James Varani

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)




Herpes simplex virus is the classic example of a virus which persists indefinitely in the host following the initial infection. Recurrent episodes of active virus growth characterize the infection in some individuals . In addition to the periodic flair-ups of active virus growth, the persistence of virus may be expressed in a more subtle way. It is possible that one or more types of human cancer result from the persistence of this virus in the host.

The exact mechanism by which the virus is maintained in the host between outbreaks of active growth is not known. It is usually not possible to isolate the virus between the outbreaks and the virus is said to be latent during this period. Most of the available evidence indicates that the virus exists in a nonreplicating form when it is latent.

This dissertation describes the establishment and characterization of a model latent herpes simplex virus infection in cell cultures . Cultures of rabbit kidney cells were infected with approximately one infectious unit of virus per 100 cells and incubated at 41C for 1-7 days. No evidence of virus growth was seen at 4 1C and infectious virus disappeared from the cells very rapidly at this temperature. Two-hundred infected cultures were transferred to 37C after incubation at 41C. In 43% of these cultures virus was recovered within 3 days after incubation at 3 7C. In 16% of these cultures no virus was recovered after incubation at 3 7C for up to 150 days. In the remaining 41% of the cultures, however, virus growth occurred but only after lag periods of variable length. The longest lag period was 45 days and the average lag period was 15.3 days. During the period when no evidence of virus growth was seen, treatment of the cells by freeze-thawing or by sonication failed to yield infectious virus. Once active virus growth occurred, virus was able to be isolated from the cultures and this virus was neutralized by commercial herpes simplex virus antiserum.

Quantitative studies indicated that 0.25-1% of the inoculum virus was able to survive incubation at 41C for up to 6 days and replicate after transfer to 3 7C. Immunofluorescence studies indicated that virus-specific antigens were produced at 4 1C. Fluorescence microscopy of cells stained with acridine orange indicated that the block in virus replication occurred early in the infection cycle. Characteristic features of cells infected with virus at 3 7C were absent from the cells infected at 41C.

Although virus failed to replicate in rabbit kidney cells at 41C, control cells preincubated at 41C were fully competent to replicate exogenous virus immediately after transfer to 3 7C .

An attempt was made to establish the latent infection in cells directly at 3 7C by using a very small inoculum of virus . The latent infection could not be established in this way. When virus was added directly onto cells at 3 7C, active virus growth was always seen within 3 days or no growth occurred at all in cultures maintained for up to 30 days .

A latent infection similar to that established in rabbit kidney cells was established in Wistar-38 cells. However, attempts to establish a similar infection in human kidney cells were unsuccessful. The basis for this cell-dependent difference was not investigated.

Cultures were treated with certain hormones and chemical agents in attempts to modify the latent infection. Hormones that were used include hydrocortisone, 17 B-estradiol, progesterone, L-thyroxin and L- epinephrine. Treatment of cultures with progesterone at the time that the cultures were infected with virus and incubated at 41C resulted in a significant reduction in the number of cultures from which virus was recovered after transfer to 3 7C. The other hormones did not affect the latent infection in this way.

Three chemical agents, 5-bromo-2-deoxyuridine, 5-iodo-2- deoxyuridine, and puromycin increased the reactivation rates of virus from cultures when the cultures were treated with the agent at the time of infection with virus and incubation at 41C. When cultures were treated with 5-bromo-2-deoxyuridine at the time of transfer from 41C to 3 7C (after the latent infection had already been established) the overall recovery rate was the same from treated cultures as from control cultures. However, the average lag period of the treated cultures was significantly increased.

Finally, treatment of cultures with neutral red dye and exposure of these cultures to strong visible light had no effect on the overall recovery rate but did increase the average lag period of the treated cultures.