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Virus-like particles in a Blastocystis species from the sea-snake, Lapemis hardwickii
Intemariona/ Journal/or Prrnred in Greor Brirain
Parusitology
Vol. 22. No. 7, pp. 1029-1032, 1992 0
002&7519/92 $5.00 + 0.00 Pergnmon Press Lrd 1992 Ausrrolion Socieryfor Parasilology
RESEARCHNOTE VIRUS-LIKE
PARTICLES IN A BLASTOCYSTIS SPECIES SNAKE, LAPEMIS HARD WICKII
FROM THE SEA-
W. L. TEOW, L. C. Ho, G. C. NG, Y. C. CHAN, E. H. YAP, P. P. CHAN, J. HOWE, V. ZAMAN and M. SINGH* Department of Microbiology, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 05 11, Republic of Singapore (Received
15June 1992; accepted 2 August 1992)
Abstract-Meow W. L., Ho L. C., No G. C., CHAN Y. C., YAP E. H., CHAN P. P., HOWEJ., ZAMANV. and SINGH M. 1992. Virus-like particles in a EIusfocysfis species from the sea-snake, Lapemis hardwickii. International Journalfor Parasitology 22: 1029-1032. A 5.0 kb band was consistently observed in purified nucleic acid samples from a sea-snake Blusfocysris. This band was resistant to DNase I treatment, and eliminated by 0.2 N-NaOH treatment. That it was highly susceptible to pancreatic ribonuclease A, with sensitivity increasing with decreasing ionic strength, and was undigested by mung bean nuclease indicated that it was a dsRNA band. Transmission electron microscopy of cesium chloride fractions revealed icosahedral virus-like particles 30 run in diameter. These icosahedral virus-like particles were not found in isolates of B. hominis. INDEX
KEY WORDS:
Blastocystis; sea-snake;
virus-like
PARASITIC protozoa have been shown to harbor viruses (Wang & Wang, 1991). The first report of virus-like particles (VLPs) in parasitic protozoa was by Mattern, Diamond & Daniel (1972) who showed, by electron microscopy, VLPs in certain strains of E&amoeba histolytica. These viruses were infectious to VLP-free strains of E. histolytica (see Diamond, Mattern & Bartgis, 1972). Subsequently, VLPs have been reported from Trichomonas vaginalis (see Wang & Wang, 1985), Giardia lamblia (see Wang 8c Wang, 1986), Leishmania braziliensis (see Tarr, Aline, Smiley, Scholler, Keithly & Stuart, 1988), Eimeria spp. (see Revets, Deleersnijder, Dejonckheese, Dekegel, Peeters, Leysen & Hamers, 1989; Ellis & Revets, 1990) and Bubesia bovis (see Johnstone, Farias, Gonzales, Dewes, Masuda, Termignoni, Amako & Ozaki, 1991). These are all RNA viruses (mainly dsRNA), icosahedral or spherical, with a diameter of 30-40 nm and a genomic size range of 5-7 kb. They appear to be closely related to the yeast dsRNA killer virus (Tipper & Bostian, 1984). Blastocystis infections have been found in humans and in several animals, including pigs, monkeys and chickens (Zierdt, 1988; Yamada, Yoshikawa, Tegoshi, Matsumoto, Yoshikawa, Shiota & Yoshida, 1987; Belova & Kostenko, 1990). These parasites appear to
*To whom all correspondence should be addressed.
particle.
be morphologically similar. We have described a Blastocystis sp. from the cecum of a sea-snake, Lapemis hardwickii (see Teow, Zaman, Ng, Chan, Yap, Howe, Gopalakrishnakone & Singh, 1991). During the course of the present studies on nucleic acid samples from Blastocystis, we discovered VLPs in the sea-snake Blastocystis but not in the human strains of B. hominis. The Blastocystis were cultured axenically in BoeckDrbohlav medium containing 30% horse serum and incubated in an anerobic jar (GasPak, BBL) at 24°C (for sea-snake Blastocystis) and 37°C (for B. hominis). Axenization was achieved using antibiotic mixtures (Zierdt & Williams, 1974). Total nucleic acids were extracted by the salt
1030
W. L. Row
23-l946.64.4-
2.32-o-
FIG. 1. Agarose gel electrophoretic analysis of nucleic acids from sea-snake Blastocystis before and after incubation in 0.2 N-NaOH at room temperature for 30 min. Lane 1, lambda DNA Hind111 fragments; lane 2, Blastocysiis nucleic acid sample before treatment; lane 3, Blastocystis nucleic acid sample after treatment.
nM-NaCl but not at 200 and 300 nM-NaCl) showed that the RNA was double-stranded. Furthermore, the T,,, (denaturation temperature) was high @SC), with an estimated GC content of 45%. After harvesting the sea-snake Blastocystis grown to late logarithmic phase, the parasites were washed in phosphate-buffered saline, and subjected to osmotic shock using distilled water, followed by sonication 10 times on ice for 30 s each time with a sonicator bath (Branson, U.S.A.). The crude homogenate was subjected to differential centrifugation according to the method of Wang & Wang (1986). The 5.0 kb band was detectable in the H (crude homogenate), P, and S, (pellet and supernatant after centrifugation at 3000 g), and P, and S, fractions (pellet and supernatant after centrifugation at 12,300 g), indicating that the VLPs
et al.
are found in the nuclear and cytoplasmic fractions. The exact location of the VLPs is not clear since it cannot be located by transmission electron microscopy (Philips 400T) in sectioned whole cells of seasnake Blustocystis due to the small numbers and wide dispersion within the cells. The S, fraction was subjected to cesium chloride density centrifugation and the fractions collected. The fractions containing the 5.0 kb RNA were found by electron microscopy to contain VLPs (Fig. 3). The VLPs were icosahedral, with an estimated diameter of 30 nm. Virus-like particles have been described from Entamoeba,
Giardia,
Trichomonas,
Eimeria,
Leishmania
and Babesia. Of these, Trichomonas inhabits the genital tract, Leishmania the reticuloendothelial system whilst Babesia is in the blood cells. The rest are intestinal parasites. How these VLPs infect these parasites in their various habitats is subject to conjecture, though, for the intestinal parasites, an oral-fecal route is the most likely mode of transmission. The present report is the first record of VLPs in the genus Blustocystis, an intestinal parasite of animals and humans. Thus far, we have not been able to detect VLPs in the isolates of B. hominis. Wang & Wang (1991) also showed that not all strains of Giardia and Trichomonas harbored viruses. The VLPs from Blastocystis appear to be similar to 7’. vaginalis (TVV), G. lamblia (GLV) and E. stiedae viruses (ESV) by size and their RNA. Whether they are closely related remains to be determined since, unlike the other warm-blooded hosts, Blastocystis VLP is from a snake parasite which grows optimally at 24°C and does not survive at 37°C. It is interesting to speculate what role these VLPs have in Blastocystis. Are these infectious virus particles? Do they have some regulatory role in the physiology of this protozoa? Following the lead provided by Wang & Wang (1991) we propose the abbreviation of BLV, after the host, Blastocystis lapemi sp. nov., the parasite of the sea-snake, Lapemis hardwickii. Acknowledgements-We typing the manuscript illustrations.
wish to thank Miss Siti Masnor for and Mr Loh Ah Keong for the
Research ::?
23 : 4 2 2
Note
4?
23 -1 9. 46. 4. fz
2, ,32. ‘o-
FIG. 2.(a) Agarose gel electrophoretic analysis of nucleic acids from sea-snake Blastocystis before and after treatment with RNase A (boiled previously to eliminate DNase). Incubation was carried out at 37°C for 30 min. Lane I, lambda DNA Hind111 marker; lane 2, nucleic acid sample of Blasfoc,vstis; lane 3, treated nucleic acid sample of Blastocystis; lane 4, yeast tRNA; lane 5, treated yeast tRNA. (b) Agarose gel electrophoretic analysis of sea-snake Elastocystis nucleic acids digested with mung bean nuclease. Digestion was carried out at 37’C for 10 min. Lane I, lambda DNA Hind111 fragments; lane 2, undigested B/astoc_wtis nucleic acid; lanes 3 5, Blastocystis nucleic acid digested with increasing concentration of enzyme (6, 12 and I8 units of enzyme ~1.’ DNA).
FE. 3. Electron
micrograph of an RNA-containing sample from the CsCl buoyant density gradient centrifugation. like particles (arrow) were icosahedral and about 30 nm in diameter, x 138,000.
The virus-
1032
W. L. TEow et al. REFERENCES
BELOVAL. M. & KOSTENKOL. A. 1990. Blastocystis galli sp. nov. (Protista: Rhizopoda) from the intestine of chickens. Parazitologiya 24: 164-168. DIAMOND L. S., MATTERN C. F. T. & BARTGIS I. L. 1972. Viruses of Entamoeba histolytica I. Identification of transmissible virus-like agents. Journal of Virology 9: 326-341. ELLIS J. & REVETS H. 1990. Eimeria species which infect chicken contain virus-like RNA molecules. Parasitology 101: 163-169. JOHNSTONER. C., FAR~ASN. A. R., GONZALESJ. C., DEWESH., MASUDAA., TERMIGNON~C., AMAKO K. & OZAKI L. S. 1991. A putative RNA virus in Babesia bovis. Molecular and Biochemical Parasitology 45: 155-l 58. MATTERN C. F. T., DIAMOND L. S. & DANIEL W. A. 1972. Viruses of Entamoeba histolytica II. Morphogenesis of the polyhedral particle (ABRM, - HK-9) -- > HB-301 and the filamentous agent (ABRM, --- > HK-9). Journal qf Virology 9: 342-358. MULLENBACHR., LAGODA P. J. L. & WELTER C. 1989. An efficient salt-chloroform extraction of DNA from blood and tissue. Trends in Genetics 5: 39 I. REVETS H., DEKEGEL D., DELEERSNIJDERW., DEJONCKHEESE J., PEETERSJ., LEYSENE. & HAMERS R. 1989. Identification of virus-like particles in Eimeria stiedae. Molecular and Biochemical Parasitology 36: 209-2 16. TARR P. I., ALINE R. F., JR, SMILEY B. L., SCHOLLER J., KEITHLYJ. & STUARTK. 1988. LRI: a candidate RNA virus of Leishmania. Proceedings of the National Academy of
Sciences qfthe United States of America 86: 9572-9575. TEOW W. L., ZAMAN V., NG G. C., CHAN Y. C.. YAP E. H., HOWE J., GOPALAKRISHNAKONEP. & SINCH M. 1991. A Blastocystis species from the sea-snake, Lapemis hardwickii (Serpentes, Hydrophiidae). International Journal fiw Parasitology 21: 723-726. TEOW W. L., NC G. C., CHAN P. P., CHAN Y. C., YAP E. H., ZAMAN, V. & SINGH M. (in press) A survey of Blastocystis in reptiles. Parasitology Research. TIPPER D. J. & BOSTIAN K. A. 1984. Double-stranded ribonucleic acid killer system in yeasts. Microbiological Reviews 48: 12% 156. WANG A. L. & WANG C. C. 1985. A linear double-stranded RNA in Trichomonas vaginalis. Journal qf Biological Chemistry 260: 3697-3702. WANG A. L. & WANG C. C. 1986. Discovery of a specific double-stranded RNA virus in Giardia lamblia. Molecular and Biochemical Parasitologla 21: 269-270. WANG A. L. & WANG C. C. 1991. Viruses of parasitic protozoa. Parasitology Today 7: 76-80. YAMADA M., YOSHIKAWAH., TEGOSHI T., MATSUMOTOY., YOSHIKAWA T., SHIOTA T. & YOSHIDA Y. 1987. Light microscopical study of Blastocystis spp. in monkeys and fowls. Parasitology Research 73: 527-53 1. ZIERDT C. H. & WILLIAMS R. L. 1974 Blastocystis hominis: axenic cultivation. Experimental Parasitology 36: 233243. ZIERDT C. H. 1988. Blastocystis hominis, a long-misunderstood intestinal parasite. Parasitology Today 4: 15-I 7.
Parusitology
Vol. 22. No. 7, pp. 1029-1032, 1992 0
002&7519/92 $5.00 + 0.00 Pergnmon Press Lrd 1992 Ausrrolion Socieryfor Parasilology
RESEARCHNOTE VIRUS-LIKE
PARTICLES IN A BLASTOCYSTIS SPECIES SNAKE, LAPEMIS HARD WICKII
FROM THE SEA-
W. L. TEOW, L. C. Ho, G. C. NG, Y. C. CHAN, E. H. YAP, P. P. CHAN, J. HOWE, V. ZAMAN and M. SINGH* Department of Microbiology, Faculty of Medicine, National University of Singapore, Lower Kent Ridge Road, Singapore 05 11, Republic of Singapore (Received
15June 1992; accepted 2 August 1992)
Abstract-Meow W. L., Ho L. C., No G. C., CHAN Y. C., YAP E. H., CHAN P. P., HOWEJ., ZAMANV. and SINGH M. 1992. Virus-like particles in a EIusfocysfis species from the sea-snake, Lapemis hardwickii. International Journalfor Parasitology 22: 1029-1032. A 5.0 kb band was consistently observed in purified nucleic acid samples from a sea-snake Blusfocysris. This band was resistant to DNase I treatment, and eliminated by 0.2 N-NaOH treatment. That it was highly susceptible to pancreatic ribonuclease A, with sensitivity increasing with decreasing ionic strength, and was undigested by mung bean nuclease indicated that it was a dsRNA band. Transmission electron microscopy of cesium chloride fractions revealed icosahedral virus-like particles 30 run in diameter. These icosahedral virus-like particles were not found in isolates of B. hominis. INDEX
KEY WORDS:
Blastocystis; sea-snake;
virus-like
PARASITIC protozoa have been shown to harbor viruses (Wang & Wang, 1991). The first report of virus-like particles (VLPs) in parasitic protozoa was by Mattern, Diamond & Daniel (1972) who showed, by electron microscopy, VLPs in certain strains of E&amoeba histolytica. These viruses were infectious to VLP-free strains of E. histolytica (see Diamond, Mattern & Bartgis, 1972). Subsequently, VLPs have been reported from Trichomonas vaginalis (see Wang & Wang, 1985), Giardia lamblia (see Wang 8c Wang, 1986), Leishmania braziliensis (see Tarr, Aline, Smiley, Scholler, Keithly & Stuart, 1988), Eimeria spp. (see Revets, Deleersnijder, Dejonckheese, Dekegel, Peeters, Leysen & Hamers, 1989; Ellis & Revets, 1990) and Bubesia bovis (see Johnstone, Farias, Gonzales, Dewes, Masuda, Termignoni, Amako & Ozaki, 1991). These are all RNA viruses (mainly dsRNA), icosahedral or spherical, with a diameter of 30-40 nm and a genomic size range of 5-7 kb. They appear to be closely related to the yeast dsRNA killer virus (Tipper & Bostian, 1984). Blastocystis infections have been found in humans and in several animals, including pigs, monkeys and chickens (Zierdt, 1988; Yamada, Yoshikawa, Tegoshi, Matsumoto, Yoshikawa, Shiota & Yoshida, 1987; Belova & Kostenko, 1990). These parasites appear to
*To whom all correspondence should be addressed.
particle.
be morphologically similar. We have described a Blastocystis sp. from the cecum of a sea-snake, Lapemis hardwickii (see Teow, Zaman, Ng, Chan, Yap, Howe, Gopalakrishnakone & Singh, 1991). During the course of the present studies on nucleic acid samples from Blastocystis, we discovered VLPs in the sea-snake Blastocystis but not in the human strains of B. hominis. The Blastocystis were cultured axenically in BoeckDrbohlav medium containing 30% horse serum and incubated in an anerobic jar (GasPak, BBL) at 24°C (for sea-snake Blastocystis) and 37°C (for B. hominis). Axenization was achieved using antibiotic mixtures (Zierdt & Williams, 1974). Total nucleic acids were extracted by the salt
1030
W. L. Row
23-l946.64.4-
2.32-o-
FIG. 1. Agarose gel electrophoretic analysis of nucleic acids from sea-snake Blastocystis before and after incubation in 0.2 N-NaOH at room temperature for 30 min. Lane 1, lambda DNA Hind111 fragments; lane 2, Blastocysiis nucleic acid sample before treatment; lane 3, Blastocystis nucleic acid sample after treatment.
nM-NaCl but not at 200 and 300 nM-NaCl) showed that the RNA was double-stranded. Furthermore, the T,,, (denaturation temperature) was high @SC), with an estimated GC content of 45%. After harvesting the sea-snake Blastocystis grown to late logarithmic phase, the parasites were washed in phosphate-buffered saline, and subjected to osmotic shock using distilled water, followed by sonication 10 times on ice for 30 s each time with a sonicator bath (Branson, U.S.A.). The crude homogenate was subjected to differential centrifugation according to the method of Wang & Wang (1986). The 5.0 kb band was detectable in the H (crude homogenate), P, and S, (pellet and supernatant after centrifugation at 3000 g), and P, and S, fractions (pellet and supernatant after centrifugation at 12,300 g), indicating that the VLPs
et al.
are found in the nuclear and cytoplasmic fractions. The exact location of the VLPs is not clear since it cannot be located by transmission electron microscopy (Philips 400T) in sectioned whole cells of seasnake Blustocystis due to the small numbers and wide dispersion within the cells. The S, fraction was subjected to cesium chloride density centrifugation and the fractions collected. The fractions containing the 5.0 kb RNA were found by electron microscopy to contain VLPs (Fig. 3). The VLPs were icosahedral, with an estimated diameter of 30 nm. Virus-like particles have been described from Entamoeba,
Giardia,
Trichomonas,
Eimeria,
Leishmania
and Babesia. Of these, Trichomonas inhabits the genital tract, Leishmania the reticuloendothelial system whilst Babesia is in the blood cells. The rest are intestinal parasites. How these VLPs infect these parasites in their various habitats is subject to conjecture, though, for the intestinal parasites, an oral-fecal route is the most likely mode of transmission. The present report is the first record of VLPs in the genus Blustocystis, an intestinal parasite of animals and humans. Thus far, we have not been able to detect VLPs in the isolates of B. hominis. Wang & Wang (1991) also showed that not all strains of Giardia and Trichomonas harbored viruses. The VLPs from Blastocystis appear to be similar to 7’. vaginalis (TVV), G. lamblia (GLV) and E. stiedae viruses (ESV) by size and their RNA. Whether they are closely related remains to be determined since, unlike the other warm-blooded hosts, Blastocystis VLP is from a snake parasite which grows optimally at 24°C and does not survive at 37°C. It is interesting to speculate what role these VLPs have in Blastocystis. Are these infectious virus particles? Do they have some regulatory role in the physiology of this protozoa? Following the lead provided by Wang & Wang (1991) we propose the abbreviation of BLV, after the host, Blastocystis lapemi sp. nov., the parasite of the sea-snake, Lapemis hardwickii. Acknowledgements-We typing the manuscript illustrations.
wish to thank Miss Siti Masnor for and Mr Loh Ah Keong for the
Research ::?
23 : 4 2 2
Note
4?
23 -1 9. 46. 4. fz
2, ,32. ‘o-
FIG. 2.(a) Agarose gel electrophoretic analysis of nucleic acids from sea-snake Blastocystis before and after treatment with RNase A (boiled previously to eliminate DNase). Incubation was carried out at 37°C for 30 min. Lane I, lambda DNA Hind111 marker; lane 2, nucleic acid sample of Blasfoc,vstis; lane 3, treated nucleic acid sample of Blastocystis; lane 4, yeast tRNA; lane 5, treated yeast tRNA. (b) Agarose gel electrophoretic analysis of sea-snake Elastocystis nucleic acids digested with mung bean nuclease. Digestion was carried out at 37’C for 10 min. Lane I, lambda DNA Hind111 fragments; lane 2, undigested B/astoc_wtis nucleic acid; lanes 3 5, Blastocystis nucleic acid digested with increasing concentration of enzyme (6, 12 and I8 units of enzyme ~1.’ DNA).
FE. 3. Electron
micrograph of an RNA-containing sample from the CsCl buoyant density gradient centrifugation. like particles (arrow) were icosahedral and about 30 nm in diameter, x 138,000.
The virus-
1032
W. L. TEow et al. REFERENCES
BELOVAL. M. & KOSTENKOL. A. 1990. Blastocystis galli sp. nov. (Protista: Rhizopoda) from the intestine of chickens. Parazitologiya 24: 164-168. DIAMOND L. S., MATTERN C. F. T. & BARTGIS I. L. 1972. Viruses of Entamoeba histolytica I. Identification of transmissible virus-like agents. Journal of Virology 9: 326-341. ELLIS J. & REVETS H. 1990. Eimeria species which infect chicken contain virus-like RNA molecules. Parasitology 101: 163-169. JOHNSTONER. C., FAR~ASN. A. R., GONZALESJ. C., DEWESH., MASUDAA., TERMIGNON~C., AMAKO K. & OZAKI L. S. 1991. A putative RNA virus in Babesia bovis. Molecular and Biochemical Parasitology 45: 155-l 58. MATTERN C. F. T., DIAMOND L. S. & DANIEL W. A. 1972. Viruses of Entamoeba histolytica II. Morphogenesis of the polyhedral particle (ABRM, - HK-9) -- > HB-301 and the filamentous agent (ABRM, --- > HK-9). Journal qf Virology 9: 342-358. MULLENBACHR., LAGODA P. J. L. & WELTER C. 1989. An efficient salt-chloroform extraction of DNA from blood and tissue. Trends in Genetics 5: 39 I. REVETS H., DEKEGEL D., DELEERSNIJDERW., DEJONCKHEESE J., PEETERSJ., LEYSENE. & HAMERS R. 1989. Identification of virus-like particles in Eimeria stiedae. Molecular and Biochemical Parasitology 36: 209-2 16. TARR P. I., ALINE R. F., JR, SMILEY B. L., SCHOLLER J., KEITHLYJ. & STUARTK. 1988. LRI: a candidate RNA virus of Leishmania. Proceedings of the National Academy of
Sciences qfthe United States of America 86: 9572-9575. TEOW W. L., ZAMAN V., NG G. C., CHAN Y. C.. YAP E. H., HOWE J., GOPALAKRISHNAKONEP. & SINCH M. 1991. A Blastocystis species from the sea-snake, Lapemis hardwickii (Serpentes, Hydrophiidae). International Journal fiw Parasitology 21: 723-726. TEOW W. L., NC G. C., CHAN P. P., CHAN Y. C., YAP E. H., ZAMAN, V. & SINGH M. (in press) A survey of Blastocystis in reptiles. Parasitology Research. TIPPER D. J. & BOSTIAN K. A. 1984. Double-stranded ribonucleic acid killer system in yeasts. Microbiological Reviews 48: 12% 156. WANG A. L. & WANG C. C. 1985. A linear double-stranded RNA in Trichomonas vaginalis. Journal qf Biological Chemistry 260: 3697-3702. WANG A. L. & WANG C. C. 1986. Discovery of a specific double-stranded RNA virus in Giardia lamblia. Molecular and Biochemical Parasitologla 21: 269-270. WANG A. L. & WANG C. C. 1991. Viruses of parasitic protozoa. Parasitology Today 7: 76-80. YAMADA M., YOSHIKAWAH., TEGOSHI T., MATSUMOTOY., YOSHIKAWA T., SHIOTA T. & YOSHIDA Y. 1987. Light microscopical study of Blastocystis spp. in monkeys and fowls. Parasitology Research 73: 527-53 1. ZIERDT C. H. & WILLIAMS R. L. 1974 Blastocystis hominis: axenic cultivation. Experimental Parasitology 36: 233243. ZIERDT C. H. 1988. Blastocystis hominis, a long-misunderstood intestinal parasite. Parasitology Today 4: 15-I 7.