Human Herpes Virus Type 2 ( HSV2 ), Human Cytomegalovirus (HCMV) in the Male Genital Tract and Fertilization

The possibility of infection of the human male genital tract by human herpes virus type 2 (HSV2) or human cytomegalovirus (HCMV) is well established and their sexual transmission has been the object of many studies. Moreover, medically assisted procreation, which helps in numerous fertility problems, raises the question of new viral risks linked to the application of these new technologies. In this review, we shall consider current knowledge in terms of the presence of HSV 2 and HCMV in the different parts of the genital tract of immunocompetent or immunodepressed men. We shall also consider the possibility of viral transmission by the sexual act or by the various techniques used in medically assisted procreation. We shall describe studies in human beings and in animals.

巨细胞病毒活动性感染患儿NK细胞及GGT水平与HCMV DNA的相关性

目的探讨巨细胞病毒(HCMV)活动性感染患儿自然杀伤(NK)细胞及γ-谷氨酰转肽酶(GGT)水平与HCMV DNA的相关性。方法选取2021年6月—2022年6月濉溪县医院收治的150例HCMV活动性感染患儿纳入研究对象,根据HCMV DNA水平将患儿分为低载量组(n=52)、中载量组(n=64)、高载量组(n=34),另外选取我院同期50名健康婴儿作为对照组。比较4组的肝功能指标[天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALP)、总胆汁酸(TBA)、GGT]及NK细胞水平,分析肝功能指标及NK细胞与HCMV DNA的相关性。结果4组AST、ALT、ALP、TBA比较无明显差异(P>0.05),4组GGT分别为(10.28±2.69)U/L、(15.64±3.13)U/L、(18.56±3.22)U/L、(23.77±4.17)U/L,4组NK细胞分别为(4.87±1.04)%、(6.14±1.15)%、(13.55±2.33)%、(19.84±3.27)%,GGT、NK随HCMV DNA载量升高而上升(P<0.05);经相关性分析,AST、ALT、ALP、TBA与HCMV DNA无明显相关性(P>0.05),NK细胞、GGT与HCMV DNA呈现正相关(P<0.05)。结论NK细胞及GGT水平与HCMV DNA具有相关性,检测HCMV DNA有助于HCMV活动性感染的诊断,但无法评估病情严重程度。

HIV/AIDS患者并发EBV和HCMV感染临床免疫学特征及影响因素分析

目的调查人类免疫缺陷病毒/获得性免疫缺陷综合征(human immunodeficiency virus/acquired immune deficiency syndrome,HIV/AIDS)患者感染EB病毒(Epstein-Barr virus,EBV)和人类巨细胞病毒(human Cytomegalovirus,HCMV)的情况,检测相关临床免疫学指标,分析其影响因素。方法选取2022年1~12月在长沙市第一医院住院并接受EBV和HCMV筛查的1093例HIV/AIDS患者。流式细胞术检测CD4^(+)T淋巴细胞数量;荧光定量PCR检测HIVRNA载量、EBV-DNA载量和HCMV-DNA载量。采用SPSS 27.0统计学软件进行统计分析,并通过Logistic回归分析HIV/AIDS患者并发病毒感染的危险因素。结果1093例HIV/AIDS患者中,EBV-DNA阳性率为48.22%(527/1093),HCMV-DNA阳性率为19.03%(208/1093)。随着CD4^(+)T淋巴细胞数量增加,EBV-DNA和HCMV-DNA的阳性率下降(χ^(2)=39.50,143.0,均P<0.001);随着HIV-RNA载量增加,EBV-DNA和HCMV-DNA的阳性率增加,差异具有统计学意义(χ^(2)=46.18,124.3,均P<0.001)。另外,患者接受抗逆转录病毒治疗(antiretroviral therapy,ART)也可明显降低EBV-DNA和HCMV-DNA的阳性率,差异具有统计学意义(χ^(2)=30.60,96.59,均P<0.001)。CD4^(+)T淋巴细胞数量和HIV-RNA载量有显著的负相关关系(r=-0.49,P<0.001)。Logistic回归分析显示CD4^(+)T淋巴细胞数量<200个/μl(OR=1.46,95%CI:1.02~2.08,P=0.037),HIV-RNA载量>200 copies/ml(OR=1.70,95%CI:1.18~2.44,P=0.004),年龄>30岁(OR=2.15,95%CI:1.44~3.19,P<0.001)是HIV/AIDS患者并发EB病毒感染的危险因素;未持续接受ART(OR=1.83,95%CI:1.10~3.02,P=0.019),HIV-RNA载量>200 copies/ml(OR=2.56,95%CI:1.50~4.35,P<0.001),CD4^(+)T淋巴细胞数量<200个/μl(OR=4.61,95%CI:2.57~8.28,P<0.001)是HIV/AIDS患者并发HCMV感染的危险因素。结论在艾滋病的治疗与管理中,当CD4^(+)T淋巴细胞数量下降(<200个/μl),HIV-RNA载量升高(>200 copies/ml)或者年龄>30岁时,应加强对病毒的监测和ART,减少HIV/AIDS患者机会性感染的可能。

Effect of Baicalein on the Expression of VIP in Extravillous Cytotrophoblasts Infected with Human Cytomegalovirus In Vitro

Summary： This paper aimed to study the ability of baicalein to block human cytomegalovirus （HCMV） infection in extravillous cytotrophoblasts （EVT） and its effect on the vasoactive intestinal peptide （VIP） expression in HCMV-infected EVT in vitro. A human trophoblast cell line （HPT-8） was chosen in this study. HCMV with 100 TCIDs0was added into culture medium to infect HPT-8 cells, and then HCMV pp65 antigen was assayed by immunofluorescence staining. The infection status was determined by vi- rus titration. Real-time quantitative polymerase chain reaction （qRT-PCR） was used to detect virus DNA load in the infected cells. The expression of VIP mRNA and protein in the infected cells was measured by qRT-PCR, immunocytochemistry and Western blotting. Concentration of VIP secreted in supernatants was determined by ELISA. Red-stained HCMV pp65 antigens were found in infected HPT-8 cells 48 h after infection. HCMV replicated in large quantity in infected HPT-8 cells 4 days after infection, reaching a peak at day 6 post-infection. After treatment with baicalein, virus DNA load in in- fected HPT-8 cells was decreased （P〈0.05）, and the levels of VIP mRNA and protein, and the concen- tration were raised to the normal （P〉0.05）. Our study suggested that baicalein exerts a positive effect on the VIP expression in HCMV-infected EVT at maternal-fetal interface.

Association of cytomegalovirus infection with human leukocyte antigen genotypes in recipients after allogeneic liver transplantation

BACKGROUND: Cytomegalovirus (CMV) infection is the important cause affecting the survival rate and function of the transplanted organ after transplantation. The occurrence of CMV infection after liver transplantation (LT) is associated with many factors. Lots of studies suggest that genetic mutation between hosts and CMV may play a role in the occurrence and development of CMV infection. CMV exists in an incubative state, affect or destroy the expression of human leukocyte antigen (HLA) molecules in the host cell surface, and interfere antigen's submission. This mechanism is the key of CMV to avoid immune defense mechanism of the host. To detect HLA and CMV antibody (CMV-Ab), CMV antigen (CMV-Ag) of transplantation recipients, we evaluated the association of CMV infection and the particular HLA genotypes in recipients after LT. METHODS: 277 blood samples were collected from 39 LT recipients. CMV antibody and antigen were detected by ELISA or immunohistochemical methods. The HLA types of the recipients were determined by PCR. To analyze the association of HLA alleles and the occurrence of CMV antigenemia in the patients, relative risk degree (RR) was used as the parameter for the Chi-square test. RESULTS: The LT recipients were serum CMV IgG positive (100%), but none of them was CMV IgM positive (0%). Thirty-three LT recipients (84.6%) were CMV antigenic positive with 1-50 positive leukocytes per 50000 leukocytes in extent and 7.2±4.2 positive leukocytes per 50000 leukocytes on average. Thirteen patients developed CMV pneumonia, with CMV antigenic positive (100%) and 17.7±5.5 positive leukocytes per 50000 leukocytes on average. Some HIA alleles were associated with the occurrence and extent of CMV antigenemia. HLA-A2 was the higher frequency allele for patients with antigenemia (P<0.05), and 7 patients carrying HLA-DR11 allele developed antigenemia (P<0.05). In the lower antigenemia group, HLA-A11 was higher in frequency than others (P<0.05). Besides, none of the patients carrying HLA-B16 allele developed clinical symptoms of CMV infection (P<0.05). CONCLUSIONS: The variability of HLA alleles might modulate immune response to CMV infection. HLA examination before transplantation should be made for prevention and treatment of CMV infection aider operation.

Human cytomegalovirus-encoded US28 may act as a tumor promoter in colorectal cancer

AIM: To assess human cytomegalovirus-encoded US28 gene function in colorectal cancer(CRC) pathogenesis.METHODS: Immunohistochemical analysis was performed to determine US28 expression in 103 CRC patient samples and 98 corresponding adjacent noncancerous samples. Patient data were compared by age, sex, tumor location, histological grade, Dukes' stage, and overall mean survival time. In addition, the US28 gene was transiently transfected into the CRC LOVO cell line, and cell proliferation was assessed using a cell counting kit-8 assay. Cell cycle analysis by flow cytometry and a cell invasion transwell assay were also carried out.RESULTS: US28 levels were clearly higher in CRC tissues(38.8%) than in adjacent noncancerous samples(7.1%)(P = 0.000). Interestingly, elevated US28 amounts in CRC tissues were significantly associated with histological grade, metastasis, Dukes' stage, and overall survival(all P < 0.05); meanwhile, US28 expression was not significantly correlated with age, sex or tumor location. In addition, multivariate Coxregression data revealed US28 level as an independent CRC prognostic marker(P = 0.000). LOVO cells successfully transfected with the US28 gene exhibited higher viability, greater chemotherapy resistance, accelerated cell cycle progression, and increased invasion ability.CONCLUSION: US28 expression is predictive of poor prognosis and may promote CRC.

Effect of Human Cytomegalovirus Infection on Nerve Growth Factor Expression in Human Glioma U251 Cells

Objective To explore the change of endogenic nerve growth factor （NGF） expression in human glioma cells infected with human cytomegalovirus （HCMV）. Methods U251 cells were cultured in RPMI 1640 culture medium and infected with HCMV AD 169 strain in vitro to establish a cell model of viral infection. Morphologic changes of U251 cells were observed under inverted microscope before and after infection with HCMV. Expression of NGF gene and protein of cells was detected by RT-PCR and Western blotting before and after infection with HCMV. Results The cytopathic effects of HCMV-infected cells appeared on day 5 after infection. However, differential NGF expression was evident on day 7. NGF expression was decreased significantly in U251 cells on day 7 after infection in comparison with control group （P〈0.05）. Conclusion HCMV can down-regulate endogenous NGF levels in human glioma cell line U251.

Effects of Human Cytomegalovirus Infection on Apoptosis and Expression of Apoptosis-Regulating Factors

Summary： The present study aimed to find out dynamic changes of apoptosis in human cytomegalovirus （HCMV） infected cells and the influence of HCMV infection on activation of caspase-3 and the expression of apoptosis-regulating genes, bcl-2 and fas mRNA. The sequential changes of apoptotic cell rate in high and low MOI （MOI=2.5 and 0.25 respectively） of HCMV infected human embryonic lung fibroblasts （HELFs） at 1 h, 12 h, 24 h, 36 h, 48 h, 72 h and 96 h post-infection were measured by flow cytometry. The expression levels of caspase-3 protein and bcl-2 and fas mRNA in HCMV infected cells （MOI=0.25） at 72 h post-infection were detected by Western blot and in situ hybridization methods, respectively. It was found that the ratio of apoptotic cells in normal controls was consistently lower, but the rates in low and high MOI infected cells were gradually increased with time prolonged, reached peak at 96 h （8.85 %） and 72 h （25.63 %）, respectively. By Western blot analysis, only a narrow band of 32 kD （1 kD=0. 992 1 ku） procaspase-3 was found in normal cells, but a wider procaspase-3 band and a much wider band of 17 kD proteins （p17） ap- peared in the infected cells. Meanwhile, the expression of bcl-2 mRNA was higher and that of fas mRNA was lower in the normal HELF cells, whereas there were significantly lower bcl-2 mRNA and higher fas mRNA expression levels in HCMV infected cells. It was concluded that HCMV was a stronger inducer of apoptosis in HELF cells. Caspase-3, as the marker of undergoing apoptosis, was expressed increasingly and activated in the infected cells, indicating its action in HCMV-inducing apoptosis. Down-regulating bcl-2 mRNA expression and up-regulating fas mRNA expression were also involved in the mechanism of HCMV-induced apoptosis.

Intrinsic host restriction factors of human cytomegalovirus replication and mechanisms of viral escape

Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed "restriction factors"(RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus(HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell's intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16(IFI16)(a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and h Daxx; the latter three being the keys elements of nuclear domain 10(ND10). IFI16 inhibits the synthesis of virus DNA by downregulating UL54 transcription- a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65(p UL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents.

Human Cytomegalovirus Infection Inhibits the Differentiation of Human Hippocampus Neural Precursor Cells into Astrocytes

HCMV is a major cause of congenital brain disease in humans, and its neuropathogenesis is not yet fully understood. The objective of the present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural precursor cell (NPCs) differentiation in vitro. Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. The identification and purity of the NPCs were confirmed by using immunofluorescence to detect the expression of the NPCs marker-Nestin. To drive NPCs differentiation, bFGF and EGF were withdrawn from the medium and replaced with FBS (10%). HCMV AD169 (MOI=5) was added into the differentiation medium at the onset of the differentiation. After 7 days of differentiation, in order to confirm whether NPCs are permissive for HCMV infection, immunofluorescence was used to stain for the presence of immediate early (IE) and late (pp65) HCMV proteins in the infected cells. The effects of HCMV infection on NPCs’ differentiation was observed by detecting the ratio of nestin and GFAP positive cells with confocal microscopy and immunofluorescence. The data showed that 95%±8% of the cells (passage 4-8) cultured were Nestin positive which suggested that majority of the cells were NPCs. On day 7 postinfection, most of the infected cells were IE and PP65 positive. The percentage of Nestin-positive cells were 93%±10% and 50%±19% (t=6.03, p<0.01) and those of GFAP-positive cells were 55±17% and 81%±11% (t=3.77, p<0.01) in HCMV treated and control groups respectively. These findings indicate that NPCs are HCMV permissive cells and HCMV (AD 169) infection suppresses the differentiation of Hippocampus-genetic human NPCs into astrocytes. These effects may provide part of the explanation for the abnormalities in brain development associated with congenital HCMV infection.

Recombinant Human IgG antibodies against Human Cytomegalovirus

Objective To study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus （HCMV） infection. Methods Fab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of VH and VL and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-K-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G. Results SDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein. Conclusion IgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 μg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

Human Cytomegalovirus UL138 Open Reading Frame Is Highly Conserved in Clinical Strains

Objective To investigate the variability of human cytomegalovirus(HCMV) UL138 open reading frame(ORF) in clinical strains.Methods HCMV UL138 ORF was amplified by polymerase chain reaction(PCR) and PCR amplification products were sequenced directly,and the data were analyzed in 19 clinical strains.Results UL138 ORF in all 30 clinical strains was amplified successfully.Compared with that of Toledo strain,the nucleotide and amino acid sequence identities of UL138 ORF in all strains were 97.41% to 99.41% and 98.24% to 99.42%,respectively.All of the nucleotide mutations were substitutions.The spatial structure and post-translational modification sites of UL138 encoded proteins were conserved.The result of phylogenetic tree showed that HCMV UL138 sequence variations were not definitely related with different clinical symptoms.Conclusion HCMV UL138 ORF in clinical strains is high conservation,which might be helpful for UL138 encoded protein to play a role in latent infection of HCMV.

Effect of Human Cytomegalovirus on Invasive Capability of Early Pregnant Extravillous Cytotrophoblasts

The effect of human cytomegalovirus （HCMV） on invasive capability of early pregnant extravillous cytotrophoblasts （EVTs） was investigated in vitro. Primary EVTs were obtained by complex phosphoesterasum digestion and gradient centrifugation from villous tissue aseptically taken from healthy pregnant women. Cytokeratin7 （CK7）, vimentin （Vim） and cerbB-2 were immunocytochemically detected to identify source of cells, and HCMVpp65 antigen was assayed to determine the infection state of primary EVTs by immunocytochemical staining. The EVTs were divided into two groups： control group and HCMV group, and the expression of c-erbB-2, matrix metalloproteinase-2 （MMP-2） and MMP-9 proteins was detected in two groups by immunocytochemistry and Western blotting. Enzymic activity changes of MMP-2 and MMP-9 were tested by gelatin zymography in primary EVTs infected with HCMV. The invasion of primary EVTs was detected by cell invasion assay in vitro after they were infected by HCMV. The cell source identification showed that the cells obtained were highly-pure primary EVTs, and primary EVTs could be infected by HCMV. Primary EVTs could express c-erbB-2, MMP-2 and MMP-9 proteins, and as compared with control group, the protein expression was decreased significantly in HCMV groups （P〈0.05）. Primary EVTs could secrete active MMP-2 and MMP-9 in vitro, and the activity of two MMPs was decreased sig- nificantly in HCMV groups （P〈0.05）. The in vitro cell invasion assay showed that the number of primary EVTs permeating Matrigel in HCMV group was decreased （P〈0.05）. We are led to conclude that HCMV can infect primary EVTs and inhibit their invasion capability, suggesting that the im- paired EVT＇s invasion capability might be related to the abnormal expression of c-erbB-2, MMP-2 and MMP-9 proteins.

SEQUENCE VARIABILITY OF HUMAN CYTOMEGALOVIRUS UL144 OPEN READING FRAME IN LOW-PASSAGE CLINICAL ISOLATES

Objective To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR (qPCR)]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.

Evaluation on Clinical Application of Three Testing Methods for Human Cytomegalovirus Infection in Pregnancy

The value of ELISA, N-PCR and RT-PCR in clinical practice for pregnant women with HCMV infection was investigated. 5581 pregnant women were screened by ELISA. Among them, 100 cases were positive for IgM (group 1). 69 for both IgM and serous DNA (group 2) and 69 for both IgM and mRNA (group 3). The infectious status, maternal-fetal transmission and pregnancy outcome were monitored. It was demonstrated that the accordance rate of group 3 and group 2 with group 1 was 56. 25 % and 43. 75 % , respectively. The maternal-fetal transmission rate in the group 1, 2 and 3 was 19. 00 % , 40. 58 % and 46. 15 %, respectively, with a significant difference found between group 2, 3 and group 1 (P<0. 01). Incidence of spontaneous abortion, fetal death, fetal abnormality and neonatal death in group 1, 2 and 3 was 10. 00 %, 15. 94 % and 30. 77 %, respectively, and that of group 3, 2 was 4 and 2 times as much as that of group 1, respectively (OR = 4. 00, P<0. 001; OR=2. 343, P<005, respectively). It was concluded that HCMV-IgM(+) can only be considered as an screening indicator for pregnant women with HCMV infection, while IgM(+) combined with serous DNA( + ) or mRNA( + ) indicates active infection and has a high incidence of maternal-fetal transmission and abnormal pregnancy outcome.

Seroprevalence of Cytomegalovirus Infection and Associated Risk Factors among Human Immunodeficiency Virus Infected Patients Attending Thika Level 5 Hospital, Kenya

Cytomegalovirus (CMV) is an important pathogen in immunocompromised individuals. Coupled with Human Immunodeficiency Virus (HIV), it causes end organ diseases leading to increased morbidity and mortality in the population. The prevalence of Cytomegalovirus infection is above 93% in HIV infected children in Kenya. Despite, a high Cytomegalovirus seroprevalence found in children, few studies have documented CMV in adults. This study was done to determine the seroprevalence of CMV infection and its associated risk factors among HIV patients attending Thika level 5 Hospital in Kiambu County, Kenya. The study also evaluated the effect CMV infection on the immunity of HIV infected patients. A cross-sectional study involving 163 HIV positive participants from different age groups was carried out. A questionnaire was used to assess the socio-demographic and specific risk factors associated with cytomegalovirus. Blood was collected and analyzed for CD4 counts, CMV IgG and IgM. The seroprevalence of CMV was found to be 89% (CMV IgG) while the incidence was 10.4% (CMV IgM). The study found that CMV infection leads to more suppression of the immunity among the HIV infected patients. In addition, education, economic status, having other sexual transmitted infections, sharing drinks, immune status and blood transfusion were associated with CMV infection (p < 0.05). The study recommends adoption of CMV screening services and education on CMV risk factors as CMV infection preventive strategies.

SEQUENCE CONSERVATION OF HUMAN CYTOMEGALOVIRUS UL140 OPEN READING FRAME IN CLINICAL STRAINS

Objective To investigate the variability of human cytomegalovirus(HCMV)UL140 open reading frame(ORF)in clinical strains,and to explore the relationship between the variability of UL140 ORF and different symptoms of HCMV infection.Methods HCMV UL140 ORF was amplified by polymerase chain reaction and sequenced selectedly in 30 clinical strains.Results UL140 ORF of all clinical strains was amplified successfully.Compared with that of Toledo strain,the nucleotide and amino acid sequence identities among all strains were 96.5%-100.0% and 95.2%-100.0%,respectively.All of the nucleotide changes were substitutions.The post-translational modification sites were conserved.The result of phylogenetic tree showed that the strains did not cluster according to different clinical symptoms.Conclusion HCMV UL140 ORF in clinical strains is highly conserved,which may play an important role in HCMV infection.

HIGH VARIABILITY OF HUMAN CYTOMEGALOVIRUS UL150 OPEN READING FRAME IN LOW-PASSAGED CLINICAL ISOLATES

Objective To investigate the polymorphism of human cytomegalovirus (HCMV) UL150 open reading frame (ORF) in low-passaged clinical isolates, and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection. Methods PCR was performed to amplify the entire HCMV UL150 ORF region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA using fluorescence quantitative PCR. PCR amplification products were sequenced directly, and the data were analyzed. Results Totally 25 among 29 isolates were amplified, and 18 isolates were sequenced successfully. HCMV UL150 ORF sequences derived from congenitally infected infants were high variability. The UL150 ORF in all 18 clinical isolates shifted backward by 8 nucleotides leading to frame-shift, and contained a single nucleotide deletion at nucleotide position 226 compared with that of Toledo strain. The nucleotide diversity was 0.1% to 6.8% and the amino acid diversity was 0.2% to 19.2% related to Toledo strain. However, the nucleotide diversity was 0.1% to 6.4% and amino acid diversity was 0.2% to 8.3% by compared with Merlin strain. Compared with Toledo, 4 new cysteine residues and 13 additional posttranslational modification sites were observed in UL150 putative proteins of clinical isolates. Moreover, the UL150 putative protein contained an additional transmembrane helix at position of 4-17 amino acid related to Toledo. Conclusion HCMV UL150 ORF and deduced amino acid sequences of clinical strains are hypervariability. No obvious linkage between the polymorphism and different pathogenesis of congenital HCMV infection is found.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Identification of human cytomegalovirus phosphoprotein 65 in C57BL/6 and BXSB mice as a potential trigger of systemic lupus erythematosus related serum markers

Objective:To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65(HCMV-pp65) in murine systemic lupus erythematosus(SLE).Methods:The prokaryotic plasmid pET-28b-pp65 was constructed to express the HCMVpp65 protein.BXSB mice and C57BL/6 mice were inoculated with pp65 eukarvotic plasmid pcDNA3.0-pp65 intramuscularly 5 times at 2-week intervals,and then the blood of the mice was subsequently collected via the retro-orbital vein.Indirect ELISAs were used to evaluate the concentration of anti-pp65 immunoglobulin G,anti-double-stranded DNA and antinuclear antibodies.lnterleukin-1β and tumor necrosis factor-α were also determined by competitive ELISA.At the same time,3 major SLE-related circulating microRNAs were examined by quantitative RT-PCR.Results:The early production of autoantibodies was observed in pp65-immunized male BXSB as well as C57BL/6 mice.Overexpression of interleukin-1β and tumor necrosis factor-a were detected in pp65-immunized male BXSB mice.Quantitative RT-PCR analyses showed that three SLE related microRNAs(microRNA-126,microRNA-125 a,and microRKA-146a) were dovvnrcgulatcd in peripheral blood mononuclear cells of pp65-immunizcd mice.Conclusions:Our findings indicate that HCMV-pp65 immunization strongly triggers the development and progression of" SLE-like disease in both BXSB and C57BL/6 mice,which indicates that the immune responses induced by HCMV-pp65 may be involved in the development of SLE.