Association of TLR3-hyporesponsiveness and functional TLR3 L412F polymorphism with recurrent herpes labialis

HSV-1 persistently infects almost 90% of our population; however, only 30% of the infected subjects suffer from recurrent herpes lesions, most frequently herpes labialis (HL). We hypothesized that variations in toll-like receptor (TLR) functions might contribute to HL susceptibility. In our study, the TLR-2/1,-3, and -7/8 responses of immune cell subsets derived from asymptomatic HSV-1 carriers were compared with responses of subjects with HL history. Remarkably, natural killer (NK) cells isolated from HL subjects showed significantly lower IFN-γ responses selectively to the TLR3 agonist poly(I:C). Furthermore, the TLR3 L412F genetic polymorphism was found to reduce NK cell TLR3-responsiveness and is associated with susceptibility to recurrent HL. The TLR3 response detected in HL total peripheral blood mononuclear cells (PBMCs), however, was not impaired, indicating restoration of NK cell TLR3-deficiency through co-stimulatory functions. In conclusion, our results suggest that decreased TLR3 response of NK cells is associated with HL susceptibility; and potentially explain why symptomatic outbreak of HL usually occurs after stress or prolonged UV light exposure, when host co-stimulatory functions are disturbed.     

Immune-specific interferon production by peripheral blood mononuclear leukocytes from patients with primary and recurrent oro-labial herpes simplex virus infections

Immune-specific IFN (IFN) is produced by the peripheral blood mononuclear leukocytes (PBML) of greater than 95% of HSV-seropositive humans with infrequent recurrences of herpes labialis [Green, Yeh, and Overall, 1981]. However, herpes virus-induced immune-specific IFN was produced by PBML from only 33 of 48 (68.8%) persons with frequent recurrences (2-12 episodes a year). Two of eight subjects with primary herpes gingivostomatitis also failed to produce immune-specific IFN during either the acute or convalescent phases of their initial HSV infection. These data suggest that some persons have a defective immune-specific IFN response that exists from the time of their primary oro-labial HSV type 1 infection. This defect may predispose to a higher frequency of disease in some individuals.     

Local anesthetic improves individuals affected with herpes simplex type 1 labialis

Infections caused by the herpes simplex virus 1 (HSV-1), commonly called herpes simplex labialis (HSL), are a public health problem, reaching around 40% of the world's population. Thus, the search for effective therapeutic alternatives in the control of the limitations caused by this virus during the stages of evolution of the disease, is necessary, since they have a direct impact on the quality of life of the patients. The aim of the present study was to evaluate the efficacy of the in situ film precursor semisolid composition in the treatment of herpes simplex lesions in human HSV-1. Ninety-eight (n = 98) patients with HSV-1 were used for this study. The initial exclusion criteria left 81 patients to be considered in the present study. Three applications were performed, the first at time zero (T0) and the other two at 8 and 16 hours, after initial application (T8 and T16). Photographs were taken in the first appointment and 24 and 72 hours after the last application. After the three periods, each patient received a total amount of 90 mg of anesthetic and the prognosis of the patients was followed for 6 months and 1 year after the application. Frequency analysis showed that 40.3% of patients had remission of symptoms 24 hours after the last application. For the present study, the film presented a positive therapeutic potential and an esthetic benefit that is absent in the current products (ointments and gels). The invent presents dosage convenience (only three applications in a 24-hour period) and a low production cost, with a much shorter healing time than that reported using topical antiretrovirals.     

Immunostimulatory function of herpes simplex virus isolates from patients with frequent herpes labialis and a deficiency in immune-specific interferon production

Approximately 30% of persons with frequent episodes of herpes labialis are deficient in the production of HSV-induced immune-specific interferon (IFN) (Green, 1985). Herpes simplex virus (HSV) strains isolated from persons who make immune-specific IFN and from persons who do not make it were examined for their immunostimulatory capabilities. HSV isolated from the primary oral lesions of two patients deficient in immune-specific IFN production, one person with an intact immune-specific IFN response, HSV types 1 and 2 laboratory strains, and Newcastle disease virus (NDV) were added to cultures of peripheral blood mononuclear cells (PBML) from HSV seropositive donors. All HSV-isolates induced comparable titers of immune-specific IFN. These studies suggest that failure of some patients to develop an immune-specific IFN response is determined by the host, not the virus.     

Growth of herpes simplex virus in epidermal keratinocytes determines cutaneous pathogenicity in mice

Herpes simplex viruses (HSV)-1 and -2 isolated from genital lesions were examined for cutaneous pathogenicity and its correlation with cellular tropism. HSV-1 caused vesiculation, erosion/ulcer, and zosteriform lesions successively, but skin lesions of HSV-2 developed without vesiculation in some mice, and with statistically significantly less frequent vesiculation than HSV-1. Thus, the virological type of HSV was correlated with its cutaneous pathogenicity. The growth characteristics of HSV-1 and -2 were compared in cultured human embryonic lung (HEL) fibroblasts, human lung cancer A549 cells, human neonatal epidermal keratinocytes, human neonatal dermal fibroblasts, HeLa cells, and Vero cells. HSV-2 produced plaques that were 72% times the size of HSV-1 plaques in epidermal keratinocytes but 230%-500% the size in the other cells. The difference between HSV-1 and -2 in the ratio of plaque size to virus yield in epidermal keratinocytes was much larger (502 times) than the ratio of the other cells (5.57-28.8 times). Keratinocytes are the major constituent of the epidermal layer of the skin and the cells in which vesiculation and erosion/ulceration occur histologically. Therefore, the smaller spread of HSV-2 in keratinocytes of the epidermal layer and the greater spread in other cells of the dermal layer might reflect its lesser invasiveness in the epidermal layer despite larger invasiveness in the dermal layer, which is reflected in the low incidence of erosion/ulcer of the skin compared to HSV-1. Thus, the growth of HSV in epidermal keratinocytes appeared to correlate with the cutaneous pathogenicity causing vesiculation in the skin.     

Tampon culture for quantitation of cervicovaginal herpes simplex virus.

From 10(1) to 10(6) TCID50 (mean tissue culture infective doses) per ml HSV I and II can be quantitatively recorded from vaginal tampons. Tampons and sterile cotton swabs are equally sensitive in recovering HSV I and II. Direct cotton swab cultures of the cervix and cervicovaginal tampon cultures from the same patients recovered similar quantities of HSV, ranging from 1.5 to 6.5 log10 TCID50/ml in 5 patients.     

Sensitive analytic ELISAs for subclass herpes virus IgG

The subclass distribution of antiviral antibodies to three herpes viruses was studied in a population of healthy blood donors. Subclassification by monoclonal antibodies led to the identification of certain viral IgG patterns. IgG1 appeared to be formed in response to almost all CMV, HSV-1 and VZV infections. A higher frequency of virus-specific IgG3 to CMV and HSV-1 suggested that these infections may be reactivated subclinically more often than VZV. The presence of CMV and VZV IgG4 showed a familial relationship, while IgG4 responses to HSV-1 were common. Persons with IgG4 as the only subclass-reactive antibody to CMV showed cell-related reactivities in a high frequency. Patients with leukemias, myelomas and Crohn's disease had a near-normal subclass pattern to the herpes viruses.     

The stability of herpes simplex virus on vaginal tampons.

Herpes simplex virus can be quantitatively recovered from vaginal tampons suspended in tissue culture medium and stored over a wide range of temperatures. Because herpes simplex virus is stable for several days with refrigeration or after freezing, this method of culture of cervicovaginal virus lends itself to epidemiologic studies.     

Effect of centrifugation on herpes simplex virus isolation

The effects of high-speed centrifugation on the isolation of herpes simplex virus (HSV) were studied. Aliquots of laboratory or clinical specimens were inoculated into test tubes and flat-bottomed tubes containing HEp2 monolayers. Test tubes were incubated at 35 degrees C on roller drums (standard method), and flat-bottomed tubes were centrifuged at 15,000g at 35 degrees C for 1 hr, before being incubated at 35 degrees C without rolling (centrifuged method). Centrifugation of clinical and laboratory specimens of HSV type 1 and HSV type 2 produced significantly increased isolation rates compared with the standard method. When clinical and laboratory specimens were diluted, the centrifuged method was more sensitive at all dilutions. When 20 specimens were used for end-point titrations, the centrifuged method was 10 times more sensitive for 15 specimens and 100 times more sensitive for five specimens. There was no difference in the time taken for the appearance of cytopathic effect (CPE) between the standard and centrifuged methods.     

Isolation of high-molecular-weight infectious DNA from type 1 herpes simplex virus

Isolation of DNA from type 1 herpes simplex virus (strain L2) is described; the DNA possessed the characteristics of an intact molecule: sedimentation rate, physical length, and infectivity. Data on infectivity of preparations of this DNA were obtained in cultures of chick embryonic fibroblasts.D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Solov'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 26–28, January, 1977.     

Ethnic variation in type of genital herpes simplex virus infection in a South London genitourinary medicine clinic

The aims of this study were to investigate the prevalence of herpes simplex virus (HSV) types 1 and 2 in the study population and correlate the results with clinical and demographic details. Consecutive HSV isolates from 334 clinic attendees were typed by immunofluorescence. Patient information was collected from the case notes. Overall, HSV-1 was isolated from 48 and HSV-2 from 287 samples, respectively. There was no significant difference in isolation rates according to gender. However, 33% of white patients' isolates typed as HSV-1, while only 6% of the isolates from the black population were HSV-1 (P < 0.001). Initial infections were seen in 81% of HSV-1 infections and 48% of HSV-2 infections, respectively. A wide discrepancy was observed in the prevalence of HSV-1 and HSV-2 infections between the ethnic groups in this population, which was not explained in terms of gender or age. This may reflect different exposure to HSV-1 in childhood or different sexual practices. The increased prevalence in genital HSV-1 reported in recent studies was not seen in this population. However, the differing proportions of primary and first episode infections may reflect a changing epidemiology.     

Further evidence for common antigens in herpes simplex and varicella-zoster viruses

To elucidate the mechanism of heterologous antibody responses to herpes simplex virus (HSV) and varicella-zoster virus (VZV) which occur in some patients with HSV or VZV infections, stronger evidence was sought for the existence of cross-reacting antibodies to these viruses, using antibody absorption procedures. Absorption of sera from initial HSV infections with HSV antigen was found to abolish heterologous antibody titer rises to VZV, as demonstrated in complement fixation, neutralization, and anti-complement immunofluorescence test systems. In most instances, convalescent-phase titers to heterologous VZV were reduced by HSV absorption to levels comparable to those in the acute-phase serum, indicating that cross-reacting antibodies were, in fact, responsible for the heterologous antibody titer rises. Absorption of convalescent-phase sera from HSV or VZV patients with homologous antigen also abolished or greatly diminished immunoprecipitating activity with the heterologous antigen, furnishing additional evidence of the existence of cross-reacting antibodies. Absorption of sera with insolubilized IgG to re-remove rheumatoid factor, which was present in a number of the sera studied, had no effect on either homologous or heterologous antibody titer increases. The demonstration of cross-reacting antibodies to HSV and VZV supports the concept that these two human herpesviruses share common antigen(s).     

In vitro reactivation of latent herpes simplex virus by the demethylating compound hexamethylene-bis-acetamide

We have characterized previously a model of herpes simplex virus (HSV) infection of rat dorsal root ganglia (DRG) following cutaneous infection. During acute infection HSV can be isolated from co-cultivated rat ganglia in (mean ± S.E.M) 4.8 ± 0.33 days (d) and from latently infected ganglia in 7.8 ± 0.53 (d) (P 0.0001). We treated co-cultivated rat ganglia from acute and latent infected rats with the demethylating compound hexamethylene-bis-acetamide (HEX) to see what effect, if any, it would have on HSV infection. HEX-treated ganglia from rats with acute infection did not differ significantly from controls in the proportion of rats, skin or ganglia positive for HSV. The mean time to detect virus was not different between treated (3.6 ± 0.38 d) and control (3.1 ± 0.46 d) (P> 0.05) groups. In latent infected rats there was no difference between treated and controlled groups in the proportions of rats, skin and ganglia positive for HSV. There was a significant difference in the mean time to CPE between the HEX and control groups respectively (4.5 ± 0.72 d vs 8.92 ± 1.42 d, P < 0.01). We conclude that HEX converted latent HSV infection to a productive one.     

Visualization of herpes simplex virus type 1 attachment to target cells using Staphylococcus aureus as a morphologic tag

Staphylococcus aureus was used as a morphologic tag to allow light microscopic localization of herpes simplex virus type 1 (HSV-1) binding and attachment to HEp-2 target cells. The virus was bound to S. aureus through an anti-HSV-1 linkage. The complex was stable and the attached virus still infectious.     

Suppression of generation and replication of acyclovir-resistant herpes simplex virus by a sensitive virus

The role of acyclovir-sensitive herpes simplex virus (HSV) was analyzed in the process of its replacement by a resistant virus in vitro and in vivo in the aspect of acyclovir therapy. The mode of replacement of acyclovir-sensitive HSV with acyclovir-resistant HSV was examined by the passages of acyclovir-sensitive wild type HSV in Vero cells under acyclovir-treatment. The development of resistance was monitored more adequately by counting the number of acyclovir-resistant viruses in 10,000 plaque forming units than by the conventional susceptibility assay. The resistance increased with the proportion of thymidine kinase-deficient (TK(-)) viruses, when the susceptibilities of acyclovir-treated HSV population to 5'-iodo-2'deoxyuridine and phosphonoacetic acid were examined. The increased resistance was due to the increased proportion of acyclovir-resistant virus but not intermediately resistant virus. Infection with mixtures of TK(-) and acyclovir-sensitive strains rendered TK(-) sensitive to acyclovir, and virus yields were reduced to the levels of acyclovir-sensitive virus in Vero cells. Their yield reduction depended on the proportion of acyclovir-sensitive viruses and induction of TK activity. This reduction in virus yields of the mixture of TK(-) and acyclovir-sensitive strains was confirmed by acyclovir treatment in the skin of mice with cutaneous infection. Acyclovir treatment combined with superinfection of acyclovir-sensitive virus delayed the development of herpetic skin lesions due to acyclovir-resistant virus and reduced virus yields in the infected skin. Acyclovir-sensitive virus plays an important role in suppressing the generation and replication of acyclovir-resistant virus during acyclovir therapy.     

单纯疱疹病毒I型胸苷激酶基因在大肠杆菌中的融合表达

用ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切已构建的含单纯疱疹病毒Ｉ 型胸苷激酶（ ＨＳＶ１ ＴＫ） 基因的ｐ ＵＣ１８／ＴＫ 质粒，将切出的ＴＫ 基因片段克隆入原核表达载体ｐ ＷＲ４５０１ 中，构建成ＨＳＶ１ ＴＫ 基因重组表达质粒ｐ ＷＲ４５０１／ＴＫ。以ＨＳＶ１ ＴＫ 特异性引物ＴＫ１ Ｆｏｒ 和ＴＫ２ Ｒｅｖ 进行ＰＣＲ 鉴定可扩增出预期的５０３ ｂｐ 的ＴＫ 基因编码区部分序列；ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切质粒ｐ ＷＲ４５０１／ ＴＫ， 可切出约１１５０ｂｐ 的ＤＮＡ 片段， 表明重组质粒中已插入ＨＳＶ１ ＴＫ基因片段。用ＩＰＴＧ 诱导ｐ ＷＲ４５０１／ＴＫ／ＪＭ１０９ ，表达出相对分子质量（ Ｍｒ） 约为９７ ０００ 的ＴＫ 和β半乳糖苷酶（ Ｍｒ５５ ０００） 融合蛋白。薄层扫描显示，该融合蛋白含量占菌体蛋白总量的２７ ．４７ ％ 。     

Relationship between type specific antibodies to herpes simplex virus and type specificity in human sera

A quantitative analysis was carried out on the relationship between type specific neutralizing antibodies to herpes simplex virus and the type specificity, using a mutual absorbing procedure. Forty-two samples were selected among human sera, on the basis of type specificity expressed by the value of II/I index. All sera with values of less than 90 of II/I index contained only type 1 specific antibody, while those with values over 111 contained only type 2 specific antibody. When the values were between 90 and 110, type 1 specific antibody was present in all, and type 2 specific antibody was present in some serum samples.     

A nonlytic transforming mutant of herpes simplex virus type 2.

A small-plaque mutant (NO.69) of herpes simplex virus type 2 (HSV-2) strain 333 has been previously isolated and characterized in this laboratory. This mutant was shown to produce a high ratio of noninfectious to infectious particles when grown at the nonpermissive temperature in hamster embryo fibroblasts [Westmoreland D. and Rapp F. (1976). Journal of Virology [8:92--102]. In this study, we have demonstrated that it is possible to obtain noninfectious stocks of this virus which retain transforming ability in a biochemical transformation assay specific for detection of the HSV gene for thymidine kinase. This mutant contains a DNA genome that has a density identical to the DNA of wild-type virus. Virus and cell DNA synthesis after infection with the mutant at both the permissive and nonpermissive temperature are similar to that observed in cultures infected with the parental virus. Clones of mouse cells biochemically transformed by this virus contain HSV antigens and are presently being examined for oncogenicity.     

Acute and latent herpes simplex virus neurological disease in mice immunized with purified virus-specific glycoproteins gB or gD

Groups of 5-week-old BALB/c mice were immunized intraperitoneally with approximately 10 micrograms of purified alum-precipitated glycoprotein gB or gD of either herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) origin. Control mice received injections of alum-precipitated 1% bovine serum albumin (BSA). Following a second immunization 4 weeks later, seroconversion was confirmed by demonstrating the presence of glycoprotein-specific antibody by immune precipitation. All animals were challenged with lethal doses of either HSV-1 or HSV-2 by footpad inoculation and assessed for acute virus-induced neurological disease and the development of ganglionic latency. Whereas 70% of control (BSA-immunized) HSV-1-infected animals developed ascending myelitis and died, 100% of mice immunized with either gB-1, gB-2, gD-1, or gD-2 antigens remained free of clinical illness and survived HSV-1 challenge. In contrast, gB-1-or gB-2-immunized mice were not protected against acute HSV-2-induced neurological disease and showed a mortality rate of 60-90% (equivalent to that seen in controls), although mean survival times were prolonged. However, significant protection against HSV-2 challenge was observed with gD-1 or gD-2 immunization. When sacral ganglia were removed from surviving mice 9-12 months after virus challenge, latent virus was detected in all gB- or gD-immunized animals, although the extent of latent infection was restricted. These results provide evidence that glycoprotein gD might be superior to glycoprotein gB as an immunogen for the control of acute HSV-1 and HSV-2 neurological disease in mice. However, neither glycoprotein prevents ganglionic latency, the source of virus for recurrent herpesvirus infections.