Production of Chemotactic Factor and Lymphotoxin by Human Leukocytes Stimulated with Herpes Simplex Virus

Peripheral blood leukocytes (PBL) of 41 subjects were tested for their ability to be stimulated by herpes simplex virus antigens as measured by [(3)H]thymidine incorporation and lymphokine production (i.e., lymphocyte-derived chemotactic factor and lymphotoxin). The PBL of seronegative subjects failed to respond to viral antigens as measured by stimulation or lymphokine production. In contrast, PBL from seropositive subjects without clinical lesions of herpes labialis were stimulated by herpes simplex virus antigens and produced lymphokines. PBL from seropositive patients with clinical lesions of herpes labialis also produced lymphokines, but [(3)H]thymidine incorporation was slightly depressed. These findings suggest that lymphocytes from patients with a recent herpes simplex virus infection may respond less vigorously to in vitro stimulation by herpes antigens, but our study fails to show any basic defect in the responsiveness of the lymphocytes to account for recurrent herpetic episodes.     

Production of Specific Antibodies by Cerebrospinal Fluid Lymphocytes in Patients with Herpes Zoster, Mumps Meningitis and Herpes Simplex Virus Encephalitis

We applied a new method consisting of short-term culture (18 h) of lymphocytes from cerebrospinal fluid (CSF-L) and peripheral blood (PBL) in viral antigen-coated ELISA plates and subsequent measurement of IgG and IgM antibodies bound to antigen. Utilizing mumps virus, herpes simplex virus (HSV), varicella zoster virus (VZV), and measles virus as antigens, we demonstrated production by CSF-L of antibodies against the aetiological agent only in all patients with mumps meningitis and HSV encephalitis and also in all patients with herpes zoster without central nervous system (CNS) symptoms. This might be considered as direct evidence that specific antibodies are produced within the CNS in inflammatory nervous system diseases. CSF-L usually produced higher amounts of antibodies than the corresponding number of PBL. In comparison with concentrations of free antibodies determined in parallel, our method had higher specificity and sensitivity and gave more precise information about the antibody response in infections of the nervous system.     

Host Defense Mechanisms Against Herpes Simplex Virus I. Control of Infection In Vitro by Sensitized Spleen Cells and Antibody

Sensitized mouse spleen cells decrease the spread of herpes simplex virus infection in cell culture lines derived from human and murine tissues. These washed, sensitized cells act alone and additively in combination with antibody to diminish the ability of single virus-infected cells to spread infection to contiguous cells. This control of infection is not species specific, unlike interferon, and appears to be distinct from the effect of antibody. Lymphotoxin was not detected in this lymphocyte-mediated response. This control of herpes simplex virus infection in vitro by sensitized lymphoid cells is immunologically specific; spleen cells from donor animals immunized with a heterotypic virus do not cause herpesvirus plaque size reduction. The ratio of spleen cells from immunized animals to target monolayer cells needed to produce this effect is > 4:1. Plaque size reduction of herpes simplex virus by spleen cells requires intact, immune, non-glass-adhering lymphoid cells.     

Lymphocyte transformation and interferon production in human mononuclear cell microcultures for assay of cellular immunity to herpes simplex virus.

Interferon production and transformation in response to herpes simplex virus antigen were studied in microcultures of human mononuclear cells. Mononuclear cells consisting of monocytes and both T and B lymphocytes were purified by Ficoll-Hypaque gradients. Lymphocytes, predominantly T with 5% B, were obtained by passage of buffy-coat cells through nylon fiber columns. For some experiments, autochthonous macrophages and column-purified lymphocytes were stimulated with herpesvirus antigen. The effect of specific antibody and cell concentration on reactivity is described. Crude and purified antigens were compared as cell culture stimulants. Significant differences in transformation and interferon were observed between donors with a history of herpes labialis and donors with no detectable antibody, both in cultures prepared by Ficoll-Hypaque gradients and by column purification of lymphocytes. Cultures from seronegative donors prepared by Ficoll-Hypaque gradients produced interferon but did not transform when stimulated by herpes simplex antigen. "Immune" interferon production, that is, type II as opposed to type I, occurred only with autochthonous macrophage and column-purified lymphocyte cultures. Interferon produced by Ficoll-Hypaque-purified mononuclear cultures was type I, and its production was unrelated to immune status. Similarly, column-purified lymphocytes responded to herpes simplex virus antigen with type I interferon if obtained from a seropositive donor.     

Human lymphoblastoid cell lines established from peripheral blood lymphocytes secreting immunoglobulins directed against herpes simplex virus

Peripheral blood lymphocytes (PBL) from genital herpes simplex virus type 2 (HSV-2) patients were transformed with Epstein-Barr virus. Using conditions optimized for successful transformation, two lymphoblastoid cell lines (LCL) have been established, which secrete immunoglobulin G (IgG) to HSV. These results suggest that PBL from patients suffering recurrent herpes virus infections can be used to establish LCL secreting HSV antigen specific human immunoglobulins.     

Immune responses to herpes simplex virus in patients with recurrent herpes labialis: I. Development of cell-mediated cytotoxic responses.

Groups of subjects during acute (0-3 days) and convalescent (2-3 weeks) phase of recurrent herpes labialis (RHL), and other subjects seropositive or seronegative for herpes simplex virus type 1 (HSV-1) antibody without any history of RHL, were tested for the appearance of cell-mediated cytotoxic responses by stimulating peripheral blood leukocytes (PBL) in vitro with ultraviolet-inactivated HSV-1 antigen, using the release of radiolabelled chromium (51Cr) from HSV-1-infected autologous, or allogeneic lymphocytes and K562 erythroleukemia cell line as nonspecific targets. Development of HSV specific cytotoxic response using autologous targets was essentially limited to subjects with RHL and in HSV antibody seropositive control subjects. Peak activity was observed during the acute phase of the disease, compared to the activity in the convalescent phase in seropositive subjects with RHL, and was preceded by high lymphoproliferative response to HSV. Higher cytotoxic responses against K562 cells were also observed in RHL subjects compared to the controls. Depletion of Leu-2+, Leu-3+ or Leu-11 effector lymphocytes from HSV-1-stimulated PBL cultures by treatment with complement and appropriate monoclonal antibodies resulted in significant reduction of cytotoxicity to HSV-1-infected autologous cells. However, cytotoxicity to K562 cells was reduced only after depletion of Leu-11+ cells. Low levels of allogeneic restriction were observed for cytotoxicity to HSV-1-infected targets. These observations suggest selective activation of virus specific Leu-2+ and Leu-3+ T cell subsets as well as natural killer cell mediated cytotoxic mechanisms during the active phase of recurrences of herpes simplex virus infection.     

Rapid diagnosis of varicella-zoster virus infection with a monoclonal antibody based direct immunofluorescence technique

Diagnosis of varicella-zoster virus (VZV) infection in immunocompromised patients is difficult because of the frequent atypical appearance. Accurate and early diagnosis is important to allow rapid commencement of antiviral chemotherapy, with consequent improvement in antiviral efficacy. A monoclonal based direct immunofluorescence antibody technique (VZV IFA) was assessed in parallel with viral culture in 56 patients with suspected VZV infection. A subgroup of 17 patients from this group with classical dermatomal herpes zoster all had positive VZV IFA tests. Only 6 patients (35%) were positive on viral culture. None of the 15 patients with proven herpes simplex virus infection had a positive VZV IFA, nor did any patient with positive VZV viral culture have a negative VZV IFA. The VZV IFA test is a rapid and sensitive technique for detecting infection with VZV.     

Serum antibodies to herpes simplex virus type 1 during active oral herpes infection

Subjects with oral herpes lesions at the time of serum sampling had higher-efficiency antibody (higher proportion of neutralizing antibody as determined by plaque reduction, compared with total antibody as detected by radioimmunoassay) to herpes simplex virus type 1 (HSV-1) than did subjects with no lesions at the time of serum sampling. These higher-efficiency sera also had higher antibody titers to structural components of herpes simplex virus type 1 than did the low-efficiency sera. Absorption of high- and low-efficiency sera with purified herpes simplex virus type 1 particles removed all neutralizing antibody but not all antibody detected by radioimmunoassay. High-efficiency serum was depleted of more antibody to particulate antigen that was the low-efficiency serum, indicating that the high-efficiency serum contained a higher proportion of antibody to the virus particle.     

Host Defense Mechanisms Against Infectious Bovine Rhinotracheitis Virus: In Vitro Stimulation of Sensitized Lymphocytes by Virus Antigen

Isolated peripheral blood lymphocytes (PBL) from cattle immunized or infected with infectious bovine rhinotracheitis (IBR) virus were cultured in vitro with ultraviolet light-inactivated IBR virus, and the degree of lymphocyte blastogenesis was quantitated by measurement of the uptake of [(3)H]thymidine into acid-insoluble material. Lymphocyte blastogenesis only occurred with PBL from immunized or infected animals. The optimal conditions for lymphocyte blastogenesis were defined. Blastogenesis was specific since cells from animals immunized against IBR failed to react with two other herpesvirus antigens tested, herpes simplex and equine rhinopneumonitis viruses. Blastogenesis could be prevented by reacting IBR antigen with IBR-specific antibody before adding to cultures, but incorporating IBR-specific antibody in the culture medium after adding free antigen failed to inhibit blastogenesis. With intranasally infected animals, lymphocyte blastogenesis was detectable after 5 days, reached peak levels between days 7 and 10, and then declined to low levels by day 19. In contrast, levels of neutralizing antibody were barely detectable on day 7 and reached maximal concentrations on day 19. The lymphocyte blastogenesis assay was emphasized as a convenient and useful in vitro correlate of cell-mediated immunity that should help define the role of cell-mediated immunity in immunity to herpesviruses.     

Reactivation of latent herpes simplex virus infection of the autonomic nervous system by postganglionic neurectomy.

Latent herpes simplex virus infection of the superior cervical autonomic ganglion was reactivated in vivo by postganglionic neurectomy. Two methods were used to demonstrate viral reactivation: (i) recovery of infectious herpes simplex virus in ganglion homogenates and (ii) acceleration of virus expression in ganglion explants in culture. Both the percentage of mice exhibiting reactivated ganglion infection and the viral titers detected in ganglia increased when neurectomized mice were treated with cyclophosphamide. Antithymocyte serum treatment prolonged the time course over which neurectomy-induced virus could be detected, but neither antithymocyte serum nor cyclophosphamide reactivated herpes simplex virus in the absence of neurectomy. These results demonstrate that postganlionic neurectomy provides a specific stimulus for herpes simplex virus reactivation and that cell-mediated immune defense are involved in the highly efficient elimination of reactivated virus from the ganglion in vivo.     

Susceptibility of human lymphocyte populations to infection by herpes simplex virus.

The antibody response to diphtheria toxoid by cultured tonsil cells was suppressed by herpes simplex virus during its inductive stage. Since only T lymphocytes readily supported virus replication, this immunosuppression may be attributed to a selective effect of the virus on this population of cells.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen.

An enzyme-linked immunosorbent assay (ELISA) kit for herpes simplex virus developed by Ortho Diagnostic Systems, Inc., was evaluated. In phase I experiments, 263 clinical specimens from genital lesions were extracted into serum-free medium and then tested by ELISA for herpes simplex virus antigen. The results were compared with those obtained by conventional viral culture. Of 83 specimens, 65 were positive by ELISA (sensitivity, 78.3%). In phase II experiments, 249 clinical specimens were tested for herpes simplex virus antigen in direct specimen and in cell cultures (MRC-5 and rabbit kidney) incubated for 2, 4, and 7 days. Of 63 specimens, 40 were positive by ELISA in the direct specimen (sensitivity, 63.5%), and by 7 days incubation, 100% of the cultures positive by viral cell culture were also positive by ELISA. The ELISA was reproducible, and when both the direct detection and amplification culture were used, the sensitivity of ELISA paralleled the diagnosis of herpes simplex virus infections by viral cytopathic effect.     

Correlation of herpes simplex virus antibody titers and specific lymphocyte stimulation in adult blood donors

Antibody titers to herpes simplex virus type 1 in sera from healthy adult donors were assayed by complement fixation, microneutralization, and an enzyme immunoassay (ELISA). This last test proved to be the most sensitive method for antibody detection. It was estimated that ELISA antibody titers were up to 40-fold higher than neutralizing antibody titers and up to 100-fold higher than complement fixation antibody titers. Due to the higher sensitivity of ELISA, only 3 of 36 blood donors tested in this assay were shown to be seronegative, whereas 6 additional persons of the same group were termed seronegative by the microneutralization assay. Furthermore, four of the latter also did not respond in the complement fixation test. In vitro stimulation of peripheral lymphocytes by using a partially purified herpes simplex virus type 1 particle antigen was achieved for all seropositive blood donors. Only those three donors who were ELISA negative reacted negatively in this stimulation assay. From these results it may be concluded that ELISA is an appropriate method not only for rapid and sensitive antibody determination but also for selecting herpes simplex virus-negative patients.     

我国部分地区病毒性脑炎标本的实验室检测

目的 初步了解我国病毒性脑炎的病原种类及其分布特征.方法用ELISA方法对2004年至2006年从我国6个省份收集的771例临床诊断为病毒性脑炎患者的急性期血清和脑脊液标本检测乙脑病毒IgM抗体,然后对乙脑IgM抗体阴性的所有血清标本检测其他7种常见病毒IgM抗体.此外,用PCR方法对54例脑脊液标本检测肠道病毒、版纳病毒和辽宁病毒的基因.结果经血清学检测,771例患者中的567例(73.5%)检测出病毒特异性IgM抗体,构成顺序为乙脑病毒(47.0%)、腮腺炎病毒(10.6%)、肠道病毒(8.8%)、单纯疱疹病毒(5.7%)、麻疹病毒(0.4%)、水痘-带状疱疹病毒(0.4%)、EB病毒(0.4%)、巨细胞病毒(0.3%);经分子生物学检测,在54例脑脊液中检测到8例(14.8%)肠道病毒基因阳性标本,未检测到版纳病毒与辽宁病毒基因阳性标本.结论乙脑病毒是我国病毒性脑炎的首要病原,腮腺炎病毒次之,肠道病毒和单纯疱疹病毒也是重要的病原.     

Electron microscopy of herpes simplex hepatitis with hepatocyte pulmonary embolization in kwashiorkor.

We report a case of herpes simplex hepatitis in a child with edematous malnutrition. Electron microscopy showed virus in parenchymal cells, with pulmonary embolization of necrotic, infected hepatic cell fragments. Systemic dissemination of herpes simplex may be related both to the profound immunoincompetence associated with kwashiorkor and to a reduction in the circulating and fixed polyanions that normally inhibit viral attachment to cells.     

Serotyping herpes simplex virus isolated by enzyme-linked immunosorbent assays.

An enzyme-linked immunosorbent assay (ELISA) using a double-antibody sandwich tecnhique has been developed to serotype isolates of herpes simplex virus from clinical sources. The results obtained using this procedure were in agreement with those obtained with a standard neutralization test in typing stock cultures and 32 clinical isolates of herpes simplex virus. Clear differentiation between the two viral serotypes was obtained using rabbit immunoglobulin cross-absorbed with heterologous virus antigen. The ELISA procedure described appears to be a convenient and accurate substitute for the neutralization test in typing herpes simplex viruses. ELISA techniques require relatively small amounts of antigen and antibody and can be performed with very simple equipment.     

Effects of lectins on peripheral infections by herpes simplex virus of rat sensory neurons in culture.

Concanavalin A and wheat germ agglutinin are capable of preventing a productive peripheral infection of dissociated rat sensory neurons in culture by herpes simplex virus type 1. Concanavalin A binds to the herpes simplex virion, rendering it inactive, whereas wheat germ agglutinin binds to the peripheral neuritic extensions of the sensory neurons, rendering them incapable of initiating a productive viral infection. This latter effect (i) seems to be specific for wheat germ agglutinin since other lectins have no effect, (ii) is not the result of cellular cytotoxicity, (iii) is dependent on an N-acetylneuraminic acid moiety, and (iv) may be due either to viral receptor site masking or to binding of wheat germ agglutinin to the neuritic receptor molecule for herpes simplex virus.     

Kinetics and genetics of herpes simplex virus-induced antibody formation in mice.

The kinetics of antibody synthesis was investigated after intraperitoneal, subcutaneous, and footpad infection of various strains of mice with herpes simplex virus. Immunoglobulin M antibodies appeared 5 days after and immunoglobulin G antibodies appeared 10 to 12 days after intraperitoneal infection with herpes simplex virus type 1. The major histocompatibility complex and the background genome of inbred mice were not found to have a systematical influence on antibody synthesis. Female mice, however, consistently produced more antibodies than did male if the infection was done intraperitoneally, but not if it was done subcutaneously or into footpads. Castration considerably increased the amount of antibodies produced by male mice. The difference in antibody formation between females and males could be abolished by injection of silica; moreover, antibody titers were enhanced by this treatment. This has also been found by immunization with a Formalin-inactivated herpes simplex virus vaccine. The effect of silica in enhancing antibody formation could be observed up to 12 days after infection. Infectious virus could be detected up to 2 days after infection, and herpes simplex virus type 1 antibody-stimulating antigens could be detected up to 4 days in ultrasonicates of macrophages. The assumption is made that androgen-sensitive cell populations, including macrophages and their soluble products, are involved in antibody-depressing mechanisms.     

Herpetic keratitis in athymic (nude) mice.

The inflammatory response to herpes simplex virus infection of the cornea was studied in athymic nude (nu/nu) and heterozygote (nu/+) BALB/c mice. Although athymic mice were highly susceptible to HSV infection and died 13 to 17 days after corneal inoculation, they failed to develop necrotizing keratitis of the cornea. Heterozygote mice survived the initial virual infection, but many of these mice developed necrotizing keratitis and permanent corneal scarring. Light and electron microscopy showed numerous inflammatory cells (polymorphonuclear leukocytes and lymphocytes) in the corneas of heterozygote mice, but not in the athymic mice. These studies suggest that the immune system plays a dual role in herpes simplex virus infection of the cornea: protection against dissemination of the virus and immunopathogenesis of necrotizing keratitis in the cornea.