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First- and third-generation cloned progeny viruses were derived from clinical isolates of herpes simplex virus and examined for their sensitivities to human interferon by inhibition of plaque formation in Vero cells. The dose-response curves obtained with the first- and third-generation clones were similar to those obtained with the parental isolates, and both the parent and the clones showed similar sensitivities to interferon. These results suggest that clinical isolates of herpes simplex virus consist of a homogeneous population of virus particles with respect to interferon sensitivity. The dose-response curves obtained with herpes simplex virus demonstrated a shallower slope than those obtained with vesicular stomatitis virus. Vesicular stomatitis virus plaque formation as completely inhibited at high concentrations of interferon, whereas complete inhibition of plaque formation by herpes simplex virus did not occur at the highest concentration of interferon used. Cloned progeny were derived from plaques appearing in the presence of high concentrations of interferon. The dose-response curves and interferon sensitivities of these clones were similar to those of the parent and third-generation clone from which they were derived. There was no evidence for genetic heterogeneity with respect to interferon sensitivity.

Mechanisms whereby prostaglandins and other cyclic adenosine 3',5'-monophosphate (cAMP) modulators might enhance the growth of herpes simplex virus (HSV) in human skin fibroblasts were explored. Prostaglandins A1, B1, E1, E2, and F2 alpha, as well as isoproterenol, imidazole, carbamylcholine, and dibutyryl cAMP had no effect on HSV growth. On the other hand, the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine and theophylline delayed the growth, suppressed the cell-to-cell spread, but inhibited neither the adsorption nor the penetration of the virus. Although none of the cAMP-elevating reagents directly enhanced HSV growth, they were found to inhibit dose dependently the antiviral action of both type I and HSV antigen-induced human interferon preparations. Furthermore, these reagents suppressed the production of HSV antigen-induced interferon by immune human mononuclear leukocytes. These data support the hypothesis that prostaglandin elaboration in vivo could contribute to exacerbations of HSV infections by compromising the host's interferon defense system.

Tissue culture cells infected with herpes simplex virus type 1 synthesize three major glycoprotein antigens (Ag-11, Ag-8, and Ag-6), which have been characterized by crossed immunoelectrophoresis. The three viral antigens have been identified as a mixture of gA and gB (Ag-11), gD (Ag-8), and gC (Ag-6). Recent findings have shown that antibodies directed to each of the three antigens individually are able to mediate antibody-dependent cell-mediated cytotoxicity when tissue culture cells late in the infectious cycle (18 h postinfection) are used. In this work, antibody-dependent cell-mediated cytotoxicity was applied to study the time postinfection at which the individual viral antigens first made their appearance at the cell surface. All three viral antigens (Ag-11, Ag-8, and Ag-6) could be demonstrated as newly synthesized from 3 to 4 h postinfection, and the quantities of the antigens at the surfaces of the infected cells increased with time postinfection. The use of cycloheximide and ultraviolet-inactivated virus demonstrated that input virus could be detected by antibody-dependent cell-mediated cytotoxicity during the first 2 h postinfection, but the cytotoxicity caused by input virus remained constant with time postinfection. In conclusion, these observations demonstrate the participation of individual herpes simplex virus surface antigens in antibody-dependent cell-mediated cytotoxicity attack on cells early in infection.

Studies were carried out to determine whether the in vitro capacity of adherent peritoneal cells to replicate herpes simplex virus type 1 (HSV-1) might correlate with the in vivo susceptibility of mice genetically resistant, moderately susceptible, or very susceptible to HSV-1 infection. Unstimulated and proteose peptone-stimulated monolayers restricted viral replication when infected immediately, but replicated HSV-1 when infected after 3 to 7 days of culture. Macrophages from resistant C57Bl/6 mice restricted HSV-1 replication significantly better than cells from susceptible mice. This function did not segregate with resistance, since macrophages from resistant F1 mice failed to restrict HSV-1 replication. Induction of peritoneal exudate cells with thioglycolate yielded cells capable of replicating HSV-1 when infected immediately after plating and after 4 days of culture.

The permissiveness of rabbit monocytes and macrophages for herpes simplex virus was examined. Peripheral blood monocytes, alveolar macrophages, and peritoneal exudate macrophages were studied for their ability to replicate herpes simplex virus strains RE and KOS. Results indicated different degrees of interaction with virus depending on the macrophage type. Only peritoneal exudate macrophages showed evidence of virus replication. Productive infection was limited, with only a small number of cells (0.02%) yielding infectious virus. Higher numbers of cells appeared to be abortively infected. Approximately 40% expressed antigens, whereas virtually all were killed by exposure to virus. Coreless particles were seen by electron microscopy in about one-third. Alveolar macrophages were also killed by virus and showed evidence of virus adsorption, but showed no indication of productive or abortive infection. Monocytes neither adsorbed nor replicated virus, and viability was unaffected. Results suggest that differences in degrees of cellular maturation or differentiation, or both, account for the spectrum of interactions seen between herpes simplex virus and rabbit macrophages.

Cytotoxic T lymphocytes (CTL) were generated in C57BL/6 mice with herpes simplex virus type 1 (HSV-1) (strains KOS, 17, HFEM, and mP) and HSV-2 (strains 186, G, and GP6). Effector lymphocytes were tested for cytotoxicity against syngeneic HSV-1- and HSV-2-infected cells in a 5-h 51Cr release assay. HSV-1 strain HFEM was found to induce CTL efficiently only when 100-fold more virus was used as compared with HSV-1 strains KOS, 17, and mP. All HSV-1 and HSV-2 strains induced cross-reactive populations of CTL. CTL generated by HSV-1 KOS and HSV-2 186 also demonstrated cross-reactivity in an ear-swelling model for delayed-type hypersensitivity. Lymphocytes generated by all HSV-2 strains were highly efficient at lysing HSV-1-infected target cells. However, HSV-2-infected target cells were found to be less susceptible to lysis by either HSV-1 or HSV-2 CTL than were HSV-1-infected target cells. The lowered susceptibility of HSV-2-infected cells was not due to an inefficient infection of BL/6 WT-3 cells as measured by standard growth assays and infectious center assays. Varying the multiplicity of infection or the time of infection did not increase the susceptibility of HSV-2-infected target cells to lysis by CTL. Increasing the effector-to-target-cell ratio resulted in an increased lysis of both HSV-1- and HSV-2-infected target cells by CTL, but the level of HSV-2-infected target cell lysis still did not approach the level of HSV-1-infected target cell lysis. HSV-2-infected cells were as efficient as HSV-1-infected cells in the cold cell competition assay employed in reducing the lysis of 51Cr-labeled, HSV-1-infected target cells. In addition, HSV-2-infected cells were susceptible to lysis by HSV-immune serum and complement.

The genetics of innate resistance of mice to hepatitis induced by herpes simplex virus type 2 (HSV-2) was analyzed by crossing resistant male GR to susceptible female BALB/c mice and backcrossing females of this F1 generation to susceptible male BALB/c mice. By scoring of macroscopic liver lesions and virus isolation studies from the liver 4 days after intraperitoneal inoculation of HSV-2, it appeared that the resistance was governed by one X-linked dominant gene or closely linked gene complex, as F1 female mice were resistant and F1 male mice were susceptible and the trait segregated in a ratio close to 1:1 in the backcross mating. A cellular expression in vitro of virus resistance was found in the macrophage population of the mice as measured by differences in the restriction of HSV-2 replication in macrophage cultures prepared from individual mice. In contrast to what was seen in macrophage cultures, virus replicated equally well in embryonic fibroblast cultures from susceptible and resistant strains of mice.

Ultracentrifugation of very dilute suspensions of herpes simplex virus directly onto monolayer cells grown in centrifuge tubes was studied. Enhanced infectivity by ultracentrifugation was similar at 4 degrees C and at 35 to 37 degrees C. The high infectivity levels of cultures centrifuged at 4 degrees C were further examined by infectious center assays. At 4 degrees C, the numbers of infectious centers in control (noncentrifuged) cultures were almost 100-fold fewer than in control cultures at 37 degrees C. However, the numbers of infectious centers in cultures ultracentrifuged at 4 degrees C were similar to those ultracentrifuged at 37 degrees C. The great difference in the numbers of infectious centers between 4 and 37 degrees C control cultures, in contrast to the similarity between 4 and 37 degrees C ultracentrifuged cultures, indicated that ultracentrifugation at 4 degrees C enhanced infectivity possibly by facilitation of herpes simplex virus penetration into monolayer cells.

Experimental intracerebral infection of 4-week-old mice with the MS strain of herpes simplex virus type 2 or its derivative cold variant was compared. The infectious process was followed in both the brain tissue and the trigeminal ganglia, using hematoxylin and eosin and antigenic tracing with indirect peroxidase-antiperoxidase staining. The wild-type virus elicited a severe meningitis and necrotic lesions by 7 days post-inoculation in the brain. The cold variant produced a mild meningitis and no necrotic lesions. In both viral infections, the neuron seemed to be the target cell at the early stages. Infection with the wild-type strain was able to spread through host tissues and produce confluent lesions, whereas infection with the cold mutant seemed to be confined to individual neurons. Data suggest that the cold variant is restricted in its ability to lyse the cells and spread in the host nervous tissue.

The ganglia of rabbits infected with a relatively benign strain of herpesvirus (E-43) and challenged with either of two virulent neurotrophic strains (MP or McKrae) were found to be colonized only by the initial benign infecting strain. Primary infection with the E-43 strain resulted in milder disease when the animals were infected with MP or McKrae strains and also prevented colonization of the ganglion by these strains. Neutralization with anti-glycoprotein C, plaque morphology, cytopathic effects, reconstruction experiments, and restriction endonuclease analysis indicated that the virus recovered from the ganglion was the initial infecting E-43 strain; no traces of the challenging MP and McKrae strains were found. The challenging McKrae strain was shed for several weeks in a few animals, but the virus isolated from the trigeminal ganglia of these animals was the primary infecting E-43 strain. These results suggest that initial infection with a relatively benign strain of herpesvirus may prevent superinfection of the ganglion (but not necessarily the end organ) by highly virulent herpes simplex virus strains and could have significant implications in the consideration of immunization against this disease in humans.

The relative contributions of macrophages and lymphocytes to the induction of resistance to primary herpes simplex virus type 1 (HSV-1) infection by polyriboinosinic-polyribocytidylic acid complex [poly (I:C)] were investigated in C58 mice. The induction of resistance was found to be strongly dependent on macrophages compared to lymphocytes. Macrophage-deficient (silica-treated) mice produced less interferon and were not as responsive to prophylactic treatment of HSV-1 infections with poly (I:C) as were either normal, lymphocyte-deficient (cyclophosphamide-treated), or T-lymphocyte-deficient (anti-thymocyte serum-treated, adult-thymectomized) mice. Silica and cyclophosphamide treatments reduced the therapeutic activity of poly (I:C), whereas T-cell depletion did not have a significant effect. Similarly, the protection of mice with exogenous interferon was markedly reduced in silica-treated mice and moderately reduced in cyclophosphamide-treated mice, but unaffected in T-cell-deficient mice. Furthermore, suppression of HSV-1 plaque formation was obtained by cocultivation of infected mouse fibroblast monolayers with peritoneal (macrophage-rich) cells, but not with splenic (lymphocyte-rich) cells, from poly (I:C)-treated mice. Peritoneal cells did not protect heterologous (human) fibroblasts, suggesting that the protection of mouse embryo fibroblasts is mediated by interferon. Collectively, the data indicate that macrophages are required for the production of poly (I:C)-induced interferon and that macrophages and perhaps B-lymphocytes are important for mediating the protection against HSV-1 infection after interferon has been produced.

Natural killer cytotoxicity is an important antiviral defense mechanism. Human peripheral blood mononuclear cells cultured with herpes simplex virus (HSV)-infected cells produced a cytokine. This substance stimulated adult natural killer cytotoxicity from 53.0 +/- 10.5% to 79.8% (P less than 0.01) against HSV-infected target cells. These data resulted in a calculated cytokine-dependent cellular cytotoxicity (CDCC) value of 65.8%. Cytokine production was not stimulated by uninfected cells and was independent of the presence or absence of antibodies to HSV in sera of donors and mononuclear cells. Cells from human colostrum also produced an HSV-stimulated cytokine which mediated CDCC by using both adult (19.8 +/- 3.9%) and neonatal (18.6 +/- 3.4%) mononuclear effectors cells. Colostral cell cytokine production was also independent of donor HSV serology. Not all colostral cultures produced the cytokine, and in general colostrum-stimulated CDCC was lower than peripheral blood leukocyte-stimulated CDCC. Colostral cell cytokine stimulation of neonatal natural killer cytotoxicity may account in part for the increased nonspecific resistance of breast-fed infants to viral infection.

Young BALB/c mice inoculated intraperitoneally with herpes simplex virus type 2 develop focal necrotizing hepatitis. After infection, the livers of these mice show increasing virus titers, which reach a maximum on day 3 after infection; this is followed by a dramatic decrease in the amount of virus recovered on days 4 and 5. This decrease in virus content is accompanied by a progressive infiltration of the lesions with mononuclear leukocytes and an apparent resolution of the lesions. Adoptive transfer of immune spleen cells from mice infected 6 days earlier accelerated this process. When 50 x 10(6) to 100 x 10(6) immune spleen cells were transferred 24 h after infection, the inflammatory response and the clearance of virus from the livers were advanced by almost 2 days. As few as 12 x 10(6) immune spleen cells accelerated the healing process, whereas fewer immune cells, disrupted immune cells, or normal spleen cells did not have an effect. The protection conferred by herpes simplex virus type 2-sensitized immune spleen cells was specific since mouse cytomegalovirus- or vaccinia virus-sensitized immune spleen cells had no effect on the course of infection with herpes simplex virus type 2, whereas some cross-reactivity was observed between herpes simplex virus types 1 and 2. This model seems to be suitable for examining the immunological mechanisms that are active during recovery from visceral herpes simplex virus infections.

By their position at sites of initial infection and their wide distribution in major organs of the body, macrophages may be decisive in determining the susceptibility or resistance of the host to virus infection. Macrophage restriction of virus replication has been shown to be closely related to virus strains or virus types and to the age of the infected host. We report the effects of the development of a solid tumor in rats on intrinsic in vitro macrophage activity against herpes simplex virus type 1. The results obtained with the infectious center assays and the analysis of single-cycle growth curves of herpes simplex virus type 1 in macrophages obtained from normal and tumor-bearing rats showed a depression of antiviral activity of macrophages from tumor-bearing rats. The possibility of immunomodulation by bacterial adjuvants on tumor-bearing rats and the effects on the antiviral activity of peritoneal macrophages were furthermore demonstrated.

An age-dependent increase in the resistance of BALB/c mice to induction of focal necrotic hepatitis by herpes simplex virus type 2 was demonstrated. In 3-week-old mice inoculated intraperitoneally with virus, numerous necrotic foci developed in the liver. As the mice matured, the number of lesions declined until the age of 8 weeks, when no further increase in resistance appeared. Corresponding to this, the virus titers of livers and spleens of 3-week-old mice were higher than in 8-week-old animals throughout the infection, and the infection was apparently terminated in these organs of the adult mice by day 5. In vitro infection of peritoneal macrophages from 3-week-old and 8-week-old mice showed that this age-related resistance was concomitant with an increased restriction of virus replication in peritoneal macrophages from adult mice. Since, furthermore, the resistance of adult mice could be abolished by intravenous inoculation of the macrophage-toxic agent silica before infection, and since adoptive transfer of 2 X 10(6) syngeneic macrophages from adult mice to young ones conferred to the latter a resistance comparable to that of the adult mice, it is concluded that macrophage maturation is responsible for the age-dependent resistance seen in this infection.

Inhibition of herpesvirus plaque growth was observed when herpes simplex virus (HSV)-sensitized rabbit lymphocytes were placed in contact with an HSV-infected human foreskin monolayer. This inhibition was obtained as early as 3 h when a ratio of 6 viable lymphocytes to target cells was used, and the supernatants of these cultures also demonstrated plaque size reduction when put onto newly infected cell monolayers. Interferon, which is present in this system, had no effect on HSV when tested on human foreskin monolayers, indicating that interferon was not the mechanism for plaque size reduction. Plaque growth inhibition was attributed to the T lymphocyte, because purified T cells reduced plaque growth and anti-rabbit thymocyte serum eliminated the effect of T cells. The specificity of this assay was determined by the facts that nonsensitized lymphocytes did not reduce the size of a plaque and the recognition of an infected cell by the sensitized lymphocyte was necessary for the release of a soluble mediator into the supernatant fluid. This cytotoxic lymphocyte was detected in the peripheral blood of rabbits as early as 4 days after initial corneal infection, with a maximum response at 7 to 10 days.

Three cysteine-rich cationic peptides, designated RatNP-1, RatNP-3, and RatNP-4, were purified from an acid extract of rat polymorphonuclear neutrophils, sequenced, and tested for antimicrobial activity. The peptides ranged from 29 to 32 amino acids in length (Mr, 3,252 to 3,825), and each contained all eight invariantly conserved "framework" residues that are characteristic of defensins. Each of the peptides killed Escherichia coli ML-35, Acinetobacter calcoaceticus HON-1, Staphylococcus aureus 502A, and Candida albicans 820 in vitro. RatNP-1, the most cationic rat defensin, was also the most potent. With this report, a total of 13 distinct defensins have been characterized in the polymorphonuclear leukocytes of four mammalian species. The existence of the defensin system in rats should facilitate investigations of the in vivo role of defensins in experimental infections.

An antibody-dependent cellular cytotoxicity (ADCC) system in which herpes simplex virus-infected Chang liver cells are used was assessed for its dependency on cellular energy, ribonucleic acid and protein synthesis, and cytoskeletal structures such as microfilaments and microtubules. The cytotoxic reaction was only slightly inhibited when glycolysis was blocked in a glucose-free medium containing 10(-2) M 2-deoxy-d-glucose. It was more substantially inhibited when respiration was blocked with 10(-2) M sodium azide. The reaction was totally ablated, however, only when both glycolysis and respiration were suppressed. This inhibitory effect of energy deprivation was mediated solely at the level of the effector cell. Ribonucleic acid synthesis or protein synthesis by the effector cells was not required, as shown by the fact that neither actinomycin D, cycloheximide, nor emetine significantly inhibited ADCC. The ADCC reaction was partially inhibited by cytochalasin B, whose inhibitory effect was rapidly reversible, and was completely and irreversibly inhibited by cytochalasin A. Cytochalasin A acted on the effector cells rather than the target cells. The reaction was also partially inhibited by colchicine, whose inhibitory effect was directed solely against the effector cells and was slowly reversible. The inhibitory effects of cytochalasin B and colchicine, when used in tandem at submaximal inhibitory concentrations, were slightly more than additive. The results suggest a cooperative role for effector cell microfilaments and microtubules in mediating ADCC. Kinetic studies of ongoing herpes simplex virus ADCC reactions after initial centrifugation showed that the lytic step requires expenditure of metabolic energy as well as intact function of both microfilaments and microtubules. These findings, in concert with previous data, indicate that the ADCC process against herpes simplex virus-infected Chang liver cells can be resolved into adhesion and lytic steps. The lytic step can be readily distinguished from the adhesion step by its increased sensitivity to low ambient temperature or metabolic energy deprivation, its sensitivity to thermal inactivation, its requirements for extracellular divalent cations, and its dependence on normal function of both microfilaments and microtubules.

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.

By ultracentrifugation of 30 ml of highly dilute suspensions of herpes simplex virus (HSV) directly onto monolayer cultures grown in centrifuge tubes, infectivity was significantly greater than without centrifugation. Ultracentrifugation at 20,000 to 25,000 rpm (28,000 to 45,000 X g) for 1.5 to 2.3 h was utilized with good preservation of cultures. With low-speed centrifugation at 3,000 rpm (1,100 X g), infectivity was almost 10-fold greater than without centrifugation. With ultracentrifugal inoculation, infectivity was about 100-fold greater than without centrifugation. Ultracentrifugal inoculation permitted the detection of HSV at concentrations as low as 0.05 plaque-forming units per ml. Similarly, ultracentrifugal inoculation of cultures was almost 100-fold more sensitive a method of detecting infectious HSV than was pelleting HSV from dilute suspensions followed by resuspension and inoculation of cultures. Ultracentrifugal inoculation of cultures may permit the isolation of HSV in situations where virus cannot be detected by ordinary means and may prove applicable to the study of other viruses.

In an attempt to define the nature of the difference in the susceptibility of C57BL/6 (resistant) and A/J (susceptible) mice to herpes simplex virus type 1, we initiated a study of virus progression through the nervous system. After inoculation of virus in a rear footpad, C57BL/6 mice were found to be more than 500-fold more resistant, but resistance did not extend to pseudorabies virus. In additional investigations, it was found that the virus was selectively restricted at the level of spinal ganglia in C57BL/6 mice. No intrinsic difference in the ability of this tissue from either mouse strain to replicate virus was found. However, by 4 days after infection, morphological investigations indicated that a mononuclear cell infiltrate was present surrounding infected neurons and satellite cells both earlier and in greater numbers in the ganglia of C57BL/6 mice. Immunohistochemical methods showed that most of these cells did not express Thy 1.2 antigen, but the vast majority bore immunoglobulin G. The mechanism by which these infiltrating cells could restrict virus spread is discussed.

Infection of human fibroblasts and HEp-2 cells with herpes simplex virus type 1 (HSV-1) produced a decrease in the intracellular levels of cyclic adenosine 5'- monophosphate (cAMP) and a concomitant increase in the cyclic guanosine 5'- monophosphate (cGMP) levels. In both cell cultures, changes in cyclic nucleotide levels were first observed at 6 h after viral inoculation and were maximal at 12 h. In human fibroblasts, the addition of theophylline, dibutyryl cAMP, or papaverine (cAMP-enhancing compounds) decreased significantly the yield of HSV-1, whereas the addition of insulin or dibutyryl cGMP (cGMP-enhancing compounds) increased the viral yield. In HEp-2 cells, only theophylline decreased the yield of HSV-1, and the cGMP-enhancing compounds had no apparent effect. Cyclic nucleotide enhancing compounds exhibited their effect only if added to either cell culture within the first 3 h after inoculation with HSV-1.

We used a model involving acute and latent herpes simplex virus (HSV) infections of mouse superior cervical ganglia to assess in vivo neuronal infections with two thymidine kinase-deficient (TK-) mutants of HSV type 1. Despite replication of the TK- HSV strains at the site of inoculation in the eyes, little if any viral replication occurred in the superior cervical ganglia, as assessed by the viral titers of ganglion homogenates, viral antigens in tissue sections, and histopathological evidence of cytopathology or inflammation. Cyclophosphamide-induced immunosuppression and treatment with 6-hydroxydopamine, which enhanced productive infections of superior cervical ganglia with TK+ HSV did not induce TK- HSV ganglionic infections. Latent TK- HSV infections were not detected by cocultivation of ganglion explants. Efforts to infect ganglia in culture after they were removed from animals indicated that superior cervical ganglia, and particularly their neuronal elements, resisted productive TK- HSV infection. These results supported the hypothesis that viral thymidine kinase facilitates acute and reactivated productive HSV infections of neurons.

The role of macrophages in the difference in liver pathogenicity between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in mice was investigated by selectively blocking the macrophage function of the mice by silica. Intravenous administration of 3 mg of silica 2 h before virus inoculation partially abolished the difference between the two virus types, as judged by macroscopic and microscopic examination of the livers and by virus isolation studies. Intraperitoneal inoculation of 50 mg of silical before virus seemed more effective in suppressing the macrophage function, since this treatment almost completely eliminated the difference in hepatotropism between HSV-1 and HSV-2 as assessed by the number and size of the lesions appearing in the liver. The final outcome of the infection, death from encephalitis, was, however, not influenced by macrophage blockade.

Proteose-peptone-activated mouse macrophages can prevent productive infection by herpes simplex virus in neighboring cells in vitro whether or not those cells belong to the same animal species. The effect does not require contact between the macrophages and the infected cells, may be prevented by adding extra arginine to the medium, and may be reversed when extra arginine is added 24 h after the macrophages. Arginase activity was found both intracellularly and released from the macrophages. The extracellular enzyme is quite stable; 64% activity was found after 48 h of incubation at 37 degrees C in tissue culture medium. No evidence was found that the inefficiency of virus replication in macrophages was due to self-starvation by arginase. As might be predicted macrophages can, by the same mechanism, limit productive infection by vaccinia virus.

Herpes simplex virus (HSV) type 1 replicated and persisted in human T, B, and myeloid cell lines with different patterns of viral replication and various effects on cell growth. T cell line CEM supported the replication of HSV for over 400 days without detectable differences in cell growth as compared with uninfected cells. HSV persisted in B cell line NC37 and myeloid cell line K562 for up to 222 and 374 days, respectively, but led to a significant decrease in the number of viable cells by 7 weeks of infection. The average number of cells producing infectious virus was very low in these cell lines (range, 0.5 to 2.7+) compared with a larger proportion of cells exhibiting HSV antigens by immunofluorescence (range, 24 to 58%). In contrast, null cell line LAZ 221 failed to replicate HSV even though the viral infection led to a cessation of cell growth.

Herpes simplex virus (HSV) infection of mouse liver after partial hepatectomy was studied. Partial hepatectomy resulted in the rapid onset of cellular DNA synthesis and the appearance of many mitotic figures (peak, 3 days after surgery). Similar changes were not seen in control animals. After partial hepatectomy, the mice were infected with thymidine kinase-positive (TK+) and -negative (TK-) HSV to investigate virus titers in liver tissue during liver cell replication. In control unoperated mice, liver titers of TK+ HSV (2 X 10(3) PFU/g) were greater than those of mice inoculated with TK HSV (4 X 10(1) to 5 X 10(2) PFU/g). After partial hepatectomy, TK+ and TK- HSV titers increased, and peak TK+ and TK- HSV titers were similar (6 X 10(5) to 8 X 10(5) PFU/g). Hepatic infection was further investigated by infectious center (IC) assays. The numbers of ICs for TK+ HSV increased 50-fold after partial hepatectomy, whereas the increase was less for TK- HSV. From the results of these studies, we hypothesize that the increase in hepatic TK+ HSV after hepatectomy may have been largely due to the increase in ICs, whereas the increase in hepatic TK- HSV was due, in part, to the increase in ICs, but may also have been due to the enhanced synthesis of TK- HSV in replicating liver cells.

The activity of 2',5'-oligoadenylate synthetase, which is induced by interferon action, was detected in mouse trigeminal ganglia infected with herpes simplex virus (HSV) type 1. When HSV was inoculated on the left cheek of mice, the virus began to appear in the ipsilateral trigeminal ganglia on day 2, reached its maximum accumulation on day 4, declined thereafter, and was no longer detected in the tissue homogenate after 11 days. After this short acute productive phase, the virus entered into the latent phase of infection. 2',5'-Oligoadenylate synthetase appeard in the trigeminal ganglia shortly after the beginning of virus multiplication; the synthetase activity began to rise on day 3, reached a maxium level on day 4, and then declined. Interferon activity (type I) also appeared in the infected ganglia, but diminished more rapidly than the synthetase. The antibody against HSV in the sera began to appear at the time when the virus and synthetase were declining. From these results it may be hypothesized, although not concluded, that in the acute productive phase of HSV infection in mouse trigeminal ganglia, virus multiplication is suppressed by interferon action, including 2',5'-oligoadenylate synthetase induction.

The possibility that interferon (IFN) and antibody could act together to reduce virus yields in infected cells was suggested by the simultaneous occurrence of IFN and antibody at infected sites. In the present in vitro studies, mixtures of IFNs and antibody acted synergistically to reduce virus yields in cultures infected with coxsackievirus A24, enterovirus 70, adenovirus, and herpes simplex virus. This synergistic reduction was observed in different cells and at different concentration of IFN and antibody. Although IFN and antibody acted synergistically against all viruses tested, the degree of synergism was dependent on the type of IFN or the virus. For example, IFN-beta and antibody was 10 to 200 times more effective than IFN-gamma and antibody against coxsackievirus A24, enterovirus 70, and adenovirus. A combination of antibody and IFN-gamma was three to five times more effective than IFN-beta and antibody against herpes simplex virus. In addition, we found that endogenously produced IFN-beta could act synergistically with antibody to coxsackievirus A24 to increase the overall antiviral effect by 10(5.0)-fold. No effect of endogenous IFN was observed in herpes simplex virus-infected cultures treated with antibody to herpes simplex virus. These studies indicate the potential importance of the synergistic effect of locally produced IFN and antibody in restricting virus early in the natural infectious process. They also suggest that combinations of IFNs and antibody may be more beneficial in the treatment of certain virus infections than IFN or antibody alone.

The rabbit provides an excellent model for the study of ocular herpes because herpetic keratitis in the rabbit eye resembles human disease in its clinical features and in its propensity for spontaneous recurrence. This paper presents a method for the electrical induction of multiple episodes of in vivo reactivation of latent HSV-1 infection with peripheral shedding of virus. Physiological levels of current delivered via an electrode implanted over the trigeminal ganglion of latently infected animals has enabled us to modify and synchronize virus shedding in preocular tear film and to cause multiple episodes of reactivation in a single animal. For this reason, the model is well suited for antiviral efficacy testing and provides an excellent opportunity for investigation of virus-host cell interactions in latent and recurring herpetic disease.

Twenty-three strains of herpes simplex virus type 1 were compared for their pathogenicity in 4-week-old BALB/c mice after peripheral (footpad) or intracerebral inoculation. Among those strains examined were (i) six clinical isolates of brain or cerebrospinal fluid origin, (ii) seven clinical isolates of oral or genital origin, (iii) five prototype laboratory strains that have been passaged numerous times in culture, and (iv) five syncytial variants capable of producing cell fusion in culture. Based on comparative 50% lethal dose values, the strains appeared to segregate into one of three classes of neurovirulence. Class I strains were highly virulent by both the peripheral and intracerebral routes of inoculation, class II strains were highly virulent by the intracerebral route only, and class III strains were highly attenuated by both routes of inoculation. In vivo growth curves for whole brain homogenates infected with class III strains revealed titers of infectious virus approaching those found in the brains of animals infected with class I or II strains. These results would therefore suggest that (i) a strain-dependent variation in neural spread exists that may influence the ability of the virus to cause acute neurological disease and (ii) the amount of infectious virus present within an infected brain does not necessarily determine or reflect the clinical status of the animal. Of the clinical isolates examined, the strains recovered from brain tissue of humans after fatal episodes of encephalitis were found to be no more neurovirulent in mice than the strains isolated from nonneural sites. However, although syncytial variants were found to be highly attenuated by the peripheral route, as a group these strains proved to be among the most virulent when inoculated directly into the central nervous system.

A rounding cell-forming--GC strain, which is a variant of a syncytial giant cell-forming herpes simplex virus (+GC Miyama strain), was highly attenuated for Swiss, BALB/c nu/nu, and nu/+ mice, whereas +GC was highly virulent to all the mice tested. +GC and -GC were antigenically indistinguishable from each other by cross-neutralization and cross-immunization. Immunosuppression induced by cyclophosphamide converted the nonlethal -GC infection of mice into a fatal infection. -GC replication in tissue culture was more effectively suppressed by spleen cells immunized with either +GC or -GC than was the +GC replication. -GC replication was also inhibited more effectively by antibody or the antibody-dependent cell-mediated system than was the +GC replication. -GC is highly sensitive to mouse interferon, but +GC was relatively resistant. These findings indicate that attenuation of this avirulent -GC strain may be due to a high susceptibility of its replication to humoral and cell-mediated defense factors. The probable roles of each defense factor in recovery from the infection with virulent and attenuated herpes simplex virus are also discussed.

Chang liver (CL) cells and human embryonic lung fibroblasts (MRC-5) were infected with type 1 herpes simplex virus (HSV), ant the time postinfection at which these cells became susceptible to lysis by antiviral antibody and complement of human origin was determined in a 51Cr release assay. Using a 1:2 dilution of fresh HSV antibody-positive human serum, we initially detected specific lysis by 4 h postinfection in HSV-infected CL cells and by 3 h postinfection in HSV-infected MRC-5 cells in suspension. MRC-5 cells were more completely lysed than CL cells. Protein inhibition studies with cycloheximide showed that all of the HSV-infected CL cells and most (83%) of the HSV-infected MRC-5 cells injured early in the infectious cycle were attacked because of newly synthesized viral surface antigens rather than because of adherent input virus. Suspension cells early in the infectious cycle were less completely lysed and required higher concentrations of both antiviral antibody and complement for lysis than cells that were in the later stages of infection (18 h postinfection). Guinea pig serum was inferior to human serum as a complement source for lysis of early infectious cycle cells. Lysis early in the infectious cycle was directly proportional to the multiplicity of infection and inversely proportional to the cell concentration. Infected cells in monolayers were lysed less readily and about 1 to 2 h later in the infectious cycle than infected cells in suspension. This difference was pronounced for CL cells, but modest for MRC-5 cells. These studies demonstrate that, despite previously held notions, HSV-infected tissue culture cells can be lysed by antiviral antibody and complement early in the infectious cycle before the initial production of progeny virus particles. The demonstration of lysis was highly dependent on experimental conditions, however, including cell type, suspension versus monolayer culture, cell density, and concentration of antibody and complement as well as the source of complement.

Capsid, envelope, and nonvirion-associated soluble components of type 1 and type 2 herpes simplex virus (HSV) were obtained from infected monolayer cell cultures and used as complement fixation (CF) antigens. Capsids were prepared by treatment of cells with the nonionic detergent Nonidet-P40, envelope material by treatment of virions with ether and high pH, and soluble components were obtained from culture fluids of untreated cells. Serological studies with experimental anti-herpesvirus sera indicate that these serotypes share cross-reacting envelope, capsid, and soluble antigens with each other and with herpesvirus B but not with varicella virus. In addition, animals immunized with crude HSV preparations contain high levels of CF antibody (1:32 to 1:64) to soluble antigens, whereas sera from humans who have experienced natural infection contain low levels of antibody (