted into a vaccine vector, such as salmonella or vaccinia. In the case of salmonella, it will be possible to develop an oral vaccine. Vectors for this appHcation include salmoneUa, BCG, poHo, adenovims, and vaccinia. 

R. Ellis, "The AppHcation of rDNA Technology to Vacciues," ia S. A. Plotkin and B. Eantiui, eds.. Vaccinia, Vaccination and Vaccinology, Elsevier, Paris, 1996. 

Thiosemicarba2ones have long been used as antiviral agents, principally against pox vimses of the vaccinia family. One compound of this series, the isatin derivative (6) C HgN OS, has been used prophylacticaHy to prevent outbreaks of smallpox in humans (10) and to inhibit the protein synthesis in poxvims-infected cells. The molecular mechanics relating to this property are still not known (11), though the binding of a metal ion may be a key factor... 

Methisa2one [1910-68-5] C qH qN OS (l-methyl-3-thiosemicarba2one of 2-oxoindole, (7), one of the more active in the isatin-3-thiosernicarba2one [487-16-1] series, has been used in the treatment and prevention of smallpox and vaccinia infections that develop as complications of smallpox vaccination... 

Replacement of the sulfur atom in the thiosemicarba2one moiety by an oxygen atom leads to a loss of antiviral activity. Methisa2one has no significant effect on vaccinia vims DNA synthesis (14) but seems to inhibit late protein synthesis by a mechanism that remains to be elucidated. 

Topical apphcation of ara-HxMP, C2QH23N40gP, significantly inhibited the development of keratitis-induced HSV-1, HSV-2, or vaccinia vims in the eyes of rabbits. Ara-HxMP also significantly controlled the development of HSV-1 or vaccinia viral-induced encephaUtis in mice and was also active in preventing equine abortion viral deaths in hamsters. Clinical trials with ara-HxMP have not yet been reported. 

The cychc phosphonate of ( -HPMPA, [( -cHPMPA [106941-26-8], C H 2N50 P, 28] also is active against HSV-1 and HSV-2 infection in mice. Both (27) and (28) are reported to be efficacious against adenovims, vaccinia vims, as weU as variceUa-2oster vims infections (58). 

Rodriguez, J. F., et al. (1988). Expression of the firefly luciferase gene in vaccinia virus a highly sensitive gene marker to follow virus dissemination in tissues of infected animals. Proc. Natl. Acad. Sci. USA 85 1667-1671. 

Hamre D, Brownlee KA, Donovick R (1951) Studies on the chemotherapy of vaccinia virus. II. The activity of some thiosemicarbazones. J Immunol 67 305-312... 

Taylor JM, QuUty D, Banadyga L, Barry M (2006) The vaccinia virus protein FIL interacts with Bim and inhibits activation of the pro-apoptotic protein Bax. The Journal of biological chemistry 281 39728-39739... 

Quenelle DC, Collins DJ, Wan WB, Beadle JR, Hostetler KY, Kern ER (2004) Oral treatment of cowpox and vaccinia virus infections in mice with ether Upid esters of ddofovir. Antimicrob Agents Chemother 48 404-412... 

Verini, M.A. and M. Ghione. Activity of distamycin A on vaccinia virus infection of cell cultures. Chemotherapia 1964, 9, 144-157. 

DNA viruses Poxviruses Variola Vaccinia Large particles 200 x 250nm complex symmetry Variola is the smallpox virus. It produces a systemic infection with a characteristic vesicular rash affecting the face, arms and legs, and has a high mortality rate. Vaccinia has been derived from the cowpox virus and is used to immunize against smallpox... 

Methisazone (Fig. 5.2IB) inhibits DNA viruses (particularly vaccinia and variola) but not RNA viruses, and has been used in the prophylaxis of smallpox. It is now little used, especially as, according to the World Health Organization, smallpox has now been eradicated. 

Virus replication comprises numerous biochemieal transformations that might provide suitable targets for antiviral therapy. The antiviral effect of thiosemicarbazones was first demonstrated by Hamre et al. [53, 54], who showed that p-aminobenzaldehyde-3-thiosemicarbazone and several of its derivatives were active against vaccinia virus in mice. These studies were extended to include thiosemicarbazones of isatin, benzene, thiophene, pyridine, and quinoline derivatives, which also showed activity against vaccinia-induced encephalitis. The nature of the aldehyde/ketone moiety was not as significant as the presence of the thiosemicarbazide side chain the latter was deemed essential for antiviral activity. 

Thornton-Manning, J. Appleton, M. L. Gonzalez, F. J. Yost, G. S. Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes correlation of 3-methyleneindolenine formation and protein binding. J. Pharmacol. Exp. Ther. 1996, 276, 21-29. 

Komano J, Miyauchi K, Matsuda Z, Yamamoto N. Inhibiting the Arp2/3 complex limits infection of both intracellular mature vaccinia virus and primate lentivi-ruses. Mol Biol Cell 2004 15(12) 5197-5207. 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

Figure 4.1. Vaccinia virus topoisomerase I reaction with DNA to form a covalent adduct. Figure adapted from Heyman et al. (1999).

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

McCraith, S., Holtzman, T., Moss, B., and Fields, S. (2000). Genome-wide analysis of vaccinia virus protein-protein interactions. Proc. Natl. Acad. Sci USA 97, 4879-4884. 

Shuman, S. (1992a). DNA strand transfer reactions catalyzed by vaccinia topoisomerase I. J. Biol. Chem. 267, 8620-8627. 

---