Study on thedistribution of high titer neutralizing antibody against human cytomegalovirus in plasma donors / 中国输血杂志

【Objective】 To investigate the distribution of plasma donors with high titer neutralizing antibodies against human cytomegalovirus (HCMV) in the general plasma donor population. 【Methods】 920 plasma samples of Taibang were tested in April 2014 to investigate the distribution of anti-HCMV neutralizing antibodies. After further testing of mixed plasma, the threshold for screening plasma was determined. From October 2019 to May 2020, neutralizing anti-HCMV in 40 078 plasma samples from 11 plasma stations in Shandong province were screened by the microcytopathic method (modified high-flux neutralization test method). The proportion of neutralizing anti-HCMV enriched in high titer and the distribution in the donor population were analyzed by SPSS 26 and Minitab19 analysis software. 【Results】 Among 920 samples, 73.26%, 0.43%, and 8.69% of them had neutralization titer<1∶15, ≥1∶60 and ≥1∶30, respectively. The neutralization titer of mixed plasma was detected, and 1∶30 was determined as the high titer. The yielding rate of high titer neutralizing anti-HCMV in Shandong was 9.06% (3 633/40 078). The proportion of plasma donors with high-titer neutralizing anti-HCMV in the donation population from plasma stations was 4.95%~13.03% (9.06±2.07) %. The proportion of plasma donors with high-titer neutralizing anti-HCMV by gender was 15.67% (2 185/13 951) in women and 5.54% (1 448/26 127) in men(P<0.05). 【Conclusion】 There was a certain proportion of plasma donors wiht high titer neutralizing anti-HCMV in the population of plasma donors in Shandong, and they can constantly serve neutralizing anti-HCMV to ensure the production of anti-HCMV immunoglobulin preparations.

Major molecular events of reactivation of human cytomegalovirus after allogeneic hematopoietic stem cell transplantation / 器官移植

Immune deficiency of the host caused by allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the initial factor of reactivation of latent human cytomegalovirus (HCMV). The risk factors of reactivation of HCMV in allo-HSCT recipients consist of the serological status of HCMV in donors and recipients, the matching degree of human leukocyte antigen (HLA) and pretreatment patterns, etc. The reactivation of HCMV is associated with the expression of a series of viral cleavage and proliferation proteins induced by the overexpression of major immediate early promoter/enhancer (MIEP) in the viral genome. In this article, the risk factors of reactivation of HCMV after allo-HSCT, the molecular changes related to maintaining latent infection of HCMV, the key role of MIEP overexpression in reactivation of HCMV, and the molecular pathways involved in reactivation of HCMV after allo-HSCT were reviewed and the major molecular events of reactivation of HCMV after allo-HSCT were elucidated, aiming to provide reference for the prevention and treatment of cytomegaloviral disease (CMVD) after allo-HSCT.

Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.

Human cytomegalovirus-encoded miR-US4-1 promotes cell apoptosis and benefits discharge of infectious virus particles by targeting QARS.

Human cytomegalovirus (HCMV) can cause congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behaviour. Identification of functional target genes of miRNAs is an important step in studies on HCMV pathogenesis. In this study, Glutaminyl-tRNA Synthetase (QARS), which could regulate signal transduction pathways for cellular apoptosis, was identified as a direct target of hcmv-miR-US4-1. Apoptosis assay revealed that as silence of QARS by ectopic expression of hcmv-miR-US4-1 and specific small interference RNA of QARS can promote cell apoptosis in HCMV-infected HELF cells. Moreover, viral growth curve assays showed that hcmv-miR-US4-1 benefits the discharge of infectious virus particles. However, silence of hcmv-miR-US4-1 by its specific inhibitor overturned these effects. These results imply that hcmv-miR-US4-1 might have the same effects during HCMV nature infection. In general, hcmv-miR-US4-1 may involve in promoting cell apoptosis and benefiting discharge of infectious virus particles via down-regulation of QARS in HCMV-infected HELF cells.

Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis.

Prevalence of Erythrocytopenia & Thrombocytopenia in Eastern Indian Population & its Correlation with Acute Antibody Mediated Renal Transplant Rejection & Cytomegaloviral Nephropathy.

Objective: Our objective was to study the prevalence of thrombocytopenia & erythrocytopenia post renal transplantation in Eastern Indian population. Thrombocytopenia & erythrocytopenia are common phenomena prevailing post transplant rejection. It is found to occur in patients developing HCMV (human cytomegaloviral nephropathy), post transplantation. Whereas in the case of none rejected patients thrombocytopenia is unlikely to occur. Methods: Several methodologies such as the serological cross match, HLA-cross match DSA luminex, renal biopsy including C4d staining, hematological RBC & platelet count monitoring were adopted in the study, along with these the serum creatinine levels of the rejected patients were tested by making use of several biochemical approaches. Statistical analysis of the data obtained from the laboratories, were also done by the software prism 4.1. Results: Out of total population of 30 patients selected randomly, 24 were found to have successful transplants as for them a significant increase in both platelet & RBC count were noticed post transplantation where as for the other 6 patients a significant decrease in the RBC & platelet count was observed post transplantation along with a significant increase in the serum creatinine levels. There was also a significant decrease in the GFR (glomerular filtration rate) which was an indication of some sort of graft dysfunctioning. All the patients were checked for viral nephropathy & the above 6 patients were found to develop HCMV nephropathy. For the above mentioned 6 patients, the presence of C4d marker in their renal peritubular capillaries after performing the immunehistochemical C4d staining was a key indicator of acute antibody mediated rejection. Conclusion: Our study clearly reveals that Thrombocytopenia & erythrocytopenia was quite common in the patients with acute antibody mediated renal transplant rejection. A low RBC & platelet count persisted in them even after transplantation owing to allograft rejection, & HCMV nephropathy. Whereas for the patients with successful transplants events such as thrombocytopenia & erythrocytopenia were nevertheless unlikely.

Late emergence of A594V and L595W mutations related to ganciclovir resistance in a patient with HCMV retinitis and long-term HIV progression

The emergence of ganciclovir (GCV) resistance during the treatment of human cytomegalovirus (HCMV) infection is a serious clinical challenge, and is associated with high morbidity and mortality. In this case report, we describe the emergence of two consecutive mutations (A594V and L595W) related to GCV resistance in a patient with HCMV retinitis and long-term HIV progression after approximately 240 days of GCV use. Following the diagnosis of retinitis, the introduction of GCV did not result in viral load reduction. The detected mutations appeared late in the treatment, and we propose that other factors (high initial HCMV load, previous GCV exposure, low CD4+ cell count), in addition to the presence of resistance mutations, may have contributed to the treatment failure of HCMV infection in this patient.

Comparison of conventional, immunological and molecular techniques for the diagnosis of symptomatic congenital human cytomegalovirus infection in neonates and infants.

Purpose: Human cytomegalovirus (HCMV) is the commonest pathogen causing congenital infection globally. The diagnosis of congenital infection is based either on viral isolation (in cell culture) or demonstration of HCMV DNA from the urine. Saliva is also being used as an alternative sample to urine for the same. The objective of this study was to compare the following assays-polymerase chain reaction (PCR) from urine, saliva and blood, serology (anti-HCMV IgM) and antigen detection (HCMV pp65 antigenaemia) for the diagnosis of congenital HCMV infection. Materials and Methods: Urine and blood samples were collected from 31 infants (median age: 13 weeks) with suspected HCMV infection. For 18 infants, additional saliva samples were collected and all the above assays were compared. Results: PCR for HCMV DNA from urine and anti-HCMV IgM were performed for all 31 infants. Of these, 22 (70.9%) were positive for both assays. In 18 (of the 22) infants positive by both assays, PCR for HCMV DNA from saliva was positive in all 18 (100%), PCR from blood in 7/18 (38.8%) and HCMV pp65 antigenaemia only in 1/18 (5.5%) of the infants. Conclusion: Detection of HCMV DNA in urine combined with anti-HCMV IgM are suitable assays to diagnose HCMV infection in infants. Both PCR from the blood and HCMV pp65 antigenaemia lack sensitivity in infants. Salivary PCR combines convenience with high sensitivity and can substitute PCR from urine, especially in the outpatient and fi eld settings. To the best of our knowledge, this is the fi rst study from India to evaluate salivary PCR for the diagnosis of congenital HCMV infection.

Investigation of Human Cytomegalovirus and Human T Lymphotropic Virus Infection of Voluntary Blood Donors in Beijing Area / 现代检验医学杂志

Objective To analysis the human cytomegalovirus (HCMV)and human T lymphotropic virus(HTLV)infection status in Beijing among voluntary blood donors.Methods Randomly selected 2 010 blood samples from five districts and counties of Beijing City to screen HCMV-IgG,HCMV-IgM and HTLV-Ⅰ/Ⅱ antibody by ELISA method.The positive samples were reexamined two times,two test results of samples were positive that were determined positive by ELISA. HTLV positive samples was confirmed by nested PCR.Results The HCMV-IgM and HCMV-IgG positive rates of Beijing blood donors were 2.19% and 92.59%,screened 1 case of anti-HTLV positive by ELISA method,then confirmed to be neg-ative result by nested PCR.The statistics showed that the HCMV-IgG positive rate female blood donors was higher than male (P 0.05).Conclusion In this investigation,2 010 cases of voluntary blood donors from five districts of Beijing were not found in cases of HTLV infection,HCMV infection was prevalent.

Analysis of HCMV gN genotypes in patients with HCMV-HIV co-infection / 中华微生物学和免疫学杂志

Objective To analyze the distribution of various genotypes of human cytomegalovirus glycoprotein N ( HCMV gN) in patients with HIV infection; to investigate the effects of HCMV-HIV co-in-fection on disease progression and the relationships between HCMV gN genotypes and disease progression. Methods Patients with active HCMV infection were screened out from 359 patients with HIV infection by using the pp65 antigenemia assay.The genes encoding HCMV gN ( UL73 ) were amplified by nested PCR ( nPCR) .The amplicons were digested by restriction enzymes including MboⅠ, ScaⅠ and SalⅠ.Then, the restricted fragment length polymorphisms were further analyzed on 4%agarose gel.The relationships be-tween HCMV genotypes and the morbidity and mortality of acquired immune deficiency syndrome ( AIDS ) were investigated via a prospective study.Results Among the 359 patients with HIV infection, 28 subjects were positive for the HCMV pp65 antigenemia assay.The HCMV gN genotypes in 20 patients with active HCMV infection were distributed as: gN-3a (4/20, 20%), gN-1 (4/20, 20%), gN-4d (1/20, 5%), gN-4b (1/20, 5%) and mixed infection (10/20, 50%).Patients with HCMV-HIV co-infection were more likely to develop AIDS during the follow-up period (RR=9.78).Patients harboring HCMV gN-1 and gN-4 genotypes would seem likely to have 4.6 times of chance leading to AIDS-associated death than those harbo-ring other HCMV gN genotypes.Conclusion HCMV infection ( especially gN-1 and gN-4 genotypes) might accelerate the progression of HIV infection.

Down-regulation of human cytomegalovirus UL138, a novel latency-associated determinant, by hcmv-miR-UL36.

MicroRNAs (miRNAs) are small RNAs, 19–23 nucleotides in length, which regulate a variety of cellular processes. Human cytomegalovirus (HCMV) encodes only one intronic miRNA: human cytomegalovirus microRNA UL36 (hcmv-miR-UL36). In this study, we found that over-expression of hcmv-miR-UL36 resulted in a more than threefold increase in HCMV DNA synthesis at 24 h post infection. Fifteen putative targets of hcmv-miR-UL36 were identified using hybrid PCR, one being the HCMV UL138 gene that has previously been identified as a novel latency-associated determinant of HCMV infection. Down-regulation of UL138 expression by hcmv-miR-UL36 was validated using luciferase reporter assays and Western blot analysis in HEK293 cells. In the presence of hcmv-miR-UL36, we observed a 74.6% decrease in luciferase activity and a 46.2% decrease in HCMV UL138 protein expression. Our results indicate that hcmv-miR-UL36 may be a viral miRNA contributing to HCMV replication.

The prevalence of human cytomegalovirus DNA in gliomas of Brazilian patients

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Possible Roles of UL112-113 Proteins in Human Cytomegalovirus DNA Replication

DNA replication of human cytomegalovirus (HCMV) is a highly regulated process that requires specific interactions between cis-acting lytic origin of replication (oriLyt) and trans-acting viral proteins. Formation of the replication initiation complex is also regulated by specific interactions among viral replication proteins. HCMV replication proteins include origin-binding proteins, core proteins that work in replication forks, and regulatory proteins that modulate host cell functions. This letter describes intriguing questions regarding how HCMV origin-binding proteins interact with oriLyt to initiate DNA replication and how the regulatory UL112-113 proteins, which are found only in beta-herpesviruses, function to promote viral DNA replication.

The induction of systemic lupus erythematosus by human cytomegalovirus pp65 / 中华微生物学和免疫学杂志

Objective To investigate the potential role of human cytomegalovirus lower matrix phosphoprotein 65 (HCMV pp65) in murine systemic lupus erythematosus (SLE).Methods The prokaryotic vector pET-28b and eukaryotic vector pcDNA 3.0 were constructed to express the HCMV pp65 protein.All the C57BL/6 mice were inoculated with pp65 eukaryotic vector intramuscularly five times at 2-week intervals and then were bled via the retro-orbital vein.Subsequently,indirect ELISA was used to evaluate the concentration of anti-pp65 IgG,anti-dsDNA and ANA.At the same time,IL-1 b,IL-6,and TNF-α were determined by competitive ELISA.Results The early onset of autoantibodies and an overexpression of IL-6 were observed in immunized male C57BL/6 mice.Conclusion HCMV pp65 triggers the deregulation of humoral immunity in C57BL/6 mice,which indicates that the immune responses induced by HCMV pp65 may be involved in the development of SLE.

Interaction between human cytomegalovirus UL136 protein and ATP1B1 protein

Interplay between the host and human cytomegalovirus (HCMV) has a pivotal role in the outcome of infection. A region (referred to as UL/b’) present in the Toledo strain of HCMV and low passage clinical isolates contains 19 additional genes, which are absent in the highly passaged laboratory strain AD169. Products of the UL/b’ genes may determine the manifestations of HCMV infection in vivo. However, little is known about the host factors, which interact with UL/b’ proteins. This study was conducted to investigate the function of the HCMV UL136 protein. By yeast two-hybrid screening, the β1 subunit of the host Na+/K+-ATPase (ATP1B1) was identified to be a candidate protein, which interacts with the HCMV UL136 protein. The interaction was further evaluated both in vitro by pull-down assay and in vivo by immunofluorescent co-localization. The results showed that the UL136 protein can interact with ATP1B1 in vitro. Co-localization of UL136-EGFP and ATP1B1-DsRed in cell membranes suggests that ATP1B1 was a partner of the UL136 protein. It can be proposed that the HCMV UL136 protein may have important roles in processes such as cell-to-cell spread, and in maintaining cell osmotic pressure and intracellular ion homeostasis during HCMV infection.

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro

BACKGROUND: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. METHODS: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining. RESULTS: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. CONCLUSION: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library / 中华微生物学和免疫学杂志

Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.

Cytomegalovirus DNA dynamic monitoring on prophylaxis of human cytomegalovirus pneumonia after renal transplantation / 中华泌尿外科杂志

Objective To discuss the clinical value of dynamic monitoring the copies of human cytomegalovirus(HCMV)-DNA in prophylaxis of HCMV pneumonia after renal transplantation.Methods There were 242 cadaveric renal transplantation recipients including 144 males and 98 females,with the average age of 41(from 17 to 71).They were divided into 2 groups(experimental group 127 cases,control group 115 cases).Recipients in experimental group were routinely monitored by blood preparation and urine aliquot FQ-PCR.The therapy was initiated when HCMC-DNA>1×103 copies/ml by blood preparation and/or urine aliquot FQ-PCR with intravenous ganciclovir for 4 weeks.The dosage was calculated according to creatinine clearance rate.FQ-PCR monitoring and Preemptive therapy was not performed in the control group.The pneumonia rate, death rate and survival between the two groups were compared. Results In experimental group, the HCMV pneumonia incidence rate was 6.3 % (8/127), onset time was 46-167 d, median time was 84 d, hospitalization time was 30-57 d,median time was 36 d, death rate was 12.5 % (1/8), breathing machine using rate was 12.5 % (1/8),concurrent other pathogen infection rate was 25 % (2/8), and + year renal graft survival rate was 98.4% (125/127).One was dead with graft function and the other dysfunction was because of acute rejection.In control group, the HCMV pneumonia incidence rate was 14.8%(17/115), onset time was 34-138 d,median time was 51 d, hospitalization time was 21-67 d,median time was 40 d,breathing machine using rate was 29.4% (5/17),concurrent other pathogen infection rate was 41.2%(7/17), death rate was 23.5% (4/17), and 1 year renal graft survival rate was 93.0% (107/115).Three was dead with graft function and the other one was dead of DGF.The other 4 cases of renal dysfunction were because of acute rejection.Significant difference existed between the 2 groups (P<0.05) except for hospitalization time (P> 0.05). Conclusion The preemptive therapy of CMV pneumonia after renal transplantation by dynamic monitoring the copies of HCMV-DNA in recipients could have a good effect, and the 1 year renal graft survival rate could be higher.

HCMV Infection Depress NGF Expression in Human Glioma Cells / 中国病毒学·英文版

Human cytomegalovirus (HCMV) is the most common cause of congenital infection, resulting in birth defects such as microcephaly. In this study, RT-PCR and Western Blotting were performed to quantify the regulation of endogenic nerve growth factor expression in neuroglia cells by HCMV infection. The results showed that basal, endogenous NGF expression in U251 was unchanged during early HCMV infection. NGF expression is strongly down-regulated during the latent phase of infection. These results suggest that HCMV can depress the NGF expression in U251 cells.

Human Cytomegalovirus Induces Intercellular Adhesion Molecule-1 Expression in a Monocytic Cell Line, THP-1

It has been reported that inflammatory diseases such as pneumonitis, retinitis, and hepatitis are associated with human cytomegalovirus (HCMV). Intercellular adhesion molecule (ICAM)-1 is an important inflammatory mediator, helping monocytes adhere to endothelial cells when tissues are infected by pathogen including the HCMV. However, little is known about the mechanism of ICAM-1 stimulation by the HCMV infection in monocytes. In this study, a monocytic cell line THP-1 was used to understand ICAM-1 expression by the HCMV infection. Flow cytometric analyses demonstrated that ICAM-1 was stimulated by the HCMV in THP-1 cells with maximum at 24 hours post infection. The stimulated ICAM-1 expression was dependent on the amount of input virus. In order to understand the mechanism of ICAM-1 stimulation during the HCMV infection, cells were treated with specific inhibitors of key elements in inflammation: NF-kappaB inhibitor PDTC, cyclooxygenase 2 inhibitor NS398, and MEK inhibitor PD98059. Flow cytometric analyses revealed that ICAM-1 expression was decreased when treated with PDTC, but not with NS398 or PD98059. Thus, it is suggested that HCMV-induced ICAM-1 expression in THP-1 cells is associates with NF-kappaB.