Acute bee paralysis virus, a cytoplasmic insect virus

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Acute

29, (1Wj)

Bee Paralysis

Virus, a Cytoplasmic

Insect Virus

A paralysis of adult honey bees (Spis ruell$era L.) has been shown t,o be caused by a virus called acute bee paralysis virus (ABI’V) (1). In size, shape, and sedimentation rate, ABl’V is similar t,o saebrood virus (SBV) which causes a disease of t,he larval honey bee; however, cross infectiou studies and serological tests wit,h these two viruses indicate that tbey are not relat,ed (2). Earlier, WCreported observatliorw in situ ou SW in nat~urally infec%ed honey bee larvae (3); it was t,herefore of int’erest to st#udy ABPV in situ so t)hnt further comparisons betweeu the two viruses could be uiadc. Acute bee paralysis virus (AHI’V) inoculum was rcconstitutmcd with wat)cr froui a freeze-dried preparation kindly supplied by Dr. 1,. Bailey, Rot’hamsted Experiuieut~al Station, Harpenden, Hert,s., England. Houey bees used in the st)udy were collected froiu au apparcnt81y uomal colony and coufincd irr sriiall 4 X -l X 4 inch aut’oelaved screen cages. Each cage contained 25 bees and was supplied with a piece of comb chont,aiuiug pollen and GO?;#sucrose in a gravity feeder. Bees in two eagcs were each injected with virus t,hrough a vent,ral intersegmcntal ~uc’iubrane. The injected bees and (*ages of appropriate coutrol bees were held at 30 f 2”. Four days aft,er injection nlost of the virus-inject’ed bees were moribund. In cou trast, none of the control bees were inoribund. Ahhough other workers (1, /t) have observed paralysis 2-4 days after honey bees arc inoculated with this virus, we did riot, observe auy sign of paralysis. l\loribund bees were dissected in saline, and pieces of fat, body, mid-iut,estine, thoracie muscle, brain, t,horacic and abdouiinal ganglia were prepared for t#hin sect’ioning. Det,ails on the preparation of tissue for elect,ron microscopy have been presented elsewherc (J)

Isodimuetric part’icles measuring 28 uip were observed in thin sectious of fat tissue froui ABPV injected bets (Ngs. 1 -:3), but not in sections from t,he other t’issucs. Viruslike part,icles were found in the cyt(oplasui but uever in t’he uucleus of a cell. The par& cles were observed free in the cytoplasm (E’ig. I) or deuiarrated by a ~011 d&led menlbrane (Figs. 2 and 3). Iwlusions were coinposed of particles randomly distributed or packed iuto crystalline lattJices (Fig. 3). AIost, of the membranous vesicles were oval in shape although part,icles were’ also observed in elongated membranous structures (Fig. 2). l’art’icles were riot found in polyhedra or granules. In additiou to the viruslike particles iu t’he fat body cytoplasm, hollow oval mcmbranous bodies were observed, which may be the remnants of tuit~ochondria following pathologic breakdown (E‘ig. 1). The appcararm of ABPV irk situ ranks it among the uouowluded group of inswt viruses. The location of the particles iu the fat body distinguishes the infection from t’hat of cahronic bee paralysis virus (CBPV), in which the virus appears t,o be liuiit~cd t,o uervous tissue (5). These observations in fat tissue from AHI’V-infeetcd bees may also explain the absence of any overt paralytic symptoms in the infected speciniens. Bailey and Gibbs (/j) suggested t>hat the abnorinal behaviour of bees injected with a lethal dose of ABE’V may be associated with the dcnsc basophilic deposits they observed in light microscope sections of t)he corpora pedunculata and lunien of the midgut. If the virus is present in or associated with these basophilic deposits, it was uot apparent in t,he thin sections examined in this study. Thus, in addit#ion to size, shape, and sedimentation rate (2), the t’ype and location of inclusions present in infected host cells are characteristics eouuuon to ABPV and SRV.

1,ISCIiSSION

ANI)

PRETJILIIP~~ARY

REPORTS

347

FIG. 1. Thin section of abdominal fat) showing ABPV particles free in the cytoplasm. Foote apparent breakdown of mitochondria (M). 32,000. X FIG. 2. Small colonies of particles enclosed in elongated membranous structures (Mb). 40,000 X FIG. 3. Thin section showing oval vesicles (5’) containing isodiamet,ric particles. 80,000 X.

345

DISCUSSION AND PRELIMINARY REFERENCES L., GIBBS; A. J., and WOODS, II. I>.,

REPORTS

The sediments were resuspended in 0.03 M pH 7..i phosphate buffer (1 ml/40 g tissue), and the suspension was clarified by cm21, 390-395 (1963). 2. BAILEY, L., GIBBS, A. J., and WOODS, 12. T)., trifuging it for 3 minutes at 8000 g. The whole procedure was done at 2-Z’. Virology 23, 425-429 (1964). 3. LEE, P. E., and FURGALA, B., Virology 25, 38iElectron microscopy showed that the 392 (1965). resulting preparations contained many iso4. BAILEY, L., J. Invert. Pathol. 7, 132-140 (1965). metric particles about 30 mp in diameter, 5. LEE, P. E., and FURGALA, B., J. Inve~l. Pathol. which tended to aggregate when mountBed 7, 170-174 (1965). in pH 7.U sodium phosphotungstate. 6. BAILEY, L., and GIBBS, A. J., J. Insect. I’athol. Schlicren diagrams obt,aincd when the 6, 395-407 (1964). virus preparat#ions were examined in the B. FCI~GAIA~ analytical ultracentrifuge showed two spePETER E. 1,~s~ -. cific components with sedimentation coeffcients at infinite dilution of 95 S and 115 S, 1Entomology Research Institute the faster occurring in slight’ly larger Canada Department of Agriculture Ottuwa, Canada amount. To study the differences between &se components, the virus preparations 2 Biology Department were fract,ionated by centrifuging them in Carleton University sucrose density gradients at about, 5”; l-2 Ottawa, Canada ml of the preparation was floated on each Accepted February 10, 1966 gradient made from 4, 7, 7, and 7 ml of solutions containing 100, 200, 300, and 400 g per litre of sucrose, respectively, prcparcd Purification of Pea Enation Mosaic Virus in G.03 111 phosphate buffer (pH T..‘,), and t.hen cent’rifuged for 3 hours at 70,000 g. Only a few plant viruses are known that l’rcparwtions from infected plants gave t’wo are transmitted both by manual inoculation light-scattering layers at8 about 19 mm and of sap and by aphids that have mou!ted 23 mm from the meniscus, arid samples since last feeding on infected plants. Lettuce from these layers, together with samplrs necrotic yellows, one such virus, has been from above, between, and below them were purified and found to have large complex dialyzed at 5” against 0.01 M phosphat,e bacilliform or bullet-shaped particles (1, 2). buffer (pH 7.5) to remove the sucrose. Pea enation mosaic virus is another, and Isometric particles were abundant (I3g. our experiments, summarized here, show it 1 a-c) in the samples from in and between has particles quite different from those of the light-scat#t,ering layers, fewer iI1 samples lettuce necrotic yellows virus, and that, it, is from below, and were not, found in samples best purified in a different way. from above the layers. The inftlctivity of An isolate of pea enation mosaic virus these samples, estimated by inoculating (PEMV), obtained from a field bean plant t#hem to lc:~vcs of C’htvwpodium amarantion Rothamsted Farm, was transmitkd to color Coste wnd Reyn, (Fig. 1 d), W;IScrudely batches of field bean (Kick Jaba I,. “Herz proportional to the numb(Jr of particles they E’reya”) by the pea aphid (Rc~~*thosiph~~ contained. pisum Harris). Syst8emically infected bean Table 1 summarizes the properties of shoots were triturated mechanically in a samples taken from the two light-scattering mixture of pH 7.5 buffer (1.5 ml/g plant layers. Particles from the upper layer aptissue) containing 0.08 M phosphate and peared irregular and distorted, and tm0.05 X ethylenediamine tetraacetatc, and trastcd poorly with the phosphotungstat,e chloroform (1 ml/g tissue). The resulting emulsion was centrifuged for 13 minutes at (Wig. la). They appeared more regular and cont8rasted better when treated for 1 hollr 2000 y, and the aqueous supernatant layer v&h 1 5%fornlaldehyde before being mixrcl was poured off and centrifuged for 2 hours and mountjctl fot at 75,000 g to sediment the virus part,icles. with phosphotungstate 1. BAILEY, Virology