The expression of interleukin-32 is activated by human cytomegalovirus infection and down regulated by hcmv-miR-UL112-1.

BACKGROUND: Interleukin-32 (IL-32) is an important factor in innate and adaptive immune responses, which activates the p38MAPK, NF-kappa B and AP-1 signaling pathways. Recent reports have highlighted that IL-32 is regulated during viral infection in humans. METHODS: Enzyme-linked immunosorbent assays (ELISA) were carried out to detect IL-32 levels in serum samples. Detailed kinetics of the transcription of IL-32 mRNA and expression of IL-32 protein during human cytomegalovirus (HCMV) infection were determined by semi-quantitative RT-PCR and western blot, respectively. The expression levels of hcmv-miR-UL112-1 were detected using TaqMan® miRNA assays during a time course of 96 hours. The effects of hcmv-miR-UL112-1 on IL-32 expression were demonstrated by luciferase assay and western blot, respectively. RESULTS: Serum levels of IL-32 in HCMV-IgM positive patients (indicating an active HCMV infection) were significantly higher than those in HCMV-IgM negative controls. HCMV infection activated cellular IL-32 transcription mainly in the immediately early (IE) phase and elevated IL-32 protein levels between 6 and 72 hours post infection (hpi) in the human embryonic lung fibroblast cell line, MRC-5. The expression of hcmv-miR-UL112-1 was detected at 24 hpi and increased gradually as the HCMV-infection process was prolonged. In addition, it was demonstrated that hcmv-miR-UL112-1 targets a sequence in the IL-32 3'-UTR. The protein level of IL-32 in HEK293 cells could be functionally down-regulated by transfected hcmv-miR-UL112-1. CONCLUSIONS: IL-32 expression was induced by active HCMV infection and could be functionally down-regulated by ectopically expressed hcmv-miR-UL112-1. Our data may indicate a new strategy of immune evasion by HCMV through post-transcriptional regulation.

Transcription characteristics of the human cytomegalovirus UL13 gene.

The human cytomegalovirus (HCMV) UL13 gene is located in the unique long (UL) region of its genome. The transcript structure of UL13 gene has not been investigated to date. By using cDNA library screening, northern blot, and rapid amplification of cDNA ends (RACE), the HCMV UL13 gene was demonstrated to be transcribed from the immediate early (IE) to the late (L) phase of infection, and at least one 1602-nt unspliced transcript was identified in the present study from three clinical isolates.

Transcriptional features and transcript structure of UL145 in different strains of human cytomegalovirus.

The human cytomegalovirus UL145 gene is located between the highly variable genes UL144 and UL146 in the UL/b' region. However, unlike its neighboring genes, UL145 is relatively conserved among strains isolated from patients. The transcriptional features and transcript structure of UL145 has not yet been examined. In this study, the transcriptional features and structure of UL145 were characterized using RNA preparations from three HCMV strains isolated from patients, designated H, C, and X, respectively. Two transcripts were identified by cDNA library screening. Two main clusters of transcripts, one located between 500 and 700 nt, and the other between 1,400 and 1,700 nt, were confirmed by Northern blot analysis. Abundant transcripts were detected at 96 h post-infection by Northern blot, suggesting that UL145 was a late gene. The terminal sequences of transcripts obtained by 3' rapid amplification of cDNA ends demonstrated the presence of polyadenylation. Transcripts identified in the RNA of H, C, and X strains 96 h post-infection had the same 3' end but different 5' ends. In addition to polycistronic transcripts with upstream genes, UL145 could be transcribed as a single transcript during late strain infections.

A rapid method to screen putative mRNA targets of any known microRNA.

BACKGROUND: microRNAs (miRNAs) are a group of regulatory RNAs that regulate gene expression by binding to specific sequences on target mRNAs. However, functional identification of mRNA targets is usually difficult and time consuming. Here we report hybrid-PCR as a new and rapid approach to screen putative mRNA targets in vitro. RESULTS: Fifteen putative target mRNAs for human cytomegalovirus (HCMV) miR-UL112-1, including previously confirmed HCMV IE72, were identified from mRNA-derived cDNAs using hybrid-PCR. Moreover, we randomly validated six different target candidates by luciferase reporter assays, and confirmed that their luciferase activities were down-regulated with co-transfection of HCMV miR-UL112-1. CONCLUSIONS: Our study demonstrated that hybrid-PCR is an effective and rapid approach for screening putative miRNA targets, with much more advantage of simplicity, low cost, and ease of implementation.

Characterization of the transcripts of human cytomegalovirus UL144.

BACKGROUND: The genome of human cytomegalovirus (HCMV) has been studied extensively, particularly in the UL/b' region. In this study, transcripts of one of the UL/b' genes, UL144, were identified in 3 HCMV isolates obtained from urine samples of congenitally infected infants. METHODS: Northern blot hybridization, cDNA library screening, and RACE-PCR were used. RESULTS: We identified at least 4 differentially regulated 3'-coterminal transcripts of UL144 in infected cells of 1,300, 1,600, 1,700, and 3,500 nucleotides (nt). The 1600 nt transcript was the major form of UL144 mRNA. The largest transcript initiated from the region within the UL141 open reading frame (ORF) and included UL141, UL142, UL143, UL144, and UL145 ORFs. CONCLUSIONS: These findings reveal the complex nature of the transcription of the UL144 gene in clinical isolates.

An antisense transcript in the human cytomegalovirus UL87 gene region.

BACKGROUND: Rapid advances in research on antisense transcripts are gradually changing our comprehension of genomic and gene expression aspects of the Herpesviridae. One such herpesvirus is the human cytomegalovirus (HCMV). Although transcription of the HCMV UL87 gene has not been specifically investigated, cDNA clones of UL87 antisense transcripts were found in HCMV cDNA libraries previously. In this study, the transcription of the UL87 antisense strand was investigated in three clinically isolated HCMV strains. RESULTS: First, an 800 nucleotides transcript having an antisense orientation to the UL87 gene was found in a late HCMV cDNA library. Then, the UL87 antisense transcript was confirmed by Rapid amplification of cDNA ends (RACE) and Northern blot in three HCMV clinical strains. Two ORFs were predicted in the antisense transcript. The putative protein of ORF 1 showed a high degree of conservation among HCMV and other CMV strains. CONCLUSION: An 800nt antisense transcript in the UL87 gene region exists in HCMV clinical strains.

Characterization of 3' termini of human cytomegalovirus UL138-UL145 transcripts in a clinical strain.

The functions of some proteins encoded by human cytomegalovirus (HCMV) UL/b' genes have been studied; however, systematic analysis of the transcripts for this region is still insufficient. The results of both rapid amplification of cDNA ends (RACE) and cDNA library screening in this study proved that 3' termini of all transcripts in the UL138-UL145 region were located approximately 20 bp downstream from each potential poly (A) signal, which were at the positions of nucleotides 7184, 9954 and 12848 in the UL/b' sequence of the H strain, respectively. Thus, there were at least two large families of polycistronic transcripts in this gene region. The first family of 3'-coterminal transcripts contained UL139, UL140 and UL141 genes, and the second one consisted of UL142, UL143, UL144 and UL145 genes. The 3'-coterminal characterization further confirmed that multiple uses of polyadenylation signals were commonly used by HCMV to utilize genetic information.

Human cytomegalovirus RL13 gene transcripts in a clinical strain.

Human cytomegalovirus (HCMV) RL13 gene is a member of the RL11 gene family. To study the expression kinetics and transcript structures of the gene, screening of cDNA library, Northern blot, 3' and 5' RACE analyses were performed with a low-passaged clinical strain. The results showed that the RL13 gene was mainly transcribed in the late expression phase with at least seven forms of transcripts of 3628, 3114, 2515-2443, 1737, 1240, 1029 and 846 nt, respectively. All these transcripts were unspliced with an identical 3' terminal and the same typical polyA signal "AATAAA". Except for the transcript of 846 nt, RL13 open reading frames (ORFs) were 909 bp and completely identical in all of the transcripts. The sequence of the RL13 ORF in the examined clinical strain had a higher similarity with that of the UL153 ORF in the Towne strain than those of RL13 ORF in any other HCMV strains.

Prevalence and type distribution of human papillomavirus in women with cervical lesions in Liaoning Province, China.

INSTRUCTION: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. The distribution and prevalence of HPV genotypes depend on the geographic region and on demographic factors. METHODS: This study aimed to investigate the prevalence and distribution of HPV genotypes in uterine cervical lesions in Liaoning Province, China. A total of 1444 cervical swabs from patients with cervical cancer (CC, n = 134), cervical intraepithelial neoplasia (CIN) II/III (n = 517), and CIN I (n = 180) were detected for HPV genotypes using the PGMY09/11 primer system and HPV GenoArray test (HybriBio Ltd., Hong Kong). Age-matched samples of 613 women without cervical neoplasia were analyzed as control. RESULTS: The prevalence of HPV was 82.84% in CC, 89.56% in CIN II/III, 70.56% in CIN I, and 44.70% in control. The 5 leading genotypes in CIN II/III were, in descending order of prevalence, HPV types 16 (61.12%), 58 (14.12%), 33 (13.93%), 31 (8.32%), and 52(6.27%); whereas HPV types 16 (73.13%), 18 (7.46%), 58 (3.73%), and 31/33/39 (all were 2.24%) were in CC. Multiple HPV infections comprising 2 to 5 types were found in 17.59% of the patients. Human papillomavirus 16 was the predominant genotype in all categories. The prevalence of both HPV type 16 and single HPV infection increased with the severity of cervical lesions (P = 0.000). CONCLUSIONS: The efficacy of the prophylactic vaccine against types 16 and 18 for preventing cervical cancer would be close to 80% in Liaoning Province, China. Human papillomavirus types 16, 18, 58, 33, and 31 may be predominant high-risk factors for CC and its precursors in this region.

Diagnostic value of HCMV pp65 antigen detection by FCA for symptomatic and asymptomatic infection: compared to quantification of HCMV DNA and detection of IgM antibody in infants.

Human cytomegalovirus (HCMV) can cause symptomatic or asymptomatic infection in infants. One hundred and twenty-six infants were assessed clinically for disease in infantile period. Eighty of them were classified as symptomatic infection on the basis of physical, instrumental, and laboratory findings, 5 were demonstrated by following up to have later developed HCMV disease, and the other 41 infants were classified as asymptomatic infection. HCMV DNA was positive in all urine samples of the symptomatic infants detected by quantitative polymerase chain reaction. HCMV-IgM antibody detected by chemiluminescent immunoassay (CLIA) was positive in 62 of the 85 symptomatic infants, but was negative in all of the samples of asymptomatic infants. HCMV pp65 antigen detected by flow cytometry assay (FCA) was positive in 77 of the 85 symptomatic infants and in none of the asymptomatic infants. The coincidence to symptom of HCMV pp65 antigen detection was higher than those of HCMV DNA and HCMV-IgM antibody detection. The sensitivity, specificity, positive prognostic value and the negative prognostic value of HCMV pp65 antigen detection for diagnosis of HCMV infection was 90.6, 100, 100 and 83.7%, respectively. We concluded that detection of pp65 antigen by FCA is more sensitive for diagnosis of HCMV infection than detection of HCMV-IgM antibody and is better than HCMV DNA quantification for distinguishing the symptomatic and asymptomatic HCMV infection in infants.

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.

[The investigation of the relationship between the phenotypes of 46, XX males and the SRY gene].

OBJECTIVE: To investigate the relationship between the phenotypes in XX male patients and the sex determining region(SRY) gene. METHODS: Multiple polymerase chain reactions were carried out in 6 male patients with karyotype of 46, XX, and then the PCR products were sequenced directly. RESULTS: Three cases of male infertility were positive for the SRY gene without evident malformation in their extra genitalia, while 3 cases with testes were negative for the SRY gene, with evident malformation in their extra genitalia. CONCLUSION: The SRY gene is key in sex determination and development, yet there might be other important genes involved.

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 HC-MV 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 HC-MV infection.

MeCP2 gene mutation analysis in autistic boys with developmental regression.

Autism and Rett syndrome are both pervasive developmental disorders and share many characteristics in common. One of these features is developmental regression with loss of social, cognitive and language skills after a period of apparently normal development during the first 1-2 years of life, which raises the question of whether there is a common pathway underlying regression in these two disorders. The Rett syndrome gene was identified as MeCP2 gene on Xq28, a powerful transcriptional repressor. To explore its possible role in the etiology of autism and involvement in regression, we searched for MeCP2 gene mutations in a well characterized sample of 31 autistic boys with developmental regression by direct sequencing. One sequence variant in 3' untranslated region was observed. The patient inherited the variant from his unaffected mother, so it may be a rare polymorphism. No coding sequence variant was found in any of the patients tested. We conclude that mutations in the coding sequence of MeCP2 are not a frequent cause of regression in autism. The long 3' untranslated region of MeCP2 is highly conserved across species, suggesting that they are important for the post-transcriptional regulation of MeCP2 gene. It may be worthwhile extending the mutation screening, with a larger sample of strictly defined phenotype, to regulatory elements and untranslated regions of this gene, to explore to what degree MeCP2 gene is involved in the etiology of autism and its possible role in the regression of autism.

Human cytomegalovirus UL145 gene is highly conserved among clinical strains.

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b') present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes,which are absent in the highly passaged laboratory strain AD169. One of these genes,UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene,the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9 -100% and 96.6-100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved,comprising the protein kinase C phosphorylation motif (PKC)and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.

Genetic polymorphism of UL144 open reading frame of human cytomegalovirus DNA detected in colon samples from infants with Hirschsprung's disease.

AIM: To explore the genetic diversities of UL144 open reading frame (ORF) of cytomegalovirus DNA detected in colon tissue from infants with Hirschsprung's disease (HD) by sequencing UL144 DNA in 23 aganglionic colon tissue and 4 urine samples from 25 HD infants. METHODS: Nest PCR was performed for amplification of the UL144 gene. The UL144 gene was analyzed with softwares, such as DNAclub, BioEdit, PROSITE database, and DNAstar. RESULTS: The strains from HD patients were distributed among three genotypes of UL144: group 1A (64%), group 2 (24%), and group 3 (12%). The UL144 genotypes between strains from HD and control group were compared by chi square test (c2 = 1.870, P = 0.393). Strains from the colon were sporadically distributed in UL144 genotypes. CONCLUSION: There are genetic diversities of UL144 ORF in colon tissue of infants with HD. However, cytomegalovirus UL144 genotypes are not associated with clinical manifestations of HD.

[Polymorphism of human cytomegalovirus UL134 gene in low-passage clinical isolates].

OBJECTIVE: Human cytomegalovirus (HCMV) displays genetic polymorphisms. Nineteen open reading frames (ORFs, UL133-UL151) found in the Toledo strain of HCMV and other low-passage clinical isolates may be essential for viral infection. This study aimed to analyze the polymorphism of HCMV UL134 gene in clinical isolates and explore the relationship between the polymorphism and HCMV infection. METHODS: PCR was performed to amplify entire UL134 region in 32 clinical isolates, which had been proven as HCMV-DNA positive by FQ-PCR. PCR products were sequenced. RESULTS: All of the 32 isolates were amplified and sequenced successfully. HCMV UL134 gene was highly conserved in the clinical isolates. UL134 ORF and its predicted protein in the clinical strains displayed 96.4%-98.3% nucleotide identity and 92.7%-94.9% amino acid identity respectively compared to those in the Toledo strain. A new posttranslational modification site, sulfationcamp (SUL) site, was found in UL134 protein of all of the clinical isolates except 35j. CONCLUSIONS: HCMV UL134 gene in clinical isolates was highly conserved. No substantial relation was found between UL134 gene and HCMV infectious diseases.

Structure and variability of the UL149 open reading frame from low-passage clinical isolates of human cytomegalovirus.

Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. 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 passage laboratory strain AD169. One of these genes, UL149 open reading frame, was amplified by PCR and sequenced from isolates obtained from infants with congenital HCMV infection, to determine whether genetic variation of this gene could influence the signs of the virus infection. The major finding is that the UL149 is a variable gene in all 26 clinical isolates, and the sequences from clinical isolates were classified into three major groups. It is concluded that the HCMV UL149 sequence is variable at the nucleotide level and it might play an important role in HCMV infection.

Sequence variability of human cytomegalovirus UL143 in low-passage clinical isolates.

BACKGROUND: Human cytomegalovirus (HCMV) infects a number of organs and tissues in vivo. The different symptoms and tissue tropisms of HCMV infection perhaps result from genetic polymorphism. A new region of DNA containing at least 19 open reading frames (ORFs) (denoted UL133 to 151) was found in the low-passage HCMV clinical strain, Toledo, and several other low-passage clinical isolates, but not present in the HCMV laboratory strain, AD169. One of these genes, UL143, was studied to explore the sequence variability of UL143 ORF in HCMV clinical isolates and examine the possible association between gene variability and the outcome of HCMV infection. METHODS: The UL143 gene of the strains obtained from suspected congenitally HCMV-infected infants was amplified by polymerase chain reaction (PCR) and sequenced. RESULTS: Nineteen sequences of the strains were divided into 2 major groups, G(1) (n = 16) and G(2) (n = 3). All of the sequences had frame-shift mutation compared to Toledo. Nucleotide polymorphisms conferred substantial amino acid substitutions when compared with Toledo. All 16 UL143 putative proteins of the strains in G(1) had a new myristylation site and loss of two PKC sites owing to missense mutations. No convincing relationships were observed between the presence of HCMV disease and the UL143 sequence group. CONCLUSIONS: HCMV-UL143 existed in low passage isolates. Sequence variability caused by frame-shift mutation was found in all HCMV clinical strains. No obvious linkage was observed between UL143 polymorphisms and the outcome of suspected congenital 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.