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Marek's disease virus genome separation from feather tip extracts by pulsed field gel electrophoresis
Journal of Virological Methods 101 (2002) 169– 174 www.elsevier.com/locate/jviromet
Marek’s disease virus genome separation from feather tip extracts by pulsed field gel electrophoresis Rinat Borenshtain, Irit Davidson * Di6ision of A6ian Diseases, Kimron Veterinary Institute, P.O. Box 12, 50250 Bet Dagan, Israel Received 16 August 2001; received in revised form 9 November 2001; accepted 12 November 2001
Abstract Marek’s disease virus is an oncogenic herpes virus of poultry that is highly cell associated. In the infected tissues and tumors the virus replicates in a low copy number. The propagation and dissemination of the virus takes place at the feather follicle epithelium, where the viral genome is produced in high copy number. As the viral genome is a large circular DNA molecule (200 kbp), pulsed field gel electrophoresis was used for separation of the viral genome directly from the infected chicken. DNA was extracted from tumors or feather tips by the phenol:chloroform technique or by low melting agar technique. It was found that feathers, being the site of virus productive replication, are useful for separation of free Marek’s disease virus DNA from in vivo infections. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Marek’s disease virus; Feathers; Tumor; Integration; DNA separation; Pulsed field gel electrophoresis
1. Introduction Marek’s disease virus is an avian oncogenic herpesvirus that spreads horizontally, disseminates through feather dust and infects by inhalation. The virus is replicated productively in feather follicle epithelium cells and enveloped virions are shed. Serving as the virus assembly site,
* Corresponding author. Tel.: + 972-3-9681602; fax: 972-39681739/753. E-mail address: iritd – [email protected] (I. Davidson).
feathers contain massive amounts of cell-free virus (Malkinson et al., 1989; Calnek and Witter, 1997). The virus could be detected in feather tips extracts by dot blot hybridization (Davidson et al., 1986; Malkinson et al., 1989) and lately by PCR (Handberg et al., 2001). In addition, feather tips were shown to be beneficial not only for the virus detection but also for collection of free virions from an infected bird (Swayne et al., 1998). Marek’s disease virus causes tumors of the lymphatic tissue that occupy the visceral organs, central and peripheral nerve system and skin. In tumors, the virus is highly cell associated (Calnek and Witter, 1997) and replicates in a low copy number that was estimated as 15 copies per cell
0166-0934/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 4 3 4 - 7
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
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(Ross et al., 1981). The few 200 kbp copies of Marek’s disease virus may integrate into cellular chromosomes (Delecluse et al., 1993) or may be episomal in the infected cell, either in a circular or a linear status (Kaschka-Dierich et al., 1979; Tanaka et al., 1978; Rhiza and Bauer, 1982; Isfort et al., 1990). As a consequence, the purification of the free viral genome from the infected bird is considered a challenge (Isfort et al., 1990; Delecluse et al., 1993). Pulsed-field gel electrophoresis technique was introduced to analyze large DNA molecules. The pulsed-field gel electrophoresis was used for separation of Marek’s disease virus DNA from cellular genome after propagation in tissue culture (Isfort et al., 1990), where the virus is propagating productively. That technique was also optimized for evaluation of total genome size and alterations in structure, which occur during in vitro attenuation of oncogenic Marek’s disease virus (Wilson and Coussens, 1991). In the present study the separation of virulent Marek’s disease virus genome directly from the bird, without prior propagation in tissue cultures was explored. DNA was extracted from both tumors and feathers of Marek’s disease virus-infected chickens. The extracts were separated by pulsed-field gel electrophoresis and hybridized to Marek’s disease virus probe. As determined by hybridization, the separation of free Marek’s disease virus genome was approximately sixfold higher in DNA extracted from feather tips than from tumors. It was now revealed that feathers are very advantageous for the separation of Marek’s disease virus genome directly from the infected chicken.
2. Materials and methods
2.1. Collection of samples The samples originating from various visceral organs and feather tip extracts of birds were included in four categories, as detailed in Table 1. Groups A and B included samples of visceral organs of commercial chickens with natural viral infection. The chickens, of various ages and types, were submitted to molecular differential diagnosis in a 7 year survey (Davidson and Borenshtain, 2001). For the present study, only PCR positive samples for Marek’s disease virus were analyzed. Groups A and B included the same organs but differed in the DNA extraction procedure; group A consisted of phenol:chloroform extracted DNA (Sambrook et al., 1989) while the DNA of group B was purified according to the low melting agar plugs technique (Wilson and Coussens, 1991). Group C included feather samples of ten Marek’s disease virus infected chickens from a commercial flock that was infected naturally with a field strain of Marek’s disease virus, denoted FR. Group D feather samples were from chickens inoculated at hatch with the Marek’s disease virus prototype strain MD11 (received from Dr R.L. Witter, ADOL, East Lansing, MI, results will be detailed elsewhere). Feathers were collected at the 12th, 28th and 56th days post infection. Groups C and D incorporated DNA extracted from feather tips by the phenol: chloroform method. DNA was extracted by a modified combination of several procedures (Davidson et al., 1986; Handberg et al., 2001; Bello et al., 2001).
Table 1 The four groups of samples tested by PFGE Group
DNA origin
Chicken origin
DNA extraction
Infective virus
A B C D
Tumor Tumor Feather tips Feather tips
Commercial Commercial Experimental Experimental
Phenol Agar plugs Phenol Phenol
Field strains Field strains FRa MD11
a
Field isolate named FR.
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
171
Briefly, the tips of 5– 7 feathers were cut and immersed over night at 55 °C in 1ml of lysis buffer (0.5% SDS, 0.1 M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 200 mg/ml). The DNA was extracted further by the phenol: chloroform method (Sambrook et al., 1989).
2.2. DNA amplification Marek’s disease virus was detected by amplification of the 132 bp tandem repeat (BamH1-H/D genomic fragments) (Becker et al., 1992). The PCR sensitivity was up to about two plaque forming units of a well tissue-culture adapted vaccine strain of MDV, CVI988, and up to a 1:1000 dilution of total genomic DNA of an infected tissue (about 50 ng) of which the MDV genomes cannot be estimated (Davidson et al., 2001). The PCR specificity for serotype 1 viruses was evidenced previously (Becker et al., 1992).
2.3. Pulsed field gel electrophoresis Pulsed field gel electrophoresis was carried out using a BioRad CHEF-DR II unit, at 200 V for 20 h employing pulsed field certified agarose gel (BioRad). Two sets of conditions were used for separation: For group A, the switching gradient was 50\ 90 s, (Isfort et al., 1990) in a 1% gel. For groups B, C and D, the switching gradient was 60\ 120 in a 1.5% gel. For size estimation of DNA fragments, a lambda ladder DNA imbedded in 1% BioRad’s low melt preparative grade agarose was used as a marker.
2.4. Detection of Marek’s disease 6irus Following electrophoresis, the gel was immersed in 0.5 mg/ml ethidium bromide for 1hr and the DNA was visualized by transillumination at 302 nm. Southern blotting of the separated DNA onto a neutral nylon transfer membrane (Schleicher & Schuell), was carried out as described by Isfort et al. (1990). The probe for Marek’s disease virus detection was the 132 bp tandem repeat PCR amplicon. Detection of the samples included
Fig. 1. Pulsed field gel electrophoresis for 14 DNAs (lanes 2 – 15) extracted from tumors in commercial chickens by the phenol: chloroform method. Lane 1 contains CHEF DNA size standards lambda ladder (Bio Rad). Panel A, ethidium bromide staining; Panel B, Marek’s disease virus probe hybridization.
in groups A and B was made by the Rad-Free kit (Schleicher & Schuoll) and for groups C and D by the DIG High Prime DNA Labeling and Detection Starter kit II (Roche). The sensitivity level of both kits was analyzed by serial dilutions of a PCR product and a similar level of 10 − 4 of the PCR amplicon was recorded.
3. Results Figs. 1 and 2 show the pulsed field gel electrophoresis results of both the ethidium bromide staining of the gel (panel A) and the hybridization to Marek’s disease virus probe (panel B). Fig. 1 shows 14 cases (lanes 2–15) of commercial chickens (group A), where DNA was extracted by the phenol:chloroform method from tissues. Fig. 2 shows the results of 14 (lanes 2–15) feather tip extracts from artificially infected chickens with strain MD11 (group D). Lane 1 in both gels was for the lambda ladder DNA marker. To analyze the specificity of the hybridization after pulsed field gel electrophoresis, DNA of uninfected chicken embryo fibroblasts and birds, purified both by phenol:chloroform and low melting agar plugs technique were assayed and found negative.
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Fig. 2. Pulsed field gel electrophoresis for 14 DNAs extracted by phenol: chloroform from feathers of experimentally infected chickens with Marek’s disease virus strain MD11. Lane 1 contains CHEF DNA size standards Lambda ladder (Bio Rad). Panel A, ethidium bromide staining; Panel B, Marek’s disease virus probe hybridization.
Table 2 summarizes the pulsed field gel electrophoresis results of the four different groups of DNA extracts. All DNA samples that were analysed by pulsed field gel electrophoresis were PCR positive. The ethidium bromide staining enabled the size estimation of the separated DNA molecules. The size of Marek’s disease virus genome of about 200 kbp was expected to migrate half to two thirds of the gel length.
3.1. Detection of Marek’s disease 6irus genome in DNA extracted from organs DNA extracts from tumors, obtained by the
phenol: chloroform method were included in group A. According to the ethidium bromide analysis, the 200 kbp molecules were detected in 21 cases out of 55 samples. However, the hybridization to the Marek’s disease virus probe revealed that only two out of the 21 cases were recognized as Marek’s disease virus DNA. In contrast, after extraction of DNA from the same organs in agar plagues, there were only two cases of 200 kbp molecules visualized with ethidium bromide and both of them were also recognized by hybridization. These were the same samples that were detected by hybridization of group A DNA extracts. The Marek’s disease virus probe identified large DNA molecules around the gel origin in four out of 55 DNA samples of group A and in 15/54 samples of group B. The high rate of positive samples of group B, that were retained at the gel origin, could be the consequence of either integration into cellular chromosomes or imperfect penetration into the gel. On the other hand, the low rate of such samples in group A might be the consequence of large molecules shearing that might occur during the extraction procedure.
3.2. Detection of Marek’s disease 6irus genome in DNA extracted from feather tips For the ten commercial birds that were infected naturally with strain FR, DNA was extracted from feather tips by the phenol:chloroform method (group C). There were four cases of separation by ethidium bromide staining and three of
Table 2 PFGE analysis as evaluated by ethidium bromide staining and hybridization to MDV probe Group
Number of MDV-PCR positive samples
PFGE Et.Br. 200kbp
A B C D
55 54 10 51
21 2 4 20
Hybridization Gel origin
200kbp
4 15 – –
2 2 3 12
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
them were identified by the Marek’s disease virus probe. The 51 chickens, infected experimentally with the MD11 strain (group D), include sampling at three times post infection (12th, 28th and 56th days). In 20 cases (2, 12 and 6 for the three bleedings, respectively), the 200 kbp molecules were visualized by ethidium bromide staining and 12 of them were also detected by hybridization (0, 7 and 5, respectively). The probe did not detected Marek’s disease virus at the gel origin when DNA was extracted from feathers (groups C and D).
4. Discussion The present study describes for the first time the separation of Marek’s disease virus genome directly from the infected chicken and the preferred in vivo origin. While, previous studies focused on in vitro replicated (Isfort et al., 1990; Wilson and Coussens, 1991), in the present study the Marek’s disease virus genome was separated by Pulsed Field Gel Electrophoresis without the need to cultivate the virus. It is valuable to obtain intact Marek’s disease virus directly from the infected bird first, because the virus is avoided of undergoing genomic changes in tissue cultures (Wilson and Coussens, 1991) and second, because new virulent field strains tend not to propagate well in vitro (Calnek and Witter, 1997). The results of the present study confirm previous studies, as the 200 kbp Marek’s disease virus genome was detected more efficiently in DNA extracted from feather tips than from tissues. The previous findings regarding copy number of Marek’s disease virus in tissues versus feather tips, site of replication and status of chromosomal integration (Ross et al., 1981; Delecluse et al., 1993; Calnek and Witter, 1997), are also reflected in the present results. The two DNAs extracted from tissues in which Marek’s disease virus genome was separated, were from the same origin, both after phenol: chloroform extraction and after extraction in low melting agar plugs. This fact substantiate the low number of cases (2/54) where the virus was de-
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tected in DNA extracted from tissues and that tissues are not efficient for separation of the Marek’s disease virus genome, as was argued previously (Delecluse et al., 1993). Nevertheless, these two cases imply for an episomal status of the viral genome. The notably higher number of cases of Marek’s disease virus DNA separation, in DNA extracted from feathers reflected the productive replication of Marek’s disease virus in the feather follicle epithelium cells. The Marek’s disease virus probe was recognized not only by 200 kbp molecules but also by larger molecules at the gel origin. As proposed before, it might be the result of Marek’s disease virus integration into cellular chromosomes (Ross et al., 1981). Although this also could be the result of imperfect separation, the data in the present study is supportive of the possibility of integration. It was detected that hybridization to large molecules occurred only in DNA extracted from tumors, where the virus is present in the tumorogenic form, unlike in feather tips, where the virus is propagating productively and is present in a cell free form. Shearing of large molecules is obvious when comparing the results obtained after phenol extraction to those obtained after extraction in low melting agar plugs (Table 2, groups A, C and D compared to group B); following extraction by phenol:chloroform, both in DNA from organs and feathers, there were molecules of approximately 200 kbp, that were not recognized by the specific probe of Marek’s disease virus, implying that this are the products of chromosomal shearing rather than separation of Marek’s disease virus genome. It is important to be aware of the possibility to separate Marek’s disease virus genome by pulsed field gel electrophoresis both for the study of known Marek’s disease virus strains and of unknown wild type viruses where the replication in culture is problematic. The opportunity to use feathers for separation of Marek’s disease virus genome is of great value. In veterinary clinical practice, sampling feathers instead of blood or inner organs is a time saving option that also enables easy screening of a flock.
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Acknowledgements This study was supported by Grant IS-270796R, from the USA– Israel Agricultural Research and Development Fund (BARD). We thank Dr R. L. Witter, ADOL, East Lansing, MI, USA, for his cooperation in providing the samples included in group D.
References Becker, Y., Asher, Y., Tabor, E., Davidson, I., Malkinson, M., Weisman, Y., 1992. Polymerase chain reaction for differentiation between pathogenic and non-pathogenic serotype 1 Marek’s disease viruses and vaccine viruses of serotype 2 and 3. J. Virol. Methods 40, 307 –322. Bello, N., Francino, O., SanChez, A., 2001. Isolation of genomic DNA from feathers. J. Vet. Diagn. Invest. 13, 162 –164. Calnek, B.W., Witter, R.L., 1997. Marek’s disease virus. In: Calnek, B.W., Barnes, H.J., Beard, C.W., McDougald, L.R., Saif, Y.M. (Eds), Disease of Poultry, 9th Ed. Iowa State University Press, Ames, Iowa, USA, pp. 369 – 413. Davidson, I., Maray, T., Malkinson, M., Becker, Y., 1986. Detection of Marek’s disease virus DNA and antigens in feathers from infected chickens. J. Virol. Methods 13, 231– 244. Davidson, I., Borenshtain, R., 2001. In vivo events of retroviral long terminal repeat integration into Marek’s disease virus in commercial poultry: detection of chimerical molecules as a marker. Avian Dis. 45, 102 –121. Davidson, I., Borenshtain, R., Weisman, Y., 2001. Molecular identification of the Marek’s disease virus vaccine strain CVI988 in vaccinated chickens, J. Vet. Med. B., In Press. Delecluse, H.-J., Schuller, S., Hammerschmidt, W., 1993. Latent Marek’s disease virus can be activated from its chromosoma-
lly integrated state in herpesvirus-transformed cells. EMBO J. 12, 3277 – 3286. Handberg, K.J., Nielsen, O.L., Jorgensen, P.H., 2001. The use of serotype 1- and serotype 3- specific polymerase chain reaction for the detection of Marek’s disease virus in chickens. Avian Pathol. 30, 243 – 249. Isfort, R., Robinson, D., Kung, H.-J., 1990. Purification of genomic sized herpesvirus DNA using pulse-field electrophoresis. J. Virol. Meth. 27, 311 – 318. Kaschka-Dierich, C., Nazerian, K., Thomssen, R., 1979. Intracellular state of Marek’s disease virus DNA in two tumorderived chicken cell lines. J. Gen. Virol. 44, 271 – 280. Malkinson, M., Davidson, I., Strenger, C., Weisman, Y., Maray, T., Levy, H., Becker, Y., 1989. Kinetics of the appearance of Marek’s disease virus DNA and antigens in the feathers of chickens infected with virulent field isolates as measured by AGP, ELISA and dot-blot hybridization. Avian Pathol. 18, 735 – 744. Rhiza, H.-J., Bauer, B., 1982. Circular forms of viral DNA in Marek’s disease virus-transformed lymphoblastoid cells. Arch. Virol. 72, 211 – 216. Ross, N.L.J., Delorbe, W., Varmus, H.E., Bishop, M., Brahic, M., Haase, A., 1981. Persistance and expression of Marek’s disease virus DNA in Tumor cells and peripheral nerves studied by in situ hybridization. J. Gen. Virol. 57, 285 – 296. Sambrook, J., Fritch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour, Cold Spring Harbour Laboratory Press. Swayne D.E., John R. G., Jackwood M.W., Pearson J.E., Reed W.M., 1998. A Laboratory Manual for the Isolation and Identification of Avian Pathogens. The American Association of Avian Pathologists. Tanaka, A., Silver, S., Nonoyama, M., 1978. Biochemical evidence of the nonintegrated status of Marek’s disease virus DNA in virus-transfected lymphoblastoid cells of chicken. Virology 88, 19 – 24. Wilson, M.R., Coussens, P.M., 1991. Purification and characterization of infectious Marek’s disease virus genomes using pulsed field electrophoresis. Virology 85, 673 – 680.
Marek’s disease virus genome separation from feather tip extracts by pulsed field gel electrophoresis Rinat Borenshtain, Irit Davidson * Di6ision of A6ian Diseases, Kimron Veterinary Institute, P.O. Box 12, 50250 Bet Dagan, Israel Received 16 August 2001; received in revised form 9 November 2001; accepted 12 November 2001
Abstract Marek’s disease virus is an oncogenic herpes virus of poultry that is highly cell associated. In the infected tissues and tumors the virus replicates in a low copy number. The propagation and dissemination of the virus takes place at the feather follicle epithelium, where the viral genome is produced in high copy number. As the viral genome is a large circular DNA molecule (200 kbp), pulsed field gel electrophoresis was used for separation of the viral genome directly from the infected chicken. DNA was extracted from tumors or feather tips by the phenol:chloroform technique or by low melting agar technique. It was found that feathers, being the site of virus productive replication, are useful for separation of free Marek’s disease virus DNA from in vivo infections. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Marek’s disease virus; Feathers; Tumor; Integration; DNA separation; Pulsed field gel electrophoresis
1. Introduction Marek’s disease virus is an avian oncogenic herpesvirus that spreads horizontally, disseminates through feather dust and infects by inhalation. The virus is replicated productively in feather follicle epithelium cells and enveloped virions are shed. Serving as the virus assembly site,
* Corresponding author. Tel.: + 972-3-9681602; fax: 972-39681739/753. E-mail address: iritd – [email protected] (I. Davidson).
feathers contain massive amounts of cell-free virus (Malkinson et al., 1989; Calnek and Witter, 1997). The virus could be detected in feather tips extracts by dot blot hybridization (Davidson et al., 1986; Malkinson et al., 1989) and lately by PCR (Handberg et al., 2001). In addition, feather tips were shown to be beneficial not only for the virus detection but also for collection of free virions from an infected bird (Swayne et al., 1998). Marek’s disease virus causes tumors of the lymphatic tissue that occupy the visceral organs, central and peripheral nerve system and skin. In tumors, the virus is highly cell associated (Calnek and Witter, 1997) and replicates in a low copy number that was estimated as 15 copies per cell
0166-0934/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 4 3 4 - 7
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
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(Ross et al., 1981). The few 200 kbp copies of Marek’s disease virus may integrate into cellular chromosomes (Delecluse et al., 1993) or may be episomal in the infected cell, either in a circular or a linear status (Kaschka-Dierich et al., 1979; Tanaka et al., 1978; Rhiza and Bauer, 1982; Isfort et al., 1990). As a consequence, the purification of the free viral genome from the infected bird is considered a challenge (Isfort et al., 1990; Delecluse et al., 1993). Pulsed-field gel electrophoresis technique was introduced to analyze large DNA molecules. The pulsed-field gel electrophoresis was used for separation of Marek’s disease virus DNA from cellular genome after propagation in tissue culture (Isfort et al., 1990), where the virus is propagating productively. That technique was also optimized for evaluation of total genome size and alterations in structure, which occur during in vitro attenuation of oncogenic Marek’s disease virus (Wilson and Coussens, 1991). In the present study the separation of virulent Marek’s disease virus genome directly from the bird, without prior propagation in tissue cultures was explored. DNA was extracted from both tumors and feathers of Marek’s disease virus-infected chickens. The extracts were separated by pulsed-field gel electrophoresis and hybridized to Marek’s disease virus probe. As determined by hybridization, the separation of free Marek’s disease virus genome was approximately sixfold higher in DNA extracted from feather tips than from tumors. It was now revealed that feathers are very advantageous for the separation of Marek’s disease virus genome directly from the infected chicken.
2. Materials and methods
2.1. Collection of samples The samples originating from various visceral organs and feather tip extracts of birds were included in four categories, as detailed in Table 1. Groups A and B included samples of visceral organs of commercial chickens with natural viral infection. The chickens, of various ages and types, were submitted to molecular differential diagnosis in a 7 year survey (Davidson and Borenshtain, 2001). For the present study, only PCR positive samples for Marek’s disease virus were analyzed. Groups A and B included the same organs but differed in the DNA extraction procedure; group A consisted of phenol:chloroform extracted DNA (Sambrook et al., 1989) while the DNA of group B was purified according to the low melting agar plugs technique (Wilson and Coussens, 1991). Group C included feather samples of ten Marek’s disease virus infected chickens from a commercial flock that was infected naturally with a field strain of Marek’s disease virus, denoted FR. Group D feather samples were from chickens inoculated at hatch with the Marek’s disease virus prototype strain MD11 (received from Dr R.L. Witter, ADOL, East Lansing, MI, results will be detailed elsewhere). Feathers were collected at the 12th, 28th and 56th days post infection. Groups C and D incorporated DNA extracted from feather tips by the phenol: chloroform method. DNA was extracted by a modified combination of several procedures (Davidson et al., 1986; Handberg et al., 2001; Bello et al., 2001).
Table 1 The four groups of samples tested by PFGE Group
DNA origin
Chicken origin
DNA extraction
Infective virus
A B C D
Tumor Tumor Feather tips Feather tips
Commercial Commercial Experimental Experimental
Phenol Agar plugs Phenol Phenol
Field strains Field strains FRa MD11
a
Field isolate named FR.
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
171
Briefly, the tips of 5– 7 feathers were cut and immersed over night at 55 °C in 1ml of lysis buffer (0.5% SDS, 0.1 M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 200 mg/ml). The DNA was extracted further by the phenol: chloroform method (Sambrook et al., 1989).
2.2. DNA amplification Marek’s disease virus was detected by amplification of the 132 bp tandem repeat (BamH1-H/D genomic fragments) (Becker et al., 1992). The PCR sensitivity was up to about two plaque forming units of a well tissue-culture adapted vaccine strain of MDV, CVI988, and up to a 1:1000 dilution of total genomic DNA of an infected tissue (about 50 ng) of which the MDV genomes cannot be estimated (Davidson et al., 2001). The PCR specificity for serotype 1 viruses was evidenced previously (Becker et al., 1992).
2.3. Pulsed field gel electrophoresis Pulsed field gel electrophoresis was carried out using a BioRad CHEF-DR II unit, at 200 V for 20 h employing pulsed field certified agarose gel (BioRad). Two sets of conditions were used for separation: For group A, the switching gradient was 50\ 90 s, (Isfort et al., 1990) in a 1% gel. For groups B, C and D, the switching gradient was 60\ 120 in a 1.5% gel. For size estimation of DNA fragments, a lambda ladder DNA imbedded in 1% BioRad’s low melt preparative grade agarose was used as a marker.
2.4. Detection of Marek’s disease 6irus Following electrophoresis, the gel was immersed in 0.5 mg/ml ethidium bromide for 1hr and the DNA was visualized by transillumination at 302 nm. Southern blotting of the separated DNA onto a neutral nylon transfer membrane (Schleicher & Schuell), was carried out as described by Isfort et al. (1990). The probe for Marek’s disease virus detection was the 132 bp tandem repeat PCR amplicon. Detection of the samples included
Fig. 1. Pulsed field gel electrophoresis for 14 DNAs (lanes 2 – 15) extracted from tumors in commercial chickens by the phenol: chloroform method. Lane 1 contains CHEF DNA size standards lambda ladder (Bio Rad). Panel A, ethidium bromide staining; Panel B, Marek’s disease virus probe hybridization.
in groups A and B was made by the Rad-Free kit (Schleicher & Schuoll) and for groups C and D by the DIG High Prime DNA Labeling and Detection Starter kit II (Roche). The sensitivity level of both kits was analyzed by serial dilutions of a PCR product and a similar level of 10 − 4 of the PCR amplicon was recorded.
3. Results Figs. 1 and 2 show the pulsed field gel electrophoresis results of both the ethidium bromide staining of the gel (panel A) and the hybridization to Marek’s disease virus probe (panel B). Fig. 1 shows 14 cases (lanes 2–15) of commercial chickens (group A), where DNA was extracted by the phenol:chloroform method from tissues. Fig. 2 shows the results of 14 (lanes 2–15) feather tip extracts from artificially infected chickens with strain MD11 (group D). Lane 1 in both gels was for the lambda ladder DNA marker. To analyze the specificity of the hybridization after pulsed field gel electrophoresis, DNA of uninfected chicken embryo fibroblasts and birds, purified both by phenol:chloroform and low melting agar plugs technique were assayed and found negative.
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
172
Fig. 2. Pulsed field gel electrophoresis for 14 DNAs extracted by phenol: chloroform from feathers of experimentally infected chickens with Marek’s disease virus strain MD11. Lane 1 contains CHEF DNA size standards Lambda ladder (Bio Rad). Panel A, ethidium bromide staining; Panel B, Marek’s disease virus probe hybridization.
Table 2 summarizes the pulsed field gel electrophoresis results of the four different groups of DNA extracts. All DNA samples that were analysed by pulsed field gel electrophoresis were PCR positive. The ethidium bromide staining enabled the size estimation of the separated DNA molecules. The size of Marek’s disease virus genome of about 200 kbp was expected to migrate half to two thirds of the gel length.
3.1. Detection of Marek’s disease 6irus genome in DNA extracted from organs DNA extracts from tumors, obtained by the
phenol: chloroform method were included in group A. According to the ethidium bromide analysis, the 200 kbp molecules were detected in 21 cases out of 55 samples. However, the hybridization to the Marek’s disease virus probe revealed that only two out of the 21 cases were recognized as Marek’s disease virus DNA. In contrast, after extraction of DNA from the same organs in agar plagues, there were only two cases of 200 kbp molecules visualized with ethidium bromide and both of them were also recognized by hybridization. These were the same samples that were detected by hybridization of group A DNA extracts. The Marek’s disease virus probe identified large DNA molecules around the gel origin in four out of 55 DNA samples of group A and in 15/54 samples of group B. The high rate of positive samples of group B, that were retained at the gel origin, could be the consequence of either integration into cellular chromosomes or imperfect penetration into the gel. On the other hand, the low rate of such samples in group A might be the consequence of large molecules shearing that might occur during the extraction procedure.
3.2. Detection of Marek’s disease 6irus genome in DNA extracted from feather tips For the ten commercial birds that were infected naturally with strain FR, DNA was extracted from feather tips by the phenol:chloroform method (group C). There were four cases of separation by ethidium bromide staining and three of
Table 2 PFGE analysis as evaluated by ethidium bromide staining and hybridization to MDV probe Group
Number of MDV-PCR positive samples
PFGE Et.Br. 200kbp
A B C D
55 54 10 51
21 2 4 20
Hybridization Gel origin
200kbp
4 15 – –
2 2 3 12
R. Borenshtain, I. Da6idson / Journal of Virological Methods 101 (2002) 169–174
them were identified by the Marek’s disease virus probe. The 51 chickens, infected experimentally with the MD11 strain (group D), include sampling at three times post infection (12th, 28th and 56th days). In 20 cases (2, 12 and 6 for the three bleedings, respectively), the 200 kbp molecules were visualized by ethidium bromide staining and 12 of them were also detected by hybridization (0, 7 and 5, respectively). The probe did not detected Marek’s disease virus at the gel origin when DNA was extracted from feathers (groups C and D).
4. Discussion The present study describes for the first time the separation of Marek’s disease virus genome directly from the infected chicken and the preferred in vivo origin. While, previous studies focused on in vitro replicated (Isfort et al., 1990; Wilson and Coussens, 1991), in the present study the Marek’s disease virus genome was separated by Pulsed Field Gel Electrophoresis without the need to cultivate the virus. It is valuable to obtain intact Marek’s disease virus directly from the infected bird first, because the virus is avoided of undergoing genomic changes in tissue cultures (Wilson and Coussens, 1991) and second, because new virulent field strains tend not to propagate well in vitro (Calnek and Witter, 1997). The results of the present study confirm previous studies, as the 200 kbp Marek’s disease virus genome was detected more efficiently in DNA extracted from feather tips than from tissues. The previous findings regarding copy number of Marek’s disease virus in tissues versus feather tips, site of replication and status of chromosomal integration (Ross et al., 1981; Delecluse et al., 1993; Calnek and Witter, 1997), are also reflected in the present results. The two DNAs extracted from tissues in which Marek’s disease virus genome was separated, were from the same origin, both after phenol: chloroform extraction and after extraction in low melting agar plugs. This fact substantiate the low number of cases (2/54) where the virus was de-
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tected in DNA extracted from tissues and that tissues are not efficient for separation of the Marek’s disease virus genome, as was argued previously (Delecluse et al., 1993). Nevertheless, these two cases imply for an episomal status of the viral genome. The notably higher number of cases of Marek’s disease virus DNA separation, in DNA extracted from feathers reflected the productive replication of Marek’s disease virus in the feather follicle epithelium cells. The Marek’s disease virus probe was recognized not only by 200 kbp molecules but also by larger molecules at the gel origin. As proposed before, it might be the result of Marek’s disease virus integration into cellular chromosomes (Ross et al., 1981). Although this also could be the result of imperfect separation, the data in the present study is supportive of the possibility of integration. It was detected that hybridization to large molecules occurred only in DNA extracted from tumors, where the virus is present in the tumorogenic form, unlike in feather tips, where the virus is propagating productively and is present in a cell free form. Shearing of large molecules is obvious when comparing the results obtained after phenol extraction to those obtained after extraction in low melting agar plugs (Table 2, groups A, C and D compared to group B); following extraction by phenol:chloroform, both in DNA from organs and feathers, there were molecules of approximately 200 kbp, that were not recognized by the specific probe of Marek’s disease virus, implying that this are the products of chromosomal shearing rather than separation of Marek’s disease virus genome. It is important to be aware of the possibility to separate Marek’s disease virus genome by pulsed field gel electrophoresis both for the study of known Marek’s disease virus strains and of unknown wild type viruses where the replication in culture is problematic. The opportunity to use feathers for separation of Marek’s disease virus genome is of great value. In veterinary clinical practice, sampling feathers instead of blood or inner organs is a time saving option that also enables easy screening of a flock.
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Acknowledgements This study was supported by Grant IS-270796R, from the USA– Israel Agricultural Research and Development Fund (BARD). We thank Dr R. L. Witter, ADOL, East Lansing, MI, USA, for his cooperation in providing the samples included in group D.
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