Molecular piracy: the viral link to carcinogenesis

The vast majority of the human experience with viral infectons is associated with acute symptoms, such as malaise, fever, chills, rhinitis and diarrhea. With this acute or lytic phase, the immune system mounts a response and eliminates the viral agent while acquiring antibodies to that specific viral subtype. With latent or chronic infections, the viral agent becomes incorporated into the human genome. Viral agents capable of integration into the host’s genetic material are particularly dangerous and may commandeer the host’s ability to regulate normal cell growth and proliferation. The oncogenic viruses may immortalize the host cell, and facilitate malignant transformation. Cell growth and proliferation may be enhanced by viral interference with tumor suppressor gene function (p53 and pRb). Viruses may act as vectors for mutated proto-oncogenes (oncogenes). Overexpression of these oncogenes in viral-infected cells interferes with normal cell function and allows unregulated cell growth and proliferation, which may lead to malignant transformation and tumour formation. Development of oral neoplasms, both benign and malignant, has been linked to several viruses. Epstein-Barr virus is associated with oral hairy leukoplakia, lymphoproliferative disease, lymphoepithelial carcinoma, B-cell lymphomas, and nasopharyngeal carcinoma. Human herpesvirus-8 has been implicated in all forms of Kaposi’s sarcoma, primary effusion lymphomas, multiple myeloma, angioimmunoblastic lymphadenopathy, and Castleman’s disease. Human herpesvirus-6 has been detected in lymphoproliferative disease, lymphomas, Hodgkin’s disease, and oral squamous cell carcinoma. The role of human papillomavirus in benign (squamous papilloma, focal epithelial hyperplasia, condyloma acuminatum, verruca vulgaris), premalignant (oral epithelial dysplasia), and malignant (squamous cell carcinoma) neoplasms within the oral cavity is well recognized. Herpes simplex virus may participate as a cofactor in oral squamous cell carcinoma development by enhancing activation, amplification, and overexpression of pre-existing oncogenes within neoplastic tissues. Because of the integral role of viruses in malignant transformation of host cells, innovative antiviral therapy may prevent tumour development, involute neoplastic proliferations, or arrest malignant progression.     

Human Herpesvirus 6 (HHV-6) Infection and Exanthem Subitum in Thailand

Of 50 patients in Thailand suspected clinically of having exanthem subitum, 31 (62%) were serodiagnosed as HHV-6 infection. Sixteen strains of HHV-6 from 31 patients (52%) whose antibody titers had converted during convalescence were isolated during the acute phase. The disease occurred in infants from 3 months to 1 year of age and most frequently at age 4-6 months. Antibody only to HHV-6 converted in 23 of 50 patients (46%), and seroconversion to HHV-6 and dengue virus was observed in 7 patients (14%), and to HHV-6 and Coxsackie B virus in 1 case (2%). In the 23 patients in whom seroconversion only to HHV-6 was observed, all had fever and rash which appeared after subsidence of the fever. Lymphadenopathy and relative lymphocytosis were recognized, associated with diarrhea, vomiting, running nose, cough and hepatomegaly. Febrile convulsions were seen in some cases. All patients recovered completely within a week.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

Objective Human herpesvirus 6(HHV-6)isolates are classified into two variants,HHV-6A and HHV-6B,based on distinct genetic,antigenic and biological characteristics.HHV-6 has been associated with encephalitis in children recently.This study aireed to estabhsh a real time PCR assay for simultaneous detection of the two subtypes of HHV-6,and apply this new assay to children with suspected encephalitis,then analyze the relationship between the infeetion with HHV-6 and encephalitis in children.Method The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene(U38)of HHV-6.The 5'end of the probes for HHV-6A and HHV-6B was labeled with the fluoreseein reporter tetrachloro-6-carboxyfluorescein and 6-earboxyfluorescein(6-FAM),separately,while the 3'end were quenched with 6-carboxy-tetramethyl-rhedamine.The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established.Then,the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 109 to 10°copies/μl,together with controls were used for testing both sensitivity and specificity of the real time PCR assay.The cerebrospinal fluid(CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.Result Both HHV6A(strain ZJ-159)and HHV-6B (strain GS)were positive on the real time PCR assay.There were no cross-reaction with herpes simplex virus type 1,type 2(HSV-1,HSV-2),varicella-zoster virus(YZV),cytomegalovirus(CMV),EpsteinBarr virus(EBV),hepatitis B virus,Staphylococcus aureus,Mycoplasma pneumoniae and human DNA.A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6.The sensitivity threshold was 10 copies/μl for the real time PCR.HHV-6 positive rate by the real time PCR assay was 4.72%(21/445),including 4 ca8es with HHV-6A infection,16 cases of HHV-6B infeedon and l case with mixed HHV-6A and HHV-6B infeetion.The new PCR assay usually took 2 to 3 hours to provide results.Conclusion This new real time PCR assay call simultaneously detect both subtypes of HHV-6,and have high specificity and sensitivity.It will pmvide an early and sensitive diagnosis of HHV-6 encephalitis in children.     

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.     

MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific

Many herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TR_L), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96-100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function.     

Ⅰ型单纯疱疹病毒致小鼠急性期疱疹疼痛模型的建立

目的观察小鼠皮下接种Ⅰ型单纯疱疹病毒（HSV-1）产生的神经痛,建立急性期疱疹疼痛模型。方法120只Balb/c雌性小鼠,随机分为2组：对照组和实验组,对照组10只,实验组110只。对照组于左后肢胫部皮下接种灭活的HSV-1;实验组于同部位皮下接种HSV-1,分别于接种前、接种后第1、2、3、4、5、6、7、8、10、12天各随机取10只小鼠,于当日9：00 am观察皮肤脱毛区变化,并测定其热痛潜伏期,4：00 pm测定同批小鼠机械痛觉反应评分。测定完毕后处死小鼠,采用聚合酶链反应检测背根神经节中HSV-1 DNA。结果实验组接种后第5天小鼠均出现皮损,第7天均达高峰,以后逐渐恢复;与接种前比较,接种后第5天出现机械痛觉反应评分明显增高,直至第12天仍较高（P＜0．05）;而热痛潜伏期两组均无明显变化（P＞0．05）;接种后第2～12天在背根神经节中均可观察到HSV-1 DNA,而对照组则无此变化。结论小鼠左后肢胫部皮下接种HSV-1后产生了相应的皮肤损害和机械痛敏,此法成功建立了急性期疱疹疼痛模型。     

延边地区淋巴瘤和鼻咽癌中EB病毒类型分布特点

目的研究我国延边地区淋巴瘤与鼻咽癌中EB病毒类型的分布特点。方法采用聚合酶链反应（PCR），巢式PCR和限制性酶切技术分析检测延边地区32例淋巴瘤与31例鼻咽癌中EB病毒的类型分布。结果在延边地区，22％的淋巴瘤和81％的鼻咽癌表现为EBNA-1阳性。EBNA-1阳性的7例淋巴瘤均表现为EB病毒-1型和“F”型，其中4例（57％）为“C”变异型；而在EBNA-1阳性的25例鼻咽癌中，22例（88％）为EB病毒-1型，3例为EB病毒-2型。除1例“f”变异型以外，均为“F”型，“C”变异型占鼻咽癌的63％（10／16）。结论EB病毒-1型为延边地区的主要EB病毒株，Barn HI“f”变异型罕见。     

武汉地区水痘带状疱疹病毒基因型分析

目的 探讨武汉地区水痘带状疱疹病毒流行的主要基因型。方法 收集96例水痘、带状疱疹患者皮损标本。采用煮沸、冻融方法提取标本中的DNA。多重PCR扩增水痘带状疱疹病毒ORF22、38、54,并测序。用ClustalW软件比对和Primer premier 5软件酶切位点分析核酸序列。结果 多重PCR检测水痘、带状疱疹患者皮损标本阳性率为81.3%（78/96）。与Dumas序列进行对比,78份阳性标本ORF22在37902、38055、38081、38177存在点突变,符合基因型J型特点。ORF38和 ORF54分别存在PstI和BglI酶切位点。结论 武汉地区水痘带状疱疹病毒流行的主要基因型为J型、ORF38 （PstI+） 和 ORF54 （BglI+）。     

生殖器部位皮损的单纯疱疹病毒检测及分型

目的 探讨生殖器疱疹部位皮损的不典型表现及其与单纯疱疹病毒型别的关系。方法 对外生殖器部位及其周围有硬结或疖肿、裂隙、毛囊炎等非水疱性皮肤黏膜损害的患者进行临床资料采集和分析,并对皮损标本进行单纯疱疹病毒的分离培养、PCR检测和病毒分型。结果 105例有外生殖器部位非水疱性皮损的患者入选本研究,在硬结(或疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎皮损中,PCR检测HSV的阳性率分别33.3%(6/18)、20%(3/15)、37.5%(6/16)、28.6%(2/7)、33.3%(4/12)、20%(5/25)和50%(6/12),总的检出阳性率为30.5%(32/105)。分离培养法检测HSV的阳性率分别为22.2%(4/18)、13.3%(2/15)、25%(4/16)、14.3%(1/7)、33.3%(4/12)、8%(2/25)和41.7%(5/12),总的检出阳性率为21%(22/105)。两种方法检测HSV的总检出率差异无统计学意义(κ=0.095,P=0.114)。HSV-PCR分型结果与荧光单克隆抗体分型结果相符。在所有HSV阳性者中,HSV-1感染占9.4%(3/32),HSV-2感染占90.6%(29/32)。结论 生殖器HSV感染的皮肤黏膜损害多样,可为外生殖器部位的硬结(疖肿)、裂隙、毛囊炎、类似擦破、单个溃疡、非特异性红斑和红肿渗液性包皮龟头炎等不典型表现,而且主要由HSV-2感染引起。