人巨细胞病毒UL139、UL140、UL138基因多态性研究
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摘要
人巨细胞病毒(Human cytomegalovirus，HCMV)为独特的β—疱疹病毒，一旦发生感染，常终身带毒。目前HCMV感染在引起先天性宫内感染的微生物中居于首位。综合国内外研究结果，活产新生儿的先天性HCMV感染率约为0.3％—2.0％。其中有症状或明显改变的占5％，主要包括：黄疸性肝炎、先天性巨结肠、小头畸形，智力发育迟缓等；有轻微症状或非典型症状改变的占5％；其余90％HCMV感染表现为无症状感染，但其中5％-15％HCMV感染患儿多在生后两年内发生神经性耳聋或不同程度的神经精神运动障碍。
目前为止，HCMV的致病机制尚不清楚。一方面与宿主的免疫功能，即有效的细胞及体液免疫有关；一方面与病毒的数量、入侵部位及病毒自身的基因结构，尤其是与病毒毒力、组织嗜性和病毒逃避机体免疫攻击能力相关的基因，也密切相关。
1996年，Cha等发现HCMV低传代的Toledo株与临床分离株大约有15kb的片段(19个ORF)在实验室AD169株中不存在。它们定位于HCMV的UL／b’区，依次命名为UL133-UL151，此区为高度可变区。
AD169、Towne等实验室株在细胞培养反复传代过程中出现了UL／b’区遗传信息的丢失和重排，并导致HCMV毒力和致病性的明显减弱。根据小鼠动物模型，Toledo株比实验室株复制水平至少高三倍；随后的研究发现，Toledo株和临床分离株对上皮和内皮细胞存在嗜性，而高传代的AD169株则丧失了这种能力；并且发现这15kb的片段对于病毒能够在移植人体组织的重症联合免疫缺陷(SCID)鼠中进行复制是必需的，暗示这段基因在HCMV于人体内复制过程中的重要性。
目前，关于HCMV UL／b’区基因的多态性及其与发病机制的关系成为国际HCMV最活跃的研究领域。其中人们已经发现其在HCMV感染过程中发挥一定作用的基因包括UL141、UL142、UL144以及UL146、UL147。
HCMV UL141基因表达产物能够下调自然杀伤细胞(NK细胞)—激活配体CD155的表达，从而介导了HCMV感染细胞有效防止自然杀伤细胞的杀伤作用。HCMV UL142基因编码的包膜糖蛋白能够下调自然杀伤细胞激活性受体(NKG2D)的配体MHCⅠ相关分子A(MICA)，从而也能够有效的防止NK细胞的杀伤作用；同时UL142编码蛋白还可以抑制NK细胞的裂解。HCMV UL144编码一种Ⅰ型跨膜糖蛋白，据推测其编码产物是一种疱疹病毒穿入介子(HveA或HveM)的类似分子，HveA是肿瘤坏死因子受体超家族成员(TNFR)之一，在人类细胞和体液免疫防御中都发挥重要作用；有关UL144不同基因型是否与HCMV感染临床及预后相关尚处于争论之中。HCMV UL146、UL147基因编码产物与α-趋化因子白细胞介素(IL-8)有相同的信号肽及半胱氨酸空间位置。但到目前为止，尚未发现上述哪个基因多态性与HCMV感染致病性明确相关；这使我们推测在这个区域的其他基因是否能够在HCMV致病性方面起重要作用。
HCMV UL139、UL140和UL138基因均定位于实验室株不存在的19个ORF中，目前它们编码蛋白在理论上是存在的，但其生物学功能还不清楚，少见文献报导。本文主要研究HCMV UL139、UL140和UL138基因在低传代临床分离株中的多态性，为探讨其多态性与HCMV感染不同致病性之间的关系奠定基础；同时推测其编码蛋白的生物学功能，进而为在分子水平上揭示HCMV感染的致病机制奠定基础。
材料与方法
一、标本来源
HCMV临床分离株及4例尿标本均来自我院1988-1993年期间住院患儿，其中有黄疸患儿、小头畸形及先天性巨结肠患儿。另外3例尿标本来自门诊同年龄组无症状感染儿，均追踪至2岁无任何异常表现。所有标本于2000年进行荧光定量PCR(QPCR)测HCMV-DNA均为阳性，并排除其他病毒感染。-70℃备用。
二、实验方法
(一)标本处理，提取DNA
1、临床分离株
标本取自病毒分离实验，取分离株上清液与等体积裂解液混合，煮沸15min，离心数秒。
2、尿标本
取1ml尿标本，15000rpm／min离心15min，弃上清，加DNA裂解液50μl，混匀，离心数秒。100℃煮沸10min，15000rpm／min离心5min。
(二)PCR扩增
1、引物设计
表1，PCR扩增引物
2、PCR扩增
反应体系为50μl：10×Buffer 5μl，dNTP混合物2.5μl(2.5mmol／L)，rTaq酶2U，上下游引物各1μl(33pmol／μl)，模板3.5μl，用双蒸水补至总反应体积50μl。
95℃预变性4分钟；95℃变性45秒，54℃退火1分钟，72℃延伸1分钟，30个循环；72℃终延伸10分钟。
(三)PCR产物纯化、回收及测序
将测序标本进行凝胶电泳后，切出目的DNA片段，应用Takara公司的PCR Fragment Recovery Kit回收。PCR扩增标本应用BigDye Terminators Cycle Sequencing Kit，采用ABI公司的3700型全自动DNA测序仪进行双向测序，测序由上海联合基因公司完成。
(四)序列分析和提交
序列分析应用软件DNAClub，BioEdit，GeneDoc，DNAStar等；用Sequin软件向GenBank提交整理后序列。
结果
一、HCMV UL139、UL140和UL138基因PCR扩增结果
26例HCMV UL139标本以及30例UL140和UL138标本经PCR扩增后全部阳性，且测序结果理想。部分序列已被GenBank收录，序列号为No．AY21 8873-AY21 8887，AY601 874-AY601 877，AY8052552，AY805259，AY805260，AY805292，AY805294，AY905263，AY905264，AY218849-AY218859及AY255774-77，AY218860-218872，AY255772-73，AY601873，AY941163-65。
二、HCMV UL139、UL140和UL138基因多态性研究
(一)HCMV-UL139基因核苷酸及氨基酸序列的比较及进化树分型
各临床分离株HCMV-UL139基因序列由408-444个核苷酸构成。核苷酸的变异成簇分布形成三个主要的基因型G1(G1a、G1b、G1c)，G2(G2a、G2b)以及G3。G1a型与Toledo株同源性为≌100％；其他亚型与Toledo株相比，包含12-36个核苷酸的插入以及24-49个核苷酸的替换，其中47.8-60.7％是错义突变，导致广泛的氨基酸变异。
HCMV UL139编码蛋白的氨基酸序列进化分析结果与核苷酸序列的进化树基本一致。以Toledo株为参考株，临床分离株(除G1a型)虽于序列的5′端存在多个核苷酸的插入，由于均为3或3N个核苷酸的插入，未造成移码突变，故3′端氨基酸序列基本相似。所有UL139编码蛋白的羧基端及8个半胱氨酸是相对保守的。
(二)HCMV-UL140基因核苷酸序列及氨基酸序列的分析
Toledo株和15株临床低传代分离株HCMV-UL140的ORF均由576个核苷酸构成，预测编码191个氨基酸的蛋白。测序结果显示Toledo株与各临床低传代分离株之间的HCMV-UL140核苷酸序列及氨基酸序列比较保守，变异率分别为0—3.2％及0—4.6％。DNA序列的变异分布在整个编码区，均为碱基替换，无插入及缺失。绝大部分的核苷酸为同义突变，没有引起编码氨基酸的改变；同时绝大多数的氨基酸变异为非保守替换。
(三)HCMV-UL138基因核苷酸序列及氨基酸序列的分析
Toledo株和19株临床低传代分离株HCMV-UL138的ORF均由510个核苷酸构成，预测编码169个氨基酸的蛋白。测序结果显示Toledo株与各临床低传代分离株之间的HCMV-UL138核苷酸序列及氨基酸序列比较保守，变异率分别为0—2.4％及0—1.8％。DNA序列的变异分布在整个编码区，均为碱基替换，无插入及缺失。绝大部分的核苷酸为同义突变，没有引起编码氨基酸的改变；同时绝大多数的氨基酸变异为非保守替换。
三、HCMV UL139、UL140和UL138编码蛋白性质及功能预测
(一)HCMV UL139编码蛋白性质及功能预测
应用Editseq及DNASis软件进行UL139编码蛋白分子量(Mr)、等电点(pI)和二级结构的预测。UL139蛋白是一个分子量大约为14.2—15.3KD，等电点为4.94-5.64的弱酸性蛋白质。蛋白质二级结构的预测可以分为6个型，与UL139核苷酸分型相对应。
我们发现所有的UL139包膜糖蛋白(gpUL139)与B淋巴细胞表面的分化抗原CD24存在一定区域的同源。
HCMV临床分离株UL139基因预测的蛋白质重要功能区域，由于各株所属基因型的不同而略不同；分别包括4或5个糖基化位点(ASN，N-glycosylation site)位点，2-4个N相连豆寇酰化位点(MYR，N—myristoylation site)位点，3或4个酪蛋白激酶Ⅱ磷酸化位点(CKP，Casein kinase phosphorylationⅡsite)位点。上述3种重要功能区域在26份标本中均存在，而且相对保守，与Toledo株相比，没有明显改变。其中G1b型的UL139基因编码蛋白，在氨基酸位点34-44之间新增了一个独特的原核膜脂蛋白脂结合位点(Prokaryotic membrane lipoprotein lipidattachment site，ADL)重要功能位点。
(二)HCMV-UL140编码蛋白性质的预测
HCMV UL140编码蛋白是一个分子量约21.5KD，pI为4.73-5.04的一个酸性蛋白。蛋白二级结构预测显示，Toledo株和15株临床低传代分离株(包括黄疸、小头畸形及先天性巨结肠三种疾病类型)HCMV UL140编码蛋白的二级结构基本相同。我们预测在氨基酸位点15-25，80-90及130-150之间可能是HCMV UL140编码蛋白的亲水性区域。
所有低传代临床分离株HCMV-UL140编码蛋白的重要功能位点均比较保守，包括蛋白激酶C磷酸化位点(PKC，Protein kinase C phosphorylation site)和ScAMP磷酸化位点(SPS，SulfationcAMP phosphorylation site)以及ASN、MYR和CKP位点。各临床分离株HCMV-UL140编码蛋白的重要功能位点略有不同。临床分离株1M的UL140蛋白缺失了一个CKP功能位点，增加了一个PKC功能位点；临床分离株51C，缺失了一个ASN功能位点。而分离株27C、29C、2J、39J等UL140蛋白增加了一个CKP功能位点。
(三)HCMV—UL138编码蛋白性质的预测
HCMV UL138编码蛋白是一个分子量约19.3kD，pI 6.84的一个弱酸性蛋白。蛋白二级结构预测显示，Toledo株和19株临床低传代分离株HCMV UL138编码蛋白的二级结构基本相同。我们预测在氨基酸位点55-65，70-90及105-115之间可能是HCMV UL138编码蛋白的亲水性区域。
所有临床低传代分离株HCMV-UL138编码蛋白的重要功能位点均比较保守，包括酪氨酸激酶磷酸化位点(TKP，tyrosine kinase phosphorylation site)，以及PKC、MYR、CKP和SPS位点。与Toledo株相比，无新增的重要功能基团；各临床分离株(除20M株外)缺失了一个PKC位点。
四、UL139、UL140及UL138基因多态性与HCMV感染不同临床表现的关系
在HCMV UL139基因中，无症状的HCMV感染患儿与有症状的患儿一样分别存在于UL139的三个基因型中。不同临床表现的HCMV感染患儿在UL139基因型中的分布没有明显不同。
我们未发现不同疾病类型(黄疸、小头畸形和先天性巨结肠)的临床分离株在UL140及UL138基因中成簇分布。
结论
1．HCMV UL139、UL140和UL138基因广泛存在于临床低传代分离株中。
2．HCMV UL139基因呈现高度多态性，是目前发现的HCMV中变异最大的基因之一，存在多个核苷酸的插入和替换(多为错义突变)。而且这些变异形成了株特异性的基因型，表明生物进化选择有助于UL139变异各型的保留，从而使UL139基因不同基因型的变异在特定的环境中可能具有更大的适应性。
3．我们发现HCMV UL139编码跨膜糖蛋白与人B淋巴细胞表面的分化抗原CD24存在一定区域的同源，可能参与机体在HCMV感染后的免疫反应。
4．HCMV UL140及UL138序列高度保守。
Human Cytomegalovirus(HCMV) is a ubiquitous betaherpesvirus with a wide spectrum of infectivity, as demonstrated by several cell types that can be infected in vitro and by multiple organs observed during infection in vivo.HCMV infection can cause serious diseases, particularly in immunocompromised patients and newborns. However, the mechanisms of viral pathogenesis are not known. Differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent HCMV infections are thought to be related to genomic variability among strains.
HCMV is a genetically complicated virus, its genome consists of 230-235 kb double-strands DNA and more than 200 predicted open reading frames (ORFs). A new region of HCMV DNA containing at least 19 ORFs( UL133-UL151) was found in the Toledo strain(EMBL,AY446871) and in several other low passage clinical isolates recently, but it was deleted in laborotary strain AD169(EMBL,X17403). The fact that AD169 shows attenuated virulence and different tropisms in endothelial cell from low passage isolates,suggests that predicted products of these new ORFs possibly determine the outcome of HCMV infection in vivo.
The possibility that UL133-151 ORFs in the UL/b′region may provide genetic markers for HCMV pathogenesis prompted to investigate these genes in HCMV clinical isolates. UL141 might downregulate natural killer cell-activating ligand CD155, UL144 encodes a structural homologue of the herpesvirus entry mediator and both UL146 and UL147 encode viral CXC(α) chemokines. At present,the variabilities and functions of these genes are being investigated.However, the definitive associations between these genes and HCMV disease have not been established,which leaves open the possibility that other variable HCMV-encoded products in the region may play a role in viral pathogenesis.
The genomic variabilities of HCMV UL139、UL140 and UL138 ORF and the relationship between the variabilities of these genes and different pathogenesis of congenital HCMV infection were detailedly investigated in this report.
Materials and Methods
1.Patients and Samples
The study population of HCMV UL139 consisted of twenty-six infants with HCMV infection who were identified in the 2nd affiliated hospital of China Medical University. These infants aged less than 14 months with suspected congenital infection had different clinical manifestations: jaundice (J,n=15), Hirschsprung's disease (C,n=4), microcephaly (M,n=4)and asymptomatic infection (A,n=3). Nineteen low passage clinical isolates and seven urine samples from the infants were used to carry out PCR amplification. Isolates, recovered from abnormal colon tissue or urine, were passaged less than 10 times in human embryonic lung fibroblasts (HELF).
The study population of UL140 and UL138 consisted of thirty HCMV infected infants who exhibited different clinical manifestations: jaundice (n=18), Hirschsprung's disease (n=7),microcephaly (n=5). All sample were clinical isolates.
2.PCR Amplification
According to the corresponding Toledo sequence, primers were designed using Primer premier 5.0 software, see Table 1.
The PCR reaction mixture contained 1×buffer, 1.5mM MgCl_2, 0.2mM dNTPs, forward and reverse primers 150ng respectively , 0.5U of Taq polymerase,template 3.5μl in a final volume of 50μl.The conditions for amplification were 95℃for 4 min followed by 30 cycles of 95℃for 45 sec, 54℃for 1 min, 72℃for 1 min, and a final extension at 72℃for 10 min.
3.Purification and Sequencing
The PCR products of the appropriate size were purified for sequencing using the PCR Fragment Recovery Kit. Sequencing was carded out on both DNA strands. The sequences were analyzed on an ABI 3700 automated sequencer.
4.Sequence Analysis
All nucleotide and amino acid sequences were analyzed using the BioEdit 5.0,DNAClub,GeneDoc,DNAStar and Sequin software.
RESULTS
1.Presence of the UL139, UL140 and UL138 ORF
All samples gave positive amplification of HCMV UL139、UL140 and UL138. PCR products selected were sequenced successfully. The sequences have been assigned Genbank accession No. AY218873-AY218887, AY601874-AY601877, AY8052552, AY805259, AY805260, AY805292, AY805294, AY905263, AY905264, AY218860-AY218872, AY255772, AY255773, AY601873, AY941163-AY941165 and AY218849-AY218859, AY255774-AY255777.
2. Analysis of the UL139、UL140 and UL138 DNA and Amino Acid Sequence
(1)Analysis of the UL139 DNA and Amino Acid Sequence
The UL139 gene sequences from HCMV infected infants ranged in length from 408 to 444 bp. Using the Toledo strain as the arbitrary reference sequence, the variants were clustered clearly into three major groups G1 (Gla、G1b、G1c), G2 (G2a、G2b) and G3. The strains in Gla showed a high level of identity to the Toledo strain. However, the strains in the other subgroups contained 12-36 nucleotide insertions and 24-49 substitutions, of which 47.8-60.7% were non-synonymous with respect to the Toledo strain.
The non-synonymous nucleotide substitutions led to a wide spectrum of amino acid changes. Phylogenetic analysis of the UL139 putative proteins was mainly consistent with the results described for DNA. Although a relatively large number of nucleotides were inserted in the 5' half of the UL139 ORF in some strains, no frame-shift mutation occurred due to 3 or 3N nucleotide insertions. The C-terminal region and the eight cysteines of all UL139 putative protein appeared relatively conservative.
(2)Analysis of the UL140 DNA and Amino Acid Sequence
The UL140 ORF in clinical isolates was identical in size to that of Toledo, composed of 576 nucleotides. The identities in nucleotide were 96.8-100.0%. The UL 140 ORF was predicted to be a possible glycoprotein of 191 amino acids. Alignment of amino acid sequences showed a high level of identity from 95.4% to 100.0%.
(3)Analysis of the UL138 DNA and Amino Acid Sequence
The UL138 ORF in clinical isolates was identical in size to that of Toledo too, composed of 510 nucleotides and predicted to be a protein of 169 amino acids. The identities in nucleotide and amino acid sequences were 97.6%-100.0% and 98.2%- 100.0% respectively.
3.Structure Analysis of the Predicted UL139, UL140 and UL138
Proteins
(1)Structure Analysis of the Predicted UL 139 Protein
The UL139 putative protein was predicted to be a glycoprotein(gpUL139). All gpUL139 shared sequence homology with human CD24 mature peptide in a restricted region. The gpUL139 contained 4 or 5 N-glycosylation sites(ASN), 3 or 4 Casein kinaseⅡphosphorylation sites(CKP), 2-4 N-myristoylation sites(MYR), depending on the sequence groups which clinical strains belonged to. In addition, the sequences in the strains of Glb contained a specific attachment site of prokaryotic membrane lipoprotein lipid.
(2)Structure Analysis of the Predicted UL 140 Protein
The post-translational modification motifs of UL 140 putative proteins in clinical isolates were highly conserved. 2J, 39J, 27C and 29C added a CKP site in the same position. However, these isolates were derived from the infants with different clinical signs which were jaundice and Hirschsprung's disease respectively.
(3)Structure Analysis of the Predicted UL 138 Protein
The post-translational modification motifs of UL138 putative proteins in clinical isolates were highly conserved.
4.Relationship Between UL139、UL140、UL138 and Signs of Congenital Infections
The strains from infants with asymptomatic HCMV infection, as those from patients who suffered from HCMV disease,existed in three UL139 genotypes. The distribution of the strains from infants with different clinical manifestations in the UL139, UL140 and UL138 sequence groups was not apparently different.
Conclusions
1. HCMV UL139, UL140 and UL138 genes were extensively present in HCMV clinical strains.
2. HCMV UL139 gene is one of the most variable region in HCMV. A large number of nucleotide insertions and non-synonymous substitutions occurred in the UL 139 ORF. The clustered polymorphisms in the UL 139 sequence imply that selective pressures might favor retention of each subgroup.
3. The finding that the putative gpUL139 shared sequence homology with human CD24 suggested that the UL139 putative protein might be involved in the immune response during HCMV infection.
4. HCMV UL 138 and UL 140 genes were highly conserved.
目前为止，HCMV的致病机制尚不清楚。一方面与宿主的免疫功能，即有效的细胞及体液免疫有关；一方面与病毒的数量、入侵部位及病毒自身的基因结构，尤其是与病毒毒力、组织嗜性和病毒逃避机体免疫攻击能力相关的基因，也密切相关。
1996年，Cha等发现HCMV低传代的Toledo株与临床分离株大约有15kb的片段(19个ORF)在实验室AD169株中不存在。它们定位于HCMV的UL／b’区，依次命名为UL133-UL151，此区为高度可变区。
AD169、Towne等实验室株在细胞培养反复传代过程中出现了UL／b’区遗传信息的丢失和重排，并导致HCMV毒力和致病性的明显减弱。根据小鼠动物模型，Toledo株比实验室株复制水平至少高三倍；随后的研究发现，Toledo株和临床分离株对上皮和内皮细胞存在嗜性，而高传代的AD169株则丧失了这种能力；并且发现这15kb的片段对于病毒能够在移植人体组织的重症联合免疫缺陷(SCID)鼠中进行复制是必需的，暗示这段基因在HCMV于人体内复制过程中的重要性。
目前，关于HCMV UL／b’区基因的多态性及其与发病机制的关系成为国际HCMV最活跃的研究领域。其中人们已经发现其在HCMV感染过程中发挥一定作用的基因包括UL141、UL142、UL144以及UL146、UL147。
HCMV UL141基因表达产物能够下调自然杀伤细胞(NK细胞)—激活配体CD155的表达，从而介导了HCMV感染细胞有效防止自然杀伤细胞的杀伤作用。HCMV UL142基因编码的包膜糖蛋白能够下调自然杀伤细胞激活性受体(NKG2D)的配体MHCⅠ相关分子A(MICA)，从而也能够有效的防止NK细胞的杀伤作用；同时UL142编码蛋白还可以抑制NK细胞的裂解。HCMV UL144编码一种Ⅰ型跨膜糖蛋白，据推测其编码产物是一种疱疹病毒穿入介子(HveA或HveM)的类似分子，HveA是肿瘤坏死因子受体超家族成员(TNFR)之一，在人类细胞和体液免疫防御中都发挥重要作用；有关UL144不同基因型是否与HCMV感染临床及预后相关尚处于争论之中。HCMV UL146、UL147基因编码产物与α-趋化因子白细胞介素(IL-8)有相同的信号肽及半胱氨酸空间位置。但到目前为止，尚未发现上述哪个基因多态性与HCMV感染致病性明确相关；这使我们推测在这个区域的其他基因是否能够在HCMV致病性方面起重要作用。
HCMV UL139、UL140和UL138基因均定位于实验室株不存在的19个ORF中，目前它们编码蛋白在理论上是存在的，但其生物学功能还不清楚，少见文献报导。本文主要研究HCMV UL139、UL140和UL138基因在低传代临床分离株中的多态性，为探讨其多态性与HCMV感染不同致病性之间的关系奠定基础；同时推测其编码蛋白的生物学功能，进而为在分子水平上揭示HCMV感染的致病机制奠定基础。
材料与方法
一、标本来源
HCMV临床分离株及4例尿标本均来自我院1988-1993年期间住院患儿，其中有黄疸患儿、小头畸形及先天性巨结肠患儿。另外3例尿标本来自门诊同年龄组无症状感染儿，均追踪至2岁无任何异常表现。所有标本于2000年进行荧光定量PCR(QPCR)测HCMV-DNA均为阳性，并排除其他病毒感染。-70℃备用。
二、实验方法
(一)标本处理，提取DNA
1、临床分离株
标本取自病毒分离实验，取分离株上清液与等体积裂解液混合，煮沸15min，离心数秒。
2、尿标本
取1ml尿标本，15000rpm／min离心15min，弃上清，加DNA裂解液50μl，混匀，离心数秒。100℃煮沸10min，15000rpm／min离心5min。
(二)PCR扩增
1、引物设计
表1，PCR扩增引物
2、PCR扩增
反应体系为50μl：10×Buffer 5μl，dNTP混合物2.5μl(2.5mmol／L)，rTaq酶2U，上下游引物各1μl(33pmol／μl)，模板3.5μl，用双蒸水补至总反应体积50μl。
95℃预变性4分钟；95℃变性45秒，54℃退火1分钟，72℃延伸1分钟，30个循环；72℃终延伸10分钟。
(三)PCR产物纯化、回收及测序
将测序标本进行凝胶电泳后，切出目的DNA片段，应用Takara公司的PCR Fragment Recovery Kit回收。PCR扩增标本应用BigDye Terminators Cycle Sequencing Kit，采用ABI公司的3700型全自动DNA测序仪进行双向测序，测序由上海联合基因公司完成。
(四)序列分析和提交
序列分析应用软件DNAClub，BioEdit，GeneDoc，DNAStar等；用Sequin软件向GenBank提交整理后序列。
结果
一、HCMV UL139、UL140和UL138基因PCR扩增结果
26例HCMV UL139标本以及30例UL140和UL138标本经PCR扩增后全部阳性，且测序结果理想。部分序列已被GenBank收录，序列号为No．AY21 8873-AY21 8887，AY601 874-AY601 877，AY8052552，AY805259，AY805260，AY805292，AY805294，AY905263，AY905264，AY218849-AY218859及AY255774-77，AY218860-218872，AY255772-73，AY601873，AY941163-65。
二、HCMV UL139、UL140和UL138基因多态性研究
(一)HCMV-UL139基因核苷酸及氨基酸序列的比较及进化树分型
各临床分离株HCMV-UL139基因序列由408-444个核苷酸构成。核苷酸的变异成簇分布形成三个主要的基因型G1(G1a、G1b、G1c)，G2(G2a、G2b)以及G3。G1a型与Toledo株同源性为≌100％；其他亚型与Toledo株相比，包含12-36个核苷酸的插入以及24-49个核苷酸的替换，其中47.8-60.7％是错义突变，导致广泛的氨基酸变异。
HCMV UL139编码蛋白的氨基酸序列进化分析结果与核苷酸序列的进化树基本一致。以Toledo株为参考株，临床分离株(除G1a型)虽于序列的5′端存在多个核苷酸的插入，由于均为3或3N个核苷酸的插入，未造成移码突变，故3′端氨基酸序列基本相似。所有UL139编码蛋白的羧基端及8个半胱氨酸是相对保守的。
(二)HCMV-UL140基因核苷酸序列及氨基酸序列的分析
Toledo株和15株临床低传代分离株HCMV-UL140的ORF均由576个核苷酸构成，预测编码191个氨基酸的蛋白。测序结果显示Toledo株与各临床低传代分离株之间的HCMV-UL140核苷酸序列及氨基酸序列比较保守，变异率分别为0—3.2％及0—4.6％。DNA序列的变异分布在整个编码区，均为碱基替换，无插入及缺失。绝大部分的核苷酸为同义突变，没有引起编码氨基酸的改变；同时绝大多数的氨基酸变异为非保守替换。
(三)HCMV-UL138基因核苷酸序列及氨基酸序列的分析
Toledo株和19株临床低传代分离株HCMV-UL138的ORF均由510个核苷酸构成，预测编码169个氨基酸的蛋白。测序结果显示Toledo株与各临床低传代分离株之间的HCMV-UL138核苷酸序列及氨基酸序列比较保守，变异率分别为0—2.4％及0—1.8％。DNA序列的变异分布在整个编码区，均为碱基替换，无插入及缺失。绝大部分的核苷酸为同义突变，没有引起编码氨基酸的改变；同时绝大多数的氨基酸变异为非保守替换。
三、HCMV UL139、UL140和UL138编码蛋白性质及功能预测
(一)HCMV UL139编码蛋白性质及功能预测
应用Editseq及DNASis软件进行UL139编码蛋白分子量(Mr)、等电点(pI)和二级结构的预测。UL139蛋白是一个分子量大约为14.2—15.3KD，等电点为4.94-5.64的弱酸性蛋白质。蛋白质二级结构的预测可以分为6个型，与UL139核苷酸分型相对应。
我们发现所有的UL139包膜糖蛋白(gpUL139)与B淋巴细胞表面的分化抗原CD24存在一定区域的同源。
HCMV临床分离株UL139基因预测的蛋白质重要功能区域，由于各株所属基因型的不同而略不同；分别包括4或5个糖基化位点(ASN，N-glycosylation site)位点，2-4个N相连豆寇酰化位点(MYR，N—myristoylation site)位点，3或4个酪蛋白激酶Ⅱ磷酸化位点(CKP，Casein kinase phosphorylationⅡsite)位点。上述3种重要功能区域在26份标本中均存在，而且相对保守，与Toledo株相比，没有明显改变。其中G1b型的UL139基因编码蛋白，在氨基酸位点34-44之间新增了一个独特的原核膜脂蛋白脂结合位点(Prokaryotic membrane lipoprotein lipidattachment site，ADL)重要功能位点。
(二)HCMV-UL140编码蛋白性质的预测
HCMV UL140编码蛋白是一个分子量约21.5KD，pI为4.73-5.04的一个酸性蛋白。蛋白二级结构预测显示，Toledo株和15株临床低传代分离株(包括黄疸、小头畸形及先天性巨结肠三种疾病类型)HCMV UL140编码蛋白的二级结构基本相同。我们预测在氨基酸位点15-25，80-90及130-150之间可能是HCMV UL140编码蛋白的亲水性区域。
所有低传代临床分离株HCMV-UL140编码蛋白的重要功能位点均比较保守，包括蛋白激酶C磷酸化位点(PKC，Protein kinase C phosphorylation site)和ScAMP磷酸化位点(SPS，SulfationcAMP phosphorylation site)以及ASN、MYR和CKP位点。各临床分离株HCMV-UL140编码蛋白的重要功能位点略有不同。临床分离株1M的UL140蛋白缺失了一个CKP功能位点，增加了一个PKC功能位点；临床分离株51C，缺失了一个ASN功能位点。而分离株27C、29C、2J、39J等UL140蛋白增加了一个CKP功能位点。
(三)HCMV—UL138编码蛋白性质的预测
HCMV UL138编码蛋白是一个分子量约19.3kD，pI 6.84的一个弱酸性蛋白。蛋白二级结构预测显示，Toledo株和19株临床低传代分离株HCMV UL138编码蛋白的二级结构基本相同。我们预测在氨基酸位点55-65，70-90及105-115之间可能是HCMV UL138编码蛋白的亲水性区域。
所有临床低传代分离株HCMV-UL138编码蛋白的重要功能位点均比较保守，包括酪氨酸激酶磷酸化位点(TKP，tyrosine kinase phosphorylation site)，以及PKC、MYR、CKP和SPS位点。与Toledo株相比，无新增的重要功能基团；各临床分离株(除20M株外)缺失了一个PKC位点。
四、UL139、UL140及UL138基因多态性与HCMV感染不同临床表现的关系
在HCMV UL139基因中，无症状的HCMV感染患儿与有症状的患儿一样分别存在于UL139的三个基因型中。不同临床表现的HCMV感染患儿在UL139基因型中的分布没有明显不同。
我们未发现不同疾病类型(黄疸、小头畸形和先天性巨结肠)的临床分离株在UL140及UL138基因中成簇分布。
结论
1．HCMV UL139、UL140和UL138基因广泛存在于临床低传代分离株中。
2．HCMV UL139基因呈现高度多态性，是目前发现的HCMV中变异最大的基因之一，存在多个核苷酸的插入和替换(多为错义突变)。而且这些变异形成了株特异性的基因型，表明生物进化选择有助于UL139变异各型的保留，从而使UL139基因不同基因型的变异在特定的环境中可能具有更大的适应性。
3．我们发现HCMV UL139编码跨膜糖蛋白与人B淋巴细胞表面的分化抗原CD24存在一定区域的同源，可能参与机体在HCMV感染后的免疫反应。
4．HCMV UL140及UL138序列高度保守。
Human Cytomegalovirus(HCMV) is a ubiquitous betaherpesvirus with a wide spectrum of infectivity, as demonstrated by several cell types that can be infected in vitro and by multiple organs observed during infection in vivo.HCMV infection can cause serious diseases, particularly in immunocompromised patients and newborns. However, the mechanisms of viral pathogenesis are not known. Differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent HCMV infections are thought to be related to genomic variability among strains.
HCMV is a genetically complicated virus, its genome consists of 230-235 kb double-strands DNA and more than 200 predicted open reading frames (ORFs). A new region of HCMV DNA containing at least 19 ORFs( UL133-UL151) was found in the Toledo strain(EMBL,AY446871) and in several other low passage clinical isolates recently, but it was deleted in laborotary strain AD169(EMBL,X17403). The fact that AD169 shows attenuated virulence and different tropisms in endothelial cell from low passage isolates,suggests that predicted products of these new ORFs possibly determine the outcome of HCMV infection in vivo.
The possibility that UL133-151 ORFs in the UL/b′region may provide genetic markers for HCMV pathogenesis prompted to investigate these genes in HCMV clinical isolates. UL141 might downregulate natural killer cell-activating ligand CD155, UL144 encodes a structural homologue of the herpesvirus entry mediator and both UL146 and UL147 encode viral CXC(α) chemokines. At present,the variabilities and functions of these genes are being investigated.However, the definitive associations between these genes and HCMV disease have not been established,which leaves open the possibility that other variable HCMV-encoded products in the region may play a role in viral pathogenesis.
The genomic variabilities of HCMV UL139、UL140 and UL138 ORF and the relationship between the variabilities of these genes and different pathogenesis of congenital HCMV infection were detailedly investigated in this report.
Materials and Methods
1.Patients and Samples
The study population of HCMV UL139 consisted of twenty-six infants with HCMV infection who were identified in the 2nd affiliated hospital of China Medical University. These infants aged less than 14 months with suspected congenital infection had different clinical manifestations: jaundice (J,n=15), Hirschsprung's disease (C,n=4), microcephaly (M,n=4)and asymptomatic infection (A,n=3). Nineteen low passage clinical isolates and seven urine samples from the infants were used to carry out PCR amplification. Isolates, recovered from abnormal colon tissue or urine, were passaged less than 10 times in human embryonic lung fibroblasts (HELF).
The study population of UL140 and UL138 consisted of thirty HCMV infected infants who exhibited different clinical manifestations: jaundice (n=18), Hirschsprung's disease (n=7),microcephaly (n=5). All sample were clinical isolates.
2.PCR Amplification
According to the corresponding Toledo sequence, primers were designed using Primer premier 5.0 software, see Table 1.
The PCR reaction mixture contained 1×buffer, 1.5mM MgCl_2, 0.2mM dNTPs, forward and reverse primers 150ng respectively , 0.5U of Taq polymerase,template 3.5μl in a final volume of 50μl.The conditions for amplification were 95℃for 4 min followed by 30 cycles of 95℃for 45 sec, 54℃for 1 min, 72℃for 1 min, and a final extension at 72℃for 10 min.
3.Purification and Sequencing
The PCR products of the appropriate size were purified for sequencing using the PCR Fragment Recovery Kit. Sequencing was carded out on both DNA strands. The sequences were analyzed on an ABI 3700 automated sequencer.
4.Sequence Analysis
All nucleotide and amino acid sequences were analyzed using the BioEdit 5.0,DNAClub,GeneDoc,DNAStar and Sequin software.
RESULTS
1.Presence of the UL139, UL140 and UL138 ORF
All samples gave positive amplification of HCMV UL139、UL140 and UL138. PCR products selected were sequenced successfully. The sequences have been assigned Genbank accession No. AY218873-AY218887, AY601874-AY601877, AY8052552, AY805259, AY805260, AY805292, AY805294, AY905263, AY905264, AY218860-AY218872, AY255772, AY255773, AY601873, AY941163-AY941165 and AY218849-AY218859, AY255774-AY255777.
2. Analysis of the UL139、UL140 and UL138 DNA and Amino Acid Sequence
(1)Analysis of the UL139 DNA and Amino Acid Sequence
The UL139 gene sequences from HCMV infected infants ranged in length from 408 to 444 bp. Using the Toledo strain as the arbitrary reference sequence, the variants were clustered clearly into three major groups G1 (Gla、G1b、G1c), G2 (G2a、G2b) and G3. The strains in Gla showed a high level of identity to the Toledo strain. However, the strains in the other subgroups contained 12-36 nucleotide insertions and 24-49 substitutions, of which 47.8-60.7% were non-synonymous with respect to the Toledo strain.
The non-synonymous nucleotide substitutions led to a wide spectrum of amino acid changes. Phylogenetic analysis of the UL139 putative proteins was mainly consistent with the results described for DNA. Although a relatively large number of nucleotides were inserted in the 5' half of the UL139 ORF in some strains, no frame-shift mutation occurred due to 3 or 3N nucleotide insertions. The C-terminal region and the eight cysteines of all UL139 putative protein appeared relatively conservative.
(2)Analysis of the UL140 DNA and Amino Acid Sequence
The UL140 ORF in clinical isolates was identical in size to that of Toledo, composed of 576 nucleotides. The identities in nucleotide were 96.8-100.0%. The UL 140 ORF was predicted to be a possible glycoprotein of 191 amino acids. Alignment of amino acid sequences showed a high level of identity from 95.4% to 100.0%.
(3)Analysis of the UL138 DNA and Amino Acid Sequence
The UL138 ORF in clinical isolates was identical in size to that of Toledo too, composed of 510 nucleotides and predicted to be a protein of 169 amino acids. The identities in nucleotide and amino acid sequences were 97.6%-100.0% and 98.2%- 100.0% respectively.
3.Structure Analysis of the Predicted UL139, UL140 and UL138
Proteins
(1)Structure Analysis of the Predicted UL 139 Protein
The UL139 putative protein was predicted to be a glycoprotein(gpUL139). All gpUL139 shared sequence homology with human CD24 mature peptide in a restricted region. The gpUL139 contained 4 or 5 N-glycosylation sites(ASN), 3 or 4 Casein kinaseⅡphosphorylation sites(CKP), 2-4 N-myristoylation sites(MYR), depending on the sequence groups which clinical strains belonged to. In addition, the sequences in the strains of Glb contained a specific attachment site of prokaryotic membrane lipoprotein lipid.
(2)Structure Analysis of the Predicted UL 140 Protein
The post-translational modification motifs of UL 140 putative proteins in clinical isolates were highly conserved. 2J, 39J, 27C and 29C added a CKP site in the same position. However, these isolates were derived from the infants with different clinical signs which were jaundice and Hirschsprung's disease respectively.
(3)Structure Analysis of the Predicted UL 138 Protein
The post-translational modification motifs of UL138 putative proteins in clinical isolates were highly conserved.
4.Relationship Between UL139、UL140、UL138 and Signs of Congenital Infections
The strains from infants with asymptomatic HCMV infection, as those from patients who suffered from HCMV disease,existed in three UL139 genotypes. The distribution of the strains from infants with different clinical manifestations in the UL139, UL140 and UL138 sequence groups was not apparently different.
Conclusions
1. HCMV UL139, UL140 and UL138 genes were extensively present in HCMV clinical strains.
2. HCMV UL139 gene is one of the most variable region in HCMV. A large number of nucleotide insertions and non-synonymous substitutions occurred in the UL 139 ORF. The clustered polymorphisms in the UL 139 sequence imply that selective pressures might favor retention of each subgroup.
3. The finding that the putative gpUL139 shared sequence homology with human CD24 suggested that the UL139 putative protein might be involved in the immune response during HCMV infection.
4. HCMV UL 138 and UL 140 genes were highly conserved.
引文
1 De la Hoz RE, Stephens G, Sherlock C. Diagnosis and treatment approaches of CMV infections in adult patients. J Clin Virol. 2002; 25 Suppl(2): S1-12.
2 阮强，吕绳敏，刘兰青，等．婴儿先天性巨结肠与巨细胞病毒感染相关性的研究．中华微生物学和免疫学杂志．1992；12(2)：103-105．
3 Kobayashi N, Takata H, Yokota S, et al. Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation. Eur J Immunol. Dec 2004; 34(12): 3370-3378.
4 Scholl S, Mugge LO, Issa MC, et al. Impact of early NK cell recovery on development of GvHD and CMV reactivation in dose-reduced regimen prior to allogeneic PBSCT. Bone Marrow Transplant. Jan 2005; 35(2): 183-90.
5 Bolovan—Fritts CA, Trout RN, Spector SA. Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells. J Virol. 2004 Dec; 78(23): 13173-81.
6 Pignatelli S, Dal Monte P, Rossini G, et al. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol, 2004 Nov-Dec; 14(6): 383-410.
7 Chou SW, Dennison KW. Analysis of interstrain variation in cytomegalovirus glycoprotein B sequencs encoding neutralization-related epitopes. J Infect Dis. 1991; 163: 1229-1234.
8 Rosen HR, Corless CL, Rabkin J, et al. Association of cytomegalovirus genotype with graft rejection after liver transplantation. Transplantation. 1998; 66: 1627-1631.
9 Torok-Storb B, Boeckh M, Hoy C, et al. Association of specific cytomegalovirus genotypes with death from myelosuppression after marrow transplantation. Blood. 1997; 90: 2097-2102.
10 Vogelberg C, Meyer-Konig U, Hufert FT, et al. Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients. J Med Virol. 1996; 50: 31-34.
11 Shepp DH, Match ME, Ashraf AB, et al. Cytomegalovirus glycoprotein B groups associated with retinitis in AIDS. J Infect Dis. 1998; 174: 184-187.
12 Klein M, Schoppel K, Amvrossiadis N, et al. Strain-specific neutralization of human cytomegalovirus isolates by human sera. J Virol. 1999; 73: 878-886.
13 Chern KH, Chandler DB, Martin DF, et al. Glycoprotein B subtyping of cytomegalovirus (CMV) in the vitreous of patients with AIDS and CMV retinitis. J Infect Dis.1998; 178: 1149-1152.
14 Binder T, Siegert W, Kruse A, et al. Identification of human cytomegalovirus variants by analysis of single strand conformation polymorphism and DNA sequencing of the envelope glycoprotein B gene region-distribution frequency in liver transplant recipients. J Virol Meth. 1999; 78:153-162.
15 Gilbert C, Handfield J, Tome E, et al. Human cytomegalovirus glycoprotein gB genotypes in blood of AIDS patients: lack of association with either the viral DNA load in leukocytes or presence of retinitis. J Med Virol. 1999; 59: 98-103.
16 Cha TA,Tom E,Kemble GW,et al. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996;70 : 78-83.
17 Berstein,D.L.&Plotkin,S.A.Cytomegalovirus vaccines in New Generation Vaccines . 649-659.(Marcel Dekker, New York,2004).
18 Brown JM, Kaneshima H, Mocarski ES. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J Infect Dis. 1995; 171: 1599-1603.
19 MacCormac LP and Grundy JE. Two clinical isolates and the Toledo strain of cytomegalovirus contain endothelial cell tropic variants that are not present in the AD169, Towne, or Davis strains.J Med Virol. Mar 1999; 57(3): 298-307.
20 Ryckman BJ, Jarvis MA, Drummond DD, et al.Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.J Virol. 2006 Jan;80(2):710-22.
21 Wang W, Taylor SL, Leisenfelder SA,et al. Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.J Virol. 2005 Feb;79(4):2115-23.
22 Tomasec P, Wang EC, Davison AJ, et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol. 2005 Feb;6(2):181-8. Epub 2005 Jan 9.
23 Ma YP, Ruan Q, He R,et al. Sequence variability of the human cytomegalovirus UL141 Open Reading Frame in clinical strains. Arch Virol. 2006 Apr;151(4):827-35.
24 Chalupny NJ, Rein-Weston A, Dosch S, et al. Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142.Biochem Biophys Res Commun. 2006 Jul 21;346(1):175-81.
25 Wills MR, Ashiru O, Reeves MB, et al. Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.J Immunol. 2005 Dec 1;175(11):7457-65.
26 Benedict CA,Butrovich KD,Lurain NS, et al. Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J Immunol. 1999, 162 : 6967-6970.
27 Lurain NS,Kapell KS,Huang DD, et al. Human cytomegalovirus UL144 open reading frame: sequence hytervariability in low-passage clinical isolates. J Virol. 1999,73 : 10040-10050.
28 Cheung TC, Humphreys IR, Potter KG, et al. Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway.Proc Natl Acad Sci USA. 2005 Sep 13;102(37):13218-23.
29 He R, Ruan Q, Xia C, et al. Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.Chin Med Sci J. 2004 Dec;19(4):293-7.
30 Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus(CMV)-Encoded Tumor Necrosis Factor- α and β -Chemokine Receptors in Congenital CMV Disease. J Infect Dis.2002,186:1057—1064.
31 Penfold ME, Dairaghi DJ, Duke GM,et al. Cytomegalovirus encodes a potent alpha chemokine.Proc Natl Acad Sci USA. 1999 Aug 17;96(17):9839-9844.
32 He R, Ruan Q, Qi Y, et al. Sequence variability of human cytomegalovirus UL146 and UL147 genes in low-passage clinical isolates.Intervirology. 2006;49(4):215-23.
33 Arav-Boger R, Foster CB, Zong JC,et al. Human cytomegalovirus-encoded alpha -chemokines exhibit high sequence variability in congenitally infected newborns.J Infect Dis. 2006 Mar 15;193(6):788-91.
34 Hassan-Walker AF, Okwuadi S, Lee L, et al. Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus.J Med Virol. 2004 Dec;74(4):573-9.
35 Peckham CS. Cytomegalovirus infection: congenital and neonatal disease. Scand J Infect Dis. 1991;80:82-87.
36 He R,Liu LQ,Lu SM,et al. Quantitative detection of HCMV-DNA from urine in infants by FQ-PCR. Clin J Pediatr. 2001;39:739-742.
37 Dolan A, Cunningham C, Hector RD, et al. Genetic content of wild-type human cytomegalovirus. J Gen Virol. 2004;85:1301-1312.
38 Rigoutsos I, Novotny J, Huynh T,et al. In silico pattern-based analysis of the human cytomegalovirus genome. J Virol.2003;77:4326-44.
39 Kay R, Rosten PM, Humphries RK. CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor. J Immunol .1991;147:1412-1416.
40 Hopp T P.protein surface analysis: methods for identifying antigenic determinants and other interaction sites.J Immunol Methods.l986;88:l-18.
41 Pignatellli S,Dal Monte P,Landini Mp,et al. gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes.J Gen Virol. 2001; Nov;82(Pt 11):2777-84
1 De la Hoz RE, Stephens G, Sherlock C. Diagnosis and treatment approaches of CMV infections in adult patients. J Clin Virol.2002 ;25 Suppl(2) :S1-12
2 Yoshihiro Tsutsui , Hideya Kawasaki , and Isao Kosugi. VIRUS-CELL INTERACTIONS: Reactivation of Latent Cytomegalovirus Infection in Mouse Brain Cells Detected after Transfer to Brain Slice Cultures. J Virol.2002 ; 76 (14) :7247-7254 .
3 Kobayashi N, Takata H, Yokota S,et al.Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation. Eur J Immunol. 2004 Dec; 34(12): 3370-3378.
4 Scholl S, Mugge LO, Issa MC, et al. Impact of early NK cell recovery on development of GvHD and CMV reactivation in dose-reduced regimen prior to allogeneic PBSCT.Bone Marrow Transplant. 2005 Jan; 35(2): 183-90.
5 Bolovan—Fritts CA, Ttout RN. Spector SA.Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells.J Virol. 2004 Dec;78(23):13173-81.
6 Pignatelli S, Dal Monte P, Rossini G, et al. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol. 2004 Nov-Dec; 14(6):383-410.
7 Smyth, R L, Sinclair, J, Scott, J P, et al. Infection and reactivation with cytomegalovirus strains in lung transplant recipients. Transplantation. 1991; 52: 480-482.
8 Larsson S, So" derberg-Naucle' r C, Wang, et al.Cytomegalovirus DNA can be detected in peripheral blood mononuclear cells from all seropositive and most seronegative healthy blood donors over time. Transfusion.l998;38: 271-278.
9 Slobedman, B. & Mocarski, E. S. Quantitative analysis of latent human cytomegalovirus. J Virol. 1999; 73: 4806-4812.
10 Taylor-Wiedeman, J, Sissons, J G P, Borysiewicz, L K, et al. Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol. 1991;72: 2059-2064.
11 Taylor-Wiedeman, J, Hayhurst, G P, Sissons, J G P, et al. Polymorphonuclear cells are not sites of persistence of human cytomegalovirus in healthy individuals. J Gen Virol. 1993;74:265-268.
12 Jarvis, M A & Nelson, J A. Mechanisms of human cytomegalovirus persistence and latency. Front Biosci. 2002; 7: d1575-d1582.
13 Reeves, M B, Coleman, H Chadderton, J, et al. Vascular endothelial and smooth muscle cells are unlikely to be major sites of latency of human cytomegalovirus in vivo. J Gen Virol. 2004; 85:3337-3341.
14 Spector, D H. Activation and regulation of human cytomegalovirus early genes. Intervirology .1996;39: 361-377.
15 Taylor-Wiedeman, J, Sissons, P & Sinclair, J. Induction of endogenous human cytomegalovirus gene expression after differentiation of monocytes from healthy carriers. J Virol.l994;68: 1597-1604.
16 Kondo, K & Mocarski, E S. Cytomegalovirus latency and latency-specific transcription in hematopoietic progenitors. Scand J Infect Dis. 1995; Suppl 99: 63-67.
17 Kondo, K, Kaneshima, H & Mocarski, E S. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors.Proc Natl Acad Sci U S A. 1994; 91: 11879-11883.
18 Jenkins, C, Abendroth, A & Slobedman, B. A novel viral transcript with homology to human interleukin-10 is expressed during latent human cytomegalovirus infection. J Virol.2004; 78: 1440-1447.
19 Bego, M, Maciejewski, J, Khaiboullina, et al.Characterization of an antisense transcript spanning the UL81-82 locus of human cytomegalovirus. J Virol.2005;79: 11022-11034.
20 Mendelson M, Monard S, Sissons P, et al. Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors.J Gen Virol. 1996 Dec;77 (Pt 12):3099-102.
21 So" derberg-Naucle' r C, Streblow D N, Fish, K N, et al .Reactivation of latent human cytomegalovirus in CD14+ monocytes is differentiation dependent.J Virol.2001; 75: 7543-7554.
22 Riegler, S, Hebart, H, Einsele, H, et al. Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus. J Gen Virol .2000; 81: 393-399.
23 Hertel, L, Lacaille, V G, Strobl, H, et al. Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus. J Virol .2003; 77: 7563-7574.
24 Weinshenker, B G, Wilton, S & Rice, G P. Phorbol esterinduced differentiation permits productive human cytomegalovirus infection in a monocytic cell line. J Immunol .1988;140: 1625-1631.
25 Gonczol, E, Andrews, P W & Plotkin, S A. Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells. Science. 1984;224: 159-161.
26 Shelbourn S L, Kothari S K, Sissons J G, et al. Repression of human cytomegalovirus gene expression associated with a novel immediate early regulatory region binding factor. Nucleic Acids Res.l989;17: 9165-9171.
27 Nelson, J A, Gnann, J W, Jr & Ghazal, P. Regulation and tissue-specific expression of human cytomegalovirus. Curr Top Microbiol Immunol. 1990, 154; 75-100.
28 Ghazal P, Lubon H, Reynolds-Kohler C, et al. Interactions between cellular regulatory proteins and a unique sequence region in the human cytomegalovirus major immediate-early promoter. Virology. 1990; 174: 18-25.
29 John S and Patrick S.Latency and reactivation of human cytomegalovirus. Journal of General Virology .2006; 87: 1763-1779
30 Reeves M B, MacAry P A, Lehner P J, et al. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci U S A.2005;102: 4140-4145.
31 Cha TA,Tom E,Kemble GW, et al 1. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996;70 : 78-83.
32 Berstein,D.L.&Plotkin,S.A.Cytomegalovirus vaccines in New Generation Vaccines , 649-659.(Marcel Dekker, New York,2004).
33 Brown JM, Kaneshima H, Mocarski ES. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J Infect Dis. 1995; 171: 1599-1603.
34 MacCormac LP and Grundy JE. Two clinical isolates and the Toledo strain of cytomegalovirus contain endothelial cell tropic variants that are not present in the AD169, Towne, or Davis strains.J Med Virol. Mar 1999; 57(3): 298-307.
35 Ryckman BJ, Jarvis MA, Drummond DD, et al.Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.J Virol. 2006 Jan;80(2):710-22.
36 Wang W, Taylor SL, Leisenfelder SA, et al. Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.J Virol. 2005 Feb;79(4):2115-23.
37 Tomasec P, Wang EC, Davison AJ, et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol. 2005 Feb;6(2):181-8.
38 Ma YP, Ruan Q, He R, et al. Sequence variability of the human cytomegalovirus UL141 Open Reading Frame in clinical strains. Arch Virol. 2006 Apr;151(4):827-35.
39 Chalupny NJ, Rein-Weston A, Dosch S, et al. Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142.Biochem Biophys Res Commun. 2006 Jul 21;346(1):175-81.
40 Wills MR, Ashiru O, Reeves MB, et al. Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.J Immunol. 2005 Dec l;175(11):7457-65.
41 Benedict CA,Butrovich KD,Lurain NS, et al. Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J Immunol. 1999; 162 : 6967-6970.
42 Lurain NS,Kapell KS,Huang DD, et al. Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates. J Virol. 1999;73 : 10040-10050.
43 Cheung TC, Humphreys IR, Potter KG, et al. Evolutionary divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway.Proc Natl Acad Sci USA. 2005 Sep 13;102(37):13218-23.
44 He R, Ruan Q, Xia C, et al. Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.Chin Med Sci J. 2004 Dec;19(4):293-7.
45 Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus(CMV)-Encoded Tumor Necrosis Factor-αandβ-Chemokine Receptors in Congenital CMV Disease. J Infect Dis.2002; 186:1057—1064.
46 Penfold ME, Dairaghi DJ, Duke GM, et al. Cytomegalovirus encodes a potent alpha chemokine.Proc Natl Acad Sci USA. 1999 Aug 17;96(17):9839-9844.
47 He R, Ruan Q, Qi Y, et al. Sequence variability of human cytomegalovirus UL146 and UL147 genes in low-passage clinical isolates.Intervirology. 2006;49(4):215-23.
48 Arav-Boger R, Foster CB, Zong JC, et al. Human cytomegalovirus-encoded alpha -chemokines exhibit high sequence variability in congenitally infected newborns.J Infect Dis. 2006 Mar 15;193(6):788-91.
49 Hassan-Walker AF, Okwuadi S, Lee L, Griffiths PD, et al. Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus.J Med Virol. 2004 Dec;74(4):573-9.
2 阮强，吕绳敏，刘兰青，等．婴儿先天性巨结肠与巨细胞病毒感染相关性的研究．中华微生物学和免疫学杂志．1992；12(2)：103-105．
3 Kobayashi N, Takata H, Yokota S, et al. Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation. Eur J Immunol. Dec 2004; 34(12): 3370-3378.
4 Scholl S, Mugge LO, Issa MC, et al. Impact of early NK cell recovery on development of GvHD and CMV reactivation in dose-reduced regimen prior to allogeneic PBSCT. Bone Marrow Transplant. Jan 2005; 35(2): 183-90.
5 Bolovan—Fritts CA, Trout RN, Spector SA. Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells. J Virol. 2004 Dec; 78(23): 13173-81.
6 Pignatelli S, Dal Monte P, Rossini G, et al. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol, 2004 Nov-Dec; 14(6): 383-410.
7 Chou SW, Dennison KW. Analysis of interstrain variation in cytomegalovirus glycoprotein B sequencs encoding neutralization-related epitopes. J Infect Dis. 1991; 163: 1229-1234.
8 Rosen HR, Corless CL, Rabkin J, et al. Association of cytomegalovirus genotype with graft rejection after liver transplantation. Transplantation. 1998; 66: 1627-1631.
9 Torok-Storb B, Boeckh M, Hoy C, et al. Association of specific cytomegalovirus genotypes with death from myelosuppression after marrow transplantation. Blood. 1997; 90: 2097-2102.
10 Vogelberg C, Meyer-Konig U, Hufert FT, et al. Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients. J Med Virol. 1996; 50: 31-34.
11 Shepp DH, Match ME, Ashraf AB, et al. Cytomegalovirus glycoprotein B groups associated with retinitis in AIDS. J Infect Dis. 1998; 174: 184-187.
12 Klein M, Schoppel K, Amvrossiadis N, et al. Strain-specific neutralization of human cytomegalovirus isolates by human sera. J Virol. 1999; 73: 878-886.
13 Chern KH, Chandler DB, Martin DF, et al. Glycoprotein B subtyping of cytomegalovirus (CMV) in the vitreous of patients with AIDS and CMV retinitis. J Infect Dis.1998; 178: 1149-1152.
14 Binder T, Siegert W, Kruse A, et al. Identification of human cytomegalovirus variants by analysis of single strand conformation polymorphism and DNA sequencing of the envelope glycoprotein B gene region-distribution frequency in liver transplant recipients. J Virol Meth. 1999; 78:153-162.
15 Gilbert C, Handfield J, Tome E, et al. Human cytomegalovirus glycoprotein gB genotypes in blood of AIDS patients: lack of association with either the viral DNA load in leukocytes or presence of retinitis. J Med Virol. 1999; 59: 98-103.
16 Cha TA,Tom E,Kemble GW,et al. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996;70 : 78-83.
17 Berstein,D.L.&Plotkin,S.A.Cytomegalovirus vaccines in New Generation Vaccines . 649-659.(Marcel Dekker, New York,2004).
18 Brown JM, Kaneshima H, Mocarski ES. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J Infect Dis. 1995; 171: 1599-1603.
19 MacCormac LP and Grundy JE. Two clinical isolates and the Toledo strain of cytomegalovirus contain endothelial cell tropic variants that are not present in the AD169, Towne, or Davis strains.J Med Virol. Mar 1999; 57(3): 298-307.
20 Ryckman BJ, Jarvis MA, Drummond DD, et al.Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.J Virol. 2006 Jan;80(2):710-22.
21 Wang W, Taylor SL, Leisenfelder SA,et al. Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.J Virol. 2005 Feb;79(4):2115-23.
22 Tomasec P, Wang EC, Davison AJ, et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol. 2005 Feb;6(2):181-8. Epub 2005 Jan 9.
23 Ma YP, Ruan Q, He R,et al. Sequence variability of the human cytomegalovirus UL141 Open Reading Frame in clinical strains. Arch Virol. 2006 Apr;151(4):827-35.
24 Chalupny NJ, Rein-Weston A, Dosch S, et al. Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142.Biochem Biophys Res Commun. 2006 Jul 21;346(1):175-81.
25 Wills MR, Ashiru O, Reeves MB, et al. Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.J Immunol. 2005 Dec 1;175(11):7457-65.
26 Benedict CA,Butrovich KD,Lurain NS, et al. Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J Immunol. 1999, 162 : 6967-6970.
27 Lurain NS,Kapell KS,Huang DD, et al. Human cytomegalovirus UL144 open reading frame: sequence hytervariability in low-passage clinical isolates. J Virol. 1999,73 : 10040-10050.
28 Cheung TC, Humphreys IR, Potter KG, et al. Evolutionarily divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway.Proc Natl Acad Sci USA. 2005 Sep 13;102(37):13218-23.
29 He R, Ruan Q, Xia C, et al. Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.Chin Med Sci J. 2004 Dec;19(4):293-7.
30 Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus(CMV)-Encoded Tumor Necrosis Factor- α and β -Chemokine Receptors in Congenital CMV Disease. J Infect Dis.2002,186:1057—1064.
31 Penfold ME, Dairaghi DJ, Duke GM,et al. Cytomegalovirus encodes a potent alpha chemokine.Proc Natl Acad Sci USA. 1999 Aug 17;96(17):9839-9844.
32 He R, Ruan Q, Qi Y, et al. Sequence variability of human cytomegalovirus UL146 and UL147 genes in low-passage clinical isolates.Intervirology. 2006;49(4):215-23.
33 Arav-Boger R, Foster CB, Zong JC,et al. Human cytomegalovirus-encoded alpha -chemokines exhibit high sequence variability in congenitally infected newborns.J Infect Dis. 2006 Mar 15;193(6):788-91.
34 Hassan-Walker AF, Okwuadi S, Lee L, et al. Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus.J Med Virol. 2004 Dec;74(4):573-9.
35 Peckham CS. Cytomegalovirus infection: congenital and neonatal disease. Scand J Infect Dis. 1991;80:82-87.
36 He R,Liu LQ,Lu SM,et al. Quantitative detection of HCMV-DNA from urine in infants by FQ-PCR. Clin J Pediatr. 2001;39:739-742.
37 Dolan A, Cunningham C, Hector RD, et al. Genetic content of wild-type human cytomegalovirus. J Gen Virol. 2004;85:1301-1312.
38 Rigoutsos I, Novotny J, Huynh T,et al. In silico pattern-based analysis of the human cytomegalovirus genome. J Virol.2003;77:4326-44.
39 Kay R, Rosten PM, Humphries RK. CD24, a signal transducer modulating B cell activation responses, is a very short peptide with a glycosyl phosphatidylinositol membrane anchor. J Immunol .1991;147:1412-1416.
40 Hopp T P.protein surface analysis: methods for identifying antigenic determinants and other interaction sites.J Immunol Methods.l986;88:l-18.
41 Pignatellli S,Dal Monte P,Landini Mp,et al. gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes.J Gen Virol. 2001; Nov;82(Pt 11):2777-84
1 De la Hoz RE, Stephens G, Sherlock C. Diagnosis and treatment approaches of CMV infections in adult patients. J Clin Virol.2002 ;25 Suppl(2) :S1-12
2 Yoshihiro Tsutsui , Hideya Kawasaki , and Isao Kosugi. VIRUS-CELL INTERACTIONS: Reactivation of Latent Cytomegalovirus Infection in Mouse Brain Cells Detected after Transfer to Brain Slice Cultures. J Virol.2002 ; 76 (14) :7247-7254 .
3 Kobayashi N, Takata H, Yokota S,et al.Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation. Eur J Immunol. 2004 Dec; 34(12): 3370-3378.
4 Scholl S, Mugge LO, Issa MC, et al. Impact of early NK cell recovery on development of GvHD and CMV reactivation in dose-reduced regimen prior to allogeneic PBSCT.Bone Marrow Transplant. 2005 Jan; 35(2): 183-90.
5 Bolovan—Fritts CA, Ttout RN. Spector SA.Human cytomegalovirus-specific CD4+-T-cell cytokine response induces fractalkine in endothelial cells.J Virol. 2004 Dec;78(23):13173-81.
6 Pignatelli S, Dal Monte P, Rossini G, et al. Genetic polymorphisms among human cytomegalovirus (HCMV) wild-type strains. Rev Med Virol. 2004 Nov-Dec; 14(6):383-410.
7 Smyth, R L, Sinclair, J, Scott, J P, et al. Infection and reactivation with cytomegalovirus strains in lung transplant recipients. Transplantation. 1991; 52: 480-482.
8 Larsson S, So" derberg-Naucle' r C, Wang, et al.Cytomegalovirus DNA can be detected in peripheral blood mononuclear cells from all seropositive and most seronegative healthy blood donors over time. Transfusion.l998;38: 271-278.
9 Slobedman, B. & Mocarski, E. S. Quantitative analysis of latent human cytomegalovirus. J Virol. 1999; 73: 4806-4812.
10 Taylor-Wiedeman, J, Sissons, J G P, Borysiewicz, L K, et al. Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J Gen Virol. 1991;72: 2059-2064.
11 Taylor-Wiedeman, J, Hayhurst, G P, Sissons, J G P, et al. Polymorphonuclear cells are not sites of persistence of human cytomegalovirus in healthy individuals. J Gen Virol. 1993;74:265-268.
12 Jarvis, M A & Nelson, J A. Mechanisms of human cytomegalovirus persistence and latency. Front Biosci. 2002; 7: d1575-d1582.
13 Reeves, M B, Coleman, H Chadderton, J, et al. Vascular endothelial and smooth muscle cells are unlikely to be major sites of latency of human cytomegalovirus in vivo. J Gen Virol. 2004; 85:3337-3341.
14 Spector, D H. Activation and regulation of human cytomegalovirus early genes. Intervirology .1996;39: 361-377.
15 Taylor-Wiedeman, J, Sissons, P & Sinclair, J. Induction of endogenous human cytomegalovirus gene expression after differentiation of monocytes from healthy carriers. J Virol.l994;68: 1597-1604.
16 Kondo, K & Mocarski, E S. Cytomegalovirus latency and latency-specific transcription in hematopoietic progenitors. Scand J Infect Dis. 1995; Suppl 99: 63-67.
17 Kondo, K, Kaneshima, H & Mocarski, E S. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors.Proc Natl Acad Sci U S A. 1994; 91: 11879-11883.
18 Jenkins, C, Abendroth, A & Slobedman, B. A novel viral transcript with homology to human interleukin-10 is expressed during latent human cytomegalovirus infection. J Virol.2004; 78: 1440-1447.
19 Bego, M, Maciejewski, J, Khaiboullina, et al.Characterization of an antisense transcript spanning the UL81-82 locus of human cytomegalovirus. J Virol.2005;79: 11022-11034.
20 Mendelson M, Monard S, Sissons P, et al. Detection of endogenous human cytomegalovirus in CD34+ bone marrow progenitors.J Gen Virol. 1996 Dec;77 (Pt 12):3099-102.
21 So" derberg-Naucle' r C, Streblow D N, Fish, K N, et al .Reactivation of latent human cytomegalovirus in CD14+ monocytes is differentiation dependent.J Virol.2001; 75: 7543-7554.
22 Riegler, S, Hebart, H, Einsele, H, et al. Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus. J Gen Virol .2000; 81: 393-399.
23 Hertel, L, Lacaille, V G, Strobl, H, et al. Susceptibility of immature and mature Langerhans cell-type dendritic cells to infection and immunomodulation by human cytomegalovirus. J Virol .2003; 77: 7563-7574.
24 Weinshenker, B G, Wilton, S & Rice, G P. Phorbol esterinduced differentiation permits productive human cytomegalovirus infection in a monocytic cell line. J Immunol .1988;140: 1625-1631.
25 Gonczol, E, Andrews, P W & Plotkin, S A. Cytomegalovirus replicates in differentiated but not in undifferentiated human embryonal carcinoma cells. Science. 1984;224: 159-161.
26 Shelbourn S L, Kothari S K, Sissons J G, et al. Repression of human cytomegalovirus gene expression associated with a novel immediate early regulatory region binding factor. Nucleic Acids Res.l989;17: 9165-9171.
27 Nelson, J A, Gnann, J W, Jr & Ghazal, P. Regulation and tissue-specific expression of human cytomegalovirus. Curr Top Microbiol Immunol. 1990, 154; 75-100.
28 Ghazal P, Lubon H, Reynolds-Kohler C, et al. Interactions between cellular regulatory proteins and a unique sequence region in the human cytomegalovirus major immediate-early promoter. Virology. 1990; 174: 18-25.
29 John S and Patrick S.Latency and reactivation of human cytomegalovirus. Journal of General Virology .2006; 87: 1763-1779
30 Reeves M B, MacAry P A, Lehner P J, et al. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci U S A.2005;102: 4140-4145.
31 Cha TA,Tom E,Kemble GW, et al 1. Human cytomegalovirus clinical isolates carry at least 19 genes not found in laboratory strains. J Virol. 1996;70 : 78-83.
32 Berstein,D.L.&Plotkin,S.A.Cytomegalovirus vaccines in New Generation Vaccines , 649-659.(Marcel Dekker, New York,2004).
33 Brown JM, Kaneshima H, Mocarski ES. Dramatic interstrain differences in the replication of human cytomegalovirus in SCID-hu mice. J Infect Dis. 1995; 171: 1599-1603.
34 MacCormac LP and Grundy JE. Two clinical isolates and the Toledo strain of cytomegalovirus contain endothelial cell tropic variants that are not present in the AD169, Towne, or Davis strains.J Med Virol. Mar 1999; 57(3): 298-307.
35 Ryckman BJ, Jarvis MA, Drummond DD, et al.Human cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to UL150 and occurs by endocytosis and low-pH fusion.J Virol. 2006 Jan;80(2):710-22.
36 Wang W, Taylor SL, Leisenfelder SA, et al. Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.J Virol. 2005 Feb;79(4):2115-23.
37 Tomasec P, Wang EC, Davison AJ, et al. Downregulation of natural killer cell-activating ligand CD155 by human cytomegalovirus UL141. Nat Immunol. 2005 Feb;6(2):181-8.
38 Ma YP, Ruan Q, He R, et al. Sequence variability of the human cytomegalovirus UL141 Open Reading Frame in clinical strains. Arch Virol. 2006 Apr;151(4):827-35.
39 Chalupny NJ, Rein-Weston A, Dosch S, et al. Down-regulation of the NKG2D ligand MICA by the human cytomegalovirus glycoprotein UL142.Biochem Biophys Res Commun. 2006 Jul 21;346(1):175-81.
40 Wills MR, Ashiru O, Reeves MB, et al. Human cytomegalovirus encodes an MHC class I-like molecule (UL142) that functions to inhibit NK cell lysis.J Immunol. 2005 Dec l;175(11):7457-65.
41 Benedict CA,Butrovich KD,Lurain NS, et al. Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus. J Immunol. 1999; 162 : 6967-6970.
42 Lurain NS,Kapell KS,Huang DD, et al. Human cytomegalovirus UL144 open reading frame: sequence hypervariability in low-passage clinical isolates. J Virol. 1999;73 : 10040-10050.
43 Cheung TC, Humphreys IR, Potter KG, et al. Evolutionary divergent herpesviruses modulate T cell activation by targeting the herpesvirus entry mediator cosignaling pathway.Proc Natl Acad Sci USA. 2005 Sep 13;102(37):13218-23.
44 He R, Ruan Q, Xia C, et al. Sequence variability of human cytomegalovirus UL144 open reading frame in low-passage clinical isolates.Chin Med Sci J. 2004 Dec;19(4):293-7.
45 Arav-Boger R, Willoughby RE, Pass RF, et al. Polymorphisms of the Cytomegalovirus(CMV)-Encoded Tumor Necrosis Factor-αandβ-Chemokine Receptors in Congenital CMV Disease. J Infect Dis.2002; 186:1057—1064.
46 Penfold ME, Dairaghi DJ, Duke GM, et al. Cytomegalovirus encodes a potent alpha chemokine.Proc Natl Acad Sci USA. 1999 Aug 17;96(17):9839-9844.
47 He R, Ruan Q, Qi Y, et al. Sequence variability of human cytomegalovirus UL146 and UL147 genes in low-passage clinical isolates.Intervirology. 2006;49(4):215-23.
48 Arav-Boger R, Foster CB, Zong JC, et al. Human cytomegalovirus-encoded alpha -chemokines exhibit high sequence variability in congenitally infected newborns.J Infect Dis. 2006 Mar 15;193(6):788-91.
49 Hassan-Walker AF, Okwuadi S, Lee L, Griffiths PD, et al. Sequence variability of the alpha-chemokine UL146 from clinical strains of human cytomegalovirus.J Med Virol. 2004 Dec;74(4):573-9.