Technique for visualizing papovaviruses in tumors and in tissue cultures.

Negative staining is used widely for the electron microscopic observation of virus particles in partially purified suspensions of virus (S. Brenner and R. W. Home, Biochim. Biophys. Acta 34:103, 1959) and in thinly spread cell preparations of infected tissue (D. F. Parsons, J. Cell Biol. 16:620, 1963). A modification of the negative-staining technique is described in the following paragraphs. This simple procedure has greatly facilitated the detection of papovavirus particles in infected tissues. A fresh or frozen-thawed tissue biopsy approximately 2 to 3 mm in diameter is rinsed in Hanks' balanced salt solution. Excess salt solution is removed with filter paper and the piece of tissue is transferred to a watch glass 1.5 inches in diameter containing 2% sodium phosphotungstate (2% phosphotungstic acid adjusted to pH 7.1 with sodium hydroxide). The piece of tissue is teased apart with sharp-pointed forceps while viewing it under a dissecting microscope. Pieces of tissue and debris that float on the surface are removed with forceps and discarded. As viewed with the microscope, only fine particulate material remains on the surface area to which the grid is to be applied. A carbon-coatzd or parlodion-coated grid is inverted onto the surface and then removed to filter paper to remove excess fluid. The freshly prepared mount is examined in an electron microscope. This technique has been used to visualize five different papovaviruses in naturally occurring and experimentally induced papillomas and in sedimented cell debris from infected tissue cultures. The results are presented in Table 1 and in Fig. 1-6. A high rate of detection of virus particles was achieved in the examination of papillomas obtained from the natural host of each of four viruses. The technique was applied to Shope papillomas of domestic rabbits in which, in the past, electron microscopy has rarely, if ever, revealed virus. It should be noted that virus was demonstrated in 1 of the 7 domestic papilloma preparations examined in the present study (Fig. 5). The papilloma containing demonstrable virus had been induced by an inoculation of Shope papilloma nucleic acid (Y. Ito, Virology 12:596, 1960). Tests for rabbit kidney vacuolating (RKV) virus in this rabbit were negative, making it unlikely that the virus seen in the one domestic rabbit papilloma was RKV virus. Rabbit kidney cell cultures were infected with intact RKV virus or with RKV virus nucleic acid (Y. Ito et al., unpublished data). After cytopathic effect was well established in the cultures, the cells were scraped from the glass surface, centrifuged lightly, and a piece of the pellet was transferred to the phosphotungstate solution. The surface material was mounted on a coated grid as described earlier. Figure 6 shows a cluster of virus particles from one of these preparations. Some of the particles are closely associated with host cell membranes. The exposed surface of the free particles is studded with well-defined capsomeres. There are several advantages in the use of this technique for detecting papovaviruses. (i) It is extremely simple and requires very little time. (ii) The only dilution of the virus is that required for suspending it in the staining solution. (iii) By eliminating centrifugation, clusters of virus particles are retained and are easily demonstrable as seen in Fig. 1, 5, and 6. The technique is especially applicable to viruses that do not readily dissociate from host cell fragments. It has been reported that as much as two-thirds of the virus in a cell extract may be lost in the pellet after low-speed centrifugation (P. H. Black et al., Virology 24:381, 1964). Because of the above advantages, this technique should prove useful in the examination of clinical specimens in which a viral etiology is known or suspected. The likelihood of mistaking cell