In 1937 a protein material was isolated (1) by ultracentrifugation from extracts of warts (2) occurring naturally in Western cottontail rabbits. Readily purified by alternate low and high speed centrifugation the material sediments in the analytical ultracentrifuge with the sharp boundary indicative of high homogeneity and with a sedimentation constant determined only approximately in previous studies to be of the order of 250 X lo-l3 cm. sec.-l dynes-l (1). Investigation of this heavy protein in the past 3 years has shown it to possess with a remarkable degree of uniformity the biological properties ascribable to the rabbit papilloma virus, as demonstrated by quantitative studies on infectivity, complement fixation, and neutralization with specific immune serum (3). Limited by the small quantities of it available, studies of the physical and chemical properties of the protein have been few. Recently, however, enough of it was obtained for studies (4) in the Tiselius apparatus, and the results showed electrophoretic homogeneity of the protein equal to that of hemocyanins (5) and low molecular weight crystalline proteins studied by this method (6). In the present work, studies have been made to obtain information relative to the molecular size, shape, and homogeneity of the protein. For this purpose two methods have been employed, consisting in the combination of diffusion measurements with (1) sedimentation and (2) viscosity data. The latter analysis is of especial interest, since it has recently been shown (7) to furnish information with respect to the size and shape of homogeneous

As a first step toward the understanding of virus particle interactions in water, we have used the modified single particle analysis test to follow the aggregation of poliovirus and reovirus as induced by low pH in suspensions containing varying amounts of dissolved salts. Salts composed of mono-, di-, and trivalent cations and mono- and divalent anions were tested for their ability to reduce or increase the aggregation of these viruses in relation to that obtained by low pH alone. Mono- and divalent cations in concentrations covering those in natural waters were generally found to cause a decrease in aggregation, with the divalent cations having a much greater effectiveness than the monovalent cations. Trivalent ions (Al3+), in micromolar concentrations, were found to cause aggregation over that at low pH alone. Anions, whether monovalent or divalent, had little ability to produce inhibition of viral aggregation, and thus the overall effects were due almost exclusively to the cation. This was true regardless of whether the overall charge on the virus particle was positive or negative, as determined by the relation between the isoelectric point and the pH at which the tests were carried out. Thus, whereas virus particles conform to classical colloid theory in many respects, there are specific exceptions which must be taken into account in the design of any experiment in which viral aggregation is a factor.

The aggregation of poliovirus and reovirus was followed in buffers at various pH values by means of a single particle analysis (SPA) test. The SPA test used here was modified from the original test reported earlier to prevent disaggregation of virus clumps from invalidating the results. The modified SPA test demonstrated that the efficiency of aggregation, which is a measure of the percentage of collisions which are effective in producing an aggregate, may vary widely depending on the conditions in which the virus is placed. The modified SPA test was also used to demonstrate that the kinetic features of viral aggregation follow the classical laws of colloid particle aggregation, which in turn are solely dependent upon diffusion of the particles as caused by brownian motion.

A fraction of the virions in a monodispersed suspension of ECHO virus is temporarily much more resistant than the rest to disinfection by chlorine (HOC1). This fraction is not genetically different. It may, however, owe its temporary resistance to a different conformation of the protein capsid that retards penetration by the HOC1.

The initial inactivation of reovirus in water containing 3 to 7 microns M bromine as HOBr was very rapid. Electron microscopy revealed extensive physical damage to the virions in as little as 1 min, but none were degraded beyond recognition. As treatment time continued, the reaction rate decreased toward a plateau of resistance, usually at about the 10-4 survival level; still no particles were lost. Progeny grown from these resistant plaque-forming units (PFU) were no more resistant to HOBr than the parent cultures. Small-number aggregation (adhering groups of two to ten virions counted by electron microscopy) had no detectable effect on the level of persistant PFU. Large aggregates seemed to be involved. Sonic treatment at 20 kHz after bromine exposure increased survival PFU titer 10- to 43-fold. Virus exposed to light centrifugation prior to bromine treatment did not show the plateau of resistance. Surviving PFU sedimented faster in a shallow sucrose gradient than single virions. Large aggregates were apparently too few to be counted by electron microscopy, but their penetration and inactivation must be achieved by any disinfectant chosen to rid water of reovirus.

SUMMARY The Rivers ‘revived’ strain of vaccinia virus (cvi), a population of virus particles produced in chorioallantoic membrane of eggs, is shown to be physically heterogeneous. Some cloned subpopulations isolated on RK cells interfere and others complement each other in mixed infection. Some are physically different, as shown by sedimentation velocity spectra, but none produce plaques on L cells. Yet pulse-inoculation (by centrifuging) at an input multiplicity of 20 virus particles per L cell produces progeny that make more plaques on L cells than on the RK cells from which they were selected. High multiplicity is essential to this process and the frequency of emerging L+ virus particles may be increased up to 400-fold if the recipient L cells are starved prior to inoculation. One cloned L+RK− virus, isolated from these progeny, is suppressed in plaque formation by antiserum to the original cvi virus but it no longer produces the characteristic necrotic lesions in rabbit skin. The sudden emergence of these large numbers of host-range variants in the absence of a mutagenic agent is similar in some respects to ‘symmetrical host-controlled modification’ observed with certain bacterial viruses.

Electron microscope counts of virus particles have usually required highly concentrated, purified, preparations with a minimum of extraneous material (2, 4, 6). Isaacs has thoroughly reviewed these results recently (5). But, the virologist frequently does not require such purity. He generally deals with crude tissue extracts, plasmas, chorioallantoic fluids, etc., in which virus activity may be 106 to 109 ID50 per ml. It is rarely greater than 1010. This paper will describe electron microscope counting of two viruses in just such crude form.

Total virus particle counts, infectivity titrations and the ratios between particles and infective units have been determined for vaccinia virus infected tissues. Growth curves of vaccinia in the chorioallantoic membrane are characterized by relatively low ratios from 1 to 4 days after inoculation and a marked rise in the ratio at more prolonged intervals. Ratio determinations of vaccinia virus passages in the egg, rabbit skin, and guinea pig skin have been made to study the phenomenon of adaptation in different hosts. The embryonated egg chorioallantoic membrane shows no variation in the ratio of particles to infectious units during passage and it is concluded that this host is completely susceptible to vaccinia. During adaptive passages on the skin of rabbits and guinea pigs relatively large amounts of non-infective virus appear as indicated by a rise in the particle-infectivity ratios. The extent of ratio increase appears related to the general resistance of the host to the virus. Finally, treatment of crude tissue extracts with sonic vibration is described as an aid in dispersing the virus particles for quantitative particle counts.

Virus of Avian Myeloblastosis. XI. Release of the Virus by Myeloblasts in Tissue Culture2 G. S. Beaudreau, G. S. Beaudreau Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar Caroline Becker, Caroline Becker Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar D. G. Sharp, D. G. Sharp Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar J. C. Painter, J. C. Painter Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar J. W. Beard J. W. Beard Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 20, Issue 2, February 1958, Pages 351–381, https://doi.org/10.1093/jnci/20.2.351 Published: 01 February 1958 Article history Received: 29 July 1957 Published: 01 February 1958

Virus of Avian Erythroblastosis. IV. pH and Thermal Stability2 Robert A. Bonar, Robert A. Bonar Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar Dorothy Beard, Dorothy Beard Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar George S. Beaudreau, George S. Beaudreau Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar D. G. Sharp, D. G. Sharp Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar J. W. Beard J. W. Beard Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 18, Issue 6, June 1957, Pages 831–842, https://doi.org/10.1093/jnci/18.6.831 Published: 01 June 1957 Article history Received: 08 February 1957 Published: 01 June 1957

Journal Article Virus of Avian Erythroblastosis. V. Adenosinetriphosphatase Activity of Blood Plasma from Chickens with the Disease Get access R. A. Bonar, R. A. Bonar Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar G. S. Beaudreau, G. S. Beaudreau Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar D. G. Sharp, D. G. Sharp Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar D. Beard, D. Beard Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar J. W. Beard J. W. Beard Department of Surgery, Duke University School of Medicine, Durham, North Carolina Search for other works by this author on: Oxford Academic PubMed Google Scholar JNCI: Journal of the National Cancer Institute, Volume 19, Issue 5, November 1957, Pages 909–922, https://doi.org/10.1093/jnci/19.5.909 Published: 01 November 1957 Article history Received: 27 May 1957 Published: 01 November 1957

A method is described whereby information regarding the degree of dispersion of a virus particle suspension may be derived from electron micrographs of the virus particles. It involves sedimentation of the particles on an agar-receiving surface and subsequent replication for electron microscopy. The pictures constitute a two-dimensional pattern from which the three-dimensional particle relationships in the liquid suspension can be derived by counting the number of free and of aggregated particles in a given area. From a consideration of the probability of chance superposition of particles when they are sedimented, a mesure of dispersion “D”, is obtained for aggregated suspensions which has been found to be substantially independent of the number of particles present in the picture. Examples are given of how “D” varies under some conditions commonly experienced in the study of purified virus suspensions.

SummarySpheroidal particles of about 100-120 mμ diameter occur characteristically in the plasma of chickens with erythroblas-tosis. With formalin-inactivated concentrates of these particles, immune serums have been produced in the chicken which effectively neutralize the infectivity of the agent. These antiserums also cause agglomeration of particles in concentrates identifiable by electron micrography as those found characteristically in this disease. The results have been interpreted to indicate that the characteristic particles agglomerated constitute the viral agent of avian erythroblastosis.

Chickens have been hyper immunized with formolized concentrates of the virus of avian erythromyeloblastic leukosis together with the untreated plasma of diseased birds containing the agent in high concentration. The resulting immune serums strongly neutralized the infectious properties of the virus and precipitated the characteristic virus particles as observed macroscopically and corroborated by electron micrographs of the precipitates. Precipitation of the virus particles was associated with proportional precipitation of the enzyme activity of the virus to dephosphorylate adenosine triphosphate. The findings constitute the critical specific criterion needed to establish the particles as the virus and the enzyme as a component inseparable, by all methods yet tried, from these virus particles.

An introductory kinetic study of the viral ATPase activity has been based on enzyme particle counts obtained from electron micrographic photographs. This approach has circumvented the necessity of determining enzyme nitrogen, purity and molecular weight.