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Els, H.J.

Erasmus, B.J.

Lecatsas, G.

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Onderste poort J. vet. Res. (1 969) , 36 (2) , 32 1-324. Printed in the Republic of South Africa by T. W . H ayne Ltd . (H . & G .) , Johannesburg fo r the G overn men t Printer. Pretoria RESEARCH NOTE ELECTRON MICROSCOPIC STUDIES ON CORRIPARTA VIRUS G. LECATSAS, B. J. ERASMUS and H . J. ELS, Veterinary Research In stitute, Onderstepoort Corr iparta virus, an unclassified arbovirus iso lated in Austra li a from Culex annulirostris (Doherty, Car-ley, Mackerras & Marks, 1963), has been likened in its struc ture to reovirus (Carley, 1967). It is relatively ether-sensi tive compared to reovirus, whi le its size has been given as 67 m p. The present investigation was ca rried out in order to com pare the virus with bluetongue virus (BTV) and A frica n horsesickness virus (AHSV) with resJ.Ject to size, structure and fine cytopa thology and to in-vestigate further its relationship to reovirus. BHK 21/C l3 cells grown in Eagle's medium supple-mented with I 0 per cent normal bovine serum were used in the investigation. The fifth suckling mouse brain passage o f the M RM 1 stra in of Corriparta vi rus was adapted to BHK 21 Cl3 cells. After three passages, the virus was seeded onto roller tubes and the cell s harvested and processed for electron micros-copy. O ur methods of preparation o f thin sections for electron microscopy have been described previously (Lecatsas & Weiss, 1969). Cells used in negative sta ining prepara tions were ruptured in di stilled water and one drop of thi s suspension mixed with one drop of phosphotungstic acid at a pH varying between 6.4 and 7. 2. Formvar film s coated with a carbon layer o n 300-mesh copper grids were used as the spec imen substrate. A Siemens Elmiskop I A electron microscope, employing a double conden sor system and operated at 80 kV, was used in the investigation. P la te 1 ( I ) shows a gra nular cytoplasmic inclusion containing embedded virus particles which appear to be inco mplete in that they contain no nucleoid and suggesting that the granular material represents capsid protein . L iberation of virus particles from such an incl usion leads to masses of progeny virus particles in the cytoplasmic matrix [Plate 1 (2)], some exhibiting dense nucleoids and others with empty or partiall y empty cores. Nucleoids with partia lly formed capsids contai ned in membrane bound bodies are depicted in Plate 1 (3 and 4), and may represent the breakdown of incomi ng virus particles in lysosome-like structures. Plate 2(5) shows a virus particle with a fine filament attached to the core, possibly representing genetic material wh ich is incompletely incorporated in the progeny part icle. Plate 2(6) shows negatively stained virus particles with no double capsid as found in reovirus a nd suggesting a low capsomere number. Our invest igations to date have indicated that the virus is pH sensitive, is not inhibited by bromode-oxyurid ine a nd is partially sensitive to chloroform , suggesti ng, together with its size, that the virus resem-bles the " Diplornaviruses" (Verwoerd , 1969a,b). In additi o n, complement fixation tests showed no sero-Received for publication on 29 August 1969.- Editor 321 logical relationship between Corriparta virus and BTV or AHSV. Size determinations in the present investigation suggest approximate values of 67m ;.t in section and 60m ;.t in negative contrast preparat io ns. The value o f 67 m,u is in close agreement with that of Carl ey (1967) and al so approximates that of AHSV (Lecatsas & Erasmus, 1967) and BTV (Els & Verwoerd, 1969). O ur measurements for reovirus give a value of 80 m ;.t in section and 75 m ;.t in negatively stained preparations. Morphologically, the virus appears to be similar to BTV, showing no double capsid as is commonly found in reovirus (Vasquez & Tournier, 1962) when negatively stained . In addition , whi le reovirus is thought to have 92 capsomeres, BTV has recently been shown to possess 32 (Els & Verwoerd, 1969). In section , the virus parti cle presen ts a dense nucleoid in " full " particles with a markedly less dense capsid . Empty particles having no visible nucleoid are com-monly found as a re particles having a fine filament attached to the core, a phenomenon cha racteristi c of BTV and AHSV when grown in BHK 21 cell s. It is suggested that this fine strand may represent genetic materi a l which has not been efficientl y in-corporated into the virus particle (Lecatsas, 1968a,b). The presence of dense, granular, cytopl asmic in-clusions containing apparently incomplete virus parti-cles is clearly demonstrated . Simila r bodies a re found in BHK 21 cells infected with BTV and AHSV, a~d are a lso reported by Holmes (1969) in m o use bram infected with Eubenangee virus. M yelin -type in-clusions containing viral nucleoids a re commonly fo und and may represent lysosomes in which particles are being degraded after initial entry into the cel l. We consider virus size a nd morphology, as well as the presence of virus-containing cytoplasmic granular inclusions as specific factors in distingu ishing BTV, AHSV and Corriparta virus from reovi rus. Other detail s of the fine cytopathology of virus-infected cells, such as the association of reovirus with spindle tubules (Dales, 1963), serve to characterize viruses more specifically. On this basis, for example, AHSV can easily be distinguished from BTV by the respective presence in BHK 21 cells of coarse cytoplasmic fil a-ments and bundles of cytoplasmic tubular elements (Lecatsas, 1968a,b ). T he lack of filaments or tubules associated with Corriparta distinguishes it fro m AHSV and BTV respectively. A CKNOWLEDGEMENT We are indebted to Dr. R . L. D oherty, Director, Queensland Institute of Medical Research fo r supply-ing us with a sample of Corriparta virus. ELECTRON MICROSCOPIC STUDIES ON CORRIPARTA VIRUS REFERENCES CARLEY, J. G., 1967. Rep. Queensland Jnst . med. Res., 22, 6. DALES, S., 1963. Association between the spindle appa-ratus and reovirus. Proc. natn. A cad. Sci. U.S.A. , 50,268- 275. DOHERTY, R . L., CARLEY, J. G ., MACKERRAS, M. J. & MARKS, E. N ., 1963 . Studies of arthropod borne virus infections in Queensland . HI. Isolation and characterization of virus strains from wild caught mosquitoes in north Queensland. Aust. J. exptl Bioi. med. Sci., 41, 17-40. ELS, H. J. & VERWOERD, D. W. , 1969. Morphology of bluetongue virus. Virology, 38 , 213- 219. HOLMES, I. H ., 1969. An electron microscopic study of Eubenangee, an Australian arbovirus. Virology , 38 , 347- 349. LECATSAS, G. & ERASMUS, B. J. , 1967. Electron mi-croscopic study of the formation of African horsesickness virus. Arch. ges. Virusforsch., 22, 442-450. LECATSAS, G., 1968a. Electron microscopic study of the formation of bluetongue virus. Onderstepoort J. vet . Res. , 35, 139- 150. LECATSAS, G ., l968b. Electron microscopic studies on reovirus type I in BHK21 cells. Onderstepoort J. vet. Res., 35, 151-158 . LECATSAS, G . & WEISS, K. E. , 1969. F ormation of Wesselsbron virus in BHK21 cells. Arch. ges. Virusforsch ., 27, 332- 338. VASQUEZ, C. & TOURNIER, P. , 1962. The morphology of reovirus. Virology , 17, 503- 510. VER WOERD, D . W., J 969a. Purification and characteri-zation of bluetongue virus. Virology, 38 , 203- 212. VERWOERD, D. W., 1969. Proc. 1st Intern . Congr. Viro-logy. Progr. Med. Viral. (in Press.) 322 G . LECATSAS, B. J. E RASMUS & H . J. ELS PLATE 1.- 1. Granular, cytoplasmic inclusion containing developing virus particles embedded in the matrix (arrows). These parti-cles show no nucleoid while particles at the periphery and centre of the inclusion show dense nucleoids. Bar equals 250 mjJ,. 2. Virus particles with and without dense nucleoids are evident in the cytoplasmic matrix. Bar equals 250 mjJ,. 3. Mem-brane-bound, lysosome-like inclusion body containing viral nucleoids. Bar equals 250 mjJ,. 4. Membrane-bound lyso-some-li ke inclusion body containing viral nucleoids with indistinct capsids. Bar equals 250 m jJ, 323 ELECTRON MICROSCOPIC STUDIES ON CORRIPARTA VIRUS PLATE 2.- 5. Virus particle with fine filament (arrow) attached to its core. 1he small core suggests that the filament represents viral nucleic acid which has not been efficiently incorporated into the virus particle. Bar equals 250 mp,. 6. Virus particles negatively stained with 2 per cent phosphotungstic acid . A low number of tubular capsomeres is suggested. " Empty particles" are also evident. Bar equals 250 mft 324 

Electron microscopic studies on corriparta virus

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Electron microscopic studies on corriparta virus

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