Community-based study on seroprevalence of herpes simplex virus type 2 infection in New Delhi

PURPOSE: To determine the seroprevalence of herpes simplex virus type 2 (HSV-2) in two urban communities in Delhi and to correlate the presence of HSV-2 seroprevalence with sociodemographic profile, risk factors and presence of other reproductive tract infections (RTIs). METHODS: Men and women aged between 15-49 years from an urban slum and an urban middle class colony were invited to participate in the study. They provided interview information; blood for HSV-2, HIV and syphilis serology; first void urine specimens for diagnosis of Neisseria gonorrhoeae and Chlamydia trachomatis infection; and genital specimens for diagnosis of bacterial vaginosis, vaginal candidiasis and trichomoniasis. RESULTS: The prevalence of HSV-2 seropositivity was found to be 7 and 8.6% in men and women, respectively. HSV-2 seropositivity was found to be significantly associated with urban middle class community and older age. No statistically significant correlation was found between HSV-2 seropositivity and other laboratory-confirmed RTIs. CONCLUSIONS: The findings of our study indicate a relatively low prevalence of HSV-2 seropositivity and other sexually transmitted infections in the two communities that were studied.     

Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes

HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2.     

Tubular structures in the cervix of mice experimentally infected with herpes simplex virus type 2

Experimental infection of the C3H/N mouse genital tract was demonstrated after intravaginal inoculation with herpes simplex virus type 2 (HSV-2). About 75% of the infected animals died by Day 7, and 75% of the surviving animals had severe vaginitis or neurological signs on Day 7. Titers of the virus recovered from vaginal secretions of infected animals reached a maximum on Day 2 and gradually decreased until Day 7. On the other hand, under the electron microscope, virus particles and tubular structures could be found in the nuclei of infected cells of the cervix in the 1st, 2nd and 4th days after infection. All cases in which virus particles could be found in the nuclei of infected cells were also positive for tubular structures and vice versa. These observations indicate that in situ diagnosis of HSV-2 infection can be made in the mouse model. The same method would be applicable for the diagnosis of human HSV-2 infection.     

Analysis of varicella-zoster virus and herpes simplex virus in various clinical samples by the use of different PCR assays

Rapid and reliable detection of varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and -2 (HSV-2) is of clinical significance in immunocompromised patients and patients with infections of the central nervous system. This paper describes the detection of VZV and HSV using the commercially available Affigene^® VZV and Affigene^® HSV 1/2 tracer kits in comparison to “in-house” polymerase chain reaction (PCR) assays. For sample preparation, Qiagen (Hilden, Germany) and Affigene^® (Cepheid AB, Bromma, Sweden) DNA extraction kits were used. 175 samples were analyzed for VZV and 352 samples for HSV-1 and -2. Generally more positive results were obtained using the Affigene^® assays compared to the “in-house” methods independent of the DNA preparation method used. There were significant differences in sensitivity between the Affigene^® HSV 1/2 tracer and the “in-house” PCR assays for the detection of both HSV-1 and -2 in cerebrospinal fluid and vesicle/skin swabs. The Affigene^® HSV 1/2 and VZV tracers are very sensitive assays for detection of VZV and HSV. A wide variety of clinical samples can be examined in combination with either the Qiagen or the Affigene^® DNA extraction kits for preparation.     

Experimental myelitis caused by herpes simplex virus type 2 in C57BL/6N and BALB/cN mice

Intraperitoneal and intracranial inoculation of herpes simplex virus type 2 (HSV 2) into BALB/cN and C57BL/6N mice was carried out to induce experimental myelitis. The myelitis was clearly observed in C57BL/6N mice following intraperitoneal inoculation. Within 24 hours before death, the mice showed urinary and rectal incontinence and paraplegia of the hind legs. Randomly distributed, severe necrosis was demonstrated in the spinal cord, mainly at the lower cord. In BALB/cN mice the clinical symptoms were not clearly observed, as the mice died shortly after their onset. Although spinal cord necrosis was more prominent in C57BL/6N mice than BALB/cN mice, brain necrosis was only found in the latter, and not in the former. Both strains of mouse showed marked nuclear pyknosis of the nerve cells and slight nuclear pyknosis of the astrocytes in the brain where HSV 2 antigen was demonstrated immunohistochemically. The antigen was also detected in the necrotic spinal cord. In contrast, intracranial inoculation of the virus into both strains did not cause myelitis. Spinal cord necrosis was not demonstrated and virus DNA was not detected, by PCR, in spinal cord samples. In the brain, however, the virus was demonstrated by both PCR and immunohistochemistry.     

DNA immunization with a herpes simplex virus 2 bacterial artificial chromosome

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.     

细胞周期蛋白依赖性激酶2活性与单纯疱疹病毒复制关系的研究

目的 探讨细胞周期蛋白依赖性激酶(CDK)2在单纯疱疹病毒(HSV)复制中的作用. 方法 以能够诱导表达CDK2显性负突变体致使CDK2功能缺陷的人结肠癌细胞株HT29Tet-Ondn-CDK2为材料,用免疫共沉淀及放射自显影法测定HSV感染后不同时间CDK2活性,并进行HSV的增殖动力学分析. 结果 HSV感染6h后CDK2活性被诱导,9h达到最大;一步生长曲线结果表明在CDK2活性被诱导前HSV处于静止状态,CDK2活性被诱导后HSV即进入快速的生长复制阶段. 结论 CDK2是HSV复制所必需的,HSV通过诱导宿主细胞CDK2活性而促进本身的复制.     

Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.     

联合靶向小干扰RNA对疱疹病毒2感染的体外抑制作用

目的 探讨联合靶向特异性小干扰RNA(siRNA)对疱疹病毒(HSV)2的体外特异抑制作用.方法 将体外合成的靶向HSV-2编码包膜糖蛋白gB的UL27.2基因、靶向DNA结合蛋白UL29.2基因的siRNA和该两种联合靶向特异性siRNA制剂,共转染Vero细胞并感染HSV-2,观察靶基因的表达情况、Vero细胞病变、空斑减数实验和子代病毒滴度并进行各组间比较:结果特异性UL27.2siRNA、UL29.2 siRNA和联合siRNA转染Vero细胞后,均能不同程度抑制各HSV-2临床株感染所导致的细胞病变,对病毒增殖抑制率分别为63.9%、86.7%和93.3%.实时定量PCR检测各组siRNA分别对UL27.2和UL29.2两个基因的表达,结果显示UL27.2 siRNA和UL29.2 siRNA对各自的靶向基因均有抑制,而联合靶向siRNA制剂对两个基因的抑制率最高.结论 联合靶向特异性siRNA制剂能有效抑制HSV-2的感染和复制.     

Effects of S-acetylglutathione in cell and animal model of herpes simplex virus type 1 infection

Intracellular glutathione (GSH) plays an important regulatory role in the host response to viral infections. Replenishment of intracellular GSH is a desirable yet challenging goal, since systemic GSH supplementation is rather inefficient due to a short half-life of GSH in blood plasma. Further, GSH is not taken up by cells directly, but needs to be broken down into amino acids and resynthesized to GSH intracellularly, this process often being impaired during viral infections. These obstacles may be overcome by a novel glutathione derivative S-acetylglutathione (S-GSH), which is more stable in plasma and taken up directly by cells with subsequent conversion to GSH. In the present study, in vitro effects of supplementation with S-GSH or GSH on intracellular GSH levels, cell survival and replication of human herpes simplex virus type 1 (HSV-1) were studied in human foreskin fibroblasts. In addition, in vivo effects of supplementation with S-GSH or GSH on HSV-1-induced mortality were studied in hr/hr mice. In cell culture, viral infection resulted in a significant decrease of intracellular GSH levels. S-GSH efficiently and dose-dependently (5 and 10 mM tested) restored intracellular GSH, and this replenishment was more efficient than with GSH supplementation. In mice, S-GSH, but not GSH, significantly decreased HSV-1-induced mortality (P<0.05). The data suggest that S-GSH is a suitable antiviral agent against HSV-1 both in vitro and in vivo, indicating that this drug may be of benefit in the adjunctive therapy of HSV-1 infections.     

栀子提取物ZG对单纯疱疹病毒1型感染后细胞膜流动性的影响

目的 观察栀子提取物ZG对单纯疱疹病毒1型感染后宿主细胞膜流动性的影响，以探讨其抗病毒作用机理。方法 采用NBD-C6-HPC特异性荧光探针标记Hep-2细胞膜脂质，以肝素钠为阳性对照，借助激光共聚焦扫描技术检测栀子提取物ZG对病毒感染后宿主细胞膜流动性的影响。结果 正常细胞膜具有良好的流动性，漂白后荧光恢复率为73．89％；病毒感染后Hep-2细胞膜流动性显著降低，荧光恢复率为18．54％；栀子提取物ZG作用后细胞膜流动性明显恢复，荧光恢复率为61．21％；肝素钠作用后细胞膜流动性明显恢复，荧光恢复率为56．62％。结论通过改善细胞膜的流动性，从而维持细胞膜的正常功能可能是栀子提取物ZG抗病毒感染的机理之一。     

Herpes simplex virus type 2 and heterosexual spread of human immunodeficiency virus infection in developing countries: hypotheses and research priorities

Herpes simplex virus type 2 (HSV-2) infection is almost always sexually transmitted, and causes genital ulceration. Significant progress in our understanding of HSV infection has occurred over the last decade, in part related to the development of accurate and sensitive laboratory tests to study HSV-2. The application of PCR and type-specific serology to individual cases and in population-based studies has enabled the identification of a potentially important role for HSV-2 infection as a cofactor in the sexual transmission of HIV. This is a particular issue in developing countries. This review describes the epidemiology of HSV-2 infection in the HIV era, the hypotheses regarding HSV–HIV interactions, and research priorities for the developing world.     

Immunization with a replication-defective herpes simplex virus 2 mutant reduces herpes simplex virus 1 infection and prevents ocular disease

Ocular infections with herpes simplex virus 1 can lead to corneal scarring and blindness, with herpes keratitis being the major infectious cause of blindness. There is currently no clinically approved vaccine and nearly all developmental vaccines are targeted against HSV-2 and genital herpes. We tested the ability of an HSV-2 replication-defective virus, a genital herpes vaccine candidate, to protect against HSV-1 corneal infection. Immunization with HSV-2 dl5-29 reduced viral replication in the cornea, prevented ocular disease and reduced latent infection by the HSV-1 strain. Therefore, this HSV-2 replication-defective mutant strain may have applications for prevention of herpes keratitis and genital herpes due to HSV-1 infection.     

单纯疱疹病毒性脑炎的脑电图分析

目的探讨脑电图（ＥＥＧ）对单纯疱疹病毒性脑炎（ＨＳＥ）提供诊断依据和评价疾病严重程度、疗效和预后的意义。方法：对４２例ＨＳＥ的ＥＥＧ行回顾性分析。结果：４２例中３７例ＥＥＧ异常,早期敏感性为８２％。结论：ＨＳＥ之ＥＥＧ早期敏感性高,对诊断有指导作用,可作为推断本病严重程度及治疗效果的依据之一     

Inhibition by acyclovir of herpes simplex virus type 2 morphologically transformed cell growth in tissue culture and tumor-bearing animals

Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.     

Expression of herpes simplex virus type 1 DNA polymerase by recombinant vaccinia virus

We have studied expression of the catalytic subunit of a phosphonoacetic acid-resistant (PAA^r) DNA polymerase (Pol) of herpes simplex virus type 1 (HSV-1) strain ANG by recombinant vaccinia virus (VV) engineered with the dominant Ecogpt selection system. In agreement with the vector construction recombinant Pol expression was regulated like a VV late function. De novo-synthesis of the 136-kDa Pol polypeptide was detectable as early as 6 h postinfection, peaked between 10 and 12 h, and correlated with specific polymerase activity. Compared with HSV-1 lytic infection, the recombinant Pol protein exhibited a reduced stability with a half-life of 7 h. Whereas the Pol-associated exonuclease activities, determined from lysates of recombinant VV- and HSV-1-infected cells, were almost identical, the polymerizing activity of recombinant Pol ceased after 10 min of incubation, in correlation with the fact that Pol depends on its cofactor for optimal chain elongation. Kinetics of cellular localization, tracked by a monospecific Pol antibody, revealed that the catalytic subunit initially assembled to a few dot-like nuclear sites, reminiscent of HSV-1 DNA replication compartments. Later during infection, the localization of recombinant Pol matched with that found in lytically HSV-1-infected cells. This study demonstrates that nuclear transport and localization of the Pol subunit is independent of herpesviral functions, and neither requires the presence of herpesviral DNA sequences. Recombinant VV provides a promising alternative to explore protein interactions of the herpesviral replication machinery in their authentic cellular environment.     

表达hIFNα-2b基因重组乳杆菌治疗HSV-2感染的研究

目的 探讨表达人干扰素α-2b的重组乳杆菌应用于阴道HSV-2感染的治疗作用。方法 建立阴道感染HSV-2的小鼠模型，使用重组菌菌液于感染前24h进行预防，或感染后48h进行治疗，记录病损程度并检测小鼠阴道分泌物中HSV-2滴度。结果使用重组菌预防或治疗的小鼠症状轻且病程缩短，病灶中病毒滴度下降很快。结论表达IFNα-2b的重组乳杆菌具有较强的局部抗病毒作用，可用于预防及治疗由HSV-2感染引起的生殖器疱疹。     

Incidence and distribution of herpes simplex virus types 1 and 2 from genital lesions in college women.

During a 9-month period, 9,772 women were treated at the student health center's gynecology clinic. Herpes simplex virus was isolated from 30 of 57 patients clinically diagnosed as suffering from a herpetic or herpetic-like genial infection for a virological incidence rate of 0.31%. Using virus plaque diameter in chick embryo cells and heat stability of viral thymidine kinase, 37% of the isolates were classified as herpes simplex virus type 1 and 63% were classified as herpes simplex virus type 2.