Association between clinical features and YMDD mutations in patients with chronic hepatitis B following lamivudine therapy.

The aim of the present study was to investigate the correlation between feature and genotype with regard to the tyrosine-methionine-aspartate-aspartate (YMDD) mutation in chronic hepatitis B patients after lamivudine (LAM) therapy. A total of 30 patients with chronic hepatitis B were recruited, who underwent one year of LAM therapy. The patients' alanine aminotransferase (ALT) level and hepatitis B envelope antigen (HBeAg) seroconversion were evaluated, hepatitis B virus (HBV) DNA was genotyped using a new genotyping method and YMDD mutations were analyzed prior to treatment and at 6 and 12 months after LAM treatment. Furthermore, the secondary protein structure of the HBV DNA polymerase gene (P gene) was analyzed. Following treatment, the results suggested that LAM therapy improved ALT normalization. There was no correlation between clinical effects and ALT level before treatment. After 12 months treatment, the rate of HBeAg loss increased and the rate of HBeAg seroconversion decreased linearly with the rise of baseline ALT level. While ALT normalization and HBeAg seroconversion were highest in patients with HBV genotype B, HBeAg loss and HBVDNA loss were highest in those with genotype C. The effect was predominant in genotype D. No YMDD mutations were identified prior to 6 months of LAM therapy. The rate of YMDD mutations after 12 months LAM therapy was 12.12%. Two patients with rtM204V + rtL180M belonged to genotype C and another patient with rtL180M alone belonged to genotype D. The turn of secondary protein structure of P gene changed to ß sheet when a rtM204V mutation occurred, and no change of secondary protein structure was associated with the rtL180M mutation. Thus, the present results indicate that one year of LAM therapy is able to improve ALT normalization. Long-term LAM therapy may induce YMDD mutation and drug resistance.

Interaction between the human cytomegalovirus­encoded UL142 and cellular Snapin proteins.

Human cytomegalovirus (HCMV) infection can cause severe illness in immunocompromised and immunodeficient individuals. As a novel HCMV­encoded major histocompatibility complex class I­related molecule, the UL142­encoded protein (pUL142) is capable of suppressing natural killer (NK) cell recognition in the course of infection. However, no host factors that directly interact with HCMV pUL142 have been reported so far. In order to understand the interactions between HCMV pUL142 and host proteins, the current study used yeast two­hybrid screening, a GST pull­down assay and an immunofluorescence assay. A host protein, the SNARE­associated protein Snapin, was identified to directly interact and colocalize with HCMV pUL142 in transfected human embryonic kidney­293 cells. Snapin is abundantly expressed in the majority of cells and mediates the release of neurotransmitters through vesicular transport in the nervous system and vesicle fusion in non­neuronal cells. It is hypothesized that HCMV pUL142 may have an impact on the neurotransmitter release process and viral dissemination via interaction with Snapin.

Validation of three splice donor and three splice acceptor sites for regulating four novel low-abundance spliced transcripts of human cytomegalovirus UL21.5 gene locus.

In a previous study, one spliced transcript of human cytomegalovirus (HCMV), named UL21.5 was identified. UL21.5 has been found to be one of the viral transcripts packaged within HCMV particles. The UL21.5 mRNA is translated into a secreted glycoprotein, which is a viral chemokine decoy receptor specifically interacting with regulated upon activation normal T cell expressed and secreted (RANTES). In the present study, four novel low-abundance 3'-coterminal spliced transcripts were identified to be transcribed from the UL21.5 gene region of a low-passage HCMV strain during the late infection phase by cDNA library screening, northern blot hybridization, reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR. Three splicing donor and three splicing acceptor sites found in the UL21.5 gene region were validated to be functional in an in vitro expression system. In addition, the determinant regulatory region that is necessary for the splice donor site at nucleotide (nt) 25533 was located in a 9-bp sequence around the site; the regulatory regions for the splice acceptor sites at nt 26597 and nt 26633 were located in a 20-bp sequence upstream of the site at nt 26597 and in a 10-bp sequence from nt 26641 to nt 26650 downstream of the site at nt 26633, respectively.

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 percent decrease in luciferase activity and a 46.2 percent decrease in HCMV UL138 protein expression. Our results indicate that hcmv-miR-UL36 may be a viral miRNA contributing to HCMV replication.

Over-expression of human cytomegalovirus miR-US25-2-3p downregulates eIF4A1 and inhibits HCMV replication.

It has been reported that human cytomegalovirus (HCMV) miR-US25-2 reduces DNA viral replication including HCMV. However, the mechanism remains unknown. In our study, eukaryotic translation initiation factor 4A1 (eIF4A1) was identified to be a direct target of miR-US25-2-3p. Small interfering RNA (siRNA) and miR-US25-2-3p mediated eIF4A1 knockdown experiments revealed that high level of miR-US25-2-3p in MRC-5 cells decreased HCMV and host genomic DNA synthesis, and inhibited cap-dependent translation and host cell proliferation. However, eIF4A1 up-regulation induced by miR-US25-2-3p inhibitor increased HCMV copy number. Therefore, the over-expression of miR-US25-2-3p and consequent lower expression of eIF4A1 may contribute to the inhibition of HCMV replication.

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.

Identification of immediate early gene X-1 as a cellular target gene of hcmv-mir-UL148D.

Human cytomegalovirus (HCMV) is a herpesvirus that causes congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behavior. The identification of functional target genes of miRNAs is an important step in the study of HCMV pathogenesis. HCMV encodes at least 14 miRNAs, including hcmv-mir-UL148D, which resides in the HCMV UL/b' region. hcmv-mir-UL148D is the only miRNA encoded by the HCMV UL/b' region; however, its targets and functional effects have not yet been eludidated. In this study, hybrid-PCR screening was used to identify target genes and dual luciferase reporter assay was used to evaluate the binding effect of hcmv-miR-UL148D to the 3' untranslated region (3'UTR) of IEX-1. In addition, western blot analysis was used to detect the expression kinetics of IEX-1 protein and apoptosis assay was used to identify the effects of hcmv-miR-UL148D on cell apoptosis. The hybrid-PCR results showed that only one binding site in the 3'UTR of the cellular gene, human immediate early gene X-1 (IEX-1), was completely complementary to an 11 nucleotide (nt) sequence in the 5' terminus of hcmv-mir-UL148D, including the entire seed region. The binding site was demonstrated to be functional by dual luciferase reporter assay with a 47% repression of the relative luciferase activity. In an in vitro system of human embryonic kidney 293 (HEK293) cells, the ectopically expressed hcmv-mir-UL148D exhibited a downregulatory effect on IEX-1 expression, and decreased the cell apoptosis induced by transfected IEX-1. Our data demonstrate that hcmv-mir-UL148D targets the cellular gene, IEX-1, downregulating its expression and thus results in anti-apoptotic effects.

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.

Characterization of human cytomegalovirus UL146 transcripts.

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, UL146, 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: All sequences of clones from a cDNA library were about 3225 bp in length with the same 5' and 3' ends. The results were accordant with that analyzed by Northen-blot and RACE-PCR in three HCMV clinical isolates. The transcript initiated from the region upstream of UL146 flanking region and terminated just downstream of UL132 including UL146, UL147, UL147A, UL148, and UL132 ORFs. Treatment of the infected cells with phosphonoacetic acid inhibited its transcription. CONCLUSIONS: UL146 ORF was transcribed with 4 downstream ORFs from UL147 to UL132 at true late kinetics. The transcript of UL146 initiated at 73nt upstream of UL146 and terminated just downstream of UL132 in the 3 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.

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.

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.

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients.

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.

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.

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 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.

[Transcription pattern of UL131A-128 mRNA in HCMV clinical strains].

OBJECTIVE: To study and research the transcription pattern of UL131A-128 mRNA in human cytomegalovirus (HCMV) clinical low passage strains. METHODS: The UL131A-128 mRNAs of from different clinical strains and kinetic periods were amplified using 3' RACE and analyzed by sequencing. Meanwhile, clones containing UL131A-128 transcripts in a HCMV cDNA library of clinical strain were selected and sequenced. RESULTS: It is successful to obtain the transcription pattern of UL131A, UL130 and UL128 gene in HCMV clinical low passage strains, the UL131A gene consisted of two exons and the coding region of UL130 gene was not interrupted by any intron in the region as reported before. However, the transcript of UL128 gene showed two patterns, one pattern consisted of the three exons, the length is 519bp, and the other one contained the three exons and the sequence of the first intron further, the length is 642bp. The quantities of UL128 transcript containing the sequence of the first intron were higher than that of transcript only containing the three exons in the studied clinical strains at all kinetic classes. It was demonstrated that the UL131A-128 mRNA were expressed with immediately early, early and late kinetics. The result of 3'RACE and HCMV cDNA library of clinical strain is conformity. CONCLUSIONS: Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with 3'-coterminal, although the initiation points of their mRNA may be different. The variation of the transcripts found in our study indicated complex nature of transcription of UL131A-128 genes in HCMV clinical strains.

Transcription pattern of UL131A-128 mRNA in clinical strains of human cytomegalovirus.

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by sequencing. Meanwhile, clones containing UL131A-128 transcripts in an HCMV cDNA library of a clinical strain were selected and sequenced. It was demonstrated that UL131A-128 mRNA was expressed with immediately early, early and late kinetics. Sequences obtained by RT-PCR showed that the UL131A gene consisted of two exons and the coding region of the UL130 gene was not interrupted by any intron in the region as reported earlier. However, the transcript of the UL128 gene showed two patterns: one pattern consisted of three exons as reported earlier; the other contained the three exons and also the first intron. Moreover, the above characteristics of UL131A-128 spliced transcripts were confirmed by the sequences of clones selected from the HCMV cDNA library. Our results demonstrated that the UL131A, UL130 and UL128 genes were transcribed with the 3'-coterminal, although the initiation points of their mRNA may be different. The variation in the transcripts found in our study indicated the complex nature of transcription of UL131A-128 genes in clinical strains of HCMV.

Characteristics of HPV prevalence among women in Liaoning province, China.

OBJECTIVES: To investigate the prevalence rates of specific human papillomavirus (HPV) types infecting women in Liaoning Province, China. METHODS: Specimens from 4780 patients with cervical disease and 165 age-matched controls were tested for HPV genotypes using a chip hybridization assay. RESULTS: The infection rates were 35.66% for patients with cervicitis, 54.61% for those with ASCUS, 64.14% for those with CIN, 83.76% for those with cervical cancer in situ, and 83.12% for those with invasive cervical cancer. The most common HPV genotype was HPV-16, followed by HPV-58, HPV-52, HPV-33, HPV-53, and HPV-31. There were 1529 single and 731 multiple infections among the 4780 patients. Single infections with high-risk genotypes were associated with various cervical diseases. HPV-16 was present in 399 of the patients with multiple infections. CONCLUSION: Compared with prevalence rates for other populations, the rates of specific HPV types infecting women are different in Liaoning Province of China.

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.