Masters Theses

Date of Award

6-1985

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Carl J. Wust

Committee Members

Arthur Brown, Richard J. Courtney

Abstract

A persistent alphavirus infection has been established in the pluripotent hematopoietic stem cell line, K-562, with Semliki Forest virus (SF) using a multiplicity of infection (MOI) of I . The infected cell cultures have been maintained for over one year.

The SF infection of K-562 cells can be characterized by three stages: 1) an early stage lasting up to 72 hours post infection (pi); 2) an intermediate stage extending from 72 hours to approximately three weeks post infection; 3) a persistent infection beginning at three weeks pi and continuing at least 15 months pi. In the last stage, small cytological changes occurred throughout the observation period.

The early stage is characterized by the production of infectious virus in high titer (2.9 x 102 pfu/cell); however, there was only a subtle cytopathic effect (CPE) seen as cytoplasmic vacuolization of the cells. Viability of SF-infected cells approached 100% during this period with cell concentration and metabolism being comparable to mock-infected controls. All cells were judged to be infected as determined by indirect immunofluorescence assay and antibody dependent complement mediated cytolysis with antiviral serum. These observations are in contrast to the obvious lytic CPE seen with this virus in other mammalian cells (including human cells).

The intermediate stage (after 72 hours pi but before three weeks) was characterized by a viability that decreased but fluctuated from 75-95%. Cell concentration decreased and then stabilized at 2 x 105 cells/ml, and cell metabolism slowed to about half as measured by uptake of 51Cr0 =4 , [3 H] thymidine, and [3 H] -amino acids. Production of infectious virus declined until it was undetectable by 21 days pi. Cells in this stage of infection were fragile to fixation for immunofluorescence studies but showed positive ADCMC reactions with anti-viral sera.

The persistent stage (after three weeks pi) was unique in that no detectable infectious virus was found but cells showed similar characteristics in respect to growth and metabolism as in the intermediate stage. These cells were especially fragile in fixation procedure for immunofluorescence (IF). By examining the cell debris in IF, it was estimated that nearly all the cells showed fluorescence. ADCMC was lower than in the other stages (48% versus 57% in intermediate, and 70% in early stage) but substantial.

An additional characteristic of persistently infected cells was that they are killed by antiviral antibody in the absence of complement. Uninfected control cells are not killed. In addition, anti-SIN hyperimmune serum could also mediate the death of SF-infected K-562 cells although to a lesser extent than the homologous anti-SF serum, suggesting that both homologous and cross-reacting antigens were present on the cell curface. This killing is analogous to that reported previously with anti-K-562 serum and unimfected K-562 cells.

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