© INSTITUTPASTEUR/ELsEVIER Paris 1992
Res. Virol. 1992, 143, 215-218
BRIEF NOTE
Electron microscopic identification of Zinga virus as a strain of Rift Valley fever virus O.D. Olaleye (i) (*), C.L. Baigent (2), G. Mueller (2), O. Tomori (z) and H. Schmitz (2) (/) Department of Virology, College of Medicine, University of Ibadan, University College Hospital, Ibadan (Nigeria), and r2~Department of Virology, Bernhard-Nocht Institute for Tropical Medicine and Hygiene, Bernhard-Nocht Street, D-2000 Hamburg (Germany) SUMMARY
Electron microscopic examination of a negatively stained suspension of Zinga virus showed particles 9 0 - 1 0 0 nm in diameter, enveloped with spikes 1 2 - 2 0 nm in length and 5 nm in diameter. Further identification of the virus by immune electron microscopy showed the reactivity of human Rift Valley fever virus-positive serum with Zinga virus. Results of this study are in agreement with earlier reports that Zinga virus is a strain of Rift Valley fever virus.
Key-words: Zinga virus, RVF; Negative staining, Immune electron microscopy, Diagnosis. INTRODUCTION
Zinga virus (Dakar B. 1976) was first isolated from a Mansonia africana mosquito in Central African Republic (A.V.I.E., 1977). It was long regarded as a distinct virus and classified as a serologically ungrouped arthropod-borne virus. Subsequently, the virus was isolated from humans and from other species of mosquitoes in Central and West Africa (Digoutte et aL, 1974; Digoutte, 1988). During studies with viruses of the phlebotomus fever group, Shope et al. (1980) detected a cross-reaction between Zinga virus and Rift Valley fever (RVF) virus. Their observation was later supported by Meegan et al. (1983) using monoclonal antibodies
prepared against three strains of RVF virus to show that Zinga virus is similar to other African strains of RVF virus. They also reported that the pathogenicity of Zinga virus for laboratory animals is similar to that described for other African RVF virus strains. Recently, we used Zinga RVF virus for a series of epidemiological and experimental studies in Nigeria. The virus used for our work was obtained courtesy of Professor Robert Shope, Yale Arbovirus Research Unit, New Haven, Connecticut, USA. The virus was tested for RVF virus identity by various techniques including electron microscopy. Herein we report results of negative staining and antigen-controlled immunodiagnosis test of the virus.
Submitted September 13, 1991, accepted March 5, 1992. (*) Correspondingauthor: O.D. Olaleye,Laboratoryof Viral Oncologyand AIDS Research, University of Southern California, Edmonson Bldg 1840, North Soto Street, Los Angeles,CA 90032-3626.
216
O.D. O L A L E Y E E T A L .
MATERIALS AND METHODS Virus preparation Zinga virus was supplied to us as a 10 % mouse brain material at the third mouse passage. It was reconstituted with 0.5 ml sterile PBS and inoculated at a dilution of 10 07o into suckling baby mice by the intracerebral route. Our stock virus was prepared with the fifth mouse brain passage. Stock virus was passed two times in Vero E6 ceils and processed for electron microscopy as described by Ellis et al. (1988).
ed 1/80 in PBS at 4°C overnight in a moist chamber. After coming to room temperature, the grids were again washed in PBS three times before being exposed to protein A/gold-10 diluted 1/20 in distilled water for 45 min. After a further washing in PBS, the reaction was fixed in 2 °70 glutaraldehyde for 30 min. Grids were finally washed in distilled water before being conventionally negatively stained with 0.8 070 sodium phosphotungstic acid. Examination was carried out using a "Philips EM300" at 80 Kv.
RESULTS AND DISCUSSION Electron microscopy For morphological identification, virus suspension was adsorbed onto glow-discharged-treated pioloform/carbon-coated nickel 300 grids for 15 rain. Grids were washed in distilled water and negatively stained with 0.8 °7o sodium phosphotungstic acid. Immune identification of the virus was carried out by a modification of the antigen-controlled immune diagnosis (ACID) test described by Mueller and Baigent (1980). Virus suspension was adsorbed onto glow-discharged-treated grids as described above. Excess virus was removed by washing three times in PBS, after which the grids were blocked with l0 07o bovine serum albumin (BSA) in distilled water to avoid non-specific reactions. The BSA was then drained off and the grids were incubated in antiserum (human polyclonal RVF-positive serum) dilut-
E x a m i n a t i o n o f the virus by transmission electron microscope using negatively stained material showed particles with an overall diameter o f 90-100 nm, enveloped with spikes about 12-20 nm in length and 5 nm in diameter. Mature particles contained ribosome-like structures within the hexagonal nucleocapsid (not well resolved on the photograph). There were capsomeres on the nucleocapsid (fig. 1). It was difficult to determine the exact n u m b e r o f capsomeres and spikes because o f variation in the orientation o f the virions. Immune electron microscopic examination o f the virus showed the specificity o f h u m a n RVFpositive serum and Zinga virus. As shown in
Fig. 1. Morphologyof Zinga virus particles negativelystained with sodium phosphotungsten. 33,000 x.
Z I N G A VIRUS A S A S T R A I N O F R I F T V A L L E Y FEVER VIRUS
Fig. 2. Positively labelled Zinga virus after overnight incubation with human positive RVF serum. Gold particles are arranged on the outline of the virus nucleocapsid. Negatively stained with sodium phosphotungsten. 33,000×.
I)"
O
,')"i:.,,..
-.
Fig. 3. Zinga virus incubated overnight with human negative RVF serum. Gold particles are not attached to virus particle. Negatively stained with sodium phosphotungsten. 33,000 x .
217
218
O.D. O L A L E Y E E T A L .
figure 2, the distinct attachment o f gold to virus panicles treated with positive serum contrasted with virus particles treated with RVF-negative serum (fig. 3).
Schmitz, Director of the Department of Virology, Bernhard-Nocht Institute for Tropical Medicine and Hygiene, Hamburg, Germany.
R V F virus is a m e m b e r o f B u n y a v i r i d a e family. According to M u r p h y and Kingsbury (1990), bunyavirus virions consist o f a lipidcontaining envelope, with fine pepIomers, within which are three loosely helical, circular nucleocapsid structures with a diameter o f 2-2.5 nm. Virions are 90-120 nm in diameter. Von B o n s d o r f f and Petterson (1975) and later Objeski and M u r p h y (1977) showed that surface projections are more ordered and form a penthexon cluster arranged in an icoshahedral lattice in R V F virus. This study shows that morphology o f Zinga virus grown in Vero E6 cells is similar to other strains o f R V F virus. Also, labelling o f Zinga virus with R V F virus-positive serum clearly demonstrates that it is a strain o f R V F virus. This report supports earlier observations o f Shope et al. (1980) and Meegan et al. (1983) that Zinga virus is a strain o f R V F virus.
References
Acknowledgements This study was supported by the German Academic Exchange Programme (DAAD) with a Visiting Fellowship grant awarded to Dr. O.D. Olaleye to work with Prof. H.
Arthropod-borne virus information exchange (1977), Catalogue. Digoutte, J.P. (1988), Assessment of the risk of Rift Valley fever and other haemorrhagic fevers in West Africa. WHO Workshop on Emergency Preparedness, Bamako, 4-9 July, 1988. Digoutte, J.P., Cordellier, R., Robin, Y., Pajot, F. & Geoffrey, B. (1974), Zinga virus (At. B. 1967), a new arbovirus isolated in the Central African Republic. Ann. Microbiol. (Instit. Pasteur), 125B, 107-118. Ellis, D.S., Shirodovia, P.V., Fleming, E. & Simpson, D.I.H. (1988), Morphology and development of Rift Valley fever virus in Vero cell culture. J. reed. Virol., 24, 161-174. Meegan, J.M., Digoutte, J.P., Peters, C.J. & Shope, R.E. (1983), Monoclonal antibodies to identify Zinga virus as Rift Valley fever virus. Lancet, I, 64. Mueller, G. & Baigent, C.L. (1980), Antigen-controlled immuno-diagnosis (ACID test). J. Immunol. Methods, 37, 185-190. Murphy, F.A. & Kingsbury, D.W. (1990), Virus taxonomy, in "Fields virology" (Fields, B.N. & Knipe, D.N.) 2rid edition, vol. 1 (p. 9-36). Raven Press, New York. Objeski, J. & Murphy, F.A. (1977), Bunyaviridae, recent biochemical developments. J. gen. Firol., 37, 1-14. Shope, R.E., Peters, C.J. & Walker, J. (1980), Serologic relationship between Rift Valley fever virus and viruses of the phlebotomus fever group. Lancet, I, 886-887. Von Bonsdorff, C.H. & Pettersson, R. (1975), Surface structure of Unkuniemi virus. J. Virol., 16, 1296-1307.
Res. Virol. 1992, 143, 215-218
BRIEF NOTE
Electron microscopic identification of Zinga virus as a strain of Rift Valley fever virus O.D. Olaleye (i) (*), C.L. Baigent (2), G. Mueller (2), O. Tomori (z) and H. Schmitz (2) (/) Department of Virology, College of Medicine, University of Ibadan, University College Hospital, Ibadan (Nigeria), and r2~Department of Virology, Bernhard-Nocht Institute for Tropical Medicine and Hygiene, Bernhard-Nocht Street, D-2000 Hamburg (Germany) SUMMARY
Electron microscopic examination of a negatively stained suspension of Zinga virus showed particles 9 0 - 1 0 0 nm in diameter, enveloped with spikes 1 2 - 2 0 nm in length and 5 nm in diameter. Further identification of the virus by immune electron microscopy showed the reactivity of human Rift Valley fever virus-positive serum with Zinga virus. Results of this study are in agreement with earlier reports that Zinga virus is a strain of Rift Valley fever virus.
Key-words: Zinga virus, RVF; Negative staining, Immune electron microscopy, Diagnosis. INTRODUCTION
Zinga virus (Dakar B. 1976) was first isolated from a Mansonia africana mosquito in Central African Republic (A.V.I.E., 1977). It was long regarded as a distinct virus and classified as a serologically ungrouped arthropod-borne virus. Subsequently, the virus was isolated from humans and from other species of mosquitoes in Central and West Africa (Digoutte et aL, 1974; Digoutte, 1988). During studies with viruses of the phlebotomus fever group, Shope et al. (1980) detected a cross-reaction between Zinga virus and Rift Valley fever (RVF) virus. Their observation was later supported by Meegan et al. (1983) using monoclonal antibodies
prepared against three strains of RVF virus to show that Zinga virus is similar to other African strains of RVF virus. They also reported that the pathogenicity of Zinga virus for laboratory animals is similar to that described for other African RVF virus strains. Recently, we used Zinga RVF virus for a series of epidemiological and experimental studies in Nigeria. The virus used for our work was obtained courtesy of Professor Robert Shope, Yale Arbovirus Research Unit, New Haven, Connecticut, USA. The virus was tested for RVF virus identity by various techniques including electron microscopy. Herein we report results of negative staining and antigen-controlled immunodiagnosis test of the virus.
Submitted September 13, 1991, accepted March 5, 1992. (*) Correspondingauthor: O.D. Olaleye,Laboratoryof Viral Oncologyand AIDS Research, University of Southern California, Edmonson Bldg 1840, North Soto Street, Los Angeles,CA 90032-3626.
216
O.D. O L A L E Y E E T A L .
MATERIALS AND METHODS Virus preparation Zinga virus was supplied to us as a 10 % mouse brain material at the third mouse passage. It was reconstituted with 0.5 ml sterile PBS and inoculated at a dilution of 10 07o into suckling baby mice by the intracerebral route. Our stock virus was prepared with the fifth mouse brain passage. Stock virus was passed two times in Vero E6 ceils and processed for electron microscopy as described by Ellis et al. (1988).
ed 1/80 in PBS at 4°C overnight in a moist chamber. After coming to room temperature, the grids were again washed in PBS three times before being exposed to protein A/gold-10 diluted 1/20 in distilled water for 45 min. After a further washing in PBS, the reaction was fixed in 2 °70 glutaraldehyde for 30 min. Grids were finally washed in distilled water before being conventionally negatively stained with 0.8 070 sodium phosphotungstic acid. Examination was carried out using a "Philips EM300" at 80 Kv.
RESULTS AND DISCUSSION Electron microscopy For morphological identification, virus suspension was adsorbed onto glow-discharged-treated pioloform/carbon-coated nickel 300 grids for 15 rain. Grids were washed in distilled water and negatively stained with 0.8 °7o sodium phosphotungstic acid. Immune identification of the virus was carried out by a modification of the antigen-controlled immune diagnosis (ACID) test described by Mueller and Baigent (1980). Virus suspension was adsorbed onto glow-discharged-treated grids as described above. Excess virus was removed by washing three times in PBS, after which the grids were blocked with l0 07o bovine serum albumin (BSA) in distilled water to avoid non-specific reactions. The BSA was then drained off and the grids were incubated in antiserum (human polyclonal RVF-positive serum) dilut-
E x a m i n a t i o n o f the virus by transmission electron microscope using negatively stained material showed particles with an overall diameter o f 90-100 nm, enveloped with spikes about 12-20 nm in length and 5 nm in diameter. Mature particles contained ribosome-like structures within the hexagonal nucleocapsid (not well resolved on the photograph). There were capsomeres on the nucleocapsid (fig. 1). It was difficult to determine the exact n u m b e r o f capsomeres and spikes because o f variation in the orientation o f the virions. Immune electron microscopic examination o f the virus showed the specificity o f h u m a n RVFpositive serum and Zinga virus. As shown in
Fig. 1. Morphologyof Zinga virus particles negativelystained with sodium phosphotungsten. 33,000 x.
Z I N G A VIRUS A S A S T R A I N O F R I F T V A L L E Y FEVER VIRUS
Fig. 2. Positively labelled Zinga virus after overnight incubation with human positive RVF serum. Gold particles are arranged on the outline of the virus nucleocapsid. Negatively stained with sodium phosphotungsten. 33,000×.
I)"
O
,')"i:.,,..
-.
Fig. 3. Zinga virus incubated overnight with human negative RVF serum. Gold particles are not attached to virus particle. Negatively stained with sodium phosphotungsten. 33,000 x .
217
218
O.D. O L A L E Y E E T A L .
figure 2, the distinct attachment o f gold to virus panicles treated with positive serum contrasted with virus particles treated with RVF-negative serum (fig. 3).
Schmitz, Director of the Department of Virology, Bernhard-Nocht Institute for Tropical Medicine and Hygiene, Hamburg, Germany.
R V F virus is a m e m b e r o f B u n y a v i r i d a e family. According to M u r p h y and Kingsbury (1990), bunyavirus virions consist o f a lipidcontaining envelope, with fine pepIomers, within which are three loosely helical, circular nucleocapsid structures with a diameter o f 2-2.5 nm. Virions are 90-120 nm in diameter. Von B o n s d o r f f and Petterson (1975) and later Objeski and M u r p h y (1977) showed that surface projections are more ordered and form a penthexon cluster arranged in an icoshahedral lattice in R V F virus. This study shows that morphology o f Zinga virus grown in Vero E6 cells is similar to other strains o f R V F virus. Also, labelling o f Zinga virus with R V F virus-positive serum clearly demonstrates that it is a strain o f R V F virus. This report supports earlier observations o f Shope et al. (1980) and Meegan et al. (1983) that Zinga virus is a strain o f R V F virus.
References
Acknowledgements This study was supported by the German Academic Exchange Programme (DAAD) with a Visiting Fellowship grant awarded to Dr. O.D. Olaleye to work with Prof. H.
Arthropod-borne virus information exchange (1977), Catalogue. Digoutte, J.P. (1988), Assessment of the risk of Rift Valley fever and other haemorrhagic fevers in West Africa. WHO Workshop on Emergency Preparedness, Bamako, 4-9 July, 1988. Digoutte, J.P., Cordellier, R., Robin, Y., Pajot, F. & Geoffrey, B. (1974), Zinga virus (At. B. 1967), a new arbovirus isolated in the Central African Republic. Ann. Microbiol. (Instit. Pasteur), 125B, 107-118. Ellis, D.S., Shirodovia, P.V., Fleming, E. & Simpson, D.I.H. (1988), Morphology and development of Rift Valley fever virus in Vero cell culture. J. reed. Virol., 24, 161-174. Meegan, J.M., Digoutte, J.P., Peters, C.J. & Shope, R.E. (1983), Monoclonal antibodies to identify Zinga virus as Rift Valley fever virus. Lancet, I, 64. Mueller, G. & Baigent, C.L. (1980), Antigen-controlled immuno-diagnosis (ACID test). J. Immunol. Methods, 37, 185-190. Murphy, F.A. & Kingsbury, D.W. (1990), Virus taxonomy, in "Fields virology" (Fields, B.N. & Knipe, D.N.) 2rid edition, vol. 1 (p. 9-36). Raven Press, New York. Objeski, J. & Murphy, F.A. (1977), Bunyaviridae, recent biochemical developments. J. gen. Firol., 37, 1-14. Shope, R.E., Peters, C.J. & Walker, J. (1980), Serologic relationship between Rift Valley fever virus and viruses of the phlebotomus fever group. Lancet, I, 886-887. Von Bonsdorff, C.H. & Pettersson, R. (1975), Surface structure of Unkuniemi virus. J. Virol., 16, 1296-1307.
