- Volume 3, Issue 2, 1968

Although the scrapie agent has for many years been considered a virus (Cuill & Chelle, 1938; Eklund, Hadlow & Kennedy, 1963; Andrewes, 1964), recent work has emphasized the need for re-examining this classification (Pattison, 1965; Alper, Haig & Clarke, 1966; Pattison & Jones, 1967; Gibbons & Hunter, 1967). We thought it worth while, therefore, to determine the effect of interferon (Isaacs & Lindenmann, 1957) on scrapie, since previous experiments had demonstrated the efficacy of this antiviral factor in inhibiting the evolution of well-defined acute (Finter, 1964a; Finter, 1967; Gresser et al. 1968a) and subacute (Gresser et al. 1967; Gresser et al. 1968b) viral diseases of mice, and because, to the best of our knowledge, the action of interferon on the scrapie agent has not previously been examined.

Swiss and ic Villejuif mice were from inbred colonies maintained at the Institut du Cancer.

In a previous paper (Abu Salih, Murant & Daft, 1968) we compared two highly sensitive serological tests, the passive haemagglutination (PHA) and bentonite flocculation (BF) tests, for detecting plant viruses. These tests depend on the attachment of either antigen or antibody to a carrier material (tanned red cells or bentonite particles). The PHA test was several hundred times more sensitive than the tubeprecipitin test and twenty to eighty times more sensitive than the BF test but had two main disadvantages. These were the short storage life of the red cells (which could be largely overcome by treating them with formalin) and the need to use high concentrations of antiserum globulin preparations to sensitize the red cells optimally. In contrast, bentonite particles could often be sensitized with lower concentrations of the serum globulin preparations and were stable for many months. Both kinds of sensitized carrier particles were time-consuming to prepare and some batches differed in behaviour from others.

Transformation of primary rat embryo cells by adenovirus types 12 and 2 has been reported by Freeman et al. (1967a, b). The transformation of primary rat kidney cells by adenovirus type 12 has also been described by van der Noordaa (1968). In the latter study, foci of transformed cells were observed 14 days after inoculation with adenovirus type 12. The rapid transformation found in this system led us to investigate the effect of other adenoviruses on these cells.

Adenovirus types 1, 2 and 3 were isolated from faeces by cultivation in dipl id human embryonic lung (HEL) cells. HEL cells were also used for viral propagation and titration. The titres of the stock viruses were respectively 105.6, 105.5 and 106.5 TCD 50 per 0.1 ml. Typing of the adenoviruses was performed in HEL cells by neutralization tests employing guinea-pig sera prepared against the prototype strains.

Serial propagation of varicella virus in tissue culture was accomplished first by Weller (1953) using human embryonic tissue in roller tubes, and later by Taylor-Robinson (1959) in stationary cultures of human fibroblasts and human amnion cells. However, the virus showed a strange anomaly in these systems in that serial transfer of the agent could not be achieved with culture fluids but only by means of intact infected cells. The virus was thus avidly cell-associated and its infectivity and stability appeared to be entirely dependent on cell integrity and viability (Gold, 1965); cell-disruption by any of a variety of procedures resulted in inactivation of the virus. In marked contrast, the virus in human skin vesicles was found to be mostly extracellular since cell-free filtrates of vesicular fluid sometimes had an infective titre almost as high as the unfiltered fluid (Taylor-Robinson, 1959).

The most widely used staining procedure in recent electron microscope studies of virus-infected plants has involved the use of uranyl acetate, either during dehydration or as a post-stain, together with lead citrate as a post-stain (Arnott & Smith, 1968; Milne & de Zoeten, 1967). Following the recent demonstration that the appearance of tobacco mosaic virus in thin section could be altered by prolonging the duration of dehydration staining with uranyl acetate (Cocking & Pojnar, 1968a), the use of other heavy-metal stains for dehydration staining, both alone and in combination with uranyl acetate, has been investigated. A double-staining procedure using phosphotungstic acid and uranyl acetate has been developed and the potential of this procedure in electron-microscope studies of virus infection is discussed.

Phosphotungstic acid has been known for many years to give high contrast when used as a tissue stain during dehydration (Huxley, 1958).