Human cytomegalovirus a sequence and UL144 variability in strains from infected children

Human cytomegalovirus (HCMV) displays genetic polymorphisms. This variability may contribute to strain-specific tissue tropism and disease expression in HCMV-infected humans. To determine strain variability in a sequence and UL144 gene regions, 51 low-passage isolates from 44 HCMV-infected children were studied. Isolates were obtained from 28 healthy children attending child care centers in Iowa and from 16 congenitally infected infants born in Texas. Isolates demonstrated substantial nucleotide variation in each gene region. Phylogenetic analysis of a sequence variability allowed 39 isolates to be grouped into six clades. The largest clade contained 16 isolates with > or = 95% nucleotide homology. Forty-eight of the 49 HCMV isolates yielding UL144 amplicons was grouped according to the clades described a few years ago [Lurain et al. (1999) Journal of Virology 73:10040-10050]. No linkage was observed among a sequence, UL144, and glycoprotein B (gB; UL55) polymorphisms. Four Texas and 11 Iowa isolates displayed > or = 95% sequence homology for a sequence and UL144 regions and possessed identical gB genotypes. No relationship between UL144 polymorphisms and outcome of congenital HCMV infection was observed. These data indicate that HCMV strains circulating among young children have UL144 polymorphisms similar to those of HCMV strains excreted by immunocompromised adults. Identification of conserved nucleotide sequences among Iowa and Texas children suggests genetic stability and biologic importance of these gene regions.     

Distribution of UL144, US28 and UL55 genotypes in Polish newborns with congenital cytomegalovirus infections

Human cytomegalovirus (HCMV) is the most common congenital infection. HCMV strains display genetic variability in different regions. Distribution of HCMV genotypes in the population of congenitally infected newborns from Central Poland and viral load in newborns' blood is described and discussed. HCMV isolates were analysed by sequencing at three sites on the genome: the UL144 tumour necrosis factor-alpha (TNFα)-like receptor gene, the US28 beta-chemokine receptor gene and the UL55 envelope glycoprotein B (gB) gene. The newborns' blood was examined for HCMV DNA with a nested (UL144, UL55) or heminested (US28) polymerase chain reaction, and the genotypes were determined by sequence analysis. HCMV DNA was detectable in 25 out of 55 examined newborns born by HCMV-infected mothers (45.5%). The blood viral load in mother-infant pairs was determined. Most of the newborns had identical virus genotype, gB2 (96%), UL144 B1 (88%) and US28 A2 (84%). These genotypes were detected in all newborns with asymptomatic congenital infection. The occurrence of UL144 B1 or US28 A2 genotypes in the babies examined was significant in comparison to other genotypes (p=0.0002 and p=0.040 respectively). There was no association between specific gB subtypes in all patients groups (p=0.463). There was no correlation between HCMV genotypes and the outcome.     

Role of human cytomegalovirus genotype polymorphisms in AIDS patients with cytomegalovirus retinitis

Although several host factors have been identified to influence the course of HCMV infection, it still remains unclear why in AIDS patients without highly active antiretroviral therapy human cytomegalovirus (HCMV) retinitis is one of the most common opportunistic infections, whereas in other immunosuppressed individuals it has a low incidence. It was suggested that HCMV glycoprotein B strains may be suitable as marker for virulence and HCMV retinitis. Moreover, UL144 ORF, a member of the TNF-α receptor superfamily, may play a crucial role in innate defences and adaptive immune response of HCMV infection. Furthermore, sequence analyses of HCMV genes UL128, UL130, and UL131A as major determinants of virus entry and replication in epithelial and other cell types were performed. To evaluate the association of sequence variability of depicted viral genes with HCMV retinitis and in vitro growth properties in retinal pigment epithelial cells (RPE) and human foreskin fibroblasts (HFF), we compared 14 HCMV isolates obtained from vitreous fluid and urine of AIDS patients with clinically proven HCMV retinitis. Isolates were analyzed by PCR cycle sequencing and phylogenetic analysis. In addition, sequences of HCMV strains AF1, U8, U11, VR1814, and its cell culture adapted derivates were included. Sequence analysis of gB yielded three genetic subtypes (gB type 1 (5 isolates), gB type 2 (12 isolates), and gB type 3 (5 Isolates)), whereas sequence analysis of UL144 showed a greater diversity (7 isolates type 1A, 2 isolates type 1C, 7 isolates type 2, and 3 isolates type 3). In contrast, the UL128, UL130, and UL131A genes of all low-passage isolates were highly conserved and showed no preferential clustering. Moreover, in HFF and RPE cells, all of our HCMV isolates replicated efficiently independently of their genetic subtype. In conclusion, beside a possible link between the gB subtype 2 and HCMV retinitis, our study found no direct evidence for a connection between UL144/UL128/UL130/UL131A genotypes and the incidence of HCMV retinitis in AIDS patients.     

Polymorphisms within human cytomegalovirus chemokine (UL146/UL147) and cytokine receptor genes (UL144) are not predictive of sequelae in congenitally infected children

Human cytomegalovirus (HCMV) viral chemokine, UL146, and TNF alpha-like receptor UL144 genes show a high degree of hypervariability in clinical isolates. These proteins are predicted to be immune modulators and may contribute to the pathogenesis of HCMV infections. We analyzed the UL146 and UL144 genetic variation of 51 HCMV isolates from congenitally infected children and 13 isolates from children in childcare. There was no statistically significant correlation between UL146 and UL144 genotypes and HCMV disease and/or sequelae. However, there were some groups that had a relatively large proportion of asymptomatic outcomes. These included UL146 group 8 (7/8 asymptomatic) and UL146 group 10 (3/3 asymptomatic). UL144 group B had 11/15 (73%) asymptomatic. UL146 and UL144 genes remained stable in serial isolates from children in daycare for intervals up to three years. These results indicate that most UL146 and UL144 genotypes do not predict clinical sequelae following congenital HCMV infections.     

Human cytomegalovirus UL144 gene polymorphisms in congenital infections

The human cytomegalovirus (HCMV) UL144 gene is a tumor necrosis factor-like receptor with the potential to affect HCMV virulence. HCMV strains display genetic variability in the UL144 region, and the analysis of a potential link between UL144 gene polymorphisms and disease severity has scarcely been studied. However, a correlation between the UL144 genotype and congenital-disease outcome has been reported in one previous study, with the observation that all asymptomatic infants had a single UL144 genotype. In order to confirm or refute this finding, we determined the UL144 polymorphisms of HCMV strains recovered from the amniotic fluids of 38 infected fetuses and compared them to HCMV strains obtained from 30 viremic adult controls. The UL144 sequences were distributed among five genotypes (A, B, C, AC, and AB), as previously described. We observed similar percentages of the three major genotypes A (37%), B (33%), and C (27%) in our population. The UL144 genotype distributions were similar among the group of infected adults and the group of infected fetuses and among symptomatic and asymptomatic fetuses (P < 0.05). In our series, all five UL144 genotypes could be vertically transmitted from mothers to fetuses, and all could cause symptomatic congenital infection. We concluded that determination of UL144 polymorphisms in cases of congenital infection is not relevant, since it is unlikely to help predict the outcome of the infection.     

HMA-SSCP方法研究人类巨细胞病毒UL144基因在临床低传代分离株中的多态性

目的 探讨人类巨细胞病毒（HCMV）UL144序列在临床患儿低传代分离株中的多态性及与临床疾病的关系。方法 对65株HCMV临床低传代分离株及7例同年龄组HCMV，DNA定量PCR方法检测阳性无症状感染儿尿液进行HCMV-ULl44 PCR扩增及HMA-SSCP分析，并对其中32份阳性标本进行测序。结果 65株分离株中有55株UL144全序列引物PCR扩增阳性，7份QPCR检测HCMV—DNA阳性无症状感染儿尿液中5份UL144全序列引物PCR扩增阳性。60份UL144扩增阳性标本HMA-SSCP（异源双链泳动及单链构象多态分析）呈现3种典型带形，巨结肠患儿分离株序列、小头畸形患儿的序列分布以1型为主，巨结肠患儿分离株序列没有2型，黄疽患儿以3型为主。结论 HCMV-UL144广泛存在于临床低传代分离株中，用HMA-SSCP检测HCMV-UL144基因在临床低传代分离株中的多态性是一种可行的方法。HCMV不同疾病类型的HCMV-UL144序列不同，提示UL144基因可能对HCMV致病性起一定作用。     

人类巨细胞病毒UL144基因在临床低传代分离株中的多态性研究

目的：探讨人类巨细胞病毒（human cytomegalovirus,HCMV)UL144序列在临床患儿低传代分离株中的多态性及其与临床疾病的关系。方法：对65株HCMV临床低传代分离及7例同年龄组HCMV－DNA定量PCR方法检测阳性健康儿尿液进行HCMV-UL144 PCR扩增分析，对HCMV-UL144扩增阳性标本进行HMA－SSCP分析，并对其中32个阳性标本进行HCMV-UL144基因全序列测定及分析。结果：65株分离株中有55株UL144全序列引物PCR扩增为阳性，7份QPCR检测HCMV-DNA阳性健康儿尿液中5份UL144全序列PCR扩增为阳性，60份UL144扩增阳性标本HMA－SSCP（heteroduplex mobility assay and single-stranded conformation polymorphism)分析呈现3种典型带形，32例个测序标本序列呈现较高的多态性，差异多位于序列的前半部，种系发生图谱分析可大致分为3组，二级结构预测表现为6种类型，在重要的蛋白质功能的序列分布以Ⅰ型为主，黄患儿以Ⅲ型为主。32个HCMV-UL144序列已被GenBank收录，结论:HCMV-UL144广泛存在于临床低传代分离株中，序列呈序高度多态性，不同疾病类型的HCMV-UL144序列不同，提示UL144基因可能在HCMV致病上起一定的作用。     

Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects a variety of cell types in vivo. 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, UL146, encodes an alpha-chemokine. PCR amplification and sequencing of this gene from serial samples obtained from transplant recipients and samples from infants with suspected congenital HCMV infection, revealed that UL146 is a hypervariable gene in vivo. However, genetic changes were highly conserved in individuals and in renal transplant recipients multiple genotypes of UL146 were present. The majority of strains characterized maintained the conserved ELRCXC motif present in the Toledo strain of HCMV. These results provide further evidence that AD169 does not represent the authentic virus in vivo and although Towne and Toledo are more representative, major genetic differences still exist. Mixed populations of HCMV strains occur in vivo so cloning of these strains is essential if an authentic genotype is to be defined.     

先天性巨结肠患者人类巨细胞病毒UL144基因多态性的研究

目的研究人类巨细胞病毒（human cytomegalovirus,HCMV）UL144基因在先天性巨结肠（Hirschsprung＇s disease,HD）临床株中的多态性,探讨HCMV UL144基因多态性与致病性之间的关系.方法随机选取53个先天性巨结肠患儿痉挛段结肠手术标本及经荧光定量PCR方法检测HCMV DNA为阳性的4个HD患儿的尿标本,对照组为无症状或仅有皮肤轻度黄疸的6个尿标本.应用巢式聚合酶链反应的方法,扩增HCMV UL144基因开放阅读框架（ORF）,扩增阳性的临床株进行双向DNA测序,最后通过DNAclub、Bioedit、DNAstar、GeneDoc等软件进行分析.结果23份HD痉挛段肠组织（46%）及4份尿标本HCMV UL144基因扩增阳性,并且完成测序.种系进化树分析结果显示25个HD患儿的DNA序列分为3个基因型,G1A型64.0%,G2型24%,G3型12%.与对照组比较,经χ^2检验,χ^2=10.93,P为0.012;其中HD临床株G1A和G3型基因经Fisher检验,P为0.015,差异具有统计学意义.全结肠型、长段型及普通型HD分散分布于UL144各个基因型中.结论HD与HCMV感染有关,HCMV可能是HD的病因之一;在HD患儿中,HCMV感染以UL144基因G1A型为主;HD的临床分型与HCMV UL144基因分型无关.     

人巨细胞病毒UL136基因在临床低传代分离株中多态性分析

目的 研究人巨细胞病毒(human cytomegalovirus，HCMV)UL136基因在临床低传代分离株中的多态性，探讨其多态性与HCMV先天性感染不同致病性之间的关系。方法 对48株经荧光定量PCR方法检测HCMV DNA为阳性的临床低传代分离株进行HCMV ULl36全序列PCR扩增，对于扩增阳性的12株PCR产物进行ULl36基因全序列测定及结果分析。结果 48株临床低传代分离株ULl36 PCR扩增，12株阳性，阳性率25％，以HCMV Toledo株作为参考株，进行序列比较分析表明，12株临床分离株ULl36开放阅读框架(open reading frame，ORF)长度均与Toledo株相同，为723bp，编码241个氨基酸的蛋白。DNA序列变异均为碱基替换，不同临床分离株ULl36基因与Toledo株进行同源性比较，结果在核苷酸水平为97．7％～99．3％，氨基酸水平为96．6％～99．1％。ULl36编码蛋白的氨基酸变异率为0．83％～3．3％。二级结构预测分为两种构象。大多数HCMV ULl36蛋白翻译后修饰位点在所有分离株中均高度保守，仅几个位点在一些分离株中存在缺失或新增。Toledo株及12株临床分离株核苷酸及氨基酸序列系统进化树分析表明：45J最接近Toledo株。结论 12株临床低传代分离株HCMV ULl36基因DNA及其编码产物的氨基酸序列比较保守，但仍存在一定多态性。未发现不同临床分离株ULl36基因多态性与HCMV先天性感染的表现关系。     

新生儿晚期黄疸人巨细胞病毒gB基因分型研究

目的了解引起新生儿晚期黄疸患者人巨细胞病毒（HCMV）gB基因型的分布,探讨HCMVgB基因多态性与黄疸之间的关系。结论采用荧光定量PCR法检测本院新生儿科98例晚期黄疸新生儿标本HCMV-DNA含量,巢式聚合酶链反应扩增阳性标本HCMVgB基因,并进行DNA测序,绘制种系进化树。利用HinfⅠ和RsaⅠ对gB基因进行限制性片段长度多态性（RFLP）分析。结果 30例晚期黄疸新生儿HCMV荧光定量PCR检测阳性,阳性率为30.6%。种系进化树分析结果显示gB基因分为4个基因型,gB1型15株（50%）,gB2型5株（16.7%）,gB3型9株（30.0%）,gB1/3混合型1株（3.3%）。以HCMV实验室标准株AD169作为参考,将序列进行同源性比较,gB1型同源性为94.7%～95.0%,gB2型同源性93.1%～93.4%,gB3型同源性94.7%,gB1/3型同源性93.7%。RFLP分析将gB基因分为4个基因型,分型结果与种系进化树分型一致。结论 HCMV感染是导致新生儿晚期黄疸的原因之一;gB基因的DNA序列比较保守,但仍存在一定的多态性,晚期黄疸新生儿中HCMV感染以gB1、gB3型为主。     

先天性巨结肠患者人类巨细胞病毒UL139基因多态性的研究

目的研究人巨细胞病毒（HCMV）UL139基因在先天性巨结肠（HD）临床株中的多态性，探讨其多态性与HD之间的关系。方法对53株HD患儿痉挛段肠组织标本和6株尿标本的临床株进行UL139开放阅读框（ORF）的扩增及测序。对照组为10个无症状HCMV感染患儿的尿标本。结果28个HD临床株完成测序，进化树分析结果显示UL139基因DNA序列分为3组5个基因型。G3为主要基因型（48．1％）。与对照组比较，X^2=7．378，P=0．194。24个临床株同时完成了HCMVUL144基因测序，HCMVUL144与UL139基因经Kendall等级相关分析r=-0．114，P=0．425。不同临床分型HD分散分布于UL139各个基因型中。结论HCMVUL139基因具有高度的多态性；UL139基因分型与HD的临床分型无关；UL139基因与UL144基因无相关性。     

The relationship between polymorphisms of HCMV UL144 ORF and clinical manifestations in 73 strains with congenital and/or perinatal HCMV infection

Summary. Human cytomegalovirus (HCMV) displays genetic variability and can cause a wide range of diseases in neonates. To explore the relationship between polymorphisms and clinical manifestations, the UL144 genes from 73 clinical strains were sequenced. All of the strains, which came from 70 infants with suspected congenital and/or perinatal HCMV infection, were non-passage strains. Among them, 23 strains were from surgery specimens, and the others were from urine samples. Clinically, 12 infants displayed asymptomatic infection and 58 patients displayed symptomatic infection. The results showed that 36 patients (49.3%) were infected with strains belonging to UL144 group G1 (G1a 33/36, G1b 3/36), 19 patients (28.8%) were infected with strains belonging to group G2, and 15 patients (21.9%) were infected with strains belonging to group G3. This result indicated that UL144 group G1 was the predominant genotype in congenital and/or perinatal HCMV infection in northern China. Compared with the distribution pattern of strains in UL144 genotypes of data from Chicago, Iowa and Texas, and Japan by chi-square test, the difference was statistically significant. This suggested that the distribution pattern of strains in UL144 genotype was related to geographic location. However, no linkage was observed between the UL144 genotypes and the severity and/or outcome of HCMV disease.     

Human cytomegalovirus UL131A, UL130 and UL128 genes are highly conserved among field isolates

Summary. Coding sequences of the UL131A, UL130, and UL128 genes of human cytomegalovirus (HCMV) were found to be highly conserved among 34 field isolates from pregnant women with primary HCMV infection and their fetuses or newborns, as well as from solid organ transplant recipients and patients with AIDS. No strain clustering was observed. In contrast, sequencing of UL55 (gB coding gene) allowed the 34 isolates to be clustered into 4 genotypes. The conservation of the UL131A-UL128 locus is consistent with the conclusion that the three encoded proteins are all essential for growth of HCMV in endothelial cells and virus transfer to leukocytes.     

Variability of gB and gH genes of human herpesvirus-6 among clinical specimens

The isolates of human herpesvirus-6 (HHV-6), a betaherpesvirus closely related to human cytomegalovirus (HCMV), are classified as either variants A (HHV-6A) or B (HHV-6B) but their intravariant variability has not been studied extensively so far. The full-length genes of envelope glycoproteins gB and gH from 40 distinct HHV-6-DNA-positive specimens and 11 laboratory strains were amplified using PCR, and their nucleotide sequence determined. Nucleotide divergences were observed at 156 (6.2%) and 98 (4.7%) positions in the case of gB and gH genes respectively. Phylogenetic analysis, including reference strain sequences, confirmed the unambiguous distinction between HHV-6A and HHV-6B for both genes. In the case of HHV-6B isolates, two subgroups of gB gene (designated as gB-B1 and gB-B2) and two subgroups of gH gene (gH-B1 and gH-B2) were identified but the phylogenetic trees of both genes were not fully congruent with each other. The analysis of gB and gH protein sequences showed that 26 and 39 critical amino acid changes respectively permitted the unambiguous distinction between HHV-6A and HHV-6B. Among HHV-6B isolates, gB and gH gene subgroups were characterized by specific amino acid signatures made of six, and two residues respectively. The linkage unbalance between amino acid signatures as well as the distribution of crucial nucleotide changes strongly suggested the occurrence of intravariant recombination within gB gene among HHV-6B isolates. These results indicate that, as in the case of HCMV, homologous recombination may contribute to the genetic variability of HHV-6.     

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.     

Analysis of the variability of CMV strains in the RL11D domain of the RL11 multigene family

Human cytomegalovirus (CMV) contains one of the largest genomes within the herpesvirus family and includes 12 multigene families. One of these is the RL11 family, whose members encode a characteristic domain, called RL11D. In the present study, the sequence variability of RL11D within the UL1, UL4, UL6, UL7, and UL10 genes of the RL11 family was investigated. For this purpose, these genes were analyzed in 70 clinical isolates obtained from urine, bronchoalveolar lavage, and blood of different patients. Substantial genetic variability among the clinical isolates was observed in all five genes analyzed. Based on phylogenetic analysis of variations in RL11D, the clinical isolates could be classified into three genotypes for UL1, 7, and 10 and, four genotypes for UL4 and 6. Further analysis showed statistically significant linkages between the following pairs of genes: UL6/UL7, UL4/UL7, UL1/UL4, and UL4/UL6. The data show that CMV strains exhibit a high interstrain variability in the RL11D domain of various RL11 family genes. Sequence variations, however, can be clearly grouped into a limited number of distinct genotypes. The genetic linkages found probably reflect a low frequency of recombination between genes that are arranged in close proximity on the viral genome.     

人巨细胞病毒UL149基因在临床低传代分离株中的多态性研究

目的研究人巨细胞病毒(humancytomegalovirus,HCMV)UL149序列在临床低传代分离株中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状之间的关系。方法对29株经荧光定量PCR方法(QPCR)检测HCMVDNA为阳性的临床低传代分离株的细胞培养上清液进行HCMVUL149全序列PCR扩增,并对PCR扩增产物进行序列测定及分析。结果29株临床低传代分离株有26株PCR扩增阳性,与HCMVToledo株进行序列比较分析,26株临床分离株UL149开放阅读框架(openreadingframe,ORF)之间存在着高度的多态性,种系进化树分析结果显示26个序列可分为3个型,黄疸患儿分布以G1型为主;小头畸形以G3型为主;巨结肠仅见于G1、G2型,G3型未见。部分临床分离株存在CKP位点的缺失及TKP位点增加。结论HCMVUL149基因在临床分离株中存在着高度的多态性。来自不同临床症状分离株的UL149基因及其编码蛋白具有一定的结构特点,并与基因型呈一定的相关性。     

Genetic variations in the gB,UL144 and UL149 genes of human cytomegalovirus strains collected from congenitally and postnatally infected Japanese children

Human cytomegalovirus (CMV) is the leading cause of intrauterine viral infection. The association of genetic polymorphisms in some particular genes with the incidence and severity of congenital infection has been controversial. To address this issue, we analyzed the genotypes of the glycoprotein B (gB), UL144 and UL149 genes of CMV clinical strains obtained from 33 congenitally and 31 postnatally infected Japanese children. Our results demonstrated that (1) CMV strains with any combination of genotypes could be vertically transmitted from mother to fetus, potentially causing neurological abnormalities, (2) the gB3 genotype was more prevalent in the congenital cases than in postnatally infected children (P < 0.05), particularly in congenital cases with sensorineural hearing loss (P = 0.009), (3) there was no relationship between gB genotype and viral load in the urine and dried umbilical cord specimens in the congenital cases, and (4) the UL144 and UL149 genotype distributions had no bias for congenial infection. In future studies, it would be interesting to see whether the gB genotypes serve as a prognostic indicator of CMV-associated diseases.     

Molecular epidemiology of primary human cytomegalovirus infection in pregnant women and their families

The source of human cytomegalovirus (HCMV) infection was investigated in 29 pregnant women with primary HCMV infection by comparing DNA sequences of UL146, UL144 and a portion of UL55 gene of HCMV strains circulating within each family. Thirteen families were identified in which the pregnant woman, the husband and/or a child were shedding HCMV. In three of these families, both the woman and the husband suffered from a concomitant primary HCMV infection. Phylogenetic analysis of UL146, UL144, and UL55 genes indicated that strains circulating within each family were identical, whereas strains from different families appeared to be distinct. However, identical UL146, UL144, and UL55 DNA sequences were observed sporadically among unrelated strains. A child rather than the husband was the virus source for the great majority of pregnant women. No association was observed between UL144 polymorphisms and intrauterine transmission.