Elisabeth Tillman and Dr. Richard A. Robison, Microbiology
Cervical cancer is the fifth most common cancer in humans as well as the second most common cause of cancer death in women [3]. In developing countries, cervical cancer is actually the most common female disease, constituting 30% of female cancers [1]. In the United States, cervical cancer is diagnosed in approximately 15,000 women and is responsible for 4,600 deaths each year [2]. At least 90% of all cervical carcinomas are related to human papilloma virus (HPV) infection. The purpose of my study is to reduce the dangerous spread of HPV through effective disinfection.
The entire disinfection study consists of three stages: virus isolation, disinfectant exposure, and infectivity analysis. Stage one, virus isolation, is the most time consuming stage because it involves complex tissue cultures called rafts. These rafts are imitation skin layers that make cultivation of HPV possible because the virus requires fully differentiated tissue to replicate. Special tissue cultures are placed on top of wire mesh in petri dishes. The area under the wire mesh is filled with media, representing the vascular system. Because of the unique diffusion of media, the cells differentiate into a skin-like tissue layer. After growing the rafts for approximately two weeks, virus particles can be extracted. This isolation process requires three days and involves tissue homogenization, a series of centrifugations, and a final purification of the virus in a cesium chloride gradient.
Stage two of the disinfection project involves the actual exposure of the virus collected in stage one to various disinfectants. Examples of disinfectants used include ortho-phthalaldehyde, gludaraldehyde, and sodium hypochlorite. This procedure is much simpler than the virus isolation and consists of drying the virus onto glass coverslips, exposing the coverslips to one of the disinfectants, neutralizing the disinfectants, sonicating the virus off the coverslips, and finally exposing the virus suspension to initially virus-free cells.
Stage three of the project, infectivity analysis, is the stage that leaves the most room for setbacks. The analysis represents a “back door” approach to assaying for infectivity. Again, because HPV only replicates in differentiated cell layers, a traditional approach to testing its infectivity is impossible. As a result, an assay must involve probing for signs of the virus at some point before a full replication cycle is completed. One approach would be to assay for the presence of transcripts of HPV’s early genes. One early transcript is the E1 transcript spliced to the E4 transcript. Probing for it would indicate whether or not the virus has infected the cells and begun transcribing its genome. The most difficult portion of this third stage, then, would be quantification. An accurate count of the transcript would represent the number of cells infected, which would in turn represent the number of active virus particles remaining after exposure to a disinfectant.
Undoubtedly, the three stages of an HPV disinfection study comprise a lengthy and involved process. I would estimate that I have progressed through approximately one half of the total project, that is, halfway through stage two. When I began the project I was encouraged by its apparent novelty as well as its straightforwardness. I was aware that a disinfection study on HPV had never before been accomplished. In addition, the original plan for executing the project called for a combined effort with researchers from another university. Apparently they had stocks of the virus and had developed a reliable assay for determining its infectivity. Essentially, my responsibility would consist only of stage two, the disinfectant exposure. I would obtain virus stocks from the other researchers and perform the disinfection process to the point of exposing the final suspension of virus to a cell monolayer. I would then freeze down those cells and send them to the other university for analysis.
However, the other group of researchers had difficulty finalizing the procedure for stage three. They were unable to obtain consistent results when checking the titers of their virus stocks, and as a result, they withdrew their involvement in the project. So I chose to assume their responsibility and isolate the virus myself as well as develop an infectivity assay.
Up to this point, I have had unexpected success in isolating HPV from raft tissue. I have had years of experience in growing rafts; however, I had never attempted isolating virus from the tissue. On my first run through the procedure I was pleased to detect a visible band of virus in the cesium chloride gradient. I isolated that fraction of the gradient and visualized the virus particles using transmission electron microscopy. The particles were consistent with the estimated 54 nanometer diameter typical of that virus type and were at an estimated concentration of 1 x 107 virus particles per milliliter.
With the virus isolation complete, my next task became developing a protocol for disinfectant exposure. I ran test runs of the procedure without the virus for the purpose of testing the toxicity of the disinfectant on the cell monolayer, and I determined the optimal time for exposing the cells to the disinfectant/virus mixture to be three hours. This would allow time for almost all the viable virus to infect the cells but keep exposure of the cells to the disinfectant at a minimum. The only portion of stage three that I have accomplished so far is isolation of RNA from a sample of the cell line to which I will expose the virus.
I estimate that one more semester will be necessary to complete stages two and three of the total disinfection project. I expect my final results to be surprising to those who assume that disinfection of HPV will be comparable to disinfection of other non-enveloped viruses. HPV has already proven to be a stable virus and is suspected to have the ability to remain active for months on an environmental surface. I look forward to settling the questions about the susceptibility of HPV to disinfectants.
References
- Munoz, N., Bosch, F.X., Shah K.V., and Mehus, A. 1992. The epidemiology of human papillomavirus and cervical cancer.
- Rubin, S.C., and Hoskins, W.J., editors. 1996. Cervical Cancer and Preinvasive Neoplasia. Lippincott-Raven Publishers.
- Sigurdsson K., Cervical cancer, Pap smear and HPV testing: and update of the role of organized Papsmear screening and HPV testing. Acta Pbstet Gynecol Scand. 1999:78 6:467-477.