Distribution of Latent Herpes Simplex Virus Type-1 and Varicella Zoster Virus DNA in Human Trigeminal Ganglia

Trigeminal ganglia removed at autopsy from immunocompetent individuals without cutaneous signs of herpesvirus infection were fixed, cut into 5-μm sections, and screened at 100-μm intervals (20 adjacent sections) by PCR for latent herpes simplex type 1(HSV-1) and varicella zoster virus (VZV) DNA. Sections that contained >5 neurons with nuclei stained by hematoxylin/eosin revealed HSV-1 DNA in most samples and VZV DNA in approximately 50% of samples. HSV-1 and VZV DNA were distributed throughout each latently infected ganglion.     

单纯疱疹病毒糖蛋白单克隆抗体对致死性腹腔感染BALB/c幼鼠的被动保护作用

用单纯疤疹病毒(HSV)Ⅰ型SM44株和Ⅱ型SaV株分别腹腔感染BALB/c小鼠,于感染前后不同时间经腹腔注射HSV单克隆抗体(McAb)观察6株McAbs对致死性腹腔感染小鼠的被动保护作用。结果4株McAbs(2C5、1A12、Mad-2、1D10)对HSV-Ⅰ感染的小鼠有保护作用,5株McAbs(2C5、1A12、2A8、1D10、CH-A9)对HSV-Ⅱ感染的小鼠有保护作用。体内保护作用与体外的中和活性相关;并分析了McAbs在中和试验中有无补体参与条件下的保护能力。还证实了HSV糖蛋白在急性感染病程中其型特异性和型共同性抗原决定簇在体内的表达。     

Effect of undernourishment on Herpes Simplex Virus Type 1 ocular infection in the Wistar rat model

We have studied the susceptibility to Herpes Simplex Virus Type 1 (HSV-1) infection in malnourished rats. Groups of 10 rats were undernourished during suckling by offspring duplication. The animals were put on commercial diet and at 1, 2, 3, 5 and 8 weeks after weaning, infected in the eye by scarification with HSV-1, strain F. Significant differences in morbidity and mortality were observed between malnourished and control groups infected three weeks after weaning. Viral titres were higher in ocular washings and brains obtained from the malnourished group. This group showed a diminution in antigen dependent lymphocyte proliferation compared to control, and significantly lower delayed type hypersensitivity reaction against inactivated virus (malnourished = 0.16 +/- 0.02 mm, control = 0.26 +/- 0.03 mm, p < 0.05). Neutralizing antibodies in serum were lower in the malnourished group and lower levels of interferon were obtained in the malnourished group 24 h post-infection. We conclude that malnutrition during suckling induces a delay in the capability to overcome HSV infection.     

Herpes Simplex Virus Type 2 and Cytomegalovirus Perigenital Ulcer in an HIV Infected Woman

We report a case of mucocutaneous Herpes Simplex Virus (HSV)-2 and Cytomegalovirus (CMV) infection in a 39-year-old female with acquired immunodeficiency syndrome, who presented with a perigenital ulcer. The patient was receiving antiretroviral treatment (ART) for 3 months before presentation. Scraping from the perigenital ulcer was positive for HSV-2 and Treponema pallidum using polymerase chain reactions (PCR). The extent and duration of the lesions led us to consider the possibility of coinfection with CMV. The patient also tested positive for CMV by PCR. On subsequent follow-up after 8 weeks, the genital lesions had healed completely. This is possibly ascribable to the ART, which led to significant immune reconstitution.     

A Functional Type I Interferon Pathway Drives Resistance to Cornea Herpes Simplex Virus Type 1 Infection by Recruitment of Leukocytes

Type I interferons are critical antiviral cytokines produced following herpes simplex virus type-1 (HSV-1) infection that act to inhibit viral spread.In the present study,we identify HSV-infected and adjacent uninfected corneal epithelial cells as the source of interferon-α.We also report mice deficient in the A1 chain of the type I IFN receptor (CD118-/) are extremely sensitive to ocular infection with low doses (100 PFU) of HSV-1 as seen by significantly elevated viral titers in the cornea compared to wild type (WT) controls.The enhanced susceptibility correlated with a loss of CD4 + and CD8 + T cell recruitment and aberrant chemokine production in the cornea despite mounting an adaptive immune response in the draining mandibular lymph node of CD118-/mice.Taken together,these results highlight the importance of IFN production in both the innate immune response as well as eliciting chemokine production required to facilitate adaptive immune cell trafficking.     

Expression Analysis of Recombinant Herpes Simplex Virus Type 1 DNase

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)¹. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, directed against the C-terminal residues 503–626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein re aching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK-21 cells at 6 h after transfection and was shown to colocalize with the cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear loca lization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seroepidemiology of Herpes Simplex Virus and Restriction Endonuclease Cleavage Analysis of Herpes Simplex Virus Type 2 in Okinawa

A seroepidemiologic study of herpes simplex virus (HSV) in Okinawa was performed. A total of 423 serum samples were collected from all over Okinawa, and the positivity rate of antibody against HSV was measured using a passive hemagglutination method. The sero positive rate for HSV in age groups of over 40 years was 100%. Seven HSV type 2 (HSV 2) isolates were obtained in Okinawa, and DNA preparations from Vero cells infected with the isolates were analyzed using five restriction endonucleases: Bam HI, Hind III, Kpn I, Bgl II and Eco Rl. Variations in the genomic region were demonstrated in five of the isolates. Such variations have not been reported previously in HSV 2 in mainland Japan. This is the first report of a seroepidemiologic study of HSV and restriction endonuclease cleavage analysis of HSV 2 in Okinawa, is a subtropical island where HSV is endemic. Acta Pathol Jpn 41: 24–30, 1991.     

单纯疱疹病毒体外诱导特异性抗体应答的实验研究

用灭活的单纯疱疹病毒（Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓ，ＨＳＶ１）为致敏原，体外诱导成人外周血淋巴细胞产生特异性抗体应答，特异性抗体诱生水平与血清抗体无相关性（Ｒ＝０．４５，Ｐ＞０．０５），抗体类型为ＩｇＧ。同法致敬新生儿淋巴细胞则不能诱生任何类型的特异抗体，表明此体外抗体应答属继发性免疫应答。特异性抗体应答有明显的ＨＳＶ１抗原剂量依赖性，且需要Ｔ、Ｂ细胞的相互作用。ＨＳＶ１体外致敏的实验研究，为探讨正常和疾病状态下体外特异性抗体应答和免疫调节提供一个有用的实验模型。     

Herpes Simplex Virus Type 1 (HSV-1) Strain HSZP Host Shutoff Gene: Nucleotide Sequence and Comparison with HSV-1 Strains Differing in Early Shutoff of Host Protein Synthesis

The UL41 gene of the HSZP strain of herpes simplex virus type 1 (HSV-1) defective with respect to the early shutoff of host protein synthesis was sequenced and compared with the corresponding HSV-1 strain KOS and 17 gene sequences. In comparison with strain 17, nine mutations (base changes) were HSZP specific, five KOS specific and four were common for both strains. Nine mutations caused codon changes. Three of these mapped to the nonconserved regions and the others to the conserved regions of the functional map of UL4l gene. One KOS specific mutation mapped to the region responsible for the binding of the virion host shutoff (vhs) protein to the alpha-transinducing factor (VP16). The possible relationship between mutations and host shutoff function is discussed. The nucleotide sequence data of the UL41 gene of HSZP and KOS have been submitted to the Genbank nucleotide database and have been assigned the accesion numbers Z72337 and Z72338. This revised version was published online in July 2006 with corrections to the Cover Date.     

Herpes Simplex Virus 1 (HSV-1) Strain HSZP Glycoprotein B Gene: Comparison of Mutations among Strains Differing in Virulence

The nonpathogenic HSZP strain of HSV-1 induces large polykaryocytes due to a syn3 mutation (His for Arg at residue 858) in the C-terminal endodomain of glycoprotein B (gB) (40). We determined the nucleotide (nt) sequence of the UL27 gene specifying the gB polypeptide of HSZP (gBHSZP) and found 3 mutations in its ectodomain at aminoacids (aa) 59, 79 and 108. The ANGpath virus, which also has a syn3 mutation in the C-terminal endodomain of gB (Val for Ala at residue 855) is pathogenic for adult mice (39), but can be made nonpathogenic by replacing the gBANGpath gene by the corresponding gBKOS sequence (21). The gBANGpath had three ectodomain mutations (at aa 62, 77 and 285), while gBKOS had at least four ectomain mutations (aa 59, 79, 313, and 553). Two mutations (aa 59 and 79) in the latter, located in the variable antigenic site IV/D1 were common for gBKOS and gBHSZP. These together with the gBANGpath mutations at aa 62 and 77 create a cluster of 4 mutations in diverse region of the N-terminal part of gB (between aa 59-79), in which the gBs of pathogenic ANGpath and 17 viruses differ from the gBs of nonpathogenic HSZP and KOS viruses. The lower pathogenicity of KOS as related to gBKOS, is furthermore associated with the change of Ser to Thr at aa 313 (locus III/D2). The possibility is discussed that mutations in both above mentioned antigenic loci could result in higher immunogenicity of the corresponding antigenic epitopes, which, in turn, would contribute to the decreased virulence of HSZP and KOS viruses.     

Protective Vaccination Against Genital Herpes Simplex Virus Type 2 (HSV-2) Infection in Mice is Associated with a Rapid Induction of Local IFN-γ-Dependent RANTES Production Following a Vaginal Viral Challenge

PROBLEM: To investigate the production of the CC chemokines RANTES. MCP-1, and MlP-1alpha in the vaginal mucosa following HSV-2 challenge in vaccinated and unvaccinated mice. METHOD OF STUDY: The concentrations of the chemokines were determined in the vagina of HSV-2-vaccinated as well as unvaccinated mice after HSV-2 challenge using a PERFEXT method combined with ELISA. RESULTS: HSV-2 infection did not induce any measurable levels of MIP-1alpha, whereas high levels of RANTES and MCP-1 were detected in unvaccinated animals at 48 hr post challenge. The vaccinated mice developed a more rapid induction of RANTES, but not of MCP-1, appearing as early as 24 hr post challenge. The local induction of RANTES production was preceded by a vaginal IFN-gamma response. Furthermore, vaccinated IFN-gamma-/- mice did not produce any enhanced levels of RANTES following HSV-2 challenge. CONCLUSIONS: The protective immune response against genital HSV-2 infection is associated with a rapid induction of local IFN-gamma-dependent RANTES production.     

Naloxone, an opioid receptor antagonist, enhances induction of protective immunity against HSV-1 infection in BALB/c mice

The immunomodulatory effects of exogenous opioids on induction of acquired immunity during microbial infection are now well known; however, our knowledge about the relationship between endogenous opioid response and microbial infections is rudimentary. Here, we report the effect of administration of Naloxone (NLX), an opioid receptor antagonist, on induction of acquired immunity during primary herpes simplex virus type 1 (HSV-1) infection. BALB/c mice received NLX, twice daily, 2 h before infection with HSV-1 until 7 days after infection. Cell-mediated immunity was assessed by evaluating lymphocyte proliferation, interferon-gamma (IFN-gamma) production, delayed type hypersensitivity (DTH) and mortality rate after acute HSV-1 challenge. The findings showed that a higher level of cell-mediated immunity was induced in the NLX-treated animals compared to the control group after induction of HSV-1 infection. However, the data indicate similar neutralizing antibody production in NLX-treated animals and control animals. This observation and further studies in this field may lead to the use of NLX as an adjuvant for designing microbial vaccines and adjunctive therapy of viral infections.     

Immunomodulation by roquinimex decreases the expression of IL-23 (p19) mRNA in the brains of herpes simplex virus type 1 infected BALB/c mice

Herpes simplex virus (HSV) is a common neurotropic virus which infects epithelial cells and subsequently the trigeminal ganglia (TG) and brain tissue. We studied how immunomodulation with roquinimex (Linomide) affects the course of corneal HSV infection in BALB/c mice. BALB/c mice have also been used in a model for HSV-based vectors in treating an autoimmune disease of the central nervous system (CNS). We addressed the questions of how immunomodulation affects the local as well as the systemic immune response and whether roquinimex could facilitate the spread of HSV to the CNS. The cytokine response in the brain and TG was studied using a quantitative rapid real-time RT-PCR method. We were interested in whether immunomodulation affects the expression of the recently described Th1-cytokine IL-23p19 in the brain and TG. The expression of IL-23 mRNA was decreased in brains of roquinimex-treated BALB/c mice. Also the expression of IL-12p35 and IFN-gamma mRNAs decreased. No significant changes were seen in IL-4 and IL-10 mRNA expression. The cytokine response was also studied using supernatants of stimulated splenocytes by EIA. Roquinimex treatment suppressed the production of IFN-gamma and also the production of IL-10 in HSV-infected BALB/c mice.     

In-Vivo Modulation of Macrophage Functions by Herpes Simplex Virus Type 2 in Resistant and Sensitive Inbred Mouse Strains

Intra-peritoneal (i.p.) infection of mice with herpes simplex virus type 2 (HSV 2) attracted macrophages into the peritoneum. Macrophages from moderately and highly HSV 2 resistant mouse strains expressed elevated phagocytosis activity 24 hours after injection. Stimulation of phagocytosis in low resistant strains was generally less effective or absent. This was, in some experiments, due to the fact that macrophages were already highly activated before the experimental infection. I.p. infection also caused HSV replication in the adherent peritoneal exudate cell (PEC) population. The capacity of macrophages supporting HSV 2 replication was low in three of four resistant mouse strains and high in all moderately and highly susceptible and in one of the resistant (SJL) strains when determined 24 hours after infection. Four different F1 hybrids between resistant and susceptible strains exhibited significantly lower yields of virus-producing macrophages than the HSV-sensitive parent. One hybrid between two HSV-susceptible lines restricted virus replication in the PEC population better than both parental strains.     

单纯疱疹-Ⅱ型病毒感染的皮损部位病毒载量与外周血Th及Tc的相关性研究

目的通过检测现症生殖器疱疹（genital herpes,GH）病人皮损部位的单纯疱疹2型病毒（HSV-2）载量,并检测其细胞免疫功能,分析GH患者HSV-2病毒载量与细胞免疫功能的相关性。方法采用荧光定量聚合酶链反应（fluorescent quantitative polymerase chain reaction,FQ-PCR）定量检测28例现症GH患者皮损部位HSV-2DNA载量,采用流式细胞计数的方法监测28例现症病人外周血具有分泌活性的Th及Tc所占比例,运用直线相关回归方法分析现症GH患者病毒载量与细胞免疫功能的相关性。结果①GH患者皮损内疱液HSV-2检出率为100%,病毒载量为（6190714±1962085）拷贝/mL。HSV-2病毒载量与患者复发次数呈显著正相关（P〈0.05）。②GH组Th1、Tc1细胞数和Th1/Th2、Tc1/Tc2比值均明显低于对照组（P〈0.01）,而Th2细胞数显著高于对照组（P〈0.01）。GH患者皮损HSV-2病毒载量与患者外周血Th1、Tc1细胞数和Th1/Th2、Tc1/Tc2比值呈显著的负相关,与Th2细胞数呈显著正相关。结论GH患者存在明显的细胞免疫功能抑制及Th、Tc细胞亚群分化失衡;GH患者的细胞免疫功能抑制可能是由于外周血T淋巴细胞亚群的变化和NK细胞减少,以及Th、Tc细胞亚群分化失衡造成,这种状态不利于机体抑制病毒复制和增殖,造成GH反复发作。     

Systemic Administration of Olygodeoxynucleotides with CpG Motifs at Priming Phase Reduces Local Th2 Response and Late Allergic Rhinitis in BALB/c Mice

Oligodeoxynucleotides (ODN) with CpG motifs (CpG ODN) induce T helper (Th)1-type reaction. We aimed to evaluate the therapeutic effect of CpG ODN in the development of late allergic rhinitis induced by ovalbumin (OVA), which is one of Th2 diseaes, in BALB/c mice. Effects of a single dose of synthetic CpG-ODN (50 μg) intraperitoneally (i.p.) at the priming phase (on day 0) by OVA on the development of late eosinophilic rhinitis at respiratory areas were compared to the control mice treated with its vehicle (ODN without CpG motifs; 50 μg). Animals were again sensitized by OVA (on day 10) i.p., and 4 days after second sensitization animals were challenged by OVA intranasally (on day 14). Four days after challenge, eosinophilic reactions, nasal lesions and local cytokine values were examined. Compared to the control group, the CpG ODN-administration increased production of OVA-specific Th1 cytokine (interferon-γ) and decreased productions of ovalubmin-specific Th2 cytokines [interleukin (IL)-5 and IL-13] in nasal cavity fluids, supernatants of splenocytes and/or sera. Also, eosinophilia and increased total IgE values were decreased in mice treated with the CpG ODN compared to the control group. Moreover, nasal lesions with infiltration of eosinophils were prominently reduced by the CpG ODN-treatment compared to the control mice. The present study suggests that the systemic administration of CpG ODN at the priming phase may reduce local OVA-specific Th2 responses, resulting in decreased nasal pathology in the late allergic eosinophilic rhinitis. The authors wish it to be known, in their opinion, Toshiharu Hayashi and Keiko Hasegawa contributed equally to this work.     

Ⅰ型单纯疱疹病毒糖蛋白D成熟肽基因毕赤酵母表达载体的构建及序列分析

目的构建单纯疱疹病毒Ⅰ型包膜糖蛋白D成熟肽基因毕赤酵母表达载体,并对序列进行分析,为进行高抗原性的真核表达重组gD蛋白奠定基础.方法PCR扩增HSV1-gD成熟肽基因,将该段基因克隆于pGEM-T克隆载体,转化鉴定后,与巴斯德毕赤酵母表达载体(pPIC9K)酶切连接,转化大肠杆菌DH5α,筛选测序确定构建了pPIC9K-gD的真核表达载体,对克隆的序列进行分析,预测表达产物的理化特性及抗原性.结果获得了重组的酵母表达载体pPIC9K-gD.测序结果证实为HSV1-gD成熟肽基因,序列分析其高度保守,预测蛋白分子量为40.52 ku,pI为7.67,包含完整成熟肽分值达1.7的多个抗原决定簇.结论成功构建了HSV1-gD成熟肽基因的毕赤酵母表达载体.     

Acute Depletion and Recovery of Peritoneal B-1 Lymphocytes in BALB/c Mice after a Single Injection of Mercury Chloride

The acute toxicity of mercury (Hg) to B cells was studied in the peritoneal cavity of BALB/c mice, a coelomic space where both B-1 and B-2 subsets of B lymphocytes are present. Up to 24 hr after a single in situ Hg injection, the peritoneal cavity became virtually devoid of lymphocytes, particularly of the B-1 subset. Lymphocyte depletion was more severe for B than T cells. This depletion was associated with partial lymphocyte activation (CD69⁺) at 6 hr of treatment and it was due to apoptosis rather than to necrosis. Partial recovery of both B and T cells was observed in the peritoneal cavity 48 hr after the Hg injection. The phenomenon was followed by a second decrease in peritoneal lymphocytes 72 hr after Hg. Neutrophils that entered the peritoneal cavity because of the Hg injection were resistant to apoptosis. No significant changes in lymphocyte number or subpopulation were found in the spleen and thymus of the mice up to 72 hr after the Hg treatment. We concluded that B lymphocytes were severely affected by the toxic effects of Hg. Our data suggest that Hg-induced unbalance in the repertoire of B cells, of the B-1 subset in particular, may result later in the secretion of the high titres of pathogenic autoantibodies that are found in the Hg-induced lupus disorder of BALB/c mice.