Persistent Herpes Simplex Virus Infection In Vitro with Cycles of Cell Destruction and Regrowth.

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.), and Mary Lou Copeland. Persistent herpes simplex virus infection in vitro with cycles of cell destruction and regrowth. J. Bacteriol. 90:205-212. 1965.-The susceptibility of two Chinese hamster cell lines to herpes simplex virus (HSV) was studied from the time of their initiation through successive subcultures. The cells' susceptibility to the cytocidal effects of HSV decreased as the number of cell passages increased. During the early cell passages, the decrease in cell susceptibility to HSV was characterized by an increased time after infection for complete cell destruction to occur, with a concomitant increase in the period when virus could be recovered from supernatant fluids. This was followed by a number of cell passages during which persistent HSV infections were established. The persistent infections were characterized by (i) cycles of virus synthesis and cell destruction followed by regrowth of the cells, (ii) initiation and maintenance under conditions optimal for cell growth in the absence of antibody, (iii) the cells' ability to be passaged while still maintaining their cycling patterns, (iv) a relationship between virus synthesis and cell proliferation, and (v) inability of long-term treatment with antibody to "cure" the persistent infections. The unique characteristics of this HSV infection were compared with other persistent in vitro viral infections.     

Characterization of nerve growth factor-dependent herpes simplex virus latency in neurons in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Treatment with acyclovir for the first 7 days after viral inoculation prevented lytic infections in 100% of the cultures and resulted in viral latency in 100% of the cultures; reactivation occurred as the result of nerve growth factor (NGF) deprivation. Treatment of the cultures with several different inhibitors of viral DNA polymerase (acyclovir, aphidicolin, and phosphonoacetic acid) for 7 days after viral inoculation did not prevent the establishment of latency, which suggests that viral DNA replication was not required. During the latent phase of the infection, viral antigens were not detected with HSV-specific immunohistochemistry. However, 24 h after NGF deprivation, viral antigens were detected in essentially all of the neurons, indicating that the majority of neurons harbored latent HSV. The establishment of latency was not strain or type specific since latency was established with HSV type 2 and four strains of HSV type 1 and reactivation occurred in response to NGF deprivation.     

Nerve growth factor deprivation results in the reactivation of latent herpes simplex virus in vitro.

Primary sympathetic neuronal cultures were maintained for up to 5 weeks after inoculation with herpes simplex virus (HSV) without evidence of viral infection. Upon deprivation of nerve growth factor, the cultures produced infectious HSV, indicating that the cultures harbored latent HSV. This study demonstrates a function of nerve growth factor in the maintenance of HSV latency.     

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.     

Specific immunity after congenital or neonatal infection with cytomegalovirus or herpes simplex virus

Infants or children who had congenital or neonatal infection with cytomegalovirus (CMV) or herpes simplex virus (HSV) have fewer than 1:30,000 mononuclear cells in their blood lymphocytes preparations that proliferate in cultures stimulated with the corresponding viral antigens. CMV and HSV responder cell frequencies in children and adults whose immunity followed postnatal infection with these viruses are 1:10,000 to 1:20,000. The low precursor frequency after congenital or neonatal infection is not associated with defective antigen processing by monocytes or nonspecific immunosuppression. Phenotypic changes in T cell subsets and the presence of antibody in the subjects suggests that the virus(es) do indeed elicit an immune response, but that this response is quantitatively deficient.     

Congenital herpes simplex virus infection diagnosed by cytology of aspirated tracheobronchial material

Aspirated tracheal secretions from a ten-day-old newborn having signs of sepsis showed small clusters of cells with cytopathic changes consistent with herpes simplex virus (HSV) infection. The presence of type 2 HSV was confirmed by an immunoperoxidase procedure on the aspirated bronchial mucus and at necropsy in most of the viscera. Since prompt antiviral chemotherapy may favorably affect the outcome of HSV infections, early cytologic studies of tracheobronchial secretions may prove useful for rapid diagnosis.     

Persistent cyclic herpes simplex virus infection in vitro. 3. Asynchrony in the progression of infection and cell regrowth

Hampar, Berge (National Institute of Dental Research, Bethesda, Md.). Persistent cyclic herpes simplex virus infection in vitro. III. Asynchrony in the progression of infection and cell regrowth. J. Bacteriol. 91:1965-1970. 1966.-The progression of virus-induced cytopathic effects (CPE) and virus synthesis was studied in localized areas of Chinese hamster cell cultures persistently infected with herpes simplex virus (HSV). CPE was initially evidenced by the presence of small multinucleated giant cells, followed by expanding plaquelike lesions with an occasional uninfected cell remaining within the infected areas. Cell detachment rapidly followed the appearance of viral antigen in infected cells. The surviving cells which proliferated to re-establish the cell sheet arose from two sources. The first was from viable cells which remained attached after expansion of localized areas of CPE, and the second was from reattachment of viable cells in the medium. CPE in localized areas was initiated at various times during the cycle irrespective of the virus titer in the medium. Cell regrowth in some areas and CPE in other areas occurred simultaneously throughout the cycle in an asynchronous fashion. Consequently, during periods of rising virus titers, most areas showed CPE while few areas displayed cell regrowth. As the virus titers declined, more areas showed cell regrowth and fewer areas displayed new cycles of CPE. CPE in localized areas was not initiated until cell regrowth had occurred. It is proposed that the proliferating cells were temporarily resistant to HSV infection, and that this resistance was ultimately lost in their progeny cells.     

Gene expression of herpes simplex virus. I. Analysis of cytoplasmic RNAs in infected xeroderma pigmentosum cells.

RNAs which are synthesized and accumulate in the cytoplasm of uninfected and herpes simplex virus type 1 (HSV-1)-infected xeroderma pigmentosum (XP) cells in the presence of cycloheximide (early RNAs) or absence of drugs (late RNAs) were analyzed by electrophoresis through denaturing polyacrylamide gradient slab gels. HSV RNAs were selected by hybridization ot HSV DNA covalently bound to cellulose. No HSV-specific low-molecular-weight (4S to 10S) RNAs were detected. However, several changes were observed in the electrophoretic pattern of the host low-molecular-weight RNAs during HSV infection. Five HSV RNAs ranging in size from 16S to 28S accumulated in the cytoplasm of infected XP cells in the presence of cycloheximide. These are of the size range predicted to encode the major early viral polypeptides. The cytoplasmic and polyadenylated early RNAs from HSV-infected XP cells were translated in vitro to produce proteins whose electrophoretic pattern resembled that of the early viral proteins synthesized in vivo.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

Cellular proteins expressed in herpes simplex virus transformed cells also accumulate on herpes simplex virus infection.

The cell proteins expressed in rat embryo cells transformed by herpes simplex virus (HSV) have been analysed by immunoprecipitation assays to determine those polypeptides which can be identified by immunoprecipitation with the sera of tumour-bearing animals and also with antisera to herpes simplex infected cells. Cell polypeptides commonly recognised by both these sera have been further characterised using a monoclonal antibody directed against a cellular polypeptide which accumulates on HSV-2 lytic infection. This monoclonal antibody recognises in HSV-transformed cells polypeptides of mol. wts. 90 000, 40 000 and 32 000. Further studies show that the accumulation of these polypeptides in HSV-transformed cells is not HSV specific but is a common feature of transformation or of cells which have been immortalised. We suggest that cellular polypeptides accumulating as a result of HSV infection may be of importance in the initiation of transformation by HSV, i.e., at the level of immortalisation of cells.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Competitive inhibition by human sera of mouse monoclonal antibody binding to glycoproteins C and D of herpes simplex virus types 1 and 2.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.     

Altering the expression kinetics of VP5 results in altered virulence and pathogenesis of herpes simplex virus type 1 in mice

While many herpes simplex virus (HSV) structural proteins are expressed with strict-late kinetics, the HSV virion protein 5 (VP5) is expressed as a "leaky-late" protein, such that appreciable amounts of VP5 are made prior to DNA replication. Our goal has been to determine if leaky-late expression of VP5 is a requirement for a normal HSV infection. It had been shown previously that recombinant viruses in which the VP5 promoter was replaced with promoters of other kinetic classes (including a strict late promoter) exhibited no alterations in replication kinetics or virus yields in vitro. In contrast, here we report that alterations in pathogenesis were observed when these recombinants were analyzed by experimental infection of mice. Following intracranial inoculation, a recombinant expressing VP5 from a strict-late promoter (U(L)38) exhibited an increased 50% lethal dose and a 10-fold decrease in virus yields in the central nervous system, while a recombinant expressing VP5 from an early (dUTPase) or another leaky-late (VP16) promoter exhibited wild-type neurovirulence. Moreover, following infection of the footpad, changing the expression kinetics of VP5 from leaky-late to strict-late resulted in 100-fold-less virus in the spinal ganglia during the acute infection than produced by either the parent virus or the rescued virus. These data indicate that the precise timing of appearance of the major capsid protein plays a role in the pathogenesis of HSV infections and that changing the expression kinetics has different effects in different cell types and tissues.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Herpes Simplex Virus-Specific IgM,IgA and IgG Subclass Antibody Responses in Primary and Nonprimary Genital Herpes Patients

To clarify the humoral immunity in herpes simplex virus (HSV) infection, HSV-specific IgM, IgA and IgG subclass antibody responses were studied in patients with genital herpes: 17 primary, 13 recurrent and 6 nonprimary first episode. A total of 181 serum samples serially collected from the patients, 5 per patient until 213 days after the onset of disease (on average), were analyzed by an enzyme-linked immunosorbent assay. IgGl, IgG3 and IgA were detected in all patients with primary and nonprimary infections, whereas IgG4 was detected in 74% of only those with nonprimary infections and IgG2 was detected in none. IgM was detected in 100% of the patients with primary infections, but also in 68% of those with nonprimary infections. IgA showed a peak similar to that of IgM in patients with primary infections. No significant difference was observed in the detection rate or pattern of antibody responses between the recurrent and nonprimary first episode infections, nor between the HSV-1 and HSV-2 infections. These findings may be useful to improve the diagnostic potential of HSV serology.     

Production of interferon by immune lymphocytes exposed to herpes simplex virus-antibody complexes.

Splenic leukocytes from rabbits immunized with herpes simplex virus (HSV) incorporated significant amounts of 3H-TdR and produced interferon after in vitro incubation with HSV antigens. In contrast, splenic leukocytes from nonimmune rabbits did not incorporate 3H-TdR or produce interferon when incubated with HSV antigens. Antigen-antibody complexes consisting of HSV antigens and anti-HSV antibody also were capable of stimulating immune leukocytes to incorporate 3H-TdR and to produce interferon.     

Use of the unlabeled antibody immunohistochemical technique for the detection of human antibody.

Two methods have been developed which permit use of the unlabeled antibody immunohistochemical technique for detection of human antibody, without the need for immunization of humans with peroxidase. Human antibody to herpes simplex virus (HSV) reacted with human cell cultures infected with HSV was the experimental system. In the first method an attempt was made to employ rabbit peroxidase-antiperoxidase (PAP) soluble complexes in connectin with human antibody. This was done by sequential addition to the HSV-infected cells of (a) human anti-HSV, (b) rabbit antihuman globulin, (c) guinea pig antirabbit globulin (the bridging reagent) and (d) rabbit PAP. Strong specific staining of HSV-infected cells was obtained; however, difficulties were encountered with nonspecific reactions on uninfected cells. In the second method PAP soluble complexes prepared with baboon antiperoxidase were bridged to the human anti-HSV antibody by rabbit antihuman globulin. Because of the phylogenetic relatedness of human and baboon globulins this resulted in firm binding which gave strong specific staining of HSV-infected cells without significant reaction in uninfected cells.