Electron Micrograph Evidence: The Ultimate Test

“During preliminary experiments to establish the proportion of virus-coded p24 protein to virus membrane-associated HLA-DR in gradient-enriched HIV-1 preparations, we became aware of a large variability between experiments. In order to determine whether HLA-DR-containing cellular material was contaminating the virus preparations, we carried out enrichment by gradient centrifugation of clarified supernatants from noninfected cells and tested this material for HLA-DR content. We found that, independently of the cell type used, gradient enrichment resulted in the isolation of large quantities of HLA-DR-containing material which banded at a density overlapping that of infectious HIV. Electron microscopy of gradient-enriched preparations from supernatants of virus-infected cells revealed an excess of vesicles with a size range of about 50-500 nm, as opposed to a minor population of virus particles of about 100 nm. Electron micrographs of infected cells showed polarized vesiculation of the cell membrane, and virus budding was frequently colocalized with nonviral membrane vesiculation. Analysis of the cellular molecules present in the fractions containing virus or exclusively cellular material demonstrated that virus and cellular vesicles share several cellular antigens, with the exception of CD43 and CD63, found mainly at the virus surface, and HLA-DQ, which was found only in the cellular vesicles.”

“The third type of smaller particle [found in cell cultures using a negative staining Transmission Electron Microscope], with surface spikes, was found…The small particles were present alongside immature and mature HIV particles but they were less numerous than HIV. The particles were usually spherical but sometimes slightly angular in shape and 65 to 90 nm in diameter [too small to be HIV]…The small spiked particles were not detected by negative staining in a preparation made from a control CEM culture not infected with HIV [so, they are not “HIV”, but are commonly associated with HIV. What are they? How is it known that some “HIV” proteins do not belong to these particles? How is it known that these are not viruses, but that the larger particles are?]”

“We have examined HIV-1 and HIV-2 infected cell cultures by negative staining and IEM [Immune Electron Microscopy], using a range of clinical and monocolonal antibodies. The study was complicated by the presence of a viruslike structure 70-80 nm in diameter, too small to be HIV…HIV-2 cultures also contained large numbers of 130-300-nm particles [too large to be HIV]. The majority of these particles had no detectable fringe and when penetrated by stain were frequently seen to contain an elongated internal core…Subsequent careful examination of the H9/RF (HIV-1) cell line revealed particles having the same morphology [structure] only at a level approaching the sensitivity of the technique”

“Lymph nodes from the PGL [persistent generalized lymphadenopathy - swollen lymph nodes] group [of people believed at risk of AIDS] were characteristically enlarged…In 11 of these 18 lymph nodes, occasional particles were identified that measured approximately 100 nm in diameter with well-delineated outer limiting membranes surrounding central to eccentric predominantly round electron-dense cores [i.e. the size, shape and structure of HIV]…[however] in 7 of 18…no cored particles were identified despite an intensive search. In two of the 20 PGL lymph nodes neither type of particle [with or without cores] were found…[we also studied] non-HIV related reactive lymph nodes [although all the people from which samples were taken were assumed to be HIV-negative, none were actually tested]. Of major significance was the finding of viral-like particles, morphologically [i.e. size, shape and structure] indistinguishable from those observed in PGL…cored particles were seen in 8 of the 15 lymph nodes…The results of this study compel us to conclude that, while the particles observed in PGL lymph nodes are indeed HIV [because 19/20 had anti-p24 staining, while none of the non-HIV lymph nodes did], morphologically similar particles can be seen in other reactive conditions, and the presence of such particles do not, by themselves, indicated infection by HIV. Clearly, techniques that demonstrate the presence of specific viral antigens or viral RNA are needed to supplement ultrastructural observations [or even actual isolation of the putative virus]”

“We used the characteristic cylindrical structure in the core [of the supposed virus particle] as an identifying characteristic for the virus to distinguish it from cellular debris and also noted that it may vary considerably in its dimensions and morphological features…We have found two basic virus particle sizes, 90 nm and 120 nm, both present in large numbers. The larger particle bears no surface projections, while the smaller particle is rarely 'naked' and usually bears projections…the existence of small, mature particles with projections remains unexplained [as does the presence of at least two different sizes when a virus should be very consistent in size]”