Molecular and Biochemical Parasitology. 45 I 1991 I 155-15.°

155

Elsevier MOLBIO 01480

A putative RNA virus in Babesia boris Rhona Carol Johnston ], Nara Amrlia Homero

R o s a F a r i a s 2, J o a o C a r l o s G o n z a l e s 2.

D e w e s I. A o i M a s u d a L, C a r l o s T e r m i g n o n i ], K a z u n o b u

Amako 3 and

Luiz Shozo Ozaki ] /Department of Biotechnoh)ey. aml -"~eterinar3 St hool. Univer~tdade Federal ~h~Rio Grande do Sul. Port~ Aleete-RS. Brazil: and -~Department t?f Bacteriology. School of Medicine. L'niverstt3' of Kyushu. Fukuoka. Japan

(Received II June 1990: accepted 4 October 1990~

Babesta boris is an intrae%.throcytic protozoan that causes bovine babesiosis. Agarose gel electrophoresis of nucleic acids extracted from t~o isolates of B. bovts reveals, besides bulk DNA, an ethidium bromide-stainable band at about 5.5 kb. Further characterization of the latter with DNase I, RNase and mung bean nuclease suggested it to be a double-stranded RNA. Sonicated parasites ~ere fractionated in a CsCI buo.~ant density gradient. A sample containing the 5.5-kb RNA ~as analysed under an electron microscope and a ,,irus-like particle was observed.

Key ~ords: Bahesia bovts: Babesta hi.eemina: Protozoan v=rus: Virus-like particle

Introduction Many reports of protozoan viruses have been published lately (for a review, see ref. 1). R N A viruses were described in association with Tric h o m o n a s vaginalis [2], Giardia lamblia [3-5], L e i s h m a n i a bra:iliensis [6-7] and A m o e b a proteus (Yazaki, K., Ishii, K. and Tsukii, Y., Isolation and characterization of virus-like panicles from A m o e b a p r o t e u s strain F, Int. Congr. Protozool., Kyoto, Japan, 1989). Babesia boris, an intraerythrocytic parasite that causes bovine babesiosis, is endemic in Rio Grande do Sul, the southernmost state of Brazil. This is a region of extensive cattle breeding. Two local isolates were used to infect splenectomised calves to a high parasitaemia. Blood was then collected and parasites purified for nucleic acids exCorrespondence a&tress." Luiz Shozo Ozaki, Department of

Biotechnology, Um~ersidade Federal do Rio Grande do Sul, P.O. Bo~i 15005, CEP 91500, Porto Alegre-RS, Brazil. .4bbtevtations: BBV. Babesia boris virus: PBS. phosphate-

buffered saline: SDS, sodium dodecyl sulphate: VLP. viruslike particle: 5.5RNA, 5.5-kb RNA molecule: DMSO. dlmeth~ I sulpho,tlde.

traction. Agarose gel electrophoresis of the preparations showed, besides the genomic DNA, an additional ethidium bromide-stainable band that migrates at about 5.5 kb, when c o m p a r e d to lambda phage D N A HindllI digest size markers. DNase I and RNase A assays on this material indicated that this molecule is an RNA. Further characterizations suggested it to be a double-stranded RNA, Electron micrographs revealed a viruslike panicle (VLP) in a 5.5-kb RNA-containing sample from a CsCI gradient of sonicated parasites.

Materials and Methods Two locally collected B. boris isolates, named b o v G l and b o v V I , were maintained in liquid nitrogen using D M S O as described [8]. Parasitaemias were induced on 3 - 6 - m o n t h - o l d splenectomised calves by inoculating cryopreserved blood or blood from another experimentally infected animal. At the peak of parasitaemia, blood was collected in trisodium citrate at a final concentration of 0 . 3 3 ~ . Blood cells were separated from plasma by centrifugation at 400 × g, resuspended Parasites.

0166-6851/91/$03.50 ,~, 1991 Elsevier Science Publishers B.V. (Biomedical Division)

m pho,,phate-buffered ,~aline tPBSI pH 7.3 and the ervthrocvte,, N sed ~ ith ().02c; , saponin. Parw, ites t~ere separated from ~hite blood cell~ u~ing differential centrifuoation and a further ad,,oq~tion on Whatman C F I I cellulose powder ~Catalogue No. 4(121 ()5()~ [9].

Nucleic acid i.~olatio~t. Nucleic acids were extracted from purified parasite~ using the phenolchloroform method as described [10] except that RNase treatment ~a~ omitted. A_oarose oel analysis, was performed in 0.7% slabs in Tris-acetate buffer, pH 7.5 [11]. C~C/ eqHilibrium density .eradient centril'ugalion am~h'Ms. Purified parasites lB. hm'L~ b o v G l iso-

late~ ~ ere sonicated in a Cole-Pahner Instrument,, Co. Ultrasonic Homogenizer with 6 bursts of 3 .,, each. The sonicated cells ~ e r e incubated with 1 /tg ml - t DNase I at room temperature for 30

rain. C,CI ~a~, added to 34(~- and centrifuged in a Sorxall AH650 rotor at 35000 re~./min for 22 h. Fraction.,, of 350 /~1 each ~ere collected u~in~ a Beckman Fraction Recover 3 System. The buoyant den~,ities of the fraction,, ~ere extrapolated from the measurements of a corresponding PBS-C,,CI gradient control. A ,,ample (20 pI) of each fraction ~a~ gel filtrated (Sephadex G-5(). Phannacia P-L Biochemicalsl in a spin cohunn [10]. I ~ e d with 1% SDS and analysed on a 0.7~7 agarose ~el slab.

Electron microscopy. Fractions of the CsCI gradient containing the 5.5-kb RNA ~ere gel filtrated and fixed ~ith 2% g.lutaraldeh.,,de and 2c) t'ornmldefivde in 50 mM cacod31ate buffer pH 7.2. Samples were negatively stained ~ith 1('~ sodium phosphotungstate and observed under a JEM 2000EX (JEOL Co. ~ electron microscope at I00 kV. Material~.

ab

c d

23.1 9.46.64.42.3--

2.0-

Fie. I. Nucleic acid,, prepared from B. hmt.~ bo~GI p,olate (b) treated ~ith I¢) ,,g ml - I DNase I in 250 mM Tri,~-HCI pH 7.5,'5 mM MnCI2,100 11~ ml - t BSA for 15 mm at 15:C cot or ~lth 0.5 l~g m l - t of pancreatic RNa,,e in I(I mM Tri,,-HCI pH 7.5. I mM EDTA for 6fl rain at 37"C td~ and anal.~,,ed m a 0.g~ a~aro,,e gel ,,lab Hmdlll fragments of phage lambda DNA c2110 ngl are used a,~ ,,]ze marker,, ;aL

Pancreatic DNase I. bovine pancreatic RNase and acridine orange were purchased from

1 2 3 a4b a ba ba b 23.1 9.4-6.6-4.4-2.3-2.0--

Fig. 2. Mung bean nuclea,.e Ireatment of DNase l-punlled 5.5RNA from B. hovi~ bo~ G I isolate anal 3 sed b~ 0.7G- agaro,,e gel electrophoresis. Phage lambda DNA Hindlll digest iI L 5.5-kb RNA ;2L ,,ingle-qranded phage lambda DNA Hmdlll d~e~t q3~ and Es('hertctm~ toh RNA t4p v, ithout fa~ and v, lth ~b~ 10 umt,, of mung bean nuclease tt~ -~ of nucleic ac=d m 30 mM ,-,odium acetate pH 4.6/50 mM NaCI/I mM ZnCI_~ at 37:C and macti~ated after IO rain x~tth 0.O1% SDS.

157

Sigma. Mung bean nuclease was from PharmaciaP-L Biochemicals. Phage A DNA (Enzibiot Cuba) cut with HindlIl (Enzibiot Cuba) was used as size markers. Glutaraldehyde and lbrmaldehyde were obtained from E. Merck.

Results and Discussion Analysis in agarose gel of total nucleic acids prepared from B. boris bovG1 isolate revealed the presence of a molecular species with an apparent size of about 5.5 kb besides bulk DNA (Fig. ib). A molecular species with the same apparent size was also detected in a second isolate (bovVl. not shown). Nucleic acids were also extracted from white blood cells of experimentally infected animals before and after parasitaemia. The 5.5-kb molecule was not observed in these preparations (results not sho~n), suggesting it to be associated with the parasite. Plasma from an animal in acute phase parasitaemia was ultracentrifuged at 109000 x g for 2 h and the pellet resuspended in

PBS with 1% SDS. Agarose gel electrophoresis of the solubilised material did not indicate the presence of any nucleic acid (not shown), but that does not eliminate the possibility of it being present in very small amounts. The 5.5-kb molecule was resistant to treatment w.ith 10 ltg ml -~ DNase I, while the bulk DNA was completely digested (Fig. lcl. It was, however, sensitive to treatment with 0.5 fig ml-~ pancreatic RNase (Fig. Id). These results indicate that 5.5-kb nucleic acid is an RNA (5.5RNAI. Further characterisations were performed on 5.SRNA purified by digesting total nucleic acid preparations from B. bori,s cells w.ith DNase I. The 5.5RNA was resistant to mung bean nuclease I Fig. 2, lane 2bl in conditions in which single-stranded nucleic acids were preferentially digested [12] (Fig. 2, lanes 3b and 4b), suggesting it to be a double strand. In formaldehyde denaturing agarose gels [11]. it runs as a 5.5-kb single-stranded molecule when compared to Escherichia coli ribosomal RNA (results not shown). Moreover,

Fig. 3. Electron micrographs of ~irus-like panicles from a 5.5RNA-containing fracuon of the CsCI buoyant densit.,, gradient cenmfugation of sonicated B. h , vts cells.

15x

in acridine orange-~tained agarose gel.,, [13]. the 5.5RNA stains green, while single-stranded DNA and RNA control.,, stain red ~re~ults not ~,ho~n). These result,, further suggest that the 5.5RNA is double-stranded, as those of other protozoan ~ iral genomes described [I-5]. Sonicated B. hovis cells were centrifuged in a C~,CI buo.~ant-densit 5 gradient at 35 000 rev./min and fraction.,, were collected from the bottom. Den.qt~ mea,,urement of the control samples ~see Materials and Methods) indicates that the structure c a r d i n g the 5.5RNA has an a~erage density of 1.3576 g ml-~. Electron microscopical examination~ of a 5.5RNA-containing CsCI gradient fraction shox~ed the pre~,ence of virus-like particles (\.'LPI ha~ing a diameter of about 38 nm ~Fig. 3~. It is presumed that the VLP ma.~ originate the 5.5RNA. The results above suggest that B. boris carries a ,~irus that is quite similar to those described in other protozoa. The kind of relationship existing between B. I~ot'is and it,,, putative ~ irus. ~hich ~ e named BBV. i~ not detemfined. Interestingl.,,, ~ e also detected a RNA of about 5.5-kb in nucleic acid preparation~ from another species of bovine babesia. B. bi,eemina. This ~as observed in one isolate, but not in two others (not shown). We have also ob.,,er~ed an additional molecule that i.,, consistently present in nucleic acid preparations of B. bovi,~ cells. It is apparently of DNA nature, since it i.,, digested b~ DNase I and not by RNase A (Fig. lc and dl, is cut b~ restriction enz.~mes and is alkali resistant (results not sho~n). In non-denaturing agarose gels it runs above that of the 5.5RNA (Fig. Ibl. It remains to be determined ~hether this molecule is related to BBV.

Acknowledgements We are greatl 5 indebted to Dr. C.C. Wang for support and helpful discussions. We also wish to thank Drs. Di6genes S. Santos and Yo-ichi Koyama and Prof. Yoshiyuki Sakaki for support. and Mr. Jos~ Zuffo Neto for technical as~,istance. Contact.,, in Japan were made possible

by the Japan International Cooperauon Agenc.~ (JIC.-k~ fl]rough its Program for Training JapaneseBrazilian Scientists in Japan. R.C.J. and N.A.R.F. are recipients of scholarships from the Brazilian National Research Council ICNPqL L.S.O., C.T. and H.D. are recipient.,, of re,,earch fellowship.,, from CNPq. Thi.,, ~ork was supported b.~ research grants from FAPERGS nos. 0045/89, 0046/89, 0048/89 and 0052/80, FINEP no. 42880668-00 and CNPq no. 700017-89.5.

References I Palterson. J,L. c 1990~ \'~ruse,, of protozoan parasite,,. Exp Para,qtol. 7(1. II I - I 13 2 \Vang. ,~.L. and Wang. C.C. c 19.~6,~ The double-~tranded RNA m Tr~h,monas va~mahs ma.~ originate from ~ wuslike pamcle~. Proc. Nail. Acad. Sol. LISA 83. 7056-7,O60. 3 Wang. A L. and Wang. C.C. q 1986~ Discove o of a ,,pecihc dot, ble-~tranded xiru,, in Giardm lamhha Mol. Biochem Para~Hol. 21, 260 276 4 De Jonckheere. J.F. and Gordb, B. I IO87~ Occurrence and tran,,fecuon of a Gtatdta ,.mJ,,. Mol. Biochem. ParasLtol 23, 85 go. 5 Mdler. RL., Wang. A.L. and Wang. C.C. 1198~ Purificanon and charactenzauon of the (_iloIdld I,mlhha double,,tranded RNA ~ tm~. Mol. Btochem. Para~alol. 28. IgO-196. (1 Tarr. P.I., Ahne. Jr., R.F., Smile~. B.L., Scholler, J.. Keithl), J. and Stuart, K. ll9ggb L R I : a candidate RNA ~tru,~ of Lelshmama. Proc Natl. Acad. Sct. LISA 85. 0572-9575. 7 Widmer. G , Comeau, A.M., Furlong. D.B., Wirth, D.F. and Patter,,on. J.L. ~ 1980l Characterization of a RNA ~ irus from the parasite Lezshmama. Proc. Nail. Acad. Sci. USA 86,, 5979-5082. g ]ame,,, E.R. q lO80D CDopreserxauon of protozoa and helminth parasites of man and animals. In: Lo~ Temperature Preserxation in Medicine and Bmlog~ ~Ashwood Smith. MJ. and Farranl, J., ed. t. pp. 155-186. Pitman Medical Lmfited, Tunbndge Wells. Kent, Li.K. 0 Baggale.~, V.C. and Atkm,,on, E.M. f 1972b U,~e of CFI I columns for preparation of DNA from rodent malarias. Tran,~. R. Soc. Trop. Med. H.~g. 66. 4-5. I0 Ozaki, L.S, and Cseko. Y.M.T. ~1984~ Genomic cloning and related techmque,. In: Genes and Anugens ol Parasite, Morel. C.M., ed. ~. pp. 16_5-185, Fundaqgo Os~ aldo Cruz. R~o de Jane~ro, Brazd. I I Maniaus. T., Fnl,~ch. E.F. and Sambrook, J. ( 1982~ Molecular Cloning. A Laboralor~ Manual. Cold Spring Harbor Laborator 3, Cold Spnng lqarbor, NY. 12 Kmeker, '~k .D. and Ko~al~ki, D. I I078~ Gene-s~zed pieces produced b~ dlgesnon of linear duplex DNA ~ith mung bean nuclease. BmchenustD' 17, 3236-3243. 13 Camuchael. G.G. and McMaster, G.K. ( 1980~ The anal},i,, of nucleic acids m gel> u,~mg gboxal and acridine orange. Method_,, Enz.vmol. 65. 380-391.