Efficacy of Kakkon-to,a traditional herb medicine,in herpes simplex virus type 1 infection in mice

Kakkon-to is one of the representative traditional herb medicines (Kampo formulae) and has been used historically for the treatment of infectious diseases in China and Japan. The efficacy of this preparation was characterised using a cutaneous herpes simplex virus type 1 (HSV-1) infection in mice as a model for human viral infection. Kakkon-to at a dose corresponding to human use reduced significantly the mortality of HSV-1-infected mice and localised skin lesions. Delayed type hypersensitivity (DTH) response to HSV-1 antigen was significantly stronger in treated mice than in untreated mice. However, no histopathological difference was noted in the skin lesions between treated and untreated mice except for the size of the lesions. Kakkon-to did not inhibit the growth of HSV-1 in vitro. Natural killer cell activity, natural cytotoxic killer cell activity, and the population of T-cell subsets in spleen cells of infected mice were not affected by the drug. Kakkon-to did not augment interferon induction and anti-HSV-1 antibody production, nor increased cytokine levels such as interleukin-1α, interleukin-2, interferon-γ, and tumour necrosis factor-α in sera of infected mice. Thus, Kakkon-to induced strong DTH to HSV-1 in infected mice, which may have caused localisation of skin lesions and reduction in the mortality of treated mice. © 1995 Wiley-Liss, Inc.     

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.     

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.     

Effect of L-lysine monohydrochloride on cutaneous herpes simplex virus in the guinea pig

The effect of topical applications of crystalline lysine therapy on cutaneous herpes simplex virus (HSV) inoculations and subsequent dorsal root ganglia (DRG) infection was studied in male Hartley guinea pigs. Although HSV-I was recovered from the inoculated sites from all animals, the L-lysine-treated skin remained clinically normal, whereas untreated controls manifested clinical symptoms up to 3 days postinoculation (p.i.). However, cocultivation of DRG (C1-S1) indicated a selective tropism of infective particles to specific DRG in the groups treated with amino acids. In lysine-treated animals, HSV was recovered from a few DRG (T-12, T-13, and L-1) at 3 days p.i. and from DRG T-10 in leucine-treated controls; yet no HSV was recovered from DRG of untreated controls. These results suggest an immunomodulatory effect of L-lysine on inoculation site infections and the possible potentiation of subsequent DRG manifestation in amino-acid-treated animals.     

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.     

Typing and subtyping of herpes simplex isolates by monoclonal fluorescence

The type specificity of 16 herpes simplex monoclonal antibodies was tested on 50 isolates previously typed by endonuclease restriction. A panel of antibodies was selected and used to type isolates from 50 primary and 50 recurrent genital infections. Sixty-four isolates from 18 patients followed up for six months were subtyped by using a larger panel of monoclonal antibodies and predominant HSV1 and HSV2 subtypes were demonstrated. In addition to confirming recurrences, four possible reinfections were identified. Type-specific differences in the location of viral antigens in infected cells were noted and a possible association of this finding with the type-specific differences in rates of recurrence of genital infections discussed.     

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.     

Detection of type 2 herpes simplex virus in cells of spermatogenic epithelium in infected testes of guinea pigs

We developed a model of herpetic orchitis in guinea pigs. Intratesticular inoculation of type 2 herpes simplex virus suspension results in infection of the testicular spermatocytes and spermatides. The possibility of viral infection dissemination from infected into intact testis is proven. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 7, pp. 79–83, July, 2007     

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.     

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.     

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.     

Intradermal doxorubicin reduces ganglionic reactivation of latent herpes simplex virus in mice after pretreatment with hypertonic saline

Recurrent lesions from herpes simplex virus (HSV) occur after reactivation of latent HSV in neurons of sensory ganglion, axonal transport of reactivated virus, and HSV replication on the skin. A potential treatment strategy is to inject epithelial sites of frequent recurrences with anti-viral or cytotoxic agents that are taken up by nerve terminals and transported by axoplasmic flow to latently infected ganglionic neurons. Doxorubicin is transported by nerves and destroys the corresponding nerve cell bodies, but earlier attempts in HSV animal models required intraneural injection to eliminate HSV infection and this treatment destroyed the nerve and large portions of the ganglion. The present study used intradermal doxorubicin in latently infected mice that had been inoculated with HSV by the lip route and passively immunized at the time of inoculation. As found previously, doxorubicin injection in the lip 2 or more months after HSV inoculation did not eliminate HSV latency as evaluated by recovery of virus from trigeminal ganglionic explants. However, when hypertonic saline was injected in the same site 24 hr prior to doxorubicin, there was a 55% reduction of positive ganglionic explant cultures. Edema from the hypertonic saline may increase access of doxorubicin to nerve terminals. This technique with hypertonic saline, which has also been used to enhance virulence of HSV skin innoculation, may have general application of increasing axoplasmic transport of drugs or biologicals. Skin toxicity may preclude doxorubicin use for HSV recurrences in patients; however, the results of this study support the concept of anti-HSV treatment via retrograde axoplasmic transport.     

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.     

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.     

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.     

Steroid hormone alteration of herpes simplex virus type 1 replication.

The effect of steroid hormones on herpes simplex virus type 1 replication was examined. Virus replication studies revealed that various concentrations of prednisolone, hydrocortisone, dexamethasone, or progesterone could decrease virus yields up to a maximum of 99%. Using isopycnic centrifugation in CsCl to separate viral from cell DNA, it was found that virus-specific DNA synthesis was decreased by 30 to 100% depending on the hormone and concentration used. Cell-specific DNA synthesis was also adversely affected, but this did not alter cell viability or plating efficiency.     

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.     

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.     

Combined chemotherapy of cutaneous herpes simplex infection of the guinea pig

Cutaneous infection of guinea pigs with HSV1 was topically treated from 2 to 6 days post infection with 7 antiherpetic substances. Phosphonoformic acid and acyclovir were found to be highly effective; trifluorothymidine, thymine arabinoside, ethyldeoxyuridine, and adenine arabinoside monophosphate all had some therapeutic effect in decreasing order, whereas iododeoxyuridine was ineffective. The efficacy of treatment was evaluated from cutaneous lesion scores by the Wilcoxon rank test. The substances were combined in marginally effective concentrations. From the 21 combinations, acyclovir + phosphonoformic acid, acyclovir + thymine arabinoside, and phosphonoformic acid + thymine arabinoside suggested a synergistic interaction, which appeared significant for acyclovir + phosphonoformic acid.