VIROLOGY 38, 347-369 (1969)
Short Communications An Electron Microscopic Study of
extremely electron-dense core (diameter 38 f 3 mµ) surrounded by a zone of modEubenangee, an Australian erate electron density, interpreted as the Arbovirus capsid (diameter 63 t 5 mg) . Release of virus from infected cells seemed to occur Many ungrouped arboviruses have been by a budding process (Fig . 1), extracellular isolated in recent years, but details of the particles were enveloped and had a mean diameter of 101 f 4 mµ (Fig . 3) . structure and development of only a few In mouse brain the virus was found in of these are known. This communication neurons, and both in brain and in BHK-21 presents a preliminary report on the struccells budding occurred only from the cell ture and possible mode of development of Eubenangee, a new ungrouped, partially margins, not into cisternae of the endoether-resistant arbovirus, isolated from mos- plasmic reticulum. This latter property could serve to distinguish Eubenangee from quitoes in northern Australia in 1963 (1) . epizootic diarrhoea of infant mice (EDIM) Tit this study, the prototype strain Invirus, which is otherwise morphologically 1074, previously passaged intracerebrally in similar (4) . 1-day-old mice, was grown in BHK-21 cells . The cells, which at no stage show Tit negative contrast preparations numerous particles with a mean diameter of 62 t 3 cytopathic effects, were harvested after 7 days and fixed in 4 % glutaraldehyde in mµ and an obvious capsomer structure 1\'Iillonig buffer . They remained in this (Fig. 4) were seen . The precise arrangement of the capsomers is still being investigated, fixative while in transit between laboratories . but the images of the particles appear to be The cells were then postfixed for 30 minutes at 4 ° in 1 % osmium tetroxide, dehydrated compatible with a 92-hole icosahedral structure . Less frequently, virus-like particles in acetone and embedded in Araldite . Sections were stained with uranyl acetate with a somewhat larger diameter (67-86 mg) then lead citrate . but without any apparent capsomer structurc were observed . Both these types of Infected mouse brain tissue, harvested after 4 days from partially paralyzed mice, particle resemble forms of bluetongue virus was fixed in 4 % fresh formaldehyde (from described by Ritchie and Bowne (5) . Enparaformaldehyde) and then postfixed and veloped particles of Eubenangee virus were not identified in our crude, negatively embedded as were the BHK-21 cells above . For negative staining, infected mouse brain stained preparation, possibly because they material was homogenized in pH 9 .0 borate could easily be mistaken for cell debris . saline, partially clarified by low speed ten- In its structure and mode of development, trifugation, and the resultant supernate Eubenangee clearly resembles not only stained with 1 % n potassium phosphotungbluctongue virus (a', 6), but also African state by a pseudo-replication technique horse sickness virus (7), Colorado tick (3) were calibrated against Magnifications fever and Chenuda viruses (8), Remerovo catalase se crystals . virus (9), and Corriparta virus (10) (as In sections of both BHK-21 cells and mouse brain tissue, virus particles appeared well as our own observations) . In a number within and around characteristic cyto- of respects, all these viruses resemble rcoplasmic foci (Figs . 1-3) . Virus particles viruses in structure and development . Differappeared approximately spherical with an ences in their heat and ether resistance may 347
Fro . 1 . Eubenangee virus budding from cytoplasm of a BILK-21 cell . Note also extracellular virions (E) . X80,000 . (The magnification markers represent 100 mg) . FIG . 2 . Cytoplasmic focus of Eubenangee virus infection in mouse brain . X80,000 . FIG . 3_ Cytoplasmic focus (F) and extraccllular virions (E) . BHK-21 . X80,000 . FIG . 4 . Negatively stained virions in crude mouse brain preparation of Eubenangee virus . Note the obvious capsomer structure and "empty" capsids penetrated by stain . X160,000. 348
SHORT COMMUNICATIONS reflect the greater or lesser importance of budding in their development . ACKNOWLEDGMENTS We thank Misses Awake van Kammen and Sue Turner for their excellent technical assistance, and the National Health and Medical Research Council of Australia for financial support . REFERENCES 1 . DOHERTY, R . L., STANDFAST, IT . A ., WETT,:Rs, R . J ., WHITEHEAD, R . H ., BARROW, G . J ., and GORMAN, B . M . (1968) . Trans . Roy . Sec. Trop . Med . Hyg . 62, 862-867 . 2 . JAMISON, R . M ., and MAYOR, H . D . (1966) . J . Bacteriol . 91, 1971-1976 . 3 . LUFTIG, R . (1967) . J . Ultrastructure Res . 20, 91-102 . 4 . ADAMS, W . R . and KRAFT, L . M . (1967) . Am . J. Pat hot . 51, 39-60 . 5 . RITCHIE, A. E ., and BOWNE, J . G . (1967) . "Proceedings of the Electron Microscopy Society of America" pp . 100-101 . Claitor, Baton Rouge, Louisiana . 6 . BOWNE, J . G ., and JONES, R . H . (1966) . Virology 30, 127-133 . 7 . LECATSAS, C ., and ERASMUS, B . J . (1967) . Arch . Ges . Virusforsch . 22, 442-450 . 8 . MURPHY, F . A ., COLEMAN, P . H ., HARRISON, A . K ., and GARY, G . W . (1968) . Virology 35, 28-40 . 9 . SHESTOPALOVA, N . M ., REINGOLD, V. N ., KARPovccH, L . U ., and CHUMAKOV, M . P . (1964) . Proc . 3rd Europ . Regional Conf . Electron Microscopy, B351-352, Czechoslovak Aced . Sci ., Prague . 10 . CARLEY, J . C . (1967) . Rep . Queensland Inst . Med . Res . 22, 6 . R . D . ScHNAGL I . H . HOLMES R . L . DOHERTY Department of Microbiology University of Melbourne Parkville, Vic., 3052, Australia ; and Queensland Institute of Medical Research Herston, Qld ., 4006, Australia Accepted March 5,1969
Strains of Phage Lambda in Current Use The strains of lambda currently in use arc not descended from a single phage isolate . Rather, these phage strains have been derived as independent isolates from a series of bacterial strains of E . coli K12 . These sepa-
rate isolations were made by several workers, after the chance discovery of a cured K12 strain (1) . It is seen that even distinct cultures of K12 prototroph yield genetically distinct lambda strains . A complication in nomenclature arises from the ambiguous use of the terms "wild type" and "reference" strain . We propose that when a lambda strain is obtained directly from a K12 strain it should be designated by the name of the bacterium from which it was derived . In contrast, lambda strains obtained by mutant selection from lysates, or by crosses between phages, should be given trivial names-preferably the name given by the person who constructed the strain . We shall summarize the history of the K12 strains which have yielded lambda phages commonly in use today . The prototrophic, F+ strain of Tatum (2) is called "K12 wild ." It is lysogenic for lambda and sensitive to virulent lambda, as is its descendent, "K12 With-in ." K12 Witkin was passed, via Lederberg, from Tatum to Witkin, and thence to Pasadena (3) . Unexpectedly, the isolate of K12 wild which Lederberg carried to Wisconsin, called "K12 Wisconsin," differs from its parent in being resistant to virulent lambda . Further, the lambda strains derived from K12 Wisconsin and K12 Witkin differ in that the former mutates at a discernible frequency to virulent lambda . Jacob and Wollman (4) found that their prototrophic F+ strain carried a lambda which contained the mutation va , known to be involved in one pathway for mutation to virulence .' The ability of K12 Wisconsin to give virulent lambda may be attributed to its carrying lambda v, . If so, one presumes that the Paris K12 was actually 1112 Wisconsin, rather than K12 Witkin . We shall designate the lambda derived from K12 Wisconsin as "AK12 ." Calef and colleagues have worked with strain 336, selected as F -trp- in two spontaneous steps (6) . This selection was carried out by Cavalli, who obtained his K12 from Lederberg . Thus, we suppose that strain 336 was derived from K12 Wisconsin (5) . The lambda produced by 336, unlike AK12, can ' For gene symbols, see the reference cited .