Electron microscopy of sacbrood virus in situ

VIROLOGY 25, 387-392 (1965)

Electron Microscopy of Sacbrood Virus in situ' PETER E. LEE

BASIL FURGALA

AND

Plant Research Institute and Entomology Research Institute, Canada Department of Agriculture, Ottawa, Canada Accepted November 14, 1964

Tsodiametric particles 28 my in size were observed in thin sections of honey bee larvae showing sacbrood disease symptoms . Particles were not occluded in polyhedra or granules . They were either randomly distributed or arranged in crystalline arrays . Some particle aggregates occurred in vesicles, while others were free within the cytoplasm . Such particles were not observed in sections from healthy bee larvae . INTRODUCTION Saebrood, a disease of honey bee larvae Linnaeus) is caused by a virus . White (1917) was the first to suggest the viral nature of sacbrood . Steinhaus (1949) and Break et at . (1963) observed spherical particles from water extracts of infected larvae with particle sizes of 60 mu and 30 Into, respectively . However, it was Bailey et at . (1964) who correlated an isodiarnetric particle measuring 28 mg to the infectivity of preparations from sacbrood larvae, thus presenting unequivocal evidence for the establishment of sacbrood as a virus disease . Larvae infected with sacbrood succumb in a few days, and the drastic changes which occur both in external and internal morphology of diseased larvae are suggestive of rapid pathologic breakdown in the host cells of diseased larvae . Nothing is known about the course of infection and of the method of viral replication . Moreover, there is no evidence that sacbrood virus particles are in polyhedra or granules, as are many of the insect viruses . Some information on these questions was obtained by studying the virus in situ in infected larval tissue with the (Apis mettifera

I Joint contribution from the Plant, Research Institute (Contribution no . 403) and the Entomology Research Institute, Research Branch, Canada Department of Agriculture, Ottawa .

election microscope . The observatiorns recorded are presented in this paper . MATERIALS AND METHODS Naturally infected larvae showing advanced symptoms of sacbrood were removed from brood cells and injected with a chromeosmic acid solution (Galton 1955) . They were held overnight at 5 ° and minced into small pieces the following day . It was not possible to identify organs from infected larvae due to the advanced disintegration of infected insects . Following fixation, the tissue was washed in distilled Hz0 and dehydrated in a graded series of ethyl alcohol with two changes in absolute alcohol . After two rinses in propylene oxide, they were embedded in Epon resin according to Luft (1961) . Embedded material was polymerized at 45 ° overnight, followed by 1 day at 63 °° . Sections were cut with a diamond knife using a Porter-Blum microtome . Sections were mounted on Formvar-carbon grids, stained for 15-30 minutes in an aqueous solution of uranyl acetate to enhance contrast, and . They were examined rinsed in distilled H,0 in a Philips model 100 electron microscope or a Siemens Elutiskop I . Photographs were taken on the latter instrument at 20,000 X or 40,000 X magnifications . Healthy larvae of a similar size to diseased ones were prepared for sectioning concurrently for comparison, 387

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Fic . 1 . Crystalline inclusion of saebrood virus which appears to he enclosed by a membrane (M) . OBSERVATIONS

Isodiametric particles 28 my in size were found in infected tissue from diseased honey bee larvae, but not in tissue from healthy larvae . The particles were not found in polyhedra or granules, although in some sections they were found in large vesicles

(Fig . 1) . In other sections, the particles were found in a cytoplasmic matrix without a surrounding membrane or vesicle (Figs . 2 and 3) . The arrangement of particles was either random or packed in crystalline arrays, and in some inclusions both random packing and

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FIG . 2 . Large inclusion lying free in the cytoplasmic matrix . Arrow points to particles showing iso , diametric, shape . Particles in highly compacted arrays form a crystal lattice with linear pattern and periodic spacing of 30 mg (eirclc) .

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Fiu . 3 . Particles distributed randomly in cytoplasm . Frn. 4 . Inclusion showing particles in crystalline array with cross-hatched pattern, as well as particles randomly oriented .

SACBROOD VIRUS IN SITU

Fic . 5 . A few particles in crystalline formation with others randomly distributed . Fw . 6 . Inclusion with particles densely compacted but in random distribution .

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crystalline lattices were observed (Figs . 1-4) . The formation of a crystalline array does not appear to be dependent on the abundance of particles . In Fig . 5, only a few particles appear in an orderly fashion; while in Fig . 6, the particles, although numerous, are packed in random fashion . The external morphology of the individual particles is evident in Fig . 2 . The hexagonal outline is clearly shown in particles located in Itie central area of the micrograph . However, fine structure of the particles is not discernible in any of the micrographs . in Figs . 2 and 4, some particles are tightly compacted in crystal lattices to give a linear pattern with a periodic spacing of 30 orµ (Fig . 2), or a cross-hatched pattern (Fig . 4) . The differences in pattern arc most likely the result of different cutting planes in the two inclusions .

Tipula iridescent virus (TIV), a large icosahedral particle (Williams and Smith, in Smith, 190:3) and sacbrood virus are similar in some respects : (1) Both viruses cause rapid pathologic breakdown of their hosts resulting in the release of fluid from infected larvae which is a rich source of virus inoculum . (2) Neither virus is occluded in polyhedra or granules . (3) Sacbrood virus like TIN' can be found in abundance when infected larvae are thin-sectioned and examined in the electron microscope . (4) Both viruses may be found in aggregates with or without a surrounding membrane or vesicle, and the particles of either virus are either arranged randomly or in crystalline arrays . These similarities are of interest, although further investigations have to be undertaken before the nature of sacbrood virus can be ascertained .

DISCUSSION The size and shape of the particles observed in infected tissue font bee larvae showing sacbrood symptoms is in agreement with the size and shape of the particles of sacbrood virus isolated by Bailey et ad .

ACKNOWLRUUttIENTS

(1964) .

The appearance of random packing and crystalline lattices of virus particles within a single inclusion may he significant . The phenomenon suggests that a random distribution of particles not only precedes crystalline formation, but may also be a feature during the breakup of a crystalline aggrogale . The absence of typical polyhedra or granule inclusions in the tissue examined sets sacbrood virus apart front the majority of insect viruses which have been studied . Although it was not possible to determine the organs sectioned in this study, the fine structure of the infected tissue resembled that of insect fat body, and the random dispersal of groups of virus particles throughout the tissues examined was at least in part located in the ground cytoplasm of the cells .

One of us (P . E . L .) wishes to thank Dr . Eric Smith, Mines Branch, Mines and Technical Surveys, Ottawa, for use of the 9iemens 1 .1minkop f electron microscope, and also Miss J . Ng Yelim for assistance in the preparation of Forntvarcarbon substrates . REFERENCES :BAS, A . J . . and Woons, 12 . 1) . BAILEY, L ., ( (1964) . Saehromcl virus of the larval honey bee (ipis mc1(ifera l .innaeus) . Virology 23, 42 Ba56K, J ., SYOBOUA, J ., and KnA, .trc, O . (1903) . Electron microscopy investigation of sacbrood of the honey bee . .1 . Insect Pathul- . S, 385399 . GALrIN, A . .! . (1955) . A chrome-osrniurn fixative fur electron inieroseopy . Ann t . keeor/ 121, 281 . Lrx-r, J . Ii . (1961) . Improvements in epoxy resin embedding materials . J . Biophgs . Biochem . ('21eal . 9, 409-414 . Sem'ru, K . M . (1963) . The cytoplasmie virus diseases . In "Insect Pathology" (E . A . Sleinhans, ed .), Vol . 1, pp . 457-497 . Academic Press, New

York . SrE,NHAi's, E . A . (19x9) . Nomenclature and clas-

sification of insectt viruses .

Boctea-iod . Her . 13,

20:3-223 .

Ww're, ( : .

F . (1917) . 1301 .-431, 1-55 .

Sachruod .

1' . S . belt,Igr .