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Growth of avian adeno-associated vims in chicken cells transfected with fowl adenovirus serotype 1 DNA
Jourml of Virologica~ Methods, 29 (1990) 335-340
335
Elsevier
VIRMET 01056
Short
Communication
Growth of avian adeno-associated virus in chicken cells transfected with fowl adenovirus serotype 1 DNA Annette
Batter,
Gerhard
Monreal
and Hans-Joachim
Batter
Institute of Poultry Diseases, Free University of Berlin, Berlin, F.R.G.
(Accepted
16 May 1990)
Summary Using the chloroquine-modified calcium phosphate coprecipitation technique, fowl adenovirus serotype 1 (FAV 1) DNA transfects efficiently chicken cell cultures. Infection of FAV 1 DNA transfected cells with helper dependent avian adenoassociated virus (AAAV) results in the production of AAAV progeny, being detected by an indirect immunofluorescence assay. These findings indicate that FAV 1 DNA introduced into the host cell promotes actively the growth of AAAV. FAV 1 DNA transfection; AAAV multiplication; Placque assay; Indirect immunofluorescence assay
Avian adeno-associated virus (AAAV) is a defective parvovirus which requires a helpervirus for active multiplication in chicken cell cultures (Yates et al., 1973). Growth kinetics illustrate that either avian adenoviruses (Bauer et al., 1986) or herpesviruses (Bauer and Monreal, 1986, 1988) provide complete helper acitivity for AAAV. Evidently, both virus species are able to express biochemical factors in the host cell which compensate for defectiveness of AAAV. In order to explore the molecular basis of the helper function, an optimal transfection method is necessary to introduce the DNA of avian helpervirus as well as defined cloned fragments of these DNA species into the chicken cell. Testing different tr~sfection methods Correspondence
to: Dr H.J. Bauer, Institute of Poultry Diseases, Free University of Berlin, Koserstrasse 21, D-1000 Berlin 33, F.R.G.
0168~8510/89/$03.500 1990 Elsevier Science Publishers B.V. (Biomedical Division)
336
for efficient transfer of fowl adenovirus serotype 1 (FAV 1) DNA in chicken cells we demonstrate that the chloroquine-modified calcium phosphate coprecipitation technique is the most suitable procedure. Furthermore, after infection of such transfected chicken cells with AAAV we find out AAAV progeny can be produced. The ATCC VR-865 strain of AAAV was obtained from the American Type Culture Collection, Rockville, MD. The virus was propagated in specific pathogen free (SPF) embryonated eggs by coinfection with FAV 1 (CELO phelps strain) and purified as described previously (Barter and Monreal, 1985). The FAV 1 was propagated in primary chicken kidney cells (CKC) (Bauer and Monreal, 1986) and purified according to a procedure from earlier studies (Laver et al., 1971). For DNA preparation, purified FAV 1 was sedimented by centrifugation (125 000 x g, I h, 4°C). The virus pellet was resuspended in lysis buffer containing 100 mM Tris-HCI, pH 9, 100 mM NaCl, 1 mM EDTA, 1% SDS and 1 mg/ml proteinase K (Boehringer, M~nheim). After incubation at 37°C for 12 h, the released DNA was phenol-extracted and precipitated with ethanol followed by dissolving in the required transfection solution. DNA concentration determined by spectrophotometric reading at a wavelength of 260 nm was adjusted to 100 ,ug/ml transfection solution. For transfection experiments with FAV 1 DNA, primary CKC and primary chicken embryo fibroblasts (CEF) (Barter and Monreal, 1988) were used. To reach subconfluency, cells were plated in 60 mm Petri dishes, 72 h and 24 h respectively prior transfection with a density of 5 x I O5 CKC/ml growth medium, and 7 x 1OS CEF/ml growth medium. Three different transfection procedures have been assayed using a FAV 1 DNA concen~ation of 1 to 3 pg per dish. First, viral DNA dissolved in Hepes buffer (20 mM Hepes, pH 7.1, 137 mM NaCl, 5 mM KCl, 0.5 mM Na2HP04, 5,5 mM glucose) was coprecipitated with calcium phosphate, final concentration being 0,125 M (Graham and van der Eb, 1973). After 0.5 h, the precipitate and DMEM containing 50 I_LMchloroquine were pipetted onto the cell monolayers, which were incubated for 4 h (Luthman and Magnusson, 1983). Second, cell monolayers were exposed to Dulbecco’s modified Eagle’s medium (DMEM) containing 1.25 mg DEAE-Dextran (average MW 5 x lo5 Da)/ml for 0.5 h. DEAE-Dextran solution was replaced by viral DNA dissolved in Tris-glucosebuffer (25 mM Tris-HCl, pH 7.2, 137 mM NaCI, 5.5 mM KCI, 0.7 mM Na2HP04, 0,5 mM glucose). After 0,5 h of incubation, fresh medium was added to the cells (Perbal et al., 1985). Third, cell monolayers were overlaid for 6 h with viral DNA dissolved in DMEM in the presence of 30 pg polybrene per dish. Culture fluid was then removed and the cells were treated with a 25% (v/v) DMSO solution for 3 min (Kawai and Nishizawa, 1984). Transfected cells were cultured in DMEM supplemented with 5% fetal calf serum and 1% chicken serum. Liquid medium was substituted for agar overlay medium 24 h after the beginning of transfection. As the comparison of the three different transfection procedures in Fig. 1 demonstrates, the efficiency of FAV 1 DNA transfection in primary CKC and CEF reaches the highest value if the chloroquine-modified calcium phosphate coprecipitation
337 35
0)
dtft;lf&f (PFU&
30
30
26
25
20
efflclancy of 20 transf.ctlon (PFU/w DNA)*,5
10
10
5
6
0
0
DNA)*,,
T T
b)
Fig. 1. Efficiency of FAV 1 DNA transfection in primary CKC (a) and CEF (b) using (f&!)calcium phosphate coprecipitation technique modified by chloroquine treatment, (8) DEAE-Dextran method and (&I) polybrene/DMSO procedure. Transfected cell monolayers overlaid with agar medium were incubated for 5 (CKC) or 8 (CEF) days at 38,5”C. Standard plaque assay (Bauer et al., 1986) was used to determine infectious FAV 1 progeny represented by plaque forming units (PFU); x f standard deviation.
technique is used. In addition, this procedure results in excellent conservation of cell monolayers compared with the standard coprecipitation technique (Graham and van der Eb, 1973), which induces important cell losses (data not shown). Using CEF as cell system, there are no optimal transfection results if cells are treated according to the polycation (DEAE-Dextran or polybrene) methods. Besides, the chloroquine modification of these procedures causes the transfection efficiency of FAV 1 DNA to be reduced significantly (data not shown). For CKC, we find out that DEAE-Dextran as well as polybrene are cell-noxious chemicals which lead to a detachment of the cell monolayers and a very low transfection efficiency. For the detection of AAAV helper activity, primary CKC and CEF grown in 60 mm Petri dishes containing two glass coverslips were transfected with FAV 1 DNA (1 to 3 pg per dish) using the chloroquine-modified calcium phosphate coprecipitation technique. 48 h (CKC) or 72 h (CEF) following transfection, cells were infected with AAAV at a multiplicity of infection (m.o.i.) of 0.5 fluorescent focus-forming units (FFU) per cell. After 1 h of adsorption, cells were overlaid with DMEM supplemented with 2% fetal calf serum and incubated again for 48 h. AAAV antigen-positive cells in the CKC as well as CEF monolayer were determined by an indirect immunofluorescence assay (Bauer and Monreal, 1986). In order to prove whether the AAAV antigen-positive CKC and CEF enclose infectious AAAV particles, the cell monolayers were frozen, thawed twice and ultrasonicated for 10 s. The cell debris was removed by centrifugation (2500 x g; 10 min) and the supematants containing AAAV antigen were stored at -2OT. Primary CKC coverslip cultures as indicator cell system (Bauer and Monreal, 1986) were then simultaneously inoculated with 0.2 ml of the supematant of CKC or CEF lysate and FAV 1 as helper virus (m.o.i. of 10 PFU per cell). Primary CKC and CEF transfected with FAV 1 DNA followed by AAAV infection produce AAAV antigen. Passaging of the supematant of CKC or CEF lysate in the CKC indicator cell system leads to a significant production of AAAV
338
progeny measured by an indirect immunofluorescence assay (Bauer and Monreal, 1986) (data not shown). At fluorescence microscopical inspection, infectious virus particles can be detected as distinct foci within the cell nucleus (Fig. 2). No fluorescence is observed on control cell cultures. Hence it follows that AAAV antigen detected in primary CKC or CEF represents infectious AAAV particles. This result demonstrates that transfected FAV 1 DNA is capable of supplying complete helper activity for AAAV growth in the chicken cell. In order to realize a helper effect, being based on the transfection of helper DNA, an essential aspect is the establishing of a suitable experimental procedure. As the data illustrated in Fig. 1 emphasize, the chloroquine modification of standard coprecipitation technique (Graham and van der Eb, 1973) is a powerful tool to render primary CKC and CEF susceptible to uptake of exogenous FAV 1 DNA molecules. There is an evidence from earlier studies (Luthman and Magnusson, 1983) that exposure to chloroquine increases the fraction of transfected mouse cells to approximately 40%. In addition, this procedure offers an optimal cell conservation due to the lack of a separate adsorption phase of DNA calcium phosphate coprecipitate to the cell monolayer. As determined by kinetic studies (Loyter et al., 1982) the entry of coprecipitated DNA molecules into mouse cells requires at least 8 h. In this period of DNA uptake it is suggested that nucleolytic
Fig.2. Indirect immunofluorescent detection of AAAV antigen in primary CEF transfected with FAV 1 DNA followed by AAAV infection. The cells on the coverslips were fixed with acetone, incubated with rabbit anti-AAAV serum and finally stained with fluorescein-labelled goat anti-rabbit IgG (Bauer and Monreal, 1986); original magnification x 500.
339
DNA degradation occurs in the cytoplasm although chloroquine may protect DNA by inactivating hydrolytic enzymes (Luthman and Magnusson, 1983). Therefore, in view of alternatives to the experimental scheme described above, it is not surprising that cotransfection of chicken cells with FAV 1 DNA as well as AAAV DNA results in a decrease of AAAV antigen-positive cells by 20 to 30 fold (data not shown). This observation eludicates that AAAV infection following FAV 1 DNA transfection remains to be established. Presently, our work is focused on the transfection of CEF with cloned restriction DNA fragments of herpesvirus of turkeys (HVT) DNA and aims at identifying DNA fragments, which encode for AAAV helper function (Bauer et al., 1989). The preliminary results confirm the pivotal role of an efficient transfection system represented by the chloroquine-modified calcium phosphate coprecipitation technique to explore regions of avian adenovirus or herpesvirus genome with helper activity for AAAV. Acknowledgements Our thanks are due to Gisela Beyer and Manuela Hoppe for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (MO 415/l-3). References Bauer, H.J. and Monreal, G. (1985) Purification method for the avian adeno-associated virus. J. Viral. Methods 11, 87-92. Bauer, H.J. and Monreal, G. (1986) Herpesviruses provide helper functions for avian adeno-associated parvovirus. J. Gen. Virol. 67, 181-185. Bauer, H.J., Hauschild, S., Logemann, K., Hehlein, K. and Monreal, G. (1986) Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): studies of viral interactions in chicken cell cultures. Avian Pathol. 15, 357-366. Bauer, H.J. and Monreal, G. (1988) Avian adeno-associated parvovirus and Marek’s disease virus: studies of viral interactions in chicken embryo fibroblasts. Arch. Viral. 98, 271-277. Bauer, H.J., Schiilier, S. and Lindenmaier, W. (1989) Identification of restriction fragments of herpesvirus of turkey’s DNA with helper activity for AAAV. In: Abstracts of the 14th international herpesvirus workshop, Nyborg Strand, Denmark, 176. Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467. Kawai, S. and Nishizawa, M. (1984) New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol. Cell. Biol. 4, 1172-I 174. Laver, W.G., Younghusband, H.B. and Wrigley, N.G. (1971) Purification and properties of chick embryo lethal orphan virus (an avian adenovirus). Virology 45, 598-614. Loyter, A., Scangos, G., Juricek, D., Keene, D. and Ruddle, F.H. (1982) Mechanisms of DNA entry into mammalian cells. Exp. Cell. Res. 139, 223-234. Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295-1308. Perbal B., Lipsick J.S., Svoboda J., Silva R.F. and Baluda, M.A. (1985) Biologically active proviral clone of myeloblastosis-associated virus type 1: implications for the genesis of avian myeloblastosis virus. J. Viral. 56, 240-244. Yates V.J., El Mishad A.M., McCormick K.J. and Trentin, J.J. (1973) Isolation and characterization of an avian adenovirus-associated virus. Infect. Immun. 7, 973-980.
335
Elsevier
VIRMET 01056
Short
Communication
Growth of avian adeno-associated virus in chicken cells transfected with fowl adenovirus serotype 1 DNA Annette
Batter,
Gerhard
Monreal
and Hans-Joachim
Batter
Institute of Poultry Diseases, Free University of Berlin, Berlin, F.R.G.
(Accepted
16 May 1990)
Summary Using the chloroquine-modified calcium phosphate coprecipitation technique, fowl adenovirus serotype 1 (FAV 1) DNA transfects efficiently chicken cell cultures. Infection of FAV 1 DNA transfected cells with helper dependent avian adenoassociated virus (AAAV) results in the production of AAAV progeny, being detected by an indirect immunofluorescence assay. These findings indicate that FAV 1 DNA introduced into the host cell promotes actively the growth of AAAV. FAV 1 DNA transfection; AAAV multiplication; Placque assay; Indirect immunofluorescence assay
Avian adeno-associated virus (AAAV) is a defective parvovirus which requires a helpervirus for active multiplication in chicken cell cultures (Yates et al., 1973). Growth kinetics illustrate that either avian adenoviruses (Bauer et al., 1986) or herpesviruses (Bauer and Monreal, 1986, 1988) provide complete helper acitivity for AAAV. Evidently, both virus species are able to express biochemical factors in the host cell which compensate for defectiveness of AAAV. In order to explore the molecular basis of the helper function, an optimal transfection method is necessary to introduce the DNA of avian helpervirus as well as defined cloned fragments of these DNA species into the chicken cell. Testing different tr~sfection methods Correspondence
to: Dr H.J. Bauer, Institute of Poultry Diseases, Free University of Berlin, Koserstrasse 21, D-1000 Berlin 33, F.R.G.
0168~8510/89/$03.500 1990 Elsevier Science Publishers B.V. (Biomedical Division)
336
for efficient transfer of fowl adenovirus serotype 1 (FAV 1) DNA in chicken cells we demonstrate that the chloroquine-modified calcium phosphate coprecipitation technique is the most suitable procedure. Furthermore, after infection of such transfected chicken cells with AAAV we find out AAAV progeny can be produced. The ATCC VR-865 strain of AAAV was obtained from the American Type Culture Collection, Rockville, MD. The virus was propagated in specific pathogen free (SPF) embryonated eggs by coinfection with FAV 1 (CELO phelps strain) and purified as described previously (Barter and Monreal, 1985). The FAV 1 was propagated in primary chicken kidney cells (CKC) (Bauer and Monreal, 1986) and purified according to a procedure from earlier studies (Laver et al., 1971). For DNA preparation, purified FAV 1 was sedimented by centrifugation (125 000 x g, I h, 4°C). The virus pellet was resuspended in lysis buffer containing 100 mM Tris-HCI, pH 9, 100 mM NaCl, 1 mM EDTA, 1% SDS and 1 mg/ml proteinase K (Boehringer, M~nheim). After incubation at 37°C for 12 h, the released DNA was phenol-extracted and precipitated with ethanol followed by dissolving in the required transfection solution. DNA concentration determined by spectrophotometric reading at a wavelength of 260 nm was adjusted to 100 ,ug/ml transfection solution. For transfection experiments with FAV 1 DNA, primary CKC and primary chicken embryo fibroblasts (CEF) (Barter and Monreal, 1988) were used. To reach subconfluency, cells were plated in 60 mm Petri dishes, 72 h and 24 h respectively prior transfection with a density of 5 x I O5 CKC/ml growth medium, and 7 x 1OS CEF/ml growth medium. Three different transfection procedures have been assayed using a FAV 1 DNA concen~ation of 1 to 3 pg per dish. First, viral DNA dissolved in Hepes buffer (20 mM Hepes, pH 7.1, 137 mM NaCl, 5 mM KCl, 0.5 mM Na2HP04, 5,5 mM glucose) was coprecipitated with calcium phosphate, final concentration being 0,125 M (Graham and van der Eb, 1973). After 0.5 h, the precipitate and DMEM containing 50 I_LMchloroquine were pipetted onto the cell monolayers, which were incubated for 4 h (Luthman and Magnusson, 1983). Second, cell monolayers were exposed to Dulbecco’s modified Eagle’s medium (DMEM) containing 1.25 mg DEAE-Dextran (average MW 5 x lo5 Da)/ml for 0.5 h. DEAE-Dextran solution was replaced by viral DNA dissolved in Tris-glucosebuffer (25 mM Tris-HCl, pH 7.2, 137 mM NaCI, 5.5 mM KCI, 0.7 mM Na2HP04, 0,5 mM glucose). After 0,5 h of incubation, fresh medium was added to the cells (Perbal et al., 1985). Third, cell monolayers were overlaid for 6 h with viral DNA dissolved in DMEM in the presence of 30 pg polybrene per dish. Culture fluid was then removed and the cells were treated with a 25% (v/v) DMSO solution for 3 min (Kawai and Nishizawa, 1984). Transfected cells were cultured in DMEM supplemented with 5% fetal calf serum and 1% chicken serum. Liquid medium was substituted for agar overlay medium 24 h after the beginning of transfection. As the comparison of the three different transfection procedures in Fig. 1 demonstrates, the efficiency of FAV 1 DNA transfection in primary CKC and CEF reaches the highest value if the chloroquine-modified calcium phosphate coprecipitation
337 35
0)
dtft;lf&f (PFU&
30
30
26
25
20
efflclancy of 20 transf.ctlon (PFU/w DNA)*,5
10
10
5
6
0
0
DNA)*,,
T T
b)
Fig. 1. Efficiency of FAV 1 DNA transfection in primary CKC (a) and CEF (b) using (f&!)calcium phosphate coprecipitation technique modified by chloroquine treatment, (8) DEAE-Dextran method and (&I) polybrene/DMSO procedure. Transfected cell monolayers overlaid with agar medium were incubated for 5 (CKC) or 8 (CEF) days at 38,5”C. Standard plaque assay (Bauer et al., 1986) was used to determine infectious FAV 1 progeny represented by plaque forming units (PFU); x f standard deviation.
technique is used. In addition, this procedure results in excellent conservation of cell monolayers compared with the standard coprecipitation technique (Graham and van der Eb, 1973), which induces important cell losses (data not shown). Using CEF as cell system, there are no optimal transfection results if cells are treated according to the polycation (DEAE-Dextran or polybrene) methods. Besides, the chloroquine modification of these procedures causes the transfection efficiency of FAV 1 DNA to be reduced significantly (data not shown). For CKC, we find out that DEAE-Dextran as well as polybrene are cell-noxious chemicals which lead to a detachment of the cell monolayers and a very low transfection efficiency. For the detection of AAAV helper activity, primary CKC and CEF grown in 60 mm Petri dishes containing two glass coverslips were transfected with FAV 1 DNA (1 to 3 pg per dish) using the chloroquine-modified calcium phosphate coprecipitation technique. 48 h (CKC) or 72 h (CEF) following transfection, cells were infected with AAAV at a multiplicity of infection (m.o.i.) of 0.5 fluorescent focus-forming units (FFU) per cell. After 1 h of adsorption, cells were overlaid with DMEM supplemented with 2% fetal calf serum and incubated again for 48 h. AAAV antigen-positive cells in the CKC as well as CEF monolayer were determined by an indirect immunofluorescence assay (Bauer and Monreal, 1986). In order to prove whether the AAAV antigen-positive CKC and CEF enclose infectious AAAV particles, the cell monolayers were frozen, thawed twice and ultrasonicated for 10 s. The cell debris was removed by centrifugation (2500 x g; 10 min) and the supematants containing AAAV antigen were stored at -2OT. Primary CKC coverslip cultures as indicator cell system (Bauer and Monreal, 1986) were then simultaneously inoculated with 0.2 ml of the supematant of CKC or CEF lysate and FAV 1 as helper virus (m.o.i. of 10 PFU per cell). Primary CKC and CEF transfected with FAV 1 DNA followed by AAAV infection produce AAAV antigen. Passaging of the supematant of CKC or CEF lysate in the CKC indicator cell system leads to a significant production of AAAV
338
progeny measured by an indirect immunofluorescence assay (Bauer and Monreal, 1986) (data not shown). At fluorescence microscopical inspection, infectious virus particles can be detected as distinct foci within the cell nucleus (Fig. 2). No fluorescence is observed on control cell cultures. Hence it follows that AAAV antigen detected in primary CKC or CEF represents infectious AAAV particles. This result demonstrates that transfected FAV 1 DNA is capable of supplying complete helper activity for AAAV growth in the chicken cell. In order to realize a helper effect, being based on the transfection of helper DNA, an essential aspect is the establishing of a suitable experimental procedure. As the data illustrated in Fig. 1 emphasize, the chloroquine modification of standard coprecipitation technique (Graham and van der Eb, 1973) is a powerful tool to render primary CKC and CEF susceptible to uptake of exogenous FAV 1 DNA molecules. There is an evidence from earlier studies (Luthman and Magnusson, 1983) that exposure to chloroquine increases the fraction of transfected mouse cells to approximately 40%. In addition, this procedure offers an optimal cell conservation due to the lack of a separate adsorption phase of DNA calcium phosphate coprecipitate to the cell monolayer. As determined by kinetic studies (Loyter et al., 1982) the entry of coprecipitated DNA molecules into mouse cells requires at least 8 h. In this period of DNA uptake it is suggested that nucleolytic
Fig.2. Indirect immunofluorescent detection of AAAV antigen in primary CEF transfected with FAV 1 DNA followed by AAAV infection. The cells on the coverslips were fixed with acetone, incubated with rabbit anti-AAAV serum and finally stained with fluorescein-labelled goat anti-rabbit IgG (Bauer and Monreal, 1986); original magnification x 500.
339
DNA degradation occurs in the cytoplasm although chloroquine may protect DNA by inactivating hydrolytic enzymes (Luthman and Magnusson, 1983). Therefore, in view of alternatives to the experimental scheme described above, it is not surprising that cotransfection of chicken cells with FAV 1 DNA as well as AAAV DNA results in a decrease of AAAV antigen-positive cells by 20 to 30 fold (data not shown). This observation eludicates that AAAV infection following FAV 1 DNA transfection remains to be established. Presently, our work is focused on the transfection of CEF with cloned restriction DNA fragments of herpesvirus of turkeys (HVT) DNA and aims at identifying DNA fragments, which encode for AAAV helper function (Bauer et al., 1989). The preliminary results confirm the pivotal role of an efficient transfection system represented by the chloroquine-modified calcium phosphate coprecipitation technique to explore regions of avian adenovirus or herpesvirus genome with helper activity for AAAV. Acknowledgements Our thanks are due to Gisela Beyer and Manuela Hoppe for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (MO 415/l-3). References Bauer, H.J. and Monreal, G. (1985) Purification method for the avian adeno-associated virus. J. Viral. Methods 11, 87-92. Bauer, H.J. and Monreal, G. (1986) Herpesviruses provide helper functions for avian adeno-associated parvovirus. J. Gen. Virol. 67, 181-185. Bauer, H.J., Hauschild, S., Logemann, K., Hehlein, K. and Monreal, G. (1986) Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): studies of viral interactions in chicken cell cultures. Avian Pathol. 15, 357-366. Bauer, H.J. and Monreal, G. (1988) Avian adeno-associated parvovirus and Marek’s disease virus: studies of viral interactions in chicken embryo fibroblasts. Arch. Viral. 98, 271-277. Bauer, H.J., Schiilier, S. and Lindenmaier, W. (1989) Identification of restriction fragments of herpesvirus of turkey’s DNA with helper activity for AAAV. In: Abstracts of the 14th international herpesvirus workshop, Nyborg Strand, Denmark, 176. Graham, F.L. and van der Eb, A.J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467. Kawai, S. and Nishizawa, M. (1984) New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol. Cell. Biol. 4, 1172-I 174. Laver, W.G., Younghusband, H.B. and Wrigley, N.G. (1971) Purification and properties of chick embryo lethal orphan virus (an avian adenovirus). Virology 45, 598-614. Loyter, A., Scangos, G., Juricek, D., Keene, D. and Ruddle, F.H. (1982) Mechanisms of DNA entry into mammalian cells. Exp. Cell. Res. 139, 223-234. Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res. 11, 1295-1308. Perbal B., Lipsick J.S., Svoboda J., Silva R.F. and Baluda, M.A. (1985) Biologically active proviral clone of myeloblastosis-associated virus type 1: implications for the genesis of avian myeloblastosis virus. J. Viral. 56, 240-244. Yates V.J., El Mishad A.M., McCormick K.J. and Trentin, J.J. (1973) Isolation and characterization of an avian adenovirus-associated virus. Infect. Immun. 7, 973-980.