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A simple and efficient method for purification of prawn baculovirus DNA
Journal of Virological Methods 67 (1997) 1 – 4
A simple and efficient method for purification of prawn baculovirus DNA F. Yang, W. Wang, R.Z. Chen, X. Xu * The Third Institute of Oceanography, Xiamen, 361005, People’s Republic of China Accepted 4 April 1997
Abstract A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 – 62 nm in width and 300–350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns. © 1997 Elsevier Science B.V. Keywords: Prawn baculovirus; DNA purification; Viral DNA; PWSBV
1. Introduction Viruses were widespread in cultured shimps and were responsible for most of the serious shrimp diseases which had resulted in big economic losses in China since 1993. Studies on the pathology of the disease and the shape of the virus showed that
* Corresponding author.
the pathogen is a non-inclusion body (subgroup C) baculovirus which can infect Penacus japonicus, P. monodon, P. chinesis, P. penicillatus (Peng et al., 1995; Wang and Liu, 1996) and lead to evident white spots on the inside surface of the crust. Thus, this prawn disease is always called white spot disease. Up to now, the only sources of prawn baculoviruses were infected prawns, which are limited not only by its breeding season but also by random infections period. Moreover, purification of the baculovirus and the viral DNA
0166-0934/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 0 6 0 - 8
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F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
are rather difficult. CsCl density gradient centrifugation or sucrose gradient centrifugation or phenol-chloroform extraction have been used in the current purification methods (Bruce et al., 1991; Chang et al., 1993, Mari et al., 1993; Arimoto et al., 1995; Wang et al., 1995) which are time-consuming and low yielding. Abundant virus and subsequently purified viral DNA are not accessible. A new method was developed to facilitate the isolation of nucleocapsids from PWSBV-infected prawn and the subsequent purification of viral DNA. In addition, the DNA fragments were fractionated on an agarose gel after restriction endounclease digestion, and distinct bands were observed.
2. Material and methods
2.1. Infected prawn Dead and moribund P. japonicus with evident white spots on the inside surface of the crust were collected and kept at 4°C from the prawn farm in Tongan, Xiamen, East China.
2.2. Isolation of nucleocapsids Hepatopancreata gill and intestine were removed from P. japonicus and placed in an icebathed beaker, homogenized as a 10% suspension in TESP buffer (50 mmol/l, Tris – HCl, pH 8.5, 10 mmol/l EDTA, 100 mmol/l NaCl; 1 mmol/l PMSF), then centrifuged at 6500 × g for 10 min at 4°C. The supernatant was recentrifuged at 30 000×g for 30 min at 4°C. The pellet was suspended in an appropriate two volume of TESP buffer containing 1% (v/v) Triton X-100. After centrifugation at 5000×g for 10 min, the supernatant was centrifuged again at 25 000 × g for 20 min. The pellet was suspended in TESP buffer and differential centrifugation was repeated, and then the precipitate was resuspended in TMP (100 mmol/l, Tris–HCl, pH 7.5, 10 mmol/l MgCl2/l, 1 mmol/l PMSF) buffer and treated with DNase and RNase. The mixture was incubated at 37°C for 15 min and 30 ml of TESP buffer were added. Differential centrifugation was repeated again and
the pellet was resuspended in 1 ml of TESP buffer, 1 ml of suspension was dropped on a copper grid, negatively stained with 2% (w/v) uranyl acetate, pH 7.6, and observed using JEM100CX II transmission electron microscope.
2.3. Purification of 6iral DNA The nucleocapsids suspension was lysed with 2 ml of GTE buffer (6 mol/l guanidine hydrochloride; 50 mmol/l Tris–HCl; 10 mmol/l EDTA; pH 7.0) slightly mixed and then centrifuged at 25 000×g for 10 min at 4°C. The supernatant was collected and 0.02 vol. of 1 mol/l MgCl2 and 0.6 vol. of isopropanol were added. After centrifugation, the pellet was picked out with pipette tip, washed twice with 70% ethanol and then dissolved in 1 ml of TE buffer containing 0.5% (w/v) SDS and 0.5 mg/ml proteinase K and incubated at 55°C for 2–3 h. The DNA was precipitated again with 0.01 vol. of 1 mol/l MgCl2 and 0.25 vol. of isopropanol and dissolved in 0.1× TE buffer. The viral DNA obtained was quantified by spectrophotometer.
2.4. Restriction endonuclease digestion A total of 1 mg of viral DNA was digested with 10 U, each of restriction endonucleases for 5 h at 37°C and loaded onto a 0.5% agarose gel in TAE buffer. Electrophoresis was performed at 2 V/cm for 6 h and visualized by ethidium bromide staining. 3. Results After treatment with Triton X-100, the viral envelope was disrupted and the nucleocapsids were shown to be 58–62 nm in diameter and 300–350 nm in length (Fig. 1) by transmission electron microscopy. The purified viral DNA was digested with BamHI, EcoRI, HindIII, KpnI, PstI, SalI and XbaI respectively, then separated by electrophoresis and very distinct bands of DNA fragments were observed (Fig. 2). According to the mobility of the bands (compared with MW markers), the size of the viral DNA is approximately 290 kb.
F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
3
4. Discussion During homogenization of infected tissues, abundant proteinases present in the shrimp’s digestion system would be released and it inevitably lead to rapid disruption of virions and degradation of viral DNA. To avoid this, the activity of the protease would have to be inhibited. Phenylmethylsulfonyl fluoride (PMSF) was found to be an efficient inhibitor for this purpose and it greatly improved the efficiency of isolation of intact virion. Also Triton X-100 disrupted biomembranes and thus prevented the formation of particles, but still enable the prawn DNA and RNA to stay in free state. Tissue debris, prawn DNA and RNA were removed by differential centrifugation, and the remaining DNA and RNA degraded by DNase and RNase. Instead of extraction with phenol/chloroform, guanidine hydrochoride was used to lyse viruses and prevent the viral DNA from fragmentation. Several restriction endonucleases were added and after electrophoresis, distinct bands were visualized, which showed the viral DNA obtained
Fig. 1. Electron micrograph of negatively stained prawn white spot baculovirus (PWSBV) nucleocapsid.
Fig. 2. Restriction pattern of the viral DNA by 0.5% agarose gel electrophoresis after restriction endonuclease digestion. Lane 1: XbaI; Lane 2: SalI; Lane 3: PstI; Lane 4: KpnI; Lane 5: HindIII; Lane 6: EcoRI; Lane 7: BamHI; Lane 8: MW markers (l DNA digested with HindIII.
was pure. Also we can conclude that viral capsid was so tight that DNase could not penetrate and thus act on viral DNA. Based on observations using the transmisson electron microscopy, PWSBV nucleocapsid appeared to be 58–62× 300–340 nm, MBV nucleocapsid was 62–68× 250–270 nm (Mari et al., 1993), while BMNV nucleocapsid was 50× 260 nm (Arimoto et al., 1995). This means that PWSBV can pack longer DNA, which is in accordance with the estimation made of restriction endonuclease fragments, approximately 290 kb. We have worked on isolation and purification of prawn baculovirus DNA by conventional methods for 2 years, but only obtained a small amount of viral DNA which was yet obviously contaminated by prawn genomic DNA. Now we have isolated and purified viral DNA several times. The highest yield was nearly 100 mg from about 100 g tissues of hepatopancreata gill and intestine, while the others were about 40 mg, depending on the degree of infection. All the viral DNA obtained were very pure. A large quantity
F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
4
of intact viral DNA, which will greatly facilitate future research, including physical mapping of the virus genome and cloning of the viral DNA.
References Arimoto, M., Yamazaki, T., Mizuta, Y., Furusawa, I., 1995. Characterization and partial cloning of the genomic DNA of a baculovirus from Penaeus japonicus (PjNOB = BMNV). Aquaculture 132, 213–220. Bruce, L.D., Tramper, B.B., Lightner, D.V., 1991. Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus. J. Virol. Methods 34, 245–254. Chang, P.S., Lo, C.F., Kou, C.H., Lu, C.C., Chen, S.N., 1993. Purification and amplification of DNA from Penaeus
.
monodon — type baculovirus (MBV). J. Invertebr. Pathol. 62, 116 – 120. Mari, J., Bonami, J.R., Poulos, B., Lightner, D., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. aquat. Org. 16, 207 – 215. Peng, B.Z., Ren, J.M., Shen, J.Y., Zhol, G.Y., Gu, H.T., Shen, Y.L., Zheng, G.X., Gong, Z.X., 1995. The studies on baculovirus-caused disease of prawns (Penacus chinesis osbeckin) Shanghai suburb. Chin. J. Vir. 11 (2), 151 – 157. Wang, C.-H., Lo, C.-F., Leu, J.-H., Chou, C.-M., Yeh, P.-Y., Tung, M.-C., Chang, C.-F., Su, M.-S., Kou, G.-H., 1995. Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis. aquat. Org. 23 (3), 239 – 242. Wang, Z.G., Liu, R.Y., 1996. Proc. 2nd Symp. on Diseases of Cultured Penaeid Shrimp. Qingdao Ocean University Press, China.
A simple and efficient method for purification of prawn baculovirus DNA F. Yang, W. Wang, R.Z. Chen, X. Xu * The Third Institute of Oceanography, Xiamen, 361005, People’s Republic of China Accepted 4 April 1997
Abstract A new method for isolation of prawn baculovirus and subsequent extraction of viral DNA was developed. No density gradient centrifugation, ultracentrifugation or phenol-chloroform extraction steps were involved. Phenylmethylsulfonyl fluoride (PMSF) was used to prevent proteinase degradation, DNase and RNase were used to degrade prawn DNA and RNA respectively. The nucleocapsid was a bacilliform virion, about 58 – 62 nm in width and 300–350 nm in length as observed by transmission electron microscopy. Intact viral DNA was obtained by lysing nucleocapsids with guanidine hydrochloride and degrading protein with proteinase K. As the viral DNA was digested with restriction endonuclease and separated by electrophoresis, restriction fragments were clearly shown on the agarose gel. The size of the DNA was estimated approximately to be 290 kb. The virus which appeared to be a prawn baculovirus was named prawn white spot baculovirus (PWSBV) due to the white spots which appeared on the inside surface of the crust of infected prawns. © 1997 Elsevier Science B.V. Keywords: Prawn baculovirus; DNA purification; Viral DNA; PWSBV
1. Introduction Viruses were widespread in cultured shimps and were responsible for most of the serious shrimp diseases which had resulted in big economic losses in China since 1993. Studies on the pathology of the disease and the shape of the virus showed that
* Corresponding author.
the pathogen is a non-inclusion body (subgroup C) baculovirus which can infect Penacus japonicus, P. monodon, P. chinesis, P. penicillatus (Peng et al., 1995; Wang and Liu, 1996) and lead to evident white spots on the inside surface of the crust. Thus, this prawn disease is always called white spot disease. Up to now, the only sources of prawn baculoviruses were infected prawns, which are limited not only by its breeding season but also by random infections period. Moreover, purification of the baculovirus and the viral DNA
0166-0934/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 6 - 0 9 3 4 ( 9 7 ) 0 0 0 6 0 - 8
2
F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
are rather difficult. CsCl density gradient centrifugation or sucrose gradient centrifugation or phenol-chloroform extraction have been used in the current purification methods (Bruce et al., 1991; Chang et al., 1993, Mari et al., 1993; Arimoto et al., 1995; Wang et al., 1995) which are time-consuming and low yielding. Abundant virus and subsequently purified viral DNA are not accessible. A new method was developed to facilitate the isolation of nucleocapsids from PWSBV-infected prawn and the subsequent purification of viral DNA. In addition, the DNA fragments were fractionated on an agarose gel after restriction endounclease digestion, and distinct bands were observed.
2. Material and methods
2.1. Infected prawn Dead and moribund P. japonicus with evident white spots on the inside surface of the crust were collected and kept at 4°C from the prawn farm in Tongan, Xiamen, East China.
2.2. Isolation of nucleocapsids Hepatopancreata gill and intestine were removed from P. japonicus and placed in an icebathed beaker, homogenized as a 10% suspension in TESP buffer (50 mmol/l, Tris – HCl, pH 8.5, 10 mmol/l EDTA, 100 mmol/l NaCl; 1 mmol/l PMSF), then centrifuged at 6500 × g for 10 min at 4°C. The supernatant was recentrifuged at 30 000×g for 30 min at 4°C. The pellet was suspended in an appropriate two volume of TESP buffer containing 1% (v/v) Triton X-100. After centrifugation at 5000×g for 10 min, the supernatant was centrifuged again at 25 000 × g for 20 min. The pellet was suspended in TESP buffer and differential centrifugation was repeated, and then the precipitate was resuspended in TMP (100 mmol/l, Tris–HCl, pH 7.5, 10 mmol/l MgCl2/l, 1 mmol/l PMSF) buffer and treated with DNase and RNase. The mixture was incubated at 37°C for 15 min and 30 ml of TESP buffer were added. Differential centrifugation was repeated again and
the pellet was resuspended in 1 ml of TESP buffer, 1 ml of suspension was dropped on a copper grid, negatively stained with 2% (w/v) uranyl acetate, pH 7.6, and observed using JEM100CX II transmission electron microscope.
2.3. Purification of 6iral DNA The nucleocapsids suspension was lysed with 2 ml of GTE buffer (6 mol/l guanidine hydrochloride; 50 mmol/l Tris–HCl; 10 mmol/l EDTA; pH 7.0) slightly mixed and then centrifuged at 25 000×g for 10 min at 4°C. The supernatant was collected and 0.02 vol. of 1 mol/l MgCl2 and 0.6 vol. of isopropanol were added. After centrifugation, the pellet was picked out with pipette tip, washed twice with 70% ethanol and then dissolved in 1 ml of TE buffer containing 0.5% (w/v) SDS and 0.5 mg/ml proteinase K and incubated at 55°C for 2–3 h. The DNA was precipitated again with 0.01 vol. of 1 mol/l MgCl2 and 0.25 vol. of isopropanol and dissolved in 0.1× TE buffer. The viral DNA obtained was quantified by spectrophotometer.
2.4. Restriction endonuclease digestion A total of 1 mg of viral DNA was digested with 10 U, each of restriction endonucleases for 5 h at 37°C and loaded onto a 0.5% agarose gel in TAE buffer. Electrophoresis was performed at 2 V/cm for 6 h and visualized by ethidium bromide staining. 3. Results After treatment with Triton X-100, the viral envelope was disrupted and the nucleocapsids were shown to be 58–62 nm in diameter and 300–350 nm in length (Fig. 1) by transmission electron microscopy. The purified viral DNA was digested with BamHI, EcoRI, HindIII, KpnI, PstI, SalI and XbaI respectively, then separated by electrophoresis and very distinct bands of DNA fragments were observed (Fig. 2). According to the mobility of the bands (compared with MW markers), the size of the viral DNA is approximately 290 kb.
F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
3
4. Discussion During homogenization of infected tissues, abundant proteinases present in the shrimp’s digestion system would be released and it inevitably lead to rapid disruption of virions and degradation of viral DNA. To avoid this, the activity of the protease would have to be inhibited. Phenylmethylsulfonyl fluoride (PMSF) was found to be an efficient inhibitor for this purpose and it greatly improved the efficiency of isolation of intact virion. Also Triton X-100 disrupted biomembranes and thus prevented the formation of particles, but still enable the prawn DNA and RNA to stay in free state. Tissue debris, prawn DNA and RNA were removed by differential centrifugation, and the remaining DNA and RNA degraded by DNase and RNase. Instead of extraction with phenol/chloroform, guanidine hydrochoride was used to lyse viruses and prevent the viral DNA from fragmentation. Several restriction endonucleases were added and after electrophoresis, distinct bands were visualized, which showed the viral DNA obtained
Fig. 1. Electron micrograph of negatively stained prawn white spot baculovirus (PWSBV) nucleocapsid.
Fig. 2. Restriction pattern of the viral DNA by 0.5% agarose gel electrophoresis after restriction endonuclease digestion. Lane 1: XbaI; Lane 2: SalI; Lane 3: PstI; Lane 4: KpnI; Lane 5: HindIII; Lane 6: EcoRI; Lane 7: BamHI; Lane 8: MW markers (l DNA digested with HindIII.
was pure. Also we can conclude that viral capsid was so tight that DNase could not penetrate and thus act on viral DNA. Based on observations using the transmisson electron microscopy, PWSBV nucleocapsid appeared to be 58–62× 300–340 nm, MBV nucleocapsid was 62–68× 250–270 nm (Mari et al., 1993), while BMNV nucleocapsid was 50× 260 nm (Arimoto et al., 1995). This means that PWSBV can pack longer DNA, which is in accordance with the estimation made of restriction endonuclease fragments, approximately 290 kb. We have worked on isolation and purification of prawn baculovirus DNA by conventional methods for 2 years, but only obtained a small amount of viral DNA which was yet obviously contaminated by prawn genomic DNA. Now we have isolated and purified viral DNA several times. The highest yield was nearly 100 mg from about 100 g tissues of hepatopancreata gill and intestine, while the others were about 40 mg, depending on the degree of infection. All the viral DNA obtained were very pure. A large quantity
F. Yang et al. / Journal of Virological Methods 67 (1997) 1–4
4
of intact viral DNA, which will greatly facilitate future research, including physical mapping of the virus genome and cloning of the viral DNA.
References Arimoto, M., Yamazaki, T., Mizuta, Y., Furusawa, I., 1995. Characterization and partial cloning of the genomic DNA of a baculovirus from Penaeus japonicus (PjNOB = BMNV). Aquaculture 132, 213–220. Bruce, L.D., Tramper, B.B., Lightner, D.V., 1991. Methods for viral isolation and DNA extraction for a penaeid shrimp baculovirus. J. Virol. Methods 34, 245–254. Chang, P.S., Lo, C.F., Kou, C.H., Lu, C.C., Chen, S.N., 1993. Purification and amplification of DNA from Penaeus
.
monodon — type baculovirus (MBV). J. Invertebr. Pathol. 62, 116 – 120. Mari, J., Bonami, J.R., Poulos, B., Lightner, D., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. aquat. Org. 16, 207 – 215. Peng, B.Z., Ren, J.M., Shen, J.Y., Zhol, G.Y., Gu, H.T., Shen, Y.L., Zheng, G.X., Gong, Z.X., 1995. The studies on baculovirus-caused disease of prawns (Penacus chinesis osbeckin) Shanghai suburb. Chin. J. Vir. 11 (2), 151 – 157. Wang, C.-H., Lo, C.-F., Leu, J.-H., Chou, C.-M., Yeh, P.-Y., Tung, M.-C., Chang, C.-F., Su, M.-S., Kou, G.-H., 1995. Purification and genomic analysis of baculovirus associated with white spot syndrome (WSBV) of Penaeus monodon. Dis. aquat. Org. 23 (3), 239 – 242. Wang, Z.G., Liu, R.Y., 1996. Proc. 2nd Symp. on Diseases of Cultured Penaeid Shrimp. Qingdao Ocean University Press, China.