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。     

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.     

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。     

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.     

人巨细胞病毒临床分离株UL131A-128mRNA转录方式的研究

目的 研究人巨细胞病毒（ human cytomegalovirns,HCMV） UL131 A-128序列在临床低传代分离株中的转录方式.方法 用3＇RACE技术扩增HCMV不同临床株,不同时期的UL131A,UL128,UL130的mRNAs并测序分析;利用PCR技术在HCMV临床株cDNA文库中筛选分别含有UL13IA,UL130,UL128序列的阳性克隆,测序并分析结果.结果 成功在临床低传代分离株中得到UL13IA,UL128,UL130基因的转录结构,UL131A,UL130的转录方式和文献报道一致,UL131A含有两个外显子,UL130为此区域内唯一不含内含子的基因.而UL128基因的转录本呈现了两种转录方式,一种结构包含三个外显子,长度为519 bp;而另一种结构包含三个外显子和第一内含子结构,长度为642 bp,在不同病毒株的不同时期均显示了包含第一内含子结构转录本的含量明显高于仅有外显子结构的UL128转录本的含量.UL131A-128三个基因在病毒的即刻早期,早期,晚期均存在.3＇RACE得到结果与HCMV cDNA文库筛选得到的结果相一致.结论 UL131A,UL130和UL128基因的mRNA结构的起始位点不同,但他们在3’末端拥有共同的终止位点.HCMV临床株UL 131A-128基因转录方式的复杂结构可能在HCMV感染的不同时期具有重要作用.     

人巨细胞病毒UL148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(HCMV)UL148序列在临床低传代分离株中的多态性。方法 对38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的细胞培养上清液进行HCMV UL148全序列PCR扩增，并对PCR扩增产物进行序列测定及分析。结果 38株临床低传代分离株有17株PCR扩增阳性，与HCMV Toledo株进行序列比较分析，17株临床分离株ULl48开放阅读框架(ORF)长度均与Toledo株相同。其编码蛋白的氨基酸变异率为0．3％～2．3％。所有分离株均有蛋白翻译后修饰位点的新增或缺失。与Toledo株相比17株临床分离株UL148蛋白质二级结构预测结果均为第15～18位之间由α-螺旋变为β-折叠。结论 17株HCMV临床低传代分离株UL148基因及其编码产物的氨基酸序列比较保守，但仍存在一定程度的多态性。     

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.     

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. The significant clinical impact of HCMV infection and progression to HCMV disease among allogeneic hematopoietic stem cell transplant recipients has been reduced by prophylactic, preemptive, and curative treatments using ganciclovir, valganciclovir, foscarnet, and cidofovir. Resistance to (val)ganciclovir results from mutations localized in HCMV UL97 gene (encoding the pUL97 phosphotransferase), UL54 gene (encoding the pUL54 DNA polymerase), or both genes, whereas foscarnet and cidofovir resistance results from mutations localized within UL54 gene only. This review is focused on HCMV antiviral drug resistance, including the functions of target genes of antivirals, the mechanisms of antiviral resistance, the different mutations in pUL97 and pUL54 that have been identified in either clinical isolates or laboratory strains, and their impact on HCMV susceptibility to antiviral drugs. It emphasizes the importance of proving that observed genetic changes confer resistance so they can be distinguished from polymorphisms. Because of the emergence of HCMV resistance to currently available drugs, novel drugs are urgently needed for the therapeutic management of HCMV‐resistant infections in hematopoietic stem cell transplant patients. Copyright © 2016 John Wiley & Sons, Ltd.     

广州地区人巨细胞病毒临床低传代株UL136基因的序列特征及多态性

目的 研究广州地区新生儿感染人巨细胞病毒(HCMV)临床低传代株UL136基因的序列特征与基因多态性.方法 从10例广州感染新生儿体内分离获得2株(D2、D3)临床HCMV分离株,经多重PCR鉴定后进行UL136基因全序列扩增.PCR产物纯化后进行基因克隆,构建HCMV UL136-pMD18-T重组质粒.经基因测序及应用生物信息学分析方法 ,分析其核酸序列稳定性、编码蛋白质的二级结构与特征.结果 成功分离2株HCMV临床分离株,测序结果 显示,D2、D3及与GenBank中公布的11株临床分离株(4J、51C、39J、33J、63J、22M、10J、32C、29C、27C、Toledo)中,UL136序列高度保守.同源性分析显示在UL136全基因序列1019个核苷酸中,存在30个位点变异,所有的变异均为碱基替换,无插入及缺失突变.编码蛋白的氨基酸序列也高度保守,240个氨基酸残基中,不同临床分离株氨基酸变异率为1.6%～3.7%.不同分离株的UL136蛋白中参与形成二级结构的氨基酸数目及等电点不同.进化树分析结果 显示D2和D3均属于1a群.结论 广州地区临床低传代分离株HCMV UL136基因核苷酸序列及其氨基酸序列极为保守,但仍存在一定多态性.其基因的稳定性提示HCMV UL136开放阅读框(ORF)可能是一个具有重要功能的基因.其编码后修饰位点提示UL136可能与膜受体介导的细胞信号转导通路有关.     

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 genetic variability in strains isolated from Japanese children during 1983-2003

The genetic variability of 74 human cytomegalovirus (HCMV) clinical isolates from 60 Japanese infants and children during 1983-2003 was investigated, and the relevance to their clinical course was studied. The patients consisted of 10 asymptomatic congenitally infected babies, 45 infected perinatally or postnatally resulting in HCMV mononucleosis/hepatitis and 5 immunocompromised hosts. The hypervariable region of the HCMV genome, that is the a sequence and UL144 region was analyzed using the polymerase chain reaction (PCR) and unrooted phylogenetic trees. HCMV glycoprotein B (gB) polymorphism was also studied. Unrooted phylogenetic trees of a sequence and UL144 allowed the isolates to be grouped to 5 and 3 clades, respectively. Three gB genotypes were also determined. However, there was no correlation between specific genotypes of these three genes and clinical forms, except for congenital infection which fell into one of three clades of the UL144 gene. In addition, the variability of the three genes had no correlation with each other. This implies that study of a single gene is insufficient for investigating the molecular epidemiology of HCMV. This study provides basic data on the genetic variability of HCMV in an Asian population and should help to determine the strains for vaccine candidates.     

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.