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In vitro exposure of preimplantation porcine embryos to porcine parvovirus
THERIOGENOLOGY
IN VITRO
EXPOSURE OF PREIMPLANTATION EMBRYOS TO PORCINE PARVOVIRUS
0. P. Bane',
PORCINE
J. E. James', C. M. GradilZ T. W. MolitorZ
and
'Department of Veterinary Clinical Medicine College of Veterinary Medicine University of Illinois Urbana, IL 61801 2Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Minnesota St. Paul, MN 55108
Received
for publication:
Accepted:
November December
6, 1987 5, 1989
ABSTRACT Early porcine embryos at the fourto eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos Treatment of embryos by microinjection of virus or witt virus. incilbation in media with virus did not significantly inhibit in vitro when compared with untreated controls. development of the embryos used to identify the presence of virus RNA-DNA hybridization was It was found that PPV-DNA was present in associated with embryos. viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos. Key words:
preimplantation
porcine
embryos,
porcine
parvovirus
Acknowledgments The authors thank David James for his skilled embryo manipulation techniques and Ms. Mary James, Ms. Jacqueline James, Ms. Teri Stone and Mr. Martin Awerdt for their assistance in embryo collection. Support for this project came from the Illinois Department of Agr'culture.
FEBRUARY
1990 VOL.33N0.2
553
THERIOGENOLOGY INTRODUCTION Embryo transfer is commonly employed by the livestock industry to facilitate the movement of genetic material while limiting disease transmission between herds. The intact zona pellucida of the embryo is a natural barrier to disease which prevents infection of the blastomeres (1). It is known, however, that certain infectious agents adhere to and partially penetrate the zona (2). Previous research virus indicated that porcine parvovirus and pseudorabies can be transmitted between animals via attachment to the porcine zona Virus pellucida (Z-4). absorbed to embryos may be removed by extensive washes (5) but this additional handling may affect the integrity of the zona pellucida. Damage to the zona pellucida may either increase the early rate of embryonic death or lead to viral penetration of the zona and to blastomere infection. Research with zona pellucida-free bovine embryos has shown that in vitro virus infection results in embryonic death (6,71. Recent in vitro studies have indicated that numerous viruses become associated with early killing porcine after exposure without the embryos pathogen blastomeres (5,8-11). Wrathall and Mengeling (12) have reported that in vitro exposure of zona pellucida-intact porcine embryos to porcine significantly parvoviruses reduces in vitro development of the embryos. The effect of infectious viruses on early preimplantation with porcine zona embryos damaged pellucidae has not been investigated. If embryo transfer is to be used as a means of genetic preservation during disease eradication, or if this technology is to be used to export genetic material, it is first necessary to explore all possibilities of disease transfer inherent to the process. This study was designed to evaluate the potential of virulent and avirulent porcine parvovirus to invade and destroy porcine embryos with damaged zona pellucidae. Nucleic acid hybridization, a sensitive and specific method capable of detecting minute amounts of viral nucleic acids (13) within samples, was used to assess virus-embryo interaction. MATERIALS Animals
and Embryo
AND METHODS
Collection
mixed-breed gilts served as embryo Sixteen healthy, postpubertal, donors. All gilts were bred by natural service, and embryos were recovered 4 to 5 d after the onset of estrus using previously described methods (14). The experiment was accomplished by flushing four gilts per day, on four separate occasions. A modified Krebs-Ringer bicarbonate medium served as recovery and storage media (14). Flushings from the reproductive tract were collected in sterile petri dishes, and embryos were isolated under a stereomicroscope (8 to 50x magnification). From each day embryos were randomly allocated to one of six treatment groups in a 3 x 2 factorial design. Thus, the same treatment groups were repeated four times. In total, 123 embryos at the four- to eight-cell stage were collected from sixteen gilts.
554
FEBRUARY
1990 VOL. 33 NO. 2
THERIOGENOLOGY Virus The two viruses used for embryo treatment were NADL-8, a virulent isolate of PPV, and KBSH, an avirulent isolate of PPV (15.16). Virus stocks of these isolates were prepared by propagation in swine testes cells following previously described methods (17). The infectious titers of the virus stocks were 2 x lo7 pfu/ml and 5 x lo7 pfu/ml, respectively, for NADL-8 and KBSH. Prior to embryo inoculation KBSH was adjusted to 2 x lo7 pfu/ml. Expclsure of Embryos
to Virus
Within one hour of recovery from the donor, embryos were allocated into six groups and exposed to one of three treatments: NADL-8, KBSH, or media (noninfected control). Two methods of virus exposure were microinjection (virus injected through the zona employed: embryo virus. pellucida of embryo) embryo with the and incubation Microinjection was carried out with micromanipulators under 250x magrification and embryo injection glass micropipettes with a 5- to 7-pm beveled tip. The micropipettes were manufactured in our laboratory using a microforge and micropipette grinder. Embryos were viewed microinjection. with a stereomicroscope (250x) for Microinjected embryos received a virus exposure of 1 to 2 nl of stock virus. Incubated embryos were exposed to lo3 pfu/ml for 48 h. All embryos were cultured in vitro at 37.5"C in modified Brinster's media in capped glass tubes after virus exposure. After 48 h, embryos were washed in sterile Brinster's media and evaluated for progression of development under stereomicroscope (8 to 50x). Embryos were pooled (n . 3 to 5) from each treatment group and subsequently frozen at -2O'C and processed for RNA-DNA hybridization. q
RNA-DNA
Hybridization
Plasmids containing PPV DNA were constructed by cloning a 3300 bascl pair (Pst-1 to EcoRl) fragment of the replicative-form (RF) DNA NADL-8 isolate of PPV Sp65 plasmids of the into and Sp64 (Promega-Biotec, Madison, WI). The cloning sites for these two lac,mids are in the opposite orientation. High specific activity 52 P :. radiolabeled strand specific RNA probes were synthesized from these two plasmids using 32P-GTP and Sp6 RNA polymerase according to methods previously reported (18) and further applied to the detection The template for producing the radiolabeled of parvovirus DNA (19). transcripts was 1.0 pg of linearized Sp-64 or Sp-65 containing PPV inserts. The nucleic acids from porcine embryos were prepared for hybridization as follows: pooled embryos were sonicated (four, 5-set pulses at 10 kc) followed by addition of digestion buffer (0.02 M Tris K [pH 7.41, 0.02 M EDTA. and 1.0% SDS) at a 1:l ratio and protease Protease digestion was allowed to proceed for 15 h (200 pg/ml). fol.owed by extraction of nucleic acids with phenol and concentration The concentrated samples were diluted 1:l by ethanol precipitation. with 0.6 N NaOH and incubated at 60°C for 30 min to denature the An equal volume of 2 M ammonium acetate was then nuc‘eic acids. were applied to nylon membranes (Hybond N, added, and samples
FEBRUARY 1990 VOL. 33 NO. 2
555
THERIOGENOLOGY
IL) Arlington Heights, using a slot-blot Amersham, apparatus NH). (Schleicher and Schuell, Keene, samples of known Control concentrations of purified PPV RF DNA were applied to each membrane for reference standards. Conditions for hybridization were of high 50% formamide at 42°C. stringency, consisting of After prehybridization for 6 to 8 h in 10 ml of hybridization buffer (50 mM 0.8% Denhardt's with NaP04, pH 6.5, 5 X SSC, 1% SDS, 50% formamide, 250 Hg denatured salmon sperm DNA and 500 pg yeast RNA), fresh buffer was added along with 7.5 ml of 50% dextran sulfate and the radiolabeled RNA probe, which previously had been boiled for 2 min to Hybridization proceeded for 36 h at 42°C. denature it. The unbound probe was removed from the membrane with two consecutive washes each of 2.0 X SSC, 0.1% SDS, followed by 0.2 X SSC, 0.1% SDS. Washed were air-dried film with an membranes and exposed to X-ray intensifying screen at -70°C for periods of up to 7 d. Assessment
of Embryo
Development
In vitro development of embryos 48 h after exposure to viruses was evaluated as follows: embryos at the four- to eight-cell stage, or those degenerating, were classified as showing no development; embryos at the 16-cell stage were classified as showing slight development; embryos at the morula or blastocyst stages were classified as showing advanced development. Treatment differences were analyzed by the Chi-sauare test (20). RESULTS A summary of the in vitro following treatment is presented
Table
1. In vitro following
Treatment/Virus
development of porcine below (Table 1).
development of preimplantation exposure to porcine parvovirus
No. Embryos
embryos
porcine
In vitro None
48
h
embryos
developmenta (%) Slight Advanced
Microinjected/media
22
9 (40.9)
4 (18.2)
9 (40.9)
Microinjected/KBSH
21
10 (47.6)
5 (23.8)
6 (28.5)
Microinjected/NADL-gb
22
13 (59.1)
1
(4.5)
7 (31.8)
Incubated/media
19
7 (36.8)
5 (26.3)
7 (36.8)
Incubated/KBSH
19
11 (57.9)
1
(5.3)
7 (36.8)
Incubated/NADL-8
20
8 (40.0)
4 (20.0)
8 (40.0)
aNone = embryos did not progress beyond the four- to eight-cell stage; slight = embryos progressed to the 16-cell stage; advanced = embryos progressed to the morula or blastocyst stage. bOne embryo in this treatment group was lost during handling. 556
FEBRUARY
1990 VOL.33N0.2
THERIOGENOLOGY Microinjection or did not significantly in lvitro when compared
incubation of embryos inhibit development with controls.
with either virus of the porcine
isolate embryos
Porcine parvovirus, a single-stranded DNA virus, packages greater than 90% negative strand DNA (V-strand). Once within a susceptible virus replication takes place via the synthesis of double cell, replicative form (RF) DNA. The RF-DNA contains both the stranded, V-strand and the copy or C-strand. Strand-specific DNA probes were employed to permit differentiation of virus (V-strand) and RF DNA virus synthesis (C-strand; Figure 1).
KBSH (iniected)
KBSH
-
(incubated)
NADL-B liniected)
NADL-Bliniected)
KBSH hcubatedl
NADL-8 (incubated\
Figure
1.
Slot Blot Hybridization of PPV DNA isolated from pooled Each slot represents the nucleic acid porcine embryos. Controls from a pooled sample of three to four embryos. 1.0 and 10.0 ng RF PPV DNA. Panel represent 0.1, with Panel A--Embryos hybridized V-strand probe. B--Embryos hybridized with C-strand probe.
FEBRUARY 1990 VOL. 33 NO. 2
557
THERIOGENOLOGY The presence of V-strand and/or C-strand virus DNA was identified The V-strand DNA was detected in in embryos by RNA-DNA hybridization. embryos microinjected and incubated with both KBSH and NADL-8 virus. The C-strand DNA was detected in embryos microinjected with both virus isolates and in embryos incubated with KBSH. Viral DNA was not detected in embryos incubated with NADL-8 or in either microinjected Viral DNA was also not detected in or incubated control embryos. uninoculated embryos or in embryos incubated with media alone using probes of both senses. The data show that virus was blastomeres of viable porcine embryos The PPV C-strand PPV V-strand probe. number of embryos.
in association with present after in vitro culture using a DNA was detected in a limited
DISCUSSION In vitro development of porcine embryos in our study was similar to that obtained by other workers for embryos collected at the fourImproved embryonic development in vitro to eight-cell stage (21.22). could have been achieved by collecting older eightto 16-cell stage embryos. Infection of blastomeres of zona pellucida-intact porcine embryos The potential for virus penetration has not been previously reported. of the zona pellucida exists because of the large, open tracks in the and also because of the potential traumatic zona left by spermatozoa, damage to the zona resulting from embryo manipulation. Bolin et al. (2) observed by electron microscopy that viruses partially penetrate Evidence of blastomere infection by the zona along the sperm tract. viruses in the above mentioned study was not found. Wrathall and Mengeling (12) reported that in vitro exposure of pig embryos to vitro porcine parvoviruses decreased in developmental causes Viruses were not found associated with embryonic cells progression. and Wrathall and Mengeling (12) did not propose a mechanism that would explain their findings. including identification, electron Various methods of virus microscopy, fluorescent antibody, animal inoculation, and cell culture infectivity have been used in previous studies of embryo-virus These methods all had limitations of sensitivity and interaction. The DNA hybridization techniques have the advantage of specificity. being extremely sensitive and specific, and, in the case of PPV, they were capable of differentiating input (parent virus) from copy strand (replicating virus) DNA. The results of this report show that the infection of embryo blastomeres by porcine parvovirus was not uniformly lethal up to 48 h Nearly one-third (31.8%) of the embryos infected after infection. with NADL-8 virus by microinjection proceeded to develop in vitro to Association of this virus with the the morula and blastocyst stages. blastomeres was indicated by the presence of PPV DNA in all pools of microinjected embryos showing advanced development. The result of
559
FEBRUARY 1990 VOL. 33 NO. 2
THERIOGENOLOGY
continued association of this virulent porcine parvovirus strain with blastomeres is not known, but presumably, the embryo would succumb to the infection upon further development. The significance of this apparently infected embryos could finding is that normal-appearing, possibly be transferred from donors to recipients followed by high embryonic mortality. horizontal transfer of infection to other embryos within the litter, or infection and seroconversion of the recipient. with the KBSH strain did not inhibit Incubation of embryos in vitro development in 36.8% of the embryos. The C-strand DNA was indicating that the virus penetrated associated with advanced embryos, the zona pellucida and infected the blastomeres. The ability of porcine embryos to survive this type of infection and develop further is not known and will be examined in a later study. The results of this study indicate that porcine embryos may develop normally in vitro for at least 48 h after infection of the with both virulent and avirulent porcine parvovirus. blastomeres Embryos with damaged or imperfect zona pellucidae as a result of or embryo micromanipulation are at embryo washing, embryo shipment, risk of being infected in utero or in vitro. Such infections may not be apparent microscopically for periods sufficient to allow the infectious agents, transfer of the embryo, and thus of the associated to the recipients. REFERENCES M.D., Hare, W.C.D. and Singh, E.L. Embryo transfer: a 1. Eaglesome, discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. ;:106-112 (1980). 2. Bolin, S.R., Turek, J.J., Runnels, L.J. and Gustafsson, D.P. Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo. Am. J. Vet. Res. z:1036-1039 (1983). S.R., Runnels, L.J., Sawyer, C.A. and Gustafsson, D.P. 3. Bolin, Experimental transmission of pseudorabies virus in swine by embryo transfer. Am. J. Vet. Res. %:278-280 (1982). A.E. and Mengeling, W.L. 4. Wrathall, parvovirus-infected fertilized pig eggs Br. Vet. J. 135:255-261 (1979).
transferring Effect of into seronegative gilts.
E.L. 5. Singh, Theriogenology
potential
The =:9-20
disease (1987).
control
R.W., Howard, T.H. and Pickett, B.W. 6. Bowen, bluetongue virus with preimplantation embryos cattle. Am. J. Vet. Res. Q:1907-1911 (1982).
FEBRUARY1990VOL.33NO.2
of
embryos.
Interaction from mice
of and
559
THERIOGENOLOGY 7. Bowen,
R.A., Elsden, bovine embryos with %:1095-1097 (1985).
8
R.P. and Siedel, G.E. bovine herpesvirus-1.
Infection of Am. J. Vet.
early Res.
Singh, E.L., McVicar, J.W., Hare, W.C.D. and Mebus, C.A. Embryo transfer as a means of controlling the transmission of viral infections. VII. The in vitro exposure of bovine and porcine virus. embryos to foot and mouth disease Theriogenology 3:587-593 (1986).
9. Singh, E.L., Dulac, G.C. and Hare, W.C.D. Embryo transfer as a means of controlling the transmission of viral infections. V. The in vitro exposure of zona pellucide-intact porcine embryos to African swine fever virus. Theriogenology x:693-700 (1984). 10. Bolin, S.R., Runnels, L.J., Sawyer, C.A., Atcheson, K.J. and Gustafsson, D.P. Resistance of porcine preimplantation embryos to pseudorabies virus. Am. J. Vet. Res. g:1711-1712 (1981). 11. Singh, E.L. and Thomas, F.C. Embryo transfer as a means of infections. controlling the transmission of viral IX. The in vitro exposure of zona pellucida-intact porcine embryos to swine vesicular disease virus. Theriogenology z:443-449 (1987). 12. Wrathall, A.E. and Mengeling, W.L. Effect of porcine parvovirus on the development of fertilized pig eggs in vitro. Br. Vet. J. 135:249-254 (1979). D.A., Krieg, P.A., Rebageleati, M.R., 13. Mellon, Mancatus, T., Zinn, K. and Green, M.R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage Sp6 polymerase. Nucleic Acid Res. E:7035-7056 (1984). 14. James, Pratt.
J.E. and &:191-195
Davis, D. (1984).
W.L. 15. Mengeling, and experimentally induced parvovirus. Am. J. Vet. 16. Molitor, T.W., comparison with
Embryo
transfer
in swine.
Modern
R.P. Cutlip, Reproductive by exposing pregnant gilts to Res. 3:1393-1399 (1976).
Vet.
disease porcine
Joo, H.S. and Collett, M.S. KBSH parvovirus: porcine parvovirus. J. Virol. s:257-263 (1985).
17. Molitor, T.W., Joo, H.S. and Collett, M.S. Porcine parvovirus DNA: characterization of the genome and replicative form DNA of two virus isolates. Virol. N:241-254 (1984). R.G., Maniatis, T. and Melton, D.A. Human B-globin 18. Green, pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 3:681-694 (1983).
560
FEBRUARY 1990 VOL. 33 NO. 2
THERIOGENOLOGY 19. Saarinen, U.M., Chorba, T.L., Tattersall, P., Young, N.S., Anderson, L.J., Palmer, E. and Coccia, P.F. Human parvovirus B19-induced epidermic acute red cell aplasia in patients with hereditary hemolytic anemia. Blood 67:1411-1417 (1986). R.D.G. and Torrie, J.H. 20. Steel, McGraw-Hill Book Co., Statistics. 21. Davis, D.L. and Day, B.N. pig eggs in vitro. J. Anim.
Principles and Procedures Inc., New York, 1983.
Cleavage and blastocyst formation Sci. 46:1043-1053 (1978).
of
by
22. James, J.E., James, D.M., Martin, P.A.. Reed, D.E. and Davis, D.L. Embryo transfer for conserving valuable genetic material from swine herds with pseudorabies. J. Am. Vet. Med. Assoc. 183:525-528 (1983).
FEBRUARY 5990 VOL. 33 NO. 2
561
IN VITRO
EXPOSURE OF PREIMPLANTATION EMBRYOS TO PORCINE PARVOVIRUS
0. P. Bane',
PORCINE
J. E. James', C. M. GradilZ T. W. MolitorZ
and
'Department of Veterinary Clinical Medicine College of Veterinary Medicine University of Illinois Urbana, IL 61801 2Department of Large Animal Clinical Sciences College of Veterinary Medicine University of Minnesota St. Paul, MN 55108
Received
for publication:
Accepted:
November December
6, 1987 5, 1989
ABSTRACT Early porcine embryos at the fourto eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos Treatment of embryos by microinjection of virus or witt virus. incilbation in media with virus did not significantly inhibit in vitro when compared with untreated controls. development of the embryos used to identify the presence of virus RNA-DNA hybridization was It was found that PPV-DNA was present in associated with embryos. viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos. Key words:
preimplantation
porcine
embryos,
porcine
parvovirus
Acknowledgments The authors thank David James for his skilled embryo manipulation techniques and Ms. Mary James, Ms. Jacqueline James, Ms. Teri Stone and Mr. Martin Awerdt for their assistance in embryo collection. Support for this project came from the Illinois Department of Agr'culture.
FEBRUARY
1990 VOL.33N0.2
553
THERIOGENOLOGY INTRODUCTION Embryo transfer is commonly employed by the livestock industry to facilitate the movement of genetic material while limiting disease transmission between herds. The intact zona pellucida of the embryo is a natural barrier to disease which prevents infection of the blastomeres (1). It is known, however, that certain infectious agents adhere to and partially penetrate the zona (2). Previous research virus indicated that porcine parvovirus and pseudorabies can be transmitted between animals via attachment to the porcine zona Virus pellucida (Z-4). absorbed to embryos may be removed by extensive washes (5) but this additional handling may affect the integrity of the zona pellucida. Damage to the zona pellucida may either increase the early rate of embryonic death or lead to viral penetration of the zona and to blastomere infection. Research with zona pellucida-free bovine embryos has shown that in vitro virus infection results in embryonic death (6,71. Recent in vitro studies have indicated that numerous viruses become associated with early killing porcine after exposure without the embryos pathogen blastomeres (5,8-11). Wrathall and Mengeling (12) have reported that in vitro exposure of zona pellucida-intact porcine embryos to porcine significantly parvoviruses reduces in vitro development of the embryos. The effect of infectious viruses on early preimplantation with porcine zona embryos damaged pellucidae has not been investigated. If embryo transfer is to be used as a means of genetic preservation during disease eradication, or if this technology is to be used to export genetic material, it is first necessary to explore all possibilities of disease transfer inherent to the process. This study was designed to evaluate the potential of virulent and avirulent porcine parvovirus to invade and destroy porcine embryos with damaged zona pellucidae. Nucleic acid hybridization, a sensitive and specific method capable of detecting minute amounts of viral nucleic acids (13) within samples, was used to assess virus-embryo interaction. MATERIALS Animals
and Embryo
AND METHODS
Collection
mixed-breed gilts served as embryo Sixteen healthy, postpubertal, donors. All gilts were bred by natural service, and embryos were recovered 4 to 5 d after the onset of estrus using previously described methods (14). The experiment was accomplished by flushing four gilts per day, on four separate occasions. A modified Krebs-Ringer bicarbonate medium served as recovery and storage media (14). Flushings from the reproductive tract were collected in sterile petri dishes, and embryos were isolated under a stereomicroscope (8 to 50x magnification). From each day embryos were randomly allocated to one of six treatment groups in a 3 x 2 factorial design. Thus, the same treatment groups were repeated four times. In total, 123 embryos at the four- to eight-cell stage were collected from sixteen gilts.
554
FEBRUARY
1990 VOL. 33 NO. 2
THERIOGENOLOGY Virus The two viruses used for embryo treatment were NADL-8, a virulent isolate of PPV, and KBSH, an avirulent isolate of PPV (15.16). Virus stocks of these isolates were prepared by propagation in swine testes cells following previously described methods (17). The infectious titers of the virus stocks were 2 x lo7 pfu/ml and 5 x lo7 pfu/ml, respectively, for NADL-8 and KBSH. Prior to embryo inoculation KBSH was adjusted to 2 x lo7 pfu/ml. Expclsure of Embryos
to Virus
Within one hour of recovery from the donor, embryos were allocated into six groups and exposed to one of three treatments: NADL-8, KBSH, or media (noninfected control). Two methods of virus exposure were microinjection (virus injected through the zona employed: embryo virus. pellucida of embryo) embryo with the and incubation Microinjection was carried out with micromanipulators under 250x magrification and embryo injection glass micropipettes with a 5- to 7-pm beveled tip. The micropipettes were manufactured in our laboratory using a microforge and micropipette grinder. Embryos were viewed microinjection. with a stereomicroscope (250x) for Microinjected embryos received a virus exposure of 1 to 2 nl of stock virus. Incubated embryos were exposed to lo3 pfu/ml for 48 h. All embryos were cultured in vitro at 37.5"C in modified Brinster's media in capped glass tubes after virus exposure. After 48 h, embryos were washed in sterile Brinster's media and evaluated for progression of development under stereomicroscope (8 to 50x). Embryos were pooled (n . 3 to 5) from each treatment group and subsequently frozen at -2O'C and processed for RNA-DNA hybridization. q
RNA-DNA
Hybridization
Plasmids containing PPV DNA were constructed by cloning a 3300 bascl pair (Pst-1 to EcoRl) fragment of the replicative-form (RF) DNA NADL-8 isolate of PPV Sp65 plasmids of the into and Sp64 (Promega-Biotec, Madison, WI). The cloning sites for these two lac,mids are in the opposite orientation. High specific activity 52 P :. radiolabeled strand specific RNA probes were synthesized from these two plasmids using 32P-GTP and Sp6 RNA polymerase according to methods previously reported (18) and further applied to the detection The template for producing the radiolabeled of parvovirus DNA (19). transcripts was 1.0 pg of linearized Sp-64 or Sp-65 containing PPV inserts. The nucleic acids from porcine embryos were prepared for hybridization as follows: pooled embryos were sonicated (four, 5-set pulses at 10 kc) followed by addition of digestion buffer (0.02 M Tris K [pH 7.41, 0.02 M EDTA. and 1.0% SDS) at a 1:l ratio and protease Protease digestion was allowed to proceed for 15 h (200 pg/ml). fol.owed by extraction of nucleic acids with phenol and concentration The concentrated samples were diluted 1:l by ethanol precipitation. with 0.6 N NaOH and incubated at 60°C for 30 min to denature the An equal volume of 2 M ammonium acetate was then nuc‘eic acids. were applied to nylon membranes (Hybond N, added, and samples
FEBRUARY 1990 VOL. 33 NO. 2
555
THERIOGENOLOGY
IL) Arlington Heights, using a slot-blot Amersham, apparatus NH). (Schleicher and Schuell, Keene, samples of known Control concentrations of purified PPV RF DNA were applied to each membrane for reference standards. Conditions for hybridization were of high 50% formamide at 42°C. stringency, consisting of After prehybridization for 6 to 8 h in 10 ml of hybridization buffer (50 mM 0.8% Denhardt's with NaP04, pH 6.5, 5 X SSC, 1% SDS, 50% formamide, 250 Hg denatured salmon sperm DNA and 500 pg yeast RNA), fresh buffer was added along with 7.5 ml of 50% dextran sulfate and the radiolabeled RNA probe, which previously had been boiled for 2 min to Hybridization proceeded for 36 h at 42°C. denature it. The unbound probe was removed from the membrane with two consecutive washes each of 2.0 X SSC, 0.1% SDS, followed by 0.2 X SSC, 0.1% SDS. Washed were air-dried film with an membranes and exposed to X-ray intensifying screen at -70°C for periods of up to 7 d. Assessment
of Embryo
Development
In vitro development of embryos 48 h after exposure to viruses was evaluated as follows: embryos at the four- to eight-cell stage, or those degenerating, were classified as showing no development; embryos at the 16-cell stage were classified as showing slight development; embryos at the morula or blastocyst stages were classified as showing advanced development. Treatment differences were analyzed by the Chi-sauare test (20). RESULTS A summary of the in vitro following treatment is presented
Table
1. In vitro following
Treatment/Virus
development of porcine below (Table 1).
development of preimplantation exposure to porcine parvovirus
No. Embryos
embryos
porcine
In vitro None
48
h
embryos
developmenta (%) Slight Advanced
Microinjected/media
22
9 (40.9)
4 (18.2)
9 (40.9)
Microinjected/KBSH
21
10 (47.6)
5 (23.8)
6 (28.5)
Microinjected/NADL-gb
22
13 (59.1)
1
(4.5)
7 (31.8)
Incubated/media
19
7 (36.8)
5 (26.3)
7 (36.8)
Incubated/KBSH
19
11 (57.9)
1
(5.3)
7 (36.8)
Incubated/NADL-8
20
8 (40.0)
4 (20.0)
8 (40.0)
aNone = embryos did not progress beyond the four- to eight-cell stage; slight = embryos progressed to the 16-cell stage; advanced = embryos progressed to the morula or blastocyst stage. bOne embryo in this treatment group was lost during handling. 556
FEBRUARY
1990 VOL.33N0.2
THERIOGENOLOGY Microinjection or did not significantly in lvitro when compared
incubation of embryos inhibit development with controls.
with either virus of the porcine
isolate embryos
Porcine parvovirus, a single-stranded DNA virus, packages greater than 90% negative strand DNA (V-strand). Once within a susceptible virus replication takes place via the synthesis of double cell, replicative form (RF) DNA. The RF-DNA contains both the stranded, V-strand and the copy or C-strand. Strand-specific DNA probes were employed to permit differentiation of virus (V-strand) and RF DNA virus synthesis (C-strand; Figure 1).
KBSH (iniected)
KBSH
-
(incubated)
NADL-B liniected)
NADL-Bliniected)
KBSH hcubatedl
NADL-8 (incubated\
Figure
1.
Slot Blot Hybridization of PPV DNA isolated from pooled Each slot represents the nucleic acid porcine embryos. Controls from a pooled sample of three to four embryos. 1.0 and 10.0 ng RF PPV DNA. Panel represent 0.1, with Panel A--Embryos hybridized V-strand probe. B--Embryos hybridized with C-strand probe.
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THERIOGENOLOGY The presence of V-strand and/or C-strand virus DNA was identified The V-strand DNA was detected in in embryos by RNA-DNA hybridization. embryos microinjected and incubated with both KBSH and NADL-8 virus. The C-strand DNA was detected in embryos microinjected with both virus isolates and in embryos incubated with KBSH. Viral DNA was not detected in embryos incubated with NADL-8 or in either microinjected Viral DNA was also not detected in or incubated control embryos. uninoculated embryos or in embryos incubated with media alone using probes of both senses. The data show that virus was blastomeres of viable porcine embryos The PPV C-strand PPV V-strand probe. number of embryos.
in association with present after in vitro culture using a DNA was detected in a limited
DISCUSSION In vitro development of porcine embryos in our study was similar to that obtained by other workers for embryos collected at the fourImproved embryonic development in vitro to eight-cell stage (21.22). could have been achieved by collecting older eightto 16-cell stage embryos. Infection of blastomeres of zona pellucida-intact porcine embryos The potential for virus penetration has not been previously reported. of the zona pellucida exists because of the large, open tracks in the and also because of the potential traumatic zona left by spermatozoa, damage to the zona resulting from embryo manipulation. Bolin et al. (2) observed by electron microscopy that viruses partially penetrate Evidence of blastomere infection by the zona along the sperm tract. viruses in the above mentioned study was not found. Wrathall and Mengeling (12) reported that in vitro exposure of pig embryos to vitro porcine parvoviruses decreased in developmental causes Viruses were not found associated with embryonic cells progression. and Wrathall and Mengeling (12) did not propose a mechanism that would explain their findings. including identification, electron Various methods of virus microscopy, fluorescent antibody, animal inoculation, and cell culture infectivity have been used in previous studies of embryo-virus These methods all had limitations of sensitivity and interaction. The DNA hybridization techniques have the advantage of specificity. being extremely sensitive and specific, and, in the case of PPV, they were capable of differentiating input (parent virus) from copy strand (replicating virus) DNA. The results of this report show that the infection of embryo blastomeres by porcine parvovirus was not uniformly lethal up to 48 h Nearly one-third (31.8%) of the embryos infected after infection. with NADL-8 virus by microinjection proceeded to develop in vitro to Association of this virus with the the morula and blastocyst stages. blastomeres was indicated by the presence of PPV DNA in all pools of microinjected embryos showing advanced development. The result of
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continued association of this virulent porcine parvovirus strain with blastomeres is not known, but presumably, the embryo would succumb to the infection upon further development. The significance of this apparently infected embryos could finding is that normal-appearing, possibly be transferred from donors to recipients followed by high embryonic mortality. horizontal transfer of infection to other embryos within the litter, or infection and seroconversion of the recipient. with the KBSH strain did not inhibit Incubation of embryos in vitro development in 36.8% of the embryos. The C-strand DNA was indicating that the virus penetrated associated with advanced embryos, the zona pellucida and infected the blastomeres. The ability of porcine embryos to survive this type of infection and develop further is not known and will be examined in a later study. The results of this study indicate that porcine embryos may develop normally in vitro for at least 48 h after infection of the with both virulent and avirulent porcine parvovirus. blastomeres Embryos with damaged or imperfect zona pellucidae as a result of or embryo micromanipulation are at embryo washing, embryo shipment, risk of being infected in utero or in vitro. Such infections may not be apparent microscopically for periods sufficient to allow the infectious agents, transfer of the embryo, and thus of the associated to the recipients. REFERENCES M.D., Hare, W.C.D. and Singh, E.L. Embryo transfer: a 1. Eaglesome, discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents. Can. Vet. J. ;:106-112 (1980). 2. Bolin, S.R., Turek, J.J., Runnels, L.J. and Gustafsson, D.P. Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo. Am. J. Vet. Res. z:1036-1039 (1983). S.R., Runnels, L.J., Sawyer, C.A. and Gustafsson, D.P. 3. Bolin, Experimental transmission of pseudorabies virus in swine by embryo transfer. Am. J. Vet. Res. %:278-280 (1982). A.E. and Mengeling, W.L. 4. Wrathall, parvovirus-infected fertilized pig eggs Br. Vet. J. 135:255-261 (1979).
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