HHV-6 DNA throughout the tissues of two stem cell transplant patients with chromosomally integrated HHV-6 and fatal CMV pneumonitis

Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.     

Serological Evidence of Canine Herpesvirus‐1 in Dogs of Kerman City,South‐east of Iran

Canine herpes virus‐1 (CHV‐1) is an alphaherpesvirus, which causes foetal and neonatal death as well as fertility problems in dogs. The virus is presumed to be enzootic in dogs all over the world, but no information was found about the seroprevalence of CHV‐1 from middle‐east countries. Therefore, this study was aimed to determine the seroprevalence of CHV‐1 among dogs in Kerman (south‐east of Iran). Blood samples were taken from 47 privately owned and 35 kennelled dogs, respectively. The entire sampled dogs were apparently healthy. Indirect immunofluorescence antibody (IFA) assay was used to detect antibodies against CHV‐1 in all sera. The overall CHV‐1 seroprevalence was estimated 20.7%, which was 22.9% and 19.1% for kennelled and owned dogs, respectively. Sex, parity and raising status (owned or kennels) did not differ significantly between seropositive and seronegative dogs. However, the infection rate was significantly higher in dogs older than 3 in comparison with younger groups (15.9% vs. 4.8%, P ≤ 0.05). In conclusion, this study revealed that CHV‐1 could be considered endemic in Iran, and more epidemiological researches are needed to identify the geographical distribution of diseases in Iran.     

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-ΔP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-ΔY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.     

婴儿型胆总管囊肿形成的病因分析

目的:探讨婴儿型胆总管囊肿形成的病因及炎症介质在其发生中的作用机制。方法:回顾性分析我院2000年—2006年收治的1岁以下的胆总管囊肿29例,采用免疫组化技术分别检测CD4、CD8、CMV、EBV在胆总管囊肿上皮细胞中的表达情况,并结合术中造影检查分析。结果:术中行胆道造影17例,其中显示胆胰管结合部囊肿11例,6例(54.5%)存在胆胰管合流异常。在囊肿壁上可以检测到CD4、CD8的表达,其中CD4表达11例(37.9%),CD8表达22例(75.9%)。在囊肿壁中可以检测到4例(13.8%)CMV表达,EBV表达为12例(41.4%)。结论:胰液逆流入胆道系统可以造成胆道上皮细胞破坏,病毒感染可以引起胆道上皮的炎症反应,引起CD8的高表达,胆道上皮破坏,脱落上皮形成蛋白栓阻塞胆总管远端造成胆总管囊肿的形成。     

单纯疱疹病毒1型截短糖蛋白B基因疫苗的构建及其诱导小鼠细胞免疫应答

目的:构建、制备单纯疱疹病毒1型截短糖蛋白B DNA疫苗，检测其诱导机体产生的细胞免疫应答效果。方法:利用PCR技术从HSV-1 SM44毒株基因组中扩增出编码HSV-1 gB14～507氨基酸序列的一段基因。定向插入真核表达质粒pcDNA₃载体中，构建出重组真核表达质粒pcDNA₃-gBt，并对其进行酶切分析、PCR鉴定及测序鉴定。于BALB/c鼠注射免疫3次，抗体分析CD4⁺、CD8⁺T细胞亚群的变化，羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)/碘化丙碇(PI)双标记的流式细胞计数法检测CTL活性。结果:PCR及测序鉴定结果显示，插入的克隆基因与GenBank中HSV-1F株gB基因序列一致，证实了HSV-1 gBt核酸疫苗的构建；pcDNA₃-gBt免疫组BALB/c鼠的CD4⁺T细胞数较空质粒（pcDNA₃）对照组和生理盐水对照组增加，CTL活性也较对照组明显增强，但是pcDNA₃-gBt免疫组BALB/c鼠的CD8⁺T细胞、CD4⁺/ CD8⁺T细胞比值与对照组比较差异均无显著性（P＞0.05）。结论:HSV-1 gBt核酸疫苗诱导较强的细胞免疫应答。     

HIV-1 Rev基因编码蛋白在PEL细胞系中的表达及其表达蛋白抑制HHV-8溶解性周期复制

目的:构建含人类免疫缺陷病毒1型(HIV-1)病毒颗粒蛋白表达调节因子(regulator of virion protein expression, Rev)编码基因的重组真核表达质粒并初步探索Rev基因编码蛋白对人类疱疹病毒8型(HHV-8)溶解性周期复制的影响.方法:构建pRev-Flag重组质粒并进行酶切鉴定和序列测定;将pRev-Flag重组质粒瞬时转染原发性渗出性淋巴瘤细胞系(PEL)BCBL-1细胞和小鼠胚胎成纤维细胞NIH/3T3,采用RT-PCR、Western blot分别从mRNA和蛋白水平检测Rev基因的表达情况;提取瞬时转染pRev-Flag重组质粒的BCBL-1细胞总RNA,进行RT-PCR检测HHV-8次要衣壳蛋白编码基因ORF26 mRNA转录水平.结果:核酸序列分析结果表明,克隆的Rev基因序列与GenBank中已登记的Rev序列100%同源.RT-PCR和Western blot都在Rev预期位置检测到特异性条带.RT-PCR检测显示,Rev基因对编码蛋白能够降低HHV-8 ORF26 mRNA转录水平.结论:成功构建含Rev基因序列的重组质粒并在真核细胞中获得正确表达;初步探索表明Rev蛋白能够抑制HHV-8溶解性周期复制.     

血浆EBV DNA定量分析在监测鼻咽癌患者复发和转移中的价值

目的 探讨血浆EB病毒DNA(EBV DNA)含量在监测鼻咽癌患者放疗后复发和转移中的临床价值.方法 采用荧光定量PCR方法 检测81例放疗后随诊的鼻咽癌患者血浆EBV DNA含量,比较缓解组与复发、转移组血浆EBV DNA差异.结果 放疗后缓解组患者血浆EBV DNA阳性率为15.7%,中位拷贝数为0 copy/ml;放疗后复发或转移组患者血浆EBV DNA阳性率93.3%,中位拷贝数6 432 copies/ml,差异均有显著性(P<0.001).结论 血浆EBV DNA定量检测有可能成为监测鼻咽癌患者放疗后复发、转移的肿瘤标记物.     

Concurrent oral shedding of feline calicivirus and feline herpesvirus 1 in cats with chronic gingivostomatitis

Oral mucosal salivary samples were collected from 25 cats with chronic gingivostomatitis and 24 cats with periodontal disease. Viral culture and isolation of feline calicivirus and feline herpesvirus 1 were performed. Eighty-eight per cent of cats with chronic gingivostomatitis were shedding both viruses, compared to 21% of cats without chronic oral inflammatory disease. Cats with chronic gingivostomatitis are significantly more likely to concurrently shed both feline calicivirus and feline herpesvirus 1 than are cats with classical periodontal disease.     

人类疱疹病毒8型包膜糖蛋白K8.1单克隆抗体的制备与鉴定

目的：制备人类疱疹病毒8型（HHV-8）包膜糖蛋白K8．1的单克隆抗体，并对其特异性进行鉴定。方法：用异丙基硫代一β—D一半乳糖苷（IPTG）诱导含重组质粒pGEX-6p-1 ＋K8．1的大肠杆菌BL21表达符胱甘肽-S-转移酶GST／K8．1融合蛋白，将以包涵体形式存在的融合蛋白进行变性、复性、复性后的Glutathione Sepharose 4B亲和层析柱纯化。以纯化的GST／K8．1融合蛋白为抗原免疫BALB／c小鼠．常规杂交瘤技术制备单克隆抗体。采用酶联免疫吸附试验（ELISA）筛选分泌K8．1单抗的杂交瘤细胞株。免疫组化染色（IHC）和Western blot法鉴定单抗的特异性。结果：建立了一株稳定分泌抗K8．1单抗的杂交瘤细胞株．命名为3G10：IHC和Westerfl blot法显示．3G10株单抗能特异地识别HHV-8K8．1蛋白。结论：成功制备出抗HHV-8包膜糖蛋白K8．1的单克隆抗体。     

Integration of human herpesvirus 6 in a Burkitt's lymphoma cell line

Human herpesvirus 6 (HHV-6) genome has been found in several human lymphoid malignancies, but configuration of the HHV-6 genome has not been well delineated. We established the HHV-6-positive, Epstein-Barr virus-negative Burkitt's lymphoma cell line Katata. In this study we investigated the status of the HHV-6 genome in Katata cells. Neither linear nor circular HHV-6 DNA was detected by Gardella gel analysis. The fluorescence in situ hybridization technique enabled us to directly visualize the integrated HHV-6 DNA at the single-cell level. Only one integrated site of viral DNA was detected in metaphase chromosomes and it was preferentially located at the long arm of chromosome 22 (22q13). Treatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or with calcium ionophore A23187 led to induction of the HHV-6 immediate-early gene as well as the late gene. Sodium n-butyrate also gave rise to expression of the HHV-6 genes. The TPA inducibility was synergistically enhanced when combined with A23187 or n-butyrate. Our study provides, for the first time, an in vitro model system of latent HHV-6 infection whose genome is integrated into host DNA of lymphoma cells.