Transcription pattern of UL131A-128 mRNA in HCMV clinical strains / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study and research the transcription pattern of UL131A-128 mRNA in human cytomegalovirus (HCMV) clinical low passage strains.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Human cytomegalovirus UL138 open reading frame is highly conserved in clinical strains / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the variability of human cytomegalovirus (HCMV) UL138 open reading frame (ORF) in clinical strains.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

The investigation of the relationship between the phenotypes of 46, XX males and the SRY gene / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the phenotypes in XX male patients and the sex determining region(SRY) gene.</p><p><b>METHODS</b>Multiple polymerase chain reactions were carried out in 6 male patients with karyotype of 46, XX, and then the PCR products were sequenced directly.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>The SRY gene is key in sex determination and development, yet there might be other important genes involved.</p>

Polymorphism analysis of human cytomegalovirus UL150 gene in low passage clinical isolates / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the polymorphism of human cytomegalovirus UL150 gene in low passage clinical isolates and try to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection.</p><p><b>METHODS</b>PCR was performed to amplify the entire HCMV UL150 gene region of 29 clinical isolates, which had been proven containing detectable HCMV-DNA by using FQ-PCR. PCR amplification products were sequenced directly and the data were analysed.</p><p><b>RESULTS</b>25 among 29 isolates were amplified and 18 isolates were sequenced successfully. By comparison with the sequence of Toledo and Merlin, the length of UL150 ORFs in all 18 clinical isolates was similar to that of Merlin than Toledo.</p><p><b>CONCLUSION</b>HCMV UL150 DNA and deduced amino acid sequences is hypervariability.</p>

Sequence conservation of human cytomegalovirus UL140 open reading frame in clinical strains / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>HCMV UL140 ORF was amplified by polymerase chain reaction and sequenced selectedly in 30 clinical strains.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>HCMV UL140 ORF in clinical strains is highly conserved, which may play an important role in HC-MV infection.</p>

Polymorphism of human cytomegalovirus UL134 gene in low-passage clinical isolates / 中国当代儿科杂志

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>HCMV UL134 gene in clinical isolates was highly conserved. No substantial relation was found between UL134 gene and HCMV infectious diseases.</p>

High variability of human cytomegalovirus UL150 open reading frame in low-passaged clinical isolates / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Sequence variability of human cytomegalovirus UL143 in low-passage clinical isolates / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>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.</p><p><b>METHODS</b>The UL143 gene of the strains obtained from suspected congenitally HCMV-infected infants was amplified by polymerase chain reaction (PCR) and sequenced.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSIONS</b>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.</p>

Variability of human cytomegalovirus UL144 gene in low-passage clinical isolates analyzed by HMA-SSCP / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Human cytomegalovirus (HCMV) infection is an important infectious agent that results in neonatal disease and congenital deformity. HCMV infection may affect in many organs. The different symptoms and tissue tropism of HCMV infection perhaps resulted from the genetic polymorphism of HCMV. HCMV UL144 open reading frames encode a homologue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection.</p><p><b>METHODS</b>HCMV-UL144 gene was amplified by PCR assay in 65 low-passage clinical isolates and urine from 7 healthy children who were HCMV-DNA positive by quantitative PCR. All the positive PCR products were analyzed by Heteroduplex mobility assay and single-stranded conformation polymorphism (HMA-SSCP) and 32 of them were sequenced.</p><p><b>RESULTS</b>Fifty-five isolates and 5 urine specimens were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORFs. Comparing UL144 sequences 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.</p><p><b>CONCLUSION</b>HCMV-UL144 existed in almost all the low passage isolates. HMA-SSCP assay is an easy and effective method to detect the genetype of HCMV-UL144 sequence. The characteristic of sequences in different isolates showed that UL144 gene may play an important role in HCMV infection.</p>

Polymorphisms of HCMV US28 gene of the clinical isolates from children / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To study the polymorphism of human cytomegalovirus US28 gene in children and investigate the relationship between the polymorphism and pathogenesis.</p><p><b>METHODS</b>The FQ-PCR was carried out to determine the DNA quantity of clinical isolate and then the segmental PCR and HMA-SSCP were performed to test the mutation of US28 gene. The typical isolates from different diseases were selected to clone and sequence, then the results were analyzed.</p><p><b>RESULTS</b>The nucleic acid mutation is frequent among the sequence of US28, those mutations focus on the two ends of US28, but most of them are sense mutation. The important functional groups of US28 are highly conserved. The amino acid mutation of some isolates resulted in the change of secondary structure, but the phylogenetic tree analysis did not show any clear association between the pathogenesis and the distribution of clinical isolates. The comparison of US28 sequences from AIDS patients with the sequences from children in our study showed that both sequences have their own specific high mutation points.</p><p><b>CONCLUSION</b>There is polymorphism among the HCMV-US28 gene of clinical isolates from children. There observed no clear relationship was between the pathogenesis and the distribution of clinical isolates.</p>

Polymorphism analysis of human cytomegalovirus UL148 gene in low passage clinical isolates / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To investigate the polymorphism of human cytomegalovirus UL148 gene in low passage clinical isolates and to study the relationship between the polymorphism and different pathogenesis of congenital HCMV infection.</p><p><b>METHODS</b>PCR was performed to amplify the entire HCMV UL148 gene region of 38 clinical isolates, which had been proven containing detectable HCMV-DNA by using FQ-PCR.PCR amplification products were sequenced directly and the sequence data were analysed.</p><p><b>RESULTS</b>Seventeen of 38 isolates were amplified successfully. By comparison with Toledo sequence, the length of UL148 ORFs in all 17 clinical isolates was similar to that of Toledo. Amino acid variability rate of UL148 protein was 0.3%-2.3%. There were additional or deleted sites of posttranslational modification of UL148 protein in all clinical isolates.</p><p><b>CONCLUSION</b>All DNA and deduced amino acid sequences of UL148 gene shared great similarity among HCMV clinical strains regardless of their polymorphism.</p>

Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To explore the relationship between human cytomegalovirus (HCMV) UL144 sequence variability and clinical disease.</p><p><b>METHODS</b>HCMV UL144 open reading frame (ORF) was amplified by PCR assay in 72 low-passage isolates [65 congenitally 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.</p><p><b>RESULTS</b>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 sequences 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.</p><p><b>CONCLUSIONS</b>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.</p>