Date of Award:

5-1989

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Robert W. Sidwell

Committee

Robert W. Sidwell

Committee

Bill B. Barnett

Abstract

Mouse monoclonal antibodies directed against Pichinde virus (PCV) were produced to evaluate their application as vehicles for the delivery of antiviral drugs or toxins to virus-infected cells. Four monoclonal antibodies, PC4.9A6, PC4.9D3, PC4.7C2, and PC4.8D3, were of the IgG2a subisotype and reacted with acetone-fixed and live PCV-infected Vero-76 cells. In vivo stained splenic macrophages derived from PCV-infected hamsters that had been injected with fluorescein-labeled PC4.9A6 (FITC9A6) demonstrated a 400% increase in total fluorescence over similarly treated, non-infected cells when analyzed by flow cytometry. This is an indication that FITC-9A6 does have some ability to specifically target PCV infected cells in vivo. Radioimmunoprecipitation of viral proteins showed that all the antibodies precipitated two different PCV proteins, one of 64,000 daltons and another of 38,000 daltons. These proteins are, respectively, PCV ix nucleoprotein (NP) and a breakdown product of NP that is present in PCV infected cells.

An immunofluorescent assay (IFA) for PCV was developed. This IFA was used for antiviral drug assays against PCV. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to fixed, virus infected cells at 24 h after infection and counting the fluorescent cells. The 50% effective dose (EDso) for ribavirin against PCV using this IFA was 6.0 IJ. g/ml. The EDso of ribavirin using inhibition of marginal PCV cytopathogenic effect after 12 days was 6.0 IJ. g/ml and using plaque reduction after 5 days is 2.5 IJ. g/ml, indicating that this IFA was of comparable sensitivity.

An immunotoxin (IT) was produced by the conjugation of gelonin to PC4.9A6. This IT was tested in vivo in PCV-infected MHA hamsters. It was not active against the disease at the dosage tested and by the intraperitoneal (i.p.) treatment route employed in this study. The positive control, ribavirin, administered i.p. for 14 days at a dosage of 40 mg/ ml significantly increased the number of survivors. Three of 5 IT toxicity control animals developed some humoral response that inhibited PC4.9A6 binding to infected cells. They did not show any humoral response to the gelonin moiety of the IT.

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