The risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of cumulus-enclosed oocytes (CEOs) or without cumulus cells (CDOs) in the presence of MMV-exposed (104 TCID50 [mean tissue culture infective dose]/ml MMVp [prototype strain of MMV]) spermatozoa was evaluated. Also, the time after embryo transfer to detection of MMV antibody and the presence of MMV DNA in the mesenteric lymph nodes of recipients and pups were investigated. All mice were MMV free, but two seropositive recipients and four seropositive pups were found in the group with CDOs. With regard to the CEOs, two of 11 holding drops and five of 11 groups of embryos were MMV positive using PCR, while neither holding drops nor embryos carried infectious MMVp, as evidenced by the in vitro infectivity assay. From IVF with CDOs, five of 14 holding drops and four of nine groups of embryos were MMV positive, while one of 14 holding drops and no embryos carried infectious MMVp. When 105 cumulus cells were analyzed 5 h after exposure to 104 TCID50/ml MMVp, cells had an average titer of 104 TCID50/ml MMVp. The present data show that, in contrast to CDOs, 2-cell embryos from CEOs did not transmit infectious MMVp to the holding drops and to recipients. This observation is due to the presence of cumulus cells during the IVF process that reduce entry of MMV into the zona pellucida and absorb some of the virus. These data further confirm the efficacy of the IVF procedure in producing embryos that are free of infectious virus, leading to virus-free seronegative recipients and rederived pups.

In the present study, the risk of transmission of mouse minute virus (MMV) to recipients of murine embryos arising from in vitro fertilization (IVF) of oocytes with MMV-exposed spermatozoa and to resulting pups was evaluated. Also, the time of seroconversion of recipients and pups was investigated. To achieve this goal, IVF of oocytes with cryopreserved spermatozoa from the inbred C3HeB/FeJ mouse strain was performed, and the resulting embryos were transferred to suitable Swiss recipients. Three groups were investigated: 1) oocytes or the developing embryos were continuously exposed to 10(4) TCID(50) MMVp per milliliter in the fertilization (human tubal fluid [HTF]), culture (KSOM), and embryo transfer (M2) media (positive control); 2) oocytes and spermatozoa were exposed to MMVp in the HTF medium only and transferred after a standard washing procedure with 10 washing steps in virus-free KSOM and M2; and 3) oocytes and spermatozoa were exposed to virus-free HTF, KSOM, and M2 (negative control). To detect antibodies to MMV in recipients and progeny, serological analyses were performed by ELISA on Days 14, 21, 28, and 42, and on Days 42 and 63, respectively, after embryo transfer. The presence of MMV in the washing drops was analyzed by PCR and an in vitro infectivity assay, while organs of some recipients and pups were analyzed by PCR. Using 10(4) of the tissue culture infective dose of MMVp per millilitre in the fertilization medium only, the present results demonstrate that 10 washing steps in the IVF-ET procedure are sufficient to remove the virus to a noninfectious dose, producing MMV-free seronegative recipients and pups. As such, there is minimal risk of transmission of MMV to recipients and pups if spermatozoa become contaminated with such viral loads.

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10 0 TCID 50 MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10 −5 TCID 50 MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.

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.