[Experimental study of the effect of human cytomegalovirus infection on cell cycle progression and the expression of cyclins].

OBJECTIVE: To observe the effect of human cytomegalovirus infection on the host cellular DNA synthesis and expression of cyclones. METHODS: HCMV infected cell was established in vitro by incubating passage cultured HEL and HCMV AD169 strain with different titres. The cells were synchronized in the G0/G1 stage by contact inhibition and infected with strain AD169 of HCMV at an MOI of 5 PFU per cell. We harvested infected cell at different time 0 h, 3 h, 6 h, 24 h, 72 h and 96 h post infection. Then the cell cycle progress was measured. Meanwhile, the DNA content and expression of proteins of cycline E, cycline A and cycline D1 were determined with FCM and Western Blot respectively. RESULTS: We found that the amount of S stage cell infected by HCMV had increased dramatically, and that of G2/M stage cell reduced during 24 h-96 h PI, and no G2/M stage cell was detected within 96 h PI. The content of 2N DNA maintained unchangeable for 24 h after infection and the content of total DNA in infected cells began to increase within 48 h PI, and the substantial cell with 2N DNA were observed 72 h after infection. However, DNA content was not altered in control group of normal HEL and HCMV PAA group. CyclinE protein was induced 12 h PI and peak induction occurred 24 h PI in contact-inhibited cells. CyclinA protein expression was not induced in HCMV infected density-arrested cells. The abundance of cyclinD1 decreased 24 h PI. CONCLUSION: The expression of cyclinE and activity of cyclinE/Cdk2 kinase are increased obviously in G0/G1 stage cells infected with HCMV, which may induce the cell cycle to overpass G1/S restriction point and make the cell cycle arrested in later G1 stage. HCMV can not activate cellular DNA synthesis, and increase of total DNA content in infected cells result from the viral DNA replication.

Effects of Jinye Baidu Granule on fetal growth and development with maternal active human cytomegalovirus infection.

OBJECTIVE: To evaluate the effects of Jinye Baidu Granule ( JYBDG), a traditional Chinese medicine compound prescription, on fetal growth and development with maternal active human cytomegalovirus infection. METHODS: A prospective, randomized and controlled trial was adopted during January 1996 to June 2002. From the pregnant women with an abnormal pregnant history, 240 cases were screened to be infected by human cytomegalovirus (HCMV) by enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). They were assigned according to the random number table to two groups. The 122 cases in the treatment group were administrated with JYBDG, one package each time, three times a day for two continuous weeks, while the other 118 in the control group did not receive any treatment. The negative conversion rate of both HCMV-IgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate between the two groups were compared, and fetal growth and development in partial fetuses were also observed. RESULTS: The negative conversion rate of both HCMV-lgM and HCMV late mRNA, the positive rate of HCMV-DNA in placenta and the intrauterine transmission rate in the treatment group were 77. 05% (94/122), 48. 98% (48/98) and 21.74% (10/46) respectively, while those in the control group were 38. 14% (45/118), 67.50% (54/80) and 52.63% (20/38) respectively, all showing significant difference between the two groups (P<0. 01). Totally 35 normal infants and 11 abnormal infants were born in the treatment group, and the number in the control group was 20 and 18 respectively, and comparison between the two groups showed significant difference (P<0.01). Six months of child birth, the scores of both mental development index (MDI) and psychomotor development index (PDI) of infants were higher in the treatment group (20 cases) than those in the control group (20 cases), but there was no significant difference between the two groups (P>0.05). CONCLUSION: JYBDG could decrease the intrauterine transmission of HCMV and is beneficial to fetal growth and development.

[Histologic change in human cytomegalovirus-infected explants of first trimester human placenta and expression of human cytomegalovirus gene].

OBJECTIVE: To observe histologic changes of human cytomegalovirus (hCMV)-infected explants of first trimester human placenta and expression of hCMV gene in the hCMV-infected explants, and investigate the mechanism of intrauterine transmission of hCMV from mother to fetus. METHODS: The first trimester placenta explants cultures were carried out, and they were infected with hCMV for 10 days. The expression of hCMV immediate early protein (IEP) 72-IEP86 were determined using indirect-immuno fluorescent assay, and in situ hybridization method was used to examine the hCMV late gene (LG) mRNA. For histologic evaluation of morphological changes in villi, transmission electron microscope was used. RESULTS: (1) Typical hCMV-induced lesions bearing hCMV IEP72-IEP86 were consistently localized in the trophoblast of covering placenta villi, interstitial cell and vascular endothelia cell 12 hours after infection, and were predominant in cytotrophoblast. (2) Replication of hCMV in placenta explants culture occurred from 12 hours to 24 hours and disappeared since 48 hours after infection with different concentrations of hCMV when examined by in situ hybridization. (3) Tissue integrity and viability of first trimester placenta explants were obtained in culture for 10 days and then explants were infected with different concentrations of hCMV [100 tissue culture infectious doses (TCID(50)), 200 TCID(50) and 300 TCID(50)], the progression of the infection was observed in the tissue that maintained its normal cellular organization under light microscope. But typical inflammation of cellular organization was observed under transmission electron microscope. CONCLUSIONS: (1) A flash replication of hCMV in placental explants culture occurs; IEP72-IEP86 may be in intrauterine infection of hCMV for a long time. (2) There are pathological ultrastructure changes in hCMV-infected explants.

[Experimental study on effect of jinye baidu preparation in inhibiting human cytomegalovirus protein kinase pul 97].

OBJECTIVE: To study the inhibitory effect of Jinye Baidu Preparation (JBP), a Chinese medicinal preparation, on human cytomegalovirus protein kinase pu197 and to explore its molecular mechanism in treating human cytomegalovirus (HCMV) infection. METHODS: Expression of the HCMV pu197mRNA in infected cells was measured by semi-quantitative RT-PCR before and after intervention of JBP or Ganciclovir (GCV), and effect of the two medicines on the proliferation activity of the infected cells was observed by MTT. RESULTS: Both JBP and GCV showed obvious inhibitory action on HCMV pu197mRNA. They could significantly enhance the proliferation activity of the cells 72 hours after HCMV infection. CONCLUSION: JBP could inhibit the gene expression and duplication of HCMV by inhibiting the gene expression of HCMV protein kinase pu197 to enhance the proliferation activity of the infected cells so as to achieve its anti-virus action.

[Observation on clinical efficacy of combined therapy of zinc supplement and jinye baidu granule in treating human cytomegalovirus infection].

OBJECTIVE: To explore the clinical efficacy of the combined therapy of zinc supplement and Jinye Baidu granule (JBG) on human cytomegalovirus (HCMV) infection. METHODS: One hundred and forty patients with positive HCMV-IgM were randomly divided into four groups, with 35 cases in each group, that is, the control group (only medicated with JBG), the high-, moderate- and low-dose zinc combined groups (treated with JBG combined with zinc gluconate tablet at dose of 30 mg, 20 mg, 10 mg every day respectively). The negative conversion rate of HCMV-IgM was observed. RESULTS: Insignificant difference in the negative conversion rate was shown in comparison of the control group with the low dose group (P > 0.05), and in comparison of the high dose with the moderate dose group (P > 0.05); however, the rate was significantly lower in the control group than that in the moderate dose and high dose group (P < 0.05). CONCLUSION: Combined therapy of zinc supplement and JBG can significantly increase the negative conversion rate of HCMV-IgM. The optimal dosage of zinc gluconate tablet was 20 mg once a day.

[Experimental study on antivirus activity of traditional Chinese medicine].

OBJECTIVE: To investigate the difference of anticytomegaloviral activity of three kinds of traditional Chinese medicines which are the injections of Jinye Baidu, Radix Isatidis and Indigowoa in vitro. METHOD: The inhibitory activity of three traditional Chinese medicines against human cytomeglovirus (HCMV AD169) infected human embryo lung fibroblasts (HELF) was observed by cytopathic effect method (CPE) and MTT method in vitro. According their value of A, anticytomegaloviral activity has evaluated. RESULT: Experimental study in vitro showed that the 50% toxicity dose (TD50) of Jinye Detoxifying, Radix Isatidis root and Indigowoa were 20, 10.23, 20.23 g x L(-1) respectively; the 50% inhibitory concertration (IC50) were 5.65, 3, 5.71 g x L(-1) respectively; the therapeutic index (TI) were 3.54, 3.41 and 3.54 respectively. It suggested that three traditional Chinese medicines had anticytomeglovirus activity and their effect increased with their concentration. CONCLUSION: Three traditional Chinese medicines of the parenteral solution of Jinye Detoxifying, Radix Isatidis root and Indigowoa have antiviral activity when they are diluted in 1:200. They are safe and valuable drug for inhibiting cytomeglovirus infection.

[Relationship between the pathologic changes of human embryo fibroblast cells and expression of late mRNA after human cytomegalovirus infection in vitro].

OBJECTIVE: To study the relationship between late mRNA and the cytopathic effect(CPE) and ultrastructural features after human cytomegalovirus (HCMV) infection in vitro. METHODS: Human embryo fibroblast cells(HEL) were infected with HCMV AD169 strain. The expression of the HCMV late mRNA was measured by semiquantitative RT-PCR, the cytopathic effect (CPE) and the cell ultrastructure were observed by means of light microscopy and electron microscopy, respectively. RESULTS: The HCMV late mRNA could be detected 12 hours postinfection and increased gradually, but the CPE appeared 48 hours postinfection in HEL cells. The HCMV infected cells exhibited significant mitochondrial enlargement and the number of mitochondrial ridge deletion, the cisternae lumen of endoplasmic reticulum (ER) dilation and vacuolization (at the end age). The mature nucleocapsid could be observed 96 hours postinfection. CONCLUSION: The ultrastructural changes have an intimate correlation with the expression of HCMV late mRNA and play an important role in the life circle of the virus. HCMV late mRNA may serve as a indicator of the clinical effect of treatment in active HCMV infection.

[Effect of toxoplasma infection during pregnancy to the development of fetus and infant].

OBJECTIVE: To investigate the effect of Toxoplasma (Tox) infection during pregnancy to the development of fetus and infant. METHODS: Enzyme linked immunoadsorbent assay (ELISA) was employed to detect Tox-IgM in peripheral blood of 3,908 pregnant women. To Tox-IgM positive cases, the umbilical blood of their neonates were sampled to detect Tox-IgM and abortion tissues were sampled to detect Tox-DNA with polymerase chain reaction. Infection group included 95 cases whose Tox-IgM or DNA were positive, and control group consisted of 119 cases without infection. Different supervision methods were used to periodically observe the intelligence development of infants 3 and 12 months after birth in these two groups. After initial intervention, verbal intelligence quotients (VIQs), performance intelligence quotients (PIQs) and full scale intelligence quotients (FSIQs) were assessed when the infants were 1 and 4 years old of these two groups. RESULTS: The incidences of abortion, stillbirth, pre-term delivery, FGR and malformation in infection group were 12 cases (12.6%), 5 cases (5.3%), 4 cases (4.2%), 4 cases (4.2%) and 3 cases (3.2%), while the incidences of these abnormalities in control group were 3 cases (2.7%), 1 cases (0.9%), 2 cases (1.8%), 2 cases (1.8%) and 1 cases (0.9%). Comparing with these two groups, there was significant difference (P < 0.01). Relative risk (RR) of these 5 abnormalities was 4.7, 5.9, 2.3, 2.3 and 3.6 respectively. Mental development index (MDI) and psychomotor development index (PDI) of infants in infection group were 93 +/- 13 and 101 +/- 16. They were significantly lower than that of control group, which were 107 +/- 17 and 111 +/- 13. There was significant difference between these two groups (P < 0.05). After intervention in 1 and 4 years, their VIQs, PIQs and FSIQs were apparently improved (P < 0.05). CONCLUSION: Congenital Tox infection is closely correlated with abnormal pregnancy outcomes and infantile mental retardation. Early intervention can improve intelligence development.