In vitro infection is a fundamental part of HIV research. A successful detectable infection forms the basis for most experimentation on drug design or vaccine development. Deviation from this infection gives insight into whether a particular treatment regimen may be effective or not. For example, neutralization assay used in vaccine studies to evaluate induction of neutralizing antibodies requires in vitro infection of cells and measuring the ability of serum antibodies to reduce or prevent an infection. Drug screening also uses the ability to limit infection as the proof of a successful interference with virus production. Infecting peripheral blood mononuclear cells (PBMCs) is a very important tool for generating viral stocks (cell free or PBMC-associated) which can be used to infect other cells. Generating these stocks is costly and limiting the use of large volumes of cellfree viral stocks as well as increasing virus yield makes research more cost effective. Thus, studying factors that increase in vitro infection can help us limit virus stock use and provide information on how one can in future gain higher yields from co-culture. This work focused on subtype C since it is the strain that infects most people worldwide and is most abundant in South Africa. Objectives: We wanted to evaluate methods used for enhancement of in vitro infection and possibly develop an in vitro infection protocol for consistent and persistent infection. DNA PCR is not valued as a means of detecting in vitro HIV-1 infections and measuring the secretion of p24 by specialisedELISA is preferred. We therefore wanted to evaluate whether the enhancement of in vitro infection would lead to a better detection of infection in vitro by standard DNA PCR or Real-time(RT)-PCR. Since NF-êâ (a host transcription factor) was identified as playing an essential role in the production of virus the next goal was to evaluate the effect known enhancers of this transcription factor would have on detection of in vitro infection because of a potential increase in virus production. Hypothesis: Spinoculation and NF-êâ enhancement by inducting stimulus gives a higher thus more consistent infection in vitro that can be detected using standard molecular techniques. Methods: Spinoculation and polybrene addition was applied to PM1, CEM.NKR-CCR5 or PBMC cultures to boost infection. Further increase in virus production was attempted using three NF-êâ enhancers. DNA PCR, RT-PCR and p24 ELISA was applied to detect enhancement of infection using the viral strain Du-151. Results: Spinoculation (1200 x g for 3 hours) was superior to polybrene as an enhancer of in vitro infection and this was demonstrated with p24-ELISA, DNA PCR and Real-time PCR. NF-êâ enhancement through UV-C irradiation enhanced viral production in the macrophage/T-cell tropic cell line PM1 (p<0.05) and was superior to H2O2 and LiCl. LiCl had a more pronounced effect in the case of PBMCs. (p<0.05) A viral concentration of 500 TCID50 was sufficiently DNA PCR detectable following 7 day incubation provided that spinoculation and UV-C irradiation was also applied. PM1 was persistently infected in vitro and is in our opinion better suited for experimentation due to the fact that it does not show the degree of cytotoxicity of CEM.NKR-CCR5. This cell line also is known to produce infectious virus that sustain the infection. Conclusion: Detectable PCR results were obtained with 500 TCID50 and the use of enhancing factors. One of these enhancing factors is spinoculation. Spinoculation is a better technique to use to enhance cell virus contact and lacks the toxicity of polybrene. NF-êâ enhancement by UV-C has the best effect on virus production in PM1 where LiCl was found to be better suited for PBMC. DNA PCR can be used to successfully detect infection when enhancing techniques are applied. / Dr. Debra Meyer
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:8270 |
| Date | 21 April 2008 |
| Creators | Greeff, Christiaan |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
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