The Experimental Model Research On Intrauterine Infection Of Murine Cytomegalovirus

Part One Effects of MCMV Intrauterine Infection on the Pregnancy and the Fetal Development[Objective] To establish the murine model of MCMV intrauterine infection and to observe the effects of MCMV intrauterine infection on the pregnancy and the fet al development.[Methods] (1) The Smith strain of MCMV was passaged by cell culture and the TCID50 was quantitated by the method of Reed-Muench. (2) MCMV-free BALB/C mice were screened by ELISA assay of IgM and IgG antibody to the virus. After overnight mating, the females in the 10-11^th day of pregnancy were randomly divided into three groups: 38 in the infected group, in which 3 subgroups were separated according to the viral infection dose(14 in subgroup HIGH, 12 in subgroup MID, 12 in subgroup LOW), received sterile inoculation of MCMV suspension via the placenta with different dose, namely, 1×10⁷TCID₅₀ in subgroup HIGH, 1×16⁶TCID₅₀ in subgroup MID and 1×10⁵TCID₅₀ in subgroup LOW, respectively. 12 in the model control group, were injected similarly with aseptic DMEM containing 3%FCS. 10 in the vacant control group received no special treatment except conceptus counting by surgery. â‘  the general status of the pregnant mice was recorded. â‘¡ IgM in the blood of the mothers were assayed by ELISA.â‘¢ the females of the 20^th day of pregnancy or the day of natural delivery were randomly chosen and the placentas and fetuses were dissected sterilely. The fet al organs (brain, lung, liver, kidney) received pathological tests, and the supernate in part of the tissue homogenate received viral isolation by in vitro culture. â‘£ MCMV mRNA was tested by RT-PCR in part of the placenta, and MCMV DNA was measured by PCR. ⑤pregnancy progress and sequel were recorded in detail, â‘¥growth and development of the offsprings were observed and the weight, height and head circumference of the newborns were measured.[Result] (1) The TCID₅₀ of the Smith strain of MCMV was 5.62×10⁷TCID₅₀/ml.(2) â‘  Nonspecific viremia and MCMV IgM were present in the infected subroup ubiquitously, while they were negative in both control groups. â‘¡ viral isolation was positive in the placentas and fetuses of the infected group and corresponding pathological changes took on in the organs. â‘¢ the positive rate of MCMV mRNA in the infected group was 58.49% (30/53) ; the positive rate of MCMV DNA in fet al organ was 37.73% (20/53 ) in brains, 32.07% (17/53 ) in lungs, 24.64%(17/53) in livers and 24.64%( 17/53) in kidneys, respectively. (3) â‘ anomaly pregnancy rate in the three infected subgroups was significantly higher than the model control group (P < 0.05). â‘¡ Fetal survival rate in the three infected subgroups was significantly lower than the model control group (P<0.001). â‘¢ as to the weight, head circumference and height of the live fetuses, the three infected subgroups were significantly lower than the model control group (P<0.01). â‘£ incidence of microcephaly in the live fetuses of the three subgroups was significantly higher than the model control group (P<0.01).⑤incidence of low birth weight ratio in the live fetuses of the three infected subgroups was significantly higher than the model control group (P<0.01). There was no statistical difference between the model control group and the vacant control group in respect to the parameters mentioned above.[Conclusion] The murine model of congenital intrauterine infection of MCMV could be etstablished through MCMV-inoculation via the placenta. Abnormal pregnancy rate rose due to MCMV intrauterine infection, which could severely affect intrauterine development of the fetus and hence increase the incidence of low birthweight ratio and microcephaly. Part Two Influence of MCMV Infection on the T Lymphocyte Subtypes of the Pregnant Mice[Objective] To observe the Influence of MCMV intrauterine infection on the T lymphocyte subtypes of the pregnant mice in different infection phases and to realize the change rule of the immune status of MCMV-infected pregnant mice.[Methods]MCMV-free BALB/C mice were screened by ELISA assay of IgM and IgG antibody to the virus. After overnight mating, the females in the 10-11^th day of pregnancy were randomly divided into two groups: 20 in the infected group received sterile inoculation of 1 ×10⁷TCID₅₀MCMV suspension via the placenta. 20 in the model control subgroup were injected similarly with aseptic DMEM containing 3%FCS. 20 in the vacant control subgroup received no special treatment. The pregnant mice were randomly chosen on the 14^th day, 17^th day, the 20^th day and delivery day, respectively, â‘ assaying the MCMV IgM in the blood by sampling via the heart on delivery day. â‘¡ sterilely dissecting the spleen and measuring the wet weight. â‘¢ preparing spleen cells and isolating PBMC. The lymphocyte proliferating reaction was examined by the method of CCK-8 after overnight culture and removing adherent cells. @ assaying the relative numbers of lymphocyte subtypes and the CD4 ⁺/CD8⁺ ratio by flow cytometry with anti-mouse CD3,CD4,CD8 monoclonal antibodies labeled by three-color fluorescence.[Result] â‘ MCMV-IgM was positive in all the pregnant mice in the infected group, while it was negative in both control groups. â‘¡The wet weight of the spleen of infected mice was significant ly higher than the control of corresponding phases and increased with the elongation of infection (P <0.01).There were pathologcail changes in the spleen. â‘¢The proliferating reaction of T lymphocytes of infected mice was significantly reduced compared with the control of corresponding phases (P< 0.01). â‘£ There was no significant difference in the relative percentage of CD3⁺T lymphocytes. The relative percentage of CD4⁺T lymphocytes was significantly lower in the infected group of corresponding phase from the 17^th day of pregnancy to delivery and decreased with the elongation of infection (P<0.01). The relative percentage of CD8⁺T lymphocytes was significantly higher in the infected group of corresponding phase from the 17^th day of pregnancy to delivery and increased with the elongation of infection (P <0.01). The CD4⁺/CD8⁺ ratio was significantly lower in the infected group of corresponding phase and decreased with the elongation of infection (P<0.01). There was no statistically significant difference between the model control and the vacant control group in respcet to the parameters compared above.[Conclusion] The change rule of T lymphocytes of spleens of MCMV-infected pregnant mice presented as decreasing CD4⁺T lymphocytes, increasing CD8⁺T lymphocytes, as well as lowering CD4⁺/CD8⁺ratio, which took on a time-effect relationship with infection time. And the immune function was inhibited, which might play an role in the vertical transmittion of MCMV. Part ThreeEffects of MCMV Infection on the Activity of NK Cells and CTL of the Pregnant Mice[Objective] To observe the effects of MCMV intrauterine infection on the activity of NK cells and CTL of the pregnant mice in different infection phases and realized the change of the cellmediated immunity of MCMV-infected pregnant mice.[Methods] MCMV-free BALB/C mice were screened by ELISA assay of IgM and IgG antibody to the virus. After overnight mating, the females in the 10-11^th day of pregnancy were randomly divided into two groups: 20 in the infected group received sterile inoculation of 1 ×10⁷TCID₅₀ MCMV suspension via the placenta. 20 in the model control subgroup were injected similarly with aseptic DMEM containing 3%FCS. 20 in the vacant control subgroup received no special treatment. The pregnant mice were randomly chosen on the 14^th day, 17^th day, the 20^th day and delivery day, respectively, â‘ assaying the MCMV IgM in the blood by sampling via the heart on delivery day. â‘¡ preparing spleen cells and isolating PBMC. The activity of NK cells was examined by the method of CCK-8 after overnight culture and removing adherent cells.â‘¢preparing spleen cells and isolating PBMC. The activity of CTL was examined by the method of Cell Counting Kit-8 (CCK-8) after overnight culture and removing adherent cells. Meanwhile,the activated function of CTL was assayed by Enzyme-linked Immunospot Assay (ELISPOT).[Results] â‘ MCMV-IgM was positive in all the pregnant mice in the infectedgroup, while it was negative in both control groups.â‘¡ the killing activity of NK cellswas higher in the infected group on the 14^th day of pregnancy, and then decreased andkept being lower than the model control group with pregnancy progress till delivery(P<0.001). There was no statistical difference between the two control groups. â‘¢the frequency of the spots produced by CTL was significantly lower in the infected group and declined with pregnancy progress compared to the model control group (P <0.001). The killing function of CTL was significantly lower in the infected group from the 17^th day to delivery and declined with pregnancy progress compared to the model control group (P <0.001). There was no statistical difference between the two control groups (P>0.05).[Conclusion] The killing activities of NK cells of MCMV-infected pregnant mice was inhibited, so were the activation and killing function of CTL, which presented time-effect relationship with infection time. The anti-MCMV cellmediated immunity of the pregnant mice was suppressed, which might be related with the vertical transmition of MCMV.