Within two to three days of coculture, the 293 cells from the Michigan group showed marked loss of adherence, aggregation into large clumps, and focal evidence of ballooning and degeneration (identical to the changes described by Foreman et al. (1)), whereas the cocultures containing 293 cells from the ATCC showed no gross morphologic changes. When supernatants from the grossly affected cocultures were passed through a 0.45-microm filter and added to new cultures of 293 cells (from the Michigan group), similar morphologic changes were induced. Thus, we confirm that there was a filterable and transmissible agent originating from Kaposi's sarcoma tissue that was able to induce marked cytotoxicity in a particular subgroup of 293 cells. However, it is much less clear to us that these cytotoxic effects are due to direct infection with KSHV.
To support our position, using a nested polymerase chain reaction (PCR) to detect KSHV DNA (with a lower limit of detection of 10 copies), (2) we failed to detect persistent KSHV DNA in cultures of 293 cells that showed gross morphologic changes. Furthermore, 293 cells that were clumped and forming giant cells showed no herpesviruses or other potential pathogens when we examined them extensively by transmission electron microscopy.
Similarly, we believe the evidence obtained by light microscopy and transmission electron microscopy that Foreman et al. present for the infection of 293 cells by KSHV is unsubstantiated. The chromatin changes cited in the far right-hand panel of Figure 1B of their article are not "typical of infection by a virus of the herpes group." The white arrows point to nuclei that resemble those in the control cells, which show changes that may be due to karyolysis and not to herpesvirus cytotoxicity. Intranuclear inclusions, which are typical of herpesvirus infections, are not shown.
The transmission electron microscopical features shown in their Figure 5 are even more problematic, since none of the structures purported to represent KSHV virions have the classic features of herpesviruses as we have seen them by electron microscopy in Kaposi's sarcoma (Figure 1) and body-cavity-based B-cell lymphoma cell lines. (3) Specifically, neither the size nor the morphologic features of the structures shown in 5 are consistent with those of herpesviruses; we suggest that the structures depicted may represent primary lysosomes. The "enveloped viral particle" in Panel E is much smaller than the "enveloped particle" in Panel A and the "nucleocapsid" in Panel C. Panel B shows perichromatin granules with halos, a common nonspecific nuclear finding, and not nucleocapsids. In summary, although Foreman and colleagues show good PCR evidence for the passage of KSHV in 293 cells, we strongly support the concept (as the authors themselves suggest) that the extensive cell lysis is not due to direct infection with KSHV.
Andrew Blauvelt, M.D.
National Cancer Institute
Bethesda, MD 20892-1908
Brian G. Herndier, Ph.D., M.D.
University of California, San Francisco
San Francisco, CA 94143
Jan M. Orenstein, M.D., Ph.D.
George Washington University
Washington, DC 20037
References
1. Foreman KE,
Friborg J Jr, Kong W, et al. Propagation of a human herpesvirus from AIDS-associated Kaposi's
sarcoma. N Engl J Med 1997;336:163-71.
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2. Blauvelt A, Sei S, Cook PM, Schulz TF, Jeang KT. Human
herpesvirus 8 infection occurs following adolescence in the United States. J Infect Dis (in press).
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3. Orenstein JM, Alkan S, Blauvelt A, et al. Visualization of human
herpesvirus type 8 in Kaposi's sarcoma by light and transmission electron microscopy. AIDS
1997;11:F35-F45.
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