The use of RNAi and transgenics to develop viral disease resistant livestock.

The possibility of genetically engineering poultry to make them resistant to avian influenza is attracting attention and has now become a real possibility with improved methods for genetic modification and the emergence of RNAi as an antiviral strategy. In order to test this possibility, we have generated transgenic mice that express RNAi molecules targeting a conserved region of the influenza A NP gene and are testing these mice for resistance to influenza infection. Transgenes were initially developed that express short hairpin RNAs (shRNAs) targeting multiple influenza A viral genes. The shRNAs were tested for inhibition of H1N1 PR8 virus in vitro. Two potent shRNAs that target the NP and PA genes were chosen for lentiviral mediated generation of transgenic mice. Transgenic founders for the NP shRNA construct and also a negative control shRNAtargeting EGFP were generated. The constitutive expression of the shRNA molecules in a range of tissue types including lung, was confirmed and so far stable transmission of the RNAi transgenes from the F0 to F3 generation has been observed. Resistance to influenza infection in these transgenic mice is now being confirmed.

Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression.

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.

First identification of a ranavirus from green pythons (Chondropython viridis).

Ten juvenile green pythons (Chondropython viridis) died or were euthanized shortly after having been illegally imported into Australia from Indonesia in 1998. Histologic examination of two of the three snakes that died revealed moderately severe chronic ulceration of the nasal mucosa and focal or periacinar degeneration and necrosis of the liver. In addition there was severe necrotizing inflammation of the pharyngeal submucosa accompanied by numerous macrophages, heterophils, and edema. An iridovirus was isolated in culture from several tissues and characterized by immunohistochemistry, electron microscopy, enzyme-linked immunosorbent Assay, polyacrylamide gel electrophoresis, polymerase chain reaction and sequence analysis, restriction endonuclease digestion, and DNA hybridization. This is the first report of a systemic ranavirus infection in any species of snake and is a new member of the genus, Ranavirus.

Ultrastructure of Hendra virus and Nipah virus within cultured cells and host animals.

The ultrastructure of Hendra and Nipah viruses is described in cultured cells, pigs, horses and humans. Differences in ultrastructure between the viruses are evident within infected cell cultures and lungs from infected amplifier hosts. These differences are important in viral identification and differentiation and understanding the pathogenesis of disease.

Expression of the potyvirus coat protein mediated by recombinant vaccinia virus and assembly of potyvirus-like particles in mammalian cells.

The coat protein of the potyvirus, Johnsongrass mosaic virus (JGMV), was expressed using a recombinant vaccinia virus (VV) system. Ultra-thin section electron microscopy demonstrated that the coat protein assembled into potyvirus-like particles (PVLPs) in recombinant VV infected cells. Infection of cells with two additional VV recombinants expressing coat protein plus N-terminal and N- and C-terminal extensions also resulted in the formation of PVLPs. These results suggest that the ability of VV to express the potyvirus coat protein at sufficient levels to allow PVLP formation in vitro, could make VV a suitable vector for the delivery of PVLPs displaying vaccine antigens in vivo without the need for particle purification and/or inclusion of adjuvant. Use of such a vaccine strategy would also benefit from the proven advantages of poxviruses as vaccines such as stability in a freeze dried form, resistance to environmental factors and the potential for oral administration.

Early recognition of cutaneous melanoma.

Criteria for the early recognition of malignant melanoma include appreciation of variegation in color and irregularity of lesion border and pigment pattern. Application of these criteria should result in the diagnosis of cutaneous melanoma in its premetastatic surgically curable phase.

Latent cytomegalovirus infection of BALB/c mouse spleens detected by an explant culture technique.

Latent murine cytomegalovirus (MCMV) infection of BALB/c mouse spleens was studied using several methods including an explant tissue culture technique, co-cultivation on allogeneic and syngeneic cell cultures and nucleic acid hybridization. BALB/c mice experience latent infection which persists for at least 6 months and involves only a small fraction of spleen cells. The explant culture technique proved to be much more sensitive than other methods for detecting latent infection of lymphoid tissues.

Immunogenicity and reactogenicity of influenza A/New Jersey/76 virus vaccines in normal adults.

Inactivated influenza A/New Jersey/76 virus vaccines were administered intramuscularly to 199 normal adults, aged 19-59, in doses of 200, 400, or 800 chick cell-agglutinating units in a double-blind, placebo-controlled trial. Systemic reactions (including fever) were uncommon, were mild, lasted less than 24 hr, and were more frequently associated with the largest dose. Local reactions were common but mild. A single, rapidly reversible, allergic reaction was noted in a volunteer 2 hr after vaccination. There was a trend toward fewer systemic reactions in vaccines who had preexisting hemagglutination-inhibiting (HAI) antibodies to the vaccine virus in their sera as compared with seronegative vaccines. All vaccine preparations at all three dosages evoked serum HAI titers of greater than or equal to 20 to greater than or equal to 40 in a high proportion of seronegative recipients, with significantly greater geometric mean titers at the highest dosage. Vaccines between the ages of 19 and 23 years manifested significantly lower serologic responses than did vaccinees over the age of 23. Thus, normal adults over the age of 23 can be immunized with a single, well-tolerated dose of A/New Jersey/76 vaccines.

Serologic responses and systemic reactions in adults after vaccination with bivalent A/Victoria/75-A/New Jersey/76 and monovalent B/Hong Kong/72 influenza vaccines.

Bivalent A/Victoria/75-A/New Jersey/76 and monovalent B/Hong Kong/72 influenza vaccines were given alone or together to adutls, and systemic reactions and antibody responses were determined. The rates of systemic reactivity observed varied among vaccine groups. Disrupted vaccines and whole-virus vaccines containing type B antigen only did not cause significant reactivity. Systemic reactions were observed after administration of the bivalent A whole-virus vaccines, and this reactivity was increased if the B vaccine was also administered. Reactions to the more reactive vaccines were less frequent in older subjects or in younger individuals with evidence of previous exposure to influenza antigens in the vaccine. Antibody responses in this study indicated that individuals older than 25 years responded better to A/New Jersey antigens than did younger subjects. The A/Victoria antigen produced lower antibody levels in older individuals than in younger subjects. The B/Hong Kong antibody responses were similar in all vaccine groups.

Serologic responses after two sequential doses of influenza A/New Jersey/76 virus vaccine in normal young adults.

The serologic responses after two sequential nonreactive doses of either chemically disrupted or whole-virus influenza A/New Jersey/76 virus vaccine were evaluated in 112 normal young adults. In general, levels of hemagglutination-inhibiting (HAI) antibody were low after the first dose of vaccine and increased significantly (P less than 0.05) in response to a second dose. Whereas one dose of the preparation from Merck Sharp and Dohme (West Point, Pa.) effectively vaccinated this population, two doses of the vaccines prepared by Parke, Davis and Company (Detroit, Mich.) and Merrell-National Laboratories (Cincinnati, Ohio) were required to produce a similar serologic response. The preparation from Wyeth Laboratories (Philadelphia, Pa.) produced low levels of HAI antibodies even after two doses. These different serologic responses correlated with the viral hemagglutinin content of each vaccine.

Serological responses to whole and split A/New Jersey vaccines in humans and mice following priming infection with influenza A viruses.

Experiments were performed in mice to investigate the role of previous infection on responses to A/NJ/76 vaccines. Results from human studies have demonstrated that the serological responses to A/NJ/76 vaccines varied according to the age of the vaccinee and appeared to be related to their previous exposure to the different strains of influenza A virus. Mice were infected with influenza A viruses representative of the major strains (Hsw1N1, HON1, H2N2, H3N2) and later inoculated with varying doses of whole or subunit A/NJ/ML virus vaccines. Results from these experiments demonstrated a low antigenicity in non-primed mice of the subunit vaccine compared to whole-virus vaccine, but that the antigenicity of both vaccines was enhanced in mice primed by previous infection with earlier H0 and H1 viruses. The responses of heterotypically primed mice were qualitatively similar to those of primed humans following A/New Jersey vaccination.