PREVALENCE OF HERPES SIMPLEX VIRUS TYPE 2 AND RISK FACTORS ASSOCIATED WITH THIS INFECTION IN WOMEN IN SOUTHERN BRAZIL

SUMMARY

The herpes simplex virus type 2 (HVS-2) is the most prevalent infection worldwide. It is a cofactor in the acquisition of human immunodeficiency virus (HIV) and the persistence of human papillomavirus (HPV). This study evaluated the prevalence of HSV-2, using the polymerase chain reaction (PCR), and associated factors in patients treated at the Federal University of Rio Grande (FURG) and Basic Health Units (BHU) in Rio Grande, Brazil. The observed prevalence of HSV-2 was 15.6%. Among the 302 women studied, 158 had received assistance in BHU and 144 were treated at FURG. The prevalence of HSV-2 in these groups was 10.8% and 20.8%, respectively, RR 1.9 and p = 0.012. Knowledge about the Pap smear, and the presence of lesions showed no association with HSV-2 infection. Multivariate analysis showed that the variable that most influenced the risk of HSV-2 infection was the presence of HIV infection, with a relative risk of 1.9 and p = 0.04. Discussion: Genital ulcers are an important entry point for HIV, and condom use is an important strategy to reduce transmission of HIV and HSV-2.     

1026例孕妇TORCH特异性IgM感染情况的调查分析

目的 调查分析怀孕各期的健康孕妇的和曾有异常妊娠孕妇及不同年龄孕妇弓形虫病毒、风疹病毒、巨细胞病毒、单纯疱疹病毒感染情况。方法 用酶联免疫吸附试验检测1026例血清TORCH-IgM抗体。结果 早孕感染者弓形虫-IgM、风疹病毒-IgM、巨细胞病毒-IgM、单纯疱疹病毒-IgM检出率分别为6．0％、7．3％、5．4％、4．4％；中孕结果分别为4．20,6、5．2％、3．1％、4．2％；异常怀孕分别为8．7％、17．4％、13．0％、8．7％；有上呼吸道感染者分别为7．4％、22．2％、11．1％、11．1％。结论 TORCH-IgM检测越早越好。有异常妊娠史的妇女再怀孕前一定要做TORCH检查。特别对于年龄偏大的孕妇尽早做TORCH-IgM检测尤为重要。     

人类疱疹病毒6,7型与特发性血小板减少性紫癜发病关系的探讨

目的　了解人类疱疹病毒 (HHV) 6型、7型在特发性血小板减少性紫癜发生中的作用及其相互作用。方法　聚合酶链反应 (PCR)检测特发性血小板减少性紫癜 (ITP)患者外周血中的HHV6 -DNA和HHV7-DNA ,缺铁性贫血组和健康体检组为对照。结果　 31例ITP患者、10例缺铁性贫血患者和 30例健康体检者外周血中 ,HHV6 -DNA的检出率分别为 :2 5 .8% (8/ 31) ,10 % (1/ 10 )和 3.33% (1/ 30 ) ;ITP组与健康体检组间 ,HHV6的感染率差异有显著意义 (P <0 .0 5 )。HHV7-DNA阳性的 6例中 ,有 5例与HHV6是合并阳性 ;健康体检组与缺铁性贫血组 ,HHV7与HHV6合并阳性分别为 0和 1例 ;两种病毒合并阳性 ,在ITP组与健康体检组间差异有统计学意义 (P <0 .0 5 )。ITP组与缺铁性贫血组间 ,HHV6阳性率、HHV7阳性率、HHV6与HHV7合并感染率前者均明显高于后者 ,但统计学差异无显著意义。结论　HHV6感染与ITP发生相关 ,HHV7可能通过激活HHV6引发ITP的发生。     

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.     

三种小干扰RNA干扰VP16 mRNA对单纯疱疹病毒-1复制的影响研究

目的探讨体外合成小干扰RNA(siRNA),在RNA干扰(RNAi)技术条件下,单纯疱疹病毒-1(HSV-1)感染Vero细胞过程中,病毒间层蛋白VP16的生物学功能。方法用T7RNA聚合酶体外合成针对VP16mRNA的三种siRNA。用脂质体2000转染Vero细胞后接种HSV-1,感染后不同时相点(12,24,36,48,60,72hpi)分别采取实验组(R)和对照组(C)标本,检测病毒滴度值,绘制子代病毒一步生长曲线。用同位素标记新合成的VP16进行免疫沉淀法检测病毒蛋白表达的变化。结果病毒子代一步生长曲线显示,转染siRNA的实验组Vero细胞接种HSV-1后24h(24hpi),病毒滴度明显低于对照组;12hpi也观察到病毒滴度低于对照组的现象;多位点的siRNA具有协同作用;实验组病毒滴度高峰出现时间后移大约12h。siRNA影响了病毒的蛋白表达。结论T7RNA聚合酶体外合成的siRNA可以用于RNAi技术研究;HSV-1间层蛋白VP16对病毒复制和组装、成熟起重要的生物学作用。     

异基因造血干细胞移植后人类疱疹病毒6型活化与急性移植物抗宿主病的相关性研究

目的研究造血干细胞移植（HSCT）后人类疱疹病毒6型（HHV．6）活化与急性移植物抗宿主病（aGVHD）的相关性。方法移植前以及移植后每周1次连续采集72例患者的外周血样本，采用筑巢式聚合酶链反应扩增HHV-6基因序列，HindⅢ酶切对HHV-6基因分型。结果72例患者移植后45例（62．5％）检出HHV-6血症，中位时间为第14（7—63）天；40例（55．6％）患者发生Ⅰ～Ⅳ度aGVHD，中位时间第26（9～73）天。HHV．6血症发生的中位时间显著早于Ⅰ～Ⅳ度aGVHD发生时间（P=0．018）。Ⅰ～Ⅳ度aGVHD累积发生率在HHV-6阳性组为68．9％（45例中31例），显著高于HHV-6阴性组的33．3％（27例中9例）（P=0．003）。Ⅱ-Ⅳ度aGVHD累积发生率HHV-6阳性组为35．6％，显著高于HHV-6阴性组的14．8％（P=0．027）。移植后Ⅰ-Ⅳ度aGVHD的发生率在巨细胞病毒（CMV）和HHV-6共感染（CMV^＋／HHV-6^＋）组、CMV阳性HHV-6阴性（CMV^＋／HHV-6^-）组、CMV阴性HHV-6阳性（CMV^-／HHV-6^-）组和CMV与HHV-6均阴性（CMV^-／HHV-6^-）组分别为78，9％，55．6％，14．3％和22．2％，差异有统计学意义（P=0．0001）。Ⅱ～Ⅳ度aGVHD累积发生率在CMV^＋／HHV-6^＋、CMV^＋／HHV-6^-、CMV^-／HHV-6^＋和CMV^-／HHV-6^-组分别为42．1％．22．2％，0％和11．1％，差异有统计学意义（P=0．008）。结论HSCT后HHV-6感染以及HHV-6和CMV共感染与aGVHD发生率存在显著相关性。     

Intracellular transport and stability of varicella-zoster virus glycoprotein K

VZV gK, an essential glycoprotein that is conserved among the alphaherpesviruses, is believed to participate in membrane fusion and cytoplasmic virion morphogenesis based on analogy to its HSV-1 homolog. However, the production of VZV gK-specific antibodies has proven difficult presumably due to its highly hydrophobic nature and, therefore, VZV gK has received limited study. To overcome this obstacle, we inserted a FLAG epitope into gK near its amino terminus and produced VZV recombinants expressing epitope-tagged gK (VZV gK-F). These recombinants grew indistinguishably from native VZV, and FLAG-tagged gK could be readily detected in VZV gK-F-infected cells. FACS analysis established that gK is transported to the plasma membrane of infected cells, while indirect immunofluorescence demonstrated that gK accumulates predominately in the Golgi. Using VZV gK-F-infected cells we demonstrated that VZV gK, like several other herpesvirus glycoproteins, is efficiently endocytosed from the plasma membrane. However, pulse-labeling experiments revealed that the half-life of gK is considerably shorter than that of other VZV glycoproteins including gB, gE and gH. This finding suggests that gK may be required in lower abundance than other viral glycoproteins during virion morphogenesis or viral entry.     

Kaposi's sarcoma herpesvirus C-terminal LANA concentrates at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) tethers KSHV terminal repeat (TR) DNA to mitotic chromosomes to efficiently segregate episomes to progeny nuclei. LANA contains N- and C-terminal chromosome binding regions. We now show that C-terminal LANA preferentially concentrates to paired dots at pericentromeric and peri-telomeric regions of a subset of mitotic chromosomes through residues 996-1139. Deletions within C-terminal LANA abolished both self-association and chromosome binding, consistent with a requirement for self-association to bind chromosomes. A deletion abolishing TR DNA binding did not affect chromosome targeting, indicating LANA's localization is not due to binding its recognition sequence in chromosomal DNA. LANA distributed similarly on human and non-human mitotic chromosomes. These results are consistent with C-terminal LANA interacting with a cell factor that concentrates at pericentromeric and peri-telomeric regions of mitotic chromosomes.     

In vitro and in vivo characterization of equine herpesvirus type 1 (EHV-1) mutants devoid of the viral chemokine-binding glycoprotein G (gG)

Glycoprotein G (gG) of equine herpesvirus type 1 (EHV-1), a structural component of virions and secreted from virus-infected cells, was shown to bind to a variety of different chemokines and as such might be involved in immune modulation. Little is known, however, about its role in the replication cycle and infection of EHV-1 in vivo. Here we report on the function of gG in context of virus infection in vitro and in vivo. A gG deletion mutant of pathogenic EHV-1 strain RacL11 (vL11DeltagG) was constructed and analyzed. Deletion of gG had virtually no effect on the growth properties of vL11DeltagG in cell culture when compared to parental virus or a rescuant virus vL11DeltagGR, respectively, and virus titers and plaque formation were unaffected in the absence of the glycoprotein. Similarly, in the murine model of EHV-1 infection, no significant differences in virulence between the gG deletion mutant and RacL11 or vL11DeltagGR were found at high doses of infection. However, infection of mice at lower doses revealed that the gG deletion mutant was able to replicate to higher titers in lungs of infected mice. Additionally, these mice lost significantly more weight than those infected with RacL11 and a more pronounced inflammatory response in lungs was observed. Therefore we concluded that deletion of gG in EHV-1 seems to lead to an exacerbation of respiratory disease in the mouse.     

Oncogenic Marek’s disease viruses lacking the 132 base pair repeats can still be attenuated by serial in vitro cell culture passages

Marek’s disease virus (MDV) can be attenuated by serially passing the virus in cell culture. During cell culture passage, two copies of a 132 bp repeat are expanded to over 30 copies. We deleted the two copies of the 132 bp repeat region in a pathogenic MDV and demonstrated that the virus was still pathogenic. The pattern and frequency of tumors in the parental and mutant virus were the same. Early virus replication, and the appearance of persistent neurological disease were also similar between the parental and deleted virus. Nevertheless, wild-type MDV and the deletion virus could be attenuated by serial in vitro cell culture passages. Based upon analyzing the passage 40 viruses, attenuation of the MDV lacking the 132 bp repeats appears to occur in a manner that is analogous to the process occurring wild-type MDV attenuation. Whatever process is involved in the cell culture attenuation of MDV, the mechanism does not involve the 132 bp repeat region.