Transmission of mouse minute virus (MMV) but not mouse hepatitis virus (MHV) following embryo transfer with experimentally exposed in vivo-derived embryos.

The present study investigated the presence and location of fluorescent microspheres having the size of mouse hepatitis virus (MHV) and of mouse minute virus (MMV) in the zona pellucida (ZP) of in vivo-produced murine embryos, the transmission of these viruses by embryos during embryo transfer, and the time of seroconversion of recipients and pups. To this end, fertilized oocytes and morulae were exposed to different concentrations of MMVp for 16 h, while 2-cell embryos and blastocysts were coincubated for 1 h. In addition, morulae were exposed to MHV-A59 for 16 h. One group of embryos was washed, and the remaining embryos remained unwashed before embryo transfer. Serological analyses were performed by means of ELISA to detect antibodies to MHV or MMV in recipients and in progeny on Days 14, 21, 28, 42, and 63 and on Days 42, 63, 84, 112, 133, and 154, respectively, after embryo transfer. Coincubation with a minimum of 10(5)/ml of fluorescent microspheres showed that particles with a diameter of 20 nm but not 100 nm crossed the ZP of murine blastocysts. Washing generally led to a 10-fold to 100-fold reduction of MMVp. Washed MMV-exposed but not MHV-exposed embryos led to the production of antibodies independent of embryonic stage and time of virus exposure. Recipients receiving embryos exposed to a minimum of 10(7) mean tissue culture infective dose (TCID(50))/ml of MHV-A59 and 10(2) TCID(50)/ml of MMVp seroconverted by Day 42 after embryo transfer. The results indicate that MMV but not MHV can be transmitted to recipients even after washing embryos 10 times before embryo transfer.

Seroconversion of calves following intravenous injection with embryos exposed to bovine viral diarrhea virus (BVDV) in vitro.

Two recent studies demonstrated that a high-affinity isolate of BVDV (SD-1), remained associated with a small percentage of in vivo-derived bovine embryos following artificial exposure to the virus and either washing or trypsin treatment. Further, the embryo-associated virus was infective in an in vitro environment. Therefore, the objective of this study was to determine if the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos could cause infection in vivo. Twenty zona-pellucida-intact morulae and blastocysts (MB) were collected on day 7 from superovulated cows. After collection, all MB were washed according to International Embryo Transfer Society (IETS) standards, and all but 4 MB (negative controls) were exposed for 2 h to 10(5)-10(6) cell culture infective doses (50% endpoint) per milliliter (CCID(50)/mL) of viral strain SD-1. Following exposure, according to IETS standards, one half of the MB were washed and one half were trypsin treated. All MB were then individually sonicated, and sonicate fluids were injected intravenously into calves on day 0. Blood was drawn to monitor for viremia and(or) seroconversion. Seroconversion of calves injected with sonicate fluids from washed and trypsin-treated embryos occurred 38% and 13% of the time, respectively. Therefore, the quantity of a high-affinity isolate of BVDV associated with single-washed or trypsin-treated embryos was infective in vivo.

Washing and trypsin treatment of in vitro derived bovine embryos exposed to bovine viral diarrhea virus.

Gametes, somatic cells and materials of animal origin in media are potential sources for introducing bovine viral diarrhea virus (BVDV) into systems for production of IVF bovine embryos. Further, the efficacy of washing and trypsin treatment for removal of BVDV from IVF embryos is questionable. Washing and trypsin treatments recommended by the International Embryo Transfer Society for in vivo-derived embryos were applied to in vitro-derived, virus-exposed, bovine embryos in this side-by-side comparison of treatments. Embryos for the study were produced in a virus-free system in which follicular oocytes were matured and fertilized in vitro and presumptive zygotes were co-cultured with bovine uterine tubal cells for 7 d. A total of 18 trials was performed, 9 using a noncytopathic BVDV and 9 using a cytopathic BVDV. In each trial, 4 equal groups of 10 or less, zona pellucida-intact embryos/ova were assembled, including 2 groups of morulae and blastocysts (M/B) and 2 groups of nonfertile or degenerated ova (NFD). Each group was prewashed and exposed to 10(4) to 10(6) TCID50/mL of either noncytopathic (SD-1) or cytopathic (NADL) BVDV for 2 h. Following in vitro viral exposure, one group of M/B and one group of NFD were washed. The other groups of M/B and NFD were trypsin-treated. Both treatments were consistent with IETS guidelines. After in vitro exposure to noncytopathic BVDV and washing, viral assays of 100% (9/9) and 78% (7/9) of the groups of M/B and NFD ova, respectively, were positive. After in vitro exposure to cytopathic BVDV and washing, viral assay of 33% (3/9) of the groups of both M/B and NFD ova were positive. After in vitro exposure to noncytopathic BVDV and trypsin treatment, viral assay of 44% (4/9) of groups of M/B and 67% (6/9) of groups of NFD ova were positive. Finally, after in vitro exposure to cytopathic BVDV and trypsin treatment, viral assay of 22% (2/9) of the groups of M/B and 44% (4/9) of the groups of NFD ova were positive. Contingency table analysis, in which data was stratified by embryo type and virus biotype, was used to compare results. While a difference existed between results of the 2 treatments of groups of M/B within the noncytopathic biotype (P = 0.01, Mantel Haenszel Chi-square), no difference was observed between comparison of treatment between all groups in both biotypes (P > 0.05).

Susceptibility of pig embryos to porcine circovirus type 2 infection.

The aim of the present study was to determine if porcine circovirus type 2 (PCV2) is able to infect embryonic cells of in vivo produced porcine embryos with and without zona pellucida (ZP). ZP-intact and ZP-free morulae (6-day post-insemination) and early blastocysts (7-day post-insemination), and hatched blastocysts (8-day post-insemination) were exposed to 10(5.0) TCID50 PCV2 per ml (strain 1121, fifth passage PK15). At 48 h post-incubation, the percentage of infected embryos and the percentage of viral antigen-positive cells per embryo were determined by indirect immunofluorescence (IF). Significantly different percentages of infected embryos were detected: 15% for ZP-free morulae, 50% for ZP-free early blastocysts and 100% for hatched blastocysts. The percentage of cells that expressed viral antigens was similar for the three stages of development. PCV2 exposure did not affect the in vitro development of the embryos during the 48 h study period. All ZP-intact embryos remained negative for viral antigens. In an additional experiment the diameter of the channels in the porcine ZP was determined. After incubation of early blastocysts with fluorescent microspheres of three different sizes, beads with a diameter of 20 nm and beads with a diameter of 26 nm crossed the zona whereas beads with a diameter of 200 nm did not. In conclusion, it can be stated that PCV2 is able to replicate in in vivo produced ZP-free morulae and blastocysts and that the susceptibility increases during development. The ZP forms a barrier to PCV2 infection, but based on the size of the channels in the ZP the possibility that PCV2 particles cross the ZP cannot be excluded.

Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells.

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.

Quality controls for bovine viral diarrhea virus-free IVF embryos.

Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.

Infectivity of bovine viral diarrhea virus associated with in vivo-derived bovine embryos.

Early research indicated that bovine viral diarrhea virus (BVDV) would not adhere to zona pellucida-intact (ZP-I), in vivo-derived bovine embryos. However, in a recent study, viral association of BVDV and in vivo-derived embryos was demonstrated. These findings raised questions regarding the infectivity of the embryo-associated virus. The objectives of this study were to evaluate the infectivity of BVDV associated with in vivo-derived bovine embryos through utilization of primary cultures of uterine tubal cells (UTC) as an in vitro model of the uterine environment and to determine if washing procedures, including trypsin treatment, were adequate to remove virus from in vivo-derived embryos. One hundred and nine ZP-I morulae and blastocysts (MB) and 77 non-fertile and degenerated (NFD) ova were collected on day 7 from 34, BVDV-negative, superovulated cows. After collection, all MB and NFD ova were washed according to International Embryo Transfer Society (IETS) standards and exposed for 2h to approximately 10(6) cell culture infective doses (50% endpoint) per milliliter of viral strain SD-1. Following exposure, some groups of <10 MB or NFD ova were washed in accordance with IETS standards. In addition, an equivalent number of MB and NFD ova were subjected to IETS standards for trypsin treatment. Subsequently, NFD ova were immediately sonicated and sonicate fluids were assayed for presence of virus, while individual and groups of MB were placed in microdrops containing primary cultures of UTCs and incubated. After 3 days, embryos, media, and UTCs were harvested from each microdrop and assayed for BVDV. Virus was detected in the sonicate fluids of 56 and 43% of the groups of NFD ova that were washed and trypsin-treated, respectively. After 3 days of microdrop culture, virus was not detected in media or sonicate fluids from any individual or groups of MB, regardless of treatment. However, virus was detected in a proportion of UTC that were co-cultured with washed groups of MB (30%), washed individual MB (9%) and trypsin treated individual MB (9%), but no virus was detected in the UTC associated with groups of trypsin-treated embryos. In conclusion, virus associated with developing embryos was infective for permissive cells. Further, the quantity of virus associated with a proportion of individual embryos (both washed and trypsin treated) was sufficient to infect the UTC. In light of these results, an attempt should be made to determine if the quantity of a high-affinity isolate of BVDV associated with an individual embryo would infect recipients via the intrauterine route.

Vertical transmission to embryo and fetus in maternal infection with rat virus (RV).

The influence of maternal rat virus (RV) infection on rat embryogenesis and fetus was examined by viral reisolation, immunostaining and PCR analysis. Vertical transmission caused by the UT-1 strain of RV depended on the stage of gestation when maternal infection occurred. When females were infected at the pre-mating point, the number of fetuses was smaller than that normally obtained, possibly due to infection at the stage of the hatched blastocyst, but almost all of the fetuses obtained were free from infection and developed normally. The incidence of transplacental infection was the highest when pregnant females were infected in the middle of the gestation stage, and some of the fetuses died. In pregnant females which were infected late in the gestation stage, all fetuses developed normally. Some of them were infected transplacentally and harbored the infectious virus. Much attention should be paid to performing reliable rederivation of RV-infected rat colonies by hysterectomy and embryo transfer.

Is caprine arthritis encephalitis virus (CAEV) transmitted vertically to early embryo development stages (morulae or blastocyst) via in vitro infected frozen semen?

The aim of this study was to determine, in vivo, whether in vitro infected cryopreserved caprine sperm is capable of transmitting caprine arthritis-encephalitis virus (CAEV) vertically to early embryo development stages via artificial insemination with in vitro infected semen. Sperm was collected from CAEV-free bucks by electroejaculation. Half of each ejaculate was inoculated with CAEV-pBSCA at a viral concentration of 10(4) TCID(50)/mL. The second half of each ejaculate was used as a negative control. The semen was then frozen. On Day 13 of superovulation treatment, 14 CAEV-free does were inseminated directly into the uterus under endoscopic control with thawed infected semen. Six CAEV-free does, used as a negative control, were inseminated intrauterine with thawed CAEV-free sperm, and eight CAEV-free does were mated with naturally infected bucks. Polymerase chain reaction (PCR) was used to detect CAEV proviral-DNA in the embryos at the D7 stage, in the embryo washing media, and in the uterine secretions of recipient does. At Day 7, all the harvested embryos were PCR-negative for CAEV proviral-DNA; however, CAEV proviral-DNA was detected in 8/14 uterine smears, and 9/14 flushing media taken from does inseminated with infected sperm, and in 1/8 uterine swabs taken from the does mated with infected bucks. The results of this study confirm that (i) artificial insemination with infected semen or mating with infected bucks may result in the transmission of CAEV to the does genital tack seven days after insemination, and (ii) irrespective of the medical status of the semen or the recipient doe, it is possible to obtain CAEV-free early embryos usable for embryo transfer.

Adeno-associated virus type 2 interferes with early development of mouse embryos.

The human helper-dependent adeno-associated virus type 2 (AAV-2) has been shown to induce differentiation in various cell types in culture including pluripotent embryonic cells, in the absence of helper virus. To assess whether induction of differentiation may influence developmental processes we analysed the effect of AAV-2 on developing mouse embryos. In vitro infection of fertilized eggs induced arrest of development at the two-cell stage. Moreover, micro injection of AAV-2 DNA (comprising either the complete AAV-2 genome or a fragment containing the P5 promoter region) into one-cell embryos, blocked development at the morula stage. In vivo, AAV-2 infection of pregnant mice led to fetal death and early abortion. These results demonstrate that the human adeno-associated virus, which is thought to be non-pathogenic, can perturb embryonic development in mice. This may provide a suitable animal model system to further elucidate the biological significance of the recent detection of adeno-associated virus DNA in human abortion material.

Viral contamination of embryos cryopreserved in liquid nitrogen.

Despite the worldwide application of embryo-freezing technology as the means of preserving germplasm of mammalian species, there is no information available on the possible transmission of infectious agents to cryopreserved embryos via contaminated liquid nitrogen (LN). Recently, it has been reported that new methods of cryopreservation which employ ultrarapid freezing or vitrification require direct contact between the freezing medium containing oocytes or embryos and liquid phase nitrogen (LPN). As models for human and animal viral pathogens three bovine viruses, bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV), and bovine immunodeficiency virus (BIV), were employed to study the potential for their transmission by experimentally contaminated LN to embryos frozen and stored in open freezing containers. Bovine embryos in a mixture of 20% ethylene glycol, 20% ME(2)SO, and 0.6% sucrose were vitrified in either unsealed standard 0.25 ml or modified open pulled straws or in plastic cryovials and then plunged into contaminated LPN. After 3-5 weeks of storage in LN, embryos were thawed and sequentially washed and only those with intact ZP were pooled together and tested in batches of three for viral contamination. From this pool of 83 batches, 13 of 61 (21.3%) batches exposed to BVDV and BHV-1 tested positive for viral association while all 22 batches exposed to BIV in unsealed containers tested negative. All control embryos vitrified in sealed cryovials and straws were free from viral contamination.