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.     

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.     

Outcome of herpes simplex virus type 2 infection in guinea pigs.

Factors that influence the outcome of genital herpes simplex virus (HSV) infection were explored in a guinea pig model. The viral inoculum required to establish infection in 50% of animals (ID50) was similar for inbred (strain 2) and outbred (Hartley) guinea pigs. However, the viral inoculum required to produce clinical disease in 50% of the animals (CD50) was 10 times greater for strain 2 compared to Hartley animals. HSV infection of both inbred and outbred animals was more likely to result in death of weanling than adult animals. The duration and severity of genital disease and the magnitude of vaginal viral replication were similar for strain 2 and Hartley animals in both young and adult animals. The lethal dose for 50% of animals (LD50) was 100-fold greater than the CD50 for Hartley animals, but the LD50 and the CD50 were equal in strain 2 guinea pigs. Viral cultures of homogenized neural tissues from infected animals revealed that HSV ascended to the level of the temporal cortex in strain 2 guinea pigs while virus was never recovered above the lumbar spinal cord in Hartley animals. Endogenous peripheral blood mononuclear cell-mediated cytolytic activity against HSV-infected targets was greater prior to HSV inoculation in survivors compared to animals that died. A fatal outcome of genital HSV-2 may relate to the failure to limit CNS viral replication. Death is more common among guinea pigs that have low endogenous HSV-directed natural killer activity, such as occurs among strain 2 and young animals whether inbred or outbred.     

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.     

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.     

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.     

Effect of age and route of inoculation on outcome of neonatal herpes simplex virus infection in guinea pigs

The morbidity and mortality of neonatal herpes simplex virus infection remains unacceptably high despite antiviral therapy. A better understanding of factors that might contribute to this poor outcome is needed but has been hindered by a lack of a good animal model. The recently described guinea pig model of neonatal HSV-2 infection was used to explore the effect of age and route of inoculation on the outcome of infection. After intranasal inoculation the onset, extent, and severity of the primary disease, as well as the number of recurrent lesion days, varied inversely with age. The route of inoculation also affected the outcome. Newborn animals were inoculated either intradermally on the scalp or by the intranasal, oral or corneal route. Animals inoculated on the scalp had the best outcome with no deaths or evidence of neurologic disease while the intranasal route produced the most severe disease, 88% mortality. Neurologic disease was common after oral (41%) and corneal (56%) inoculation but resolved spontaneously whereas following intranasal (39%) inoculation all animals with neurologic disease died. Recurrent disease manifest by cutaneous lesions was observed in all survivors of each group but also differed by the route of inoculation. The guinea pig model of neonatal HSV-2 disease appears to mimic human disease. The studies presented here show that the outcome of infection is influenced by the age and route of inoculation. © 1996 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.     

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.     

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.     

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.     

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.     

A reproducible method for the optimum infection of human mononuclear cells and lymphoid cell lines by herpes simplex virus type I

Parameters for the infection of human mononuclear cells (MNC) with herpes simplex virus type 1 (HSV-1) were investigated and a procedure was established which resulted in a reproducible optimum number of cells expressing virus-specific cell surface antigens The number of cells infected was independent of the sex of the donor and independent of whether the donor was HSV- seropositive or -seronegative. On the average 18±6% of HSV-infected MNC from any given donor expressed HSV-specific cell surface antigens. When the standard procedure was applied to a variety of lymphoid cell lines, a high percentage of cells of both B and non-T/non-B lines expressed HSV-specific cell surface antigens, whereas T-cell lines appeared resistant to HSV infection.     

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.     

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.     

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 ％ 。     

Shedding of herpes simplex virus type 1 into saliva in patients with orofacial fracture

Shedding of herpes simplex virus type 1 (HSV-1) into saliva was studied in 83 patients with orofacial fractures. Infectious virus was isolated from 14 of 83 patients (16.8%) with no detectable herpetic lesion during hospitalization. Of the 83 patients, 44 (53%) had HSV-1 specific antibody. Virus shedding into saliva was observed only in the patients with antibody to HSV-1; thus, the frequency of virus shedding patients among those with antibody was 31% (14 of 44 patients). The frequency was obviously higher than that of a healthy population. The period of HSV-1 shedding had a mean of 3.7 days with range of 1 to 8 days, which is also significantly longer than that of a healthy population. These results strongly suggest that both treatment and operation lead effectively to reactivation of latently infected HSV-1.     

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.