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。     

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。     

人巨细胞病毒UL132基因在先天感染患儿临床株中的变异研究

目的研究人巨细胞病毒(human cytomegalovirus,HCMV)UL132基因序列在先天性感染患儿中的多态性,探讨HCMV基因多态性与其感染引起不同临床症状的关系。方法对30株经荧光定量PCR方法(Q-PCR)检测HCMV DNA为阳性的临床株进行HCMV UL132全序列PCR扩增,并对PCR扩增产物进行序列测定及分析。结果30株HCMV临床株的测序结果与HCMV Toledo株进行序列比较分析显示,临床株UL132开放阅读框架(open reading frame,ORF)间存在着高度的多态性,基因变异主要集中在ULl32 ORF的5’端。30株HCMV临床株种系进化树分析结果显示,UL132序列可分为3个基因型,黄疸患儿分布以G1型为主;神经损伤患儿以G2型为主;巨结肠患儿分别见于G1、G2、G3 3个基因型。所有临床株的ULl32编码蛋白是疏水性蛋白,含有信号肽及跨膜蛋白区域,并在信号肽和跨膜蛋白区域之间存在2个保守的N-糖基化位点。结论HCMV UL132基因在临床株中存在着高度的多态性。来自不同临床症状的HCMV UL132基因及其编码蛋白具有一定的结构特点,提示观132基因及其所编码的蛋白在HCMV的致病性上可能起重要作用。     

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。     

人巨细胞病毒UL138～142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)UL138～UL14 2基因在临床低传代分离株中的多态性及其与HCMV先天感染致病性之间的关系。方法　对经荧光定量PCR方法检测HCMV DNA为阳性的临床分离株进行UL138～UL14 2基因全序列PCR扩增 ,对扩增阳性的标本进行全序列的测序及结果分析。结果　HCMV临床分离株的UL138、UL14 2ORF(openreadingframe)高度保守 ;UL139ORF呈现高度多态性 ,可被明确划分为 3个基因型 ,且核苷酸及氨基酸的变异主要集中在序列的 5′端 ;所有临床分离株的UL14 0ORF在Toledo株第 174位核苷酸处插入 1个胞嘧啶核苷酸 ,其ORF较Toledo株增加了 2 31个核苷酸 ;所有临床分离株的UL14 1ORF在Toledo株第 2 2 7位核苷酸处缺失了 1个胸腺嘧啶核苷酸 ,故形成UL14 1a及UL14 1b 2个新的ORF。HCMV临床分离株的UL14 0蛋白较Toledo株新增了ScAMP磷酸化和酪蛋白激酶Ⅱ磷酸化位点 ,其它基因编码蛋白的重要功能区域相对保守。结论　HCMV临床分离株UL139ORF的 5′端呈现高度多态性 ,而且被明确地分成 3个基因型 ,故其可能在HCMV先天感染的致病性差异方面起一定作用 ;尚未发现UL138～UL14 2中某个特定基因与HCMV先天感染致病性有本质联系     

人类巨细胞病毒编码趋化因子HCMV UL 146序列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)UL146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23株HCMV临床低传代分离株及2例同年龄组HCMV-DNA定量PCR方法检测阳性健康儿尿液进行HCMV-UL146 PCR扩增及测序分析。结果UL146序列呈现较高的多态性,UL146的序列可以分为3组,所有巨结肠患儿标本均分布在G2组,无症状感染儿均在G2B组,而α化学因子功能域在各序列中保守。结论序列的变异可能会影响UL146吸附中性粒细胞,影响病毒扩散。     

人类巨细胞病毒UL145基因在临床低传代分离株中的多态性

有研究表明人巨细胞病毒(HCMV)在体内存在不同的细胞嗜性,提示来自于不同临床症状患儿的HCMV分离株的基因结构可能存在差异.我们曾报道过HCMV UL144基因多态性[1].本文着重研究了HCMV UL145序列在临床患儿低传代分离株中的多态性.     

人巨细胞病毒UL143基因在临床低传代分离株中的多态性研究

目的 探讨人巨细胞病毒(human eytomegalovirus,HCMV)UL143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19株HCMV临床低传代分离株进行HCMV-UL143 PCR扩增分析及伞序列测定分析.结果 19株HCMV感染患儿临床分离株均因碱基插入造成移码突变,开放阅读框架(ORF)比Toledo株短.根据序列变异情况可将19个序列分为2组,第1组16个序列新增一个MYRISTYL位点;缺失2个PKC磷酸化位点.未发现黄疸、小头畸形、先天性巨结肠等不同疾病类型的序列之间的差异.结论 HCMV-UL143较多存在于临床低传代分离株中,序列呈现一定多态性.     

人巨细胞病毒UL150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(human cytomegalovirns,HCMV)UL150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(Q-PCR)检测HCMV-DNA为阳性的临床低传代分离株进行HCMV UL150全序列PCR扩增,并对PCR扩增产物进行序列测定及分析.结果在29株临床低传代分离株中25株PCR扩增阳性.其中18株完成了测序.18株临床分离株HCMV UL150开放阅读框架(open reading frame,ORF)与HCMV Toledo株相应序列进行比较分析,显示18株低传代临床分离株的HCMV UL150 ORF均为1920bp,编码蛋白含有640个氨基酸,临床株的ORF及编码的氨基酸序列的长度均与Merlin株一致.结论 HCMV UL150基因在临床分离株中存在着高度的多态性,未发现其与HCMV感染不同临床症状间存在明显的关系.     

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 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.     

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.     

Human cytomegalovirus glycoprotein B genotypes in Brazilian mothers and their congenitally infected infants

A case-control study design was used in order to compare the distribution of human cytomegalovirus (HCMV) glycoprotein B (gB) genotypes in 48 mothers of 49 congenitally infected infants with that observed in 144 mothers of 146 uninfected infants to study genetic variation of HCMV strains and maternal-fetal transmission. Congenital infection with HCMV was characterized by DNA detection and virus isolation from two urine or saliva samples collected prior to the third week of life. Genotyping of HCMV was carried out by a polymerase chain reaction-restriction fragment length polymorphism analysis of the variable region of the gB gene, testing for four genotypes. Genotype frequency was similar among the 28 non-transmitting mothers who were shedding virus (gB1: 25%; gB2: 28.6%; gB3: 42.8%; gB4: 0%), the 37 transmitting mothers (gB1: 21.6%; gB2: 46%; gB3: 27%; gB4: 0%), and the 49 infected infants (gB1: 39%; gB2: 37%; gB3: 24%; gB4: 0%). The same genotype was detected at different body sites (urine, saliva, and blood) of each infected newborn and in the respective mother (breast milk, urine, and saliva). Co-infection with multiple genotypes was observed in the immediate postpartum period in two mothers of infected infants (5.4%) and one non-transmitting mother (3.6%). The gB genotype was not correlated with intrauterine HCMV transmission. The genotype distribution found reflects the overall frequency of wild strains circulating in this geographic region. A single genotype is responsible for congenital HCMV infection. Co-infection with more than one strain, as characterized by gB genotype, was infrequent in women who were presumably immunocompetent.     

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.     

Human Cytomegalovirus UL144 Is Associated with Viremia and Infant Development Sequelae in Congenital Infection

Human cytomegalovirus (HCMV) strains may be genotyped based on polymorphisms that exist within the UL144 gene, which is one of 19 viral genes lost in attenuated laboratory strains. In the present study, UL144 genotypes in congenitally infected babies (congenital cytomegalovirus [cCMV]) were determined, and the relationship between the genotype, viral load, cytokine profile, and patient developmental outcome was investigated. All cCMV infections identified during 2006 and 2007 were included (n = 29). A portion of the infants were clinically assessed at birth and at 12 to 18 months postinfection for cCMV clinical sequelae (n = 18/29). The plasma viral load (PVL) was requested for 23/29 patients, and the UL144 genotype was determined (n = 27/29). The cytokine profile in patient plasma or serum was assessed (n = 20/29). UL144 genotypes A, B, and C were detected within the cCMV population at 33.3%, 29.6%, and 25.9%, respectively. UL144 A and C were associated with a high PVL (P < 0.04). Furthermore, a significant association between the developmental outcome and UL144 A and C was observed (P < 0.04). Only patients infected with UL144 B and A/B were described as having a normal clinical outcome. In addition, a significant correlation between interleukin 10 (IL-10) levels and the PVL was observed (P < 0.04); however, there was no association between the genotype and the cytokine profile. The present study determined that the specific detection of UL144 genotypes A and C was indicative of serious cCMV infection and more likely to lead to long-term cCMV-associated clinical manifestations. The inclusion of HCMV UL144 genotyping along with the recommended PVL monitoring following cCMV diagnosis may aid prediction of the clinical outcome.Congenital cytomegalovirus (cCMV) infection is the most common congenital infection in the developed world, affecting approximately 1% of live-born neonates. It is a frequent cause of mental retardation and the leading nongenetic cause of sensorineural hearing loss (SNHL) (7, 11). cCMV disease can result from either primary maternal infection or reactivation from latency during pregnancy, although generally, the most severe clinical syndromes follow primary infection. However, at present there is no way of definitively identifying at birth those infants who will develop sequelae and those who will not.The clinical significance of human cytomegalovirus (HCMV) molecular epidemiology is unclear and controversial, as the 236-kb viral genome suggests that a large number of polymorphic strains may potentially exist (10). HCMV infects many different cell types, resulting in a diverse range of clinical manifestations and suggesting that the clinical outcome may be related to both genetic variation among HCMV strains and the host immune response(s). Previous studies have investigated genetic polymorphisms that exist within the envelope glycoprotein genes, as their encoded proteins are targets for neutralizing antibodies. However, the glycoprotein B (gB) gene, which encodes a putative target for HCMV vaccination, has shown no consistent relationship with disease outcome (3, 16, 25).HCMV strains may be genotyped based on polymorphisms that exist within the UL144 gene, which is one of 19 viral genes lost in attenuated laboratory strains (4). The majority of these deleted genes are nonessential for viral replication; however, expression in vivo may contribute to disease pathogenesis (8). Three main HCMV genotypes, based on the ectodomain of the UL144 protein, have been described: UL144 A, UL144 B, and UL144 C (1, 2, 16, 24). Conflicting reports on the association between the UL144 genotypes and the viral load, clinical presentation, and clinical outcome have been published. Arav-Boger and colleagues concluded that infection with the UL144 A and C strains was associated with unfavorable clinical outcome in neonates (2). In addition, genotype UL144 C was linked to termination of pregnancy following detection of HCMV in the amniotic fluid (1). In direct contrast, UL144 genetic polymorphisms were associated with neither clinical presentation nor viral-load levels in the amniotic fluid in a French population (18, 19). Furthermore, Bale and colleagues found no relationship between the UL144 genotype and the congenital clinical outcome (3).The present study addresses these inconsistent findings. The UL144 genotypes, detected in a well-defined, geographically distinct group of congenitally infected infants, were analyzed with respect to the viral loads, immunological cytokine profiles, and developmental outcomes of affected infants. Our findings show that the HCMV genotypes UL144 A and C are significantly associated with high plasma viral loads (PVLs) and long-term cCMV clinical sequelae.(Part of this work was presented in a 15-minute oral presentation [abstract number O-12] at the 2008 Congenital Cytomegalovirus Conference, Centers for Disease Control and Prevention, Atlanta, GA, 5 November 2008.)     

Is the mixture of human cytomegalovirus genotypes frequent in infants with congenital infection at birth in a high seroprevalence population?

It is still not well known, in a population with high human cytomegalovirus (HCMV) seroprevalence, whether a child with congenital infection harbors multiple viral strains at birth, and whether the prolonged viral excretion in these children is secondary to the persistence of the same viral strain. To verify the genomic diversity of HCMV detected in congenitally infected children, the nucleotide viral sequences from urine and/or saliva obtained at birth from 14 newborns with congenital infection and breast milk obtained from mothers of 5 of these children were analyzed. Among the 14 children, 10 had sequential samples until the median age of 10 months. The viral nucleotide sequences in the breast milk were compared with those identified in the respective children at birth. The differentiation of viral strains was based on the variability of 3 regions of viral genes (UL55/gB, UL144, and UL73/gN). In 13/14 children (92.8%), a single genotype was observed at birth. Different viral genotypes were found in 1 child (7.2%). Among the sequential samples from 10 children, the same genotype obtained at birth was detected in 9/10 (90%), and in 1 of them (10%), a genotype change in the urine was found. More than 1 HCMV strain in milk was observed in 2 mothers (2/5, 40%). In a population with high seroprevalence, a single genotype was found in the majority of infected children. Reinfection did not frequently occur in the first months of life. Maternal reinfection does not seem to be a rare event in transmitter mothers.