Growth Of Herpes Simplex Virus Research Articles

Isolation, characterization and acyclovir susceptibility of herpes simplex virus isolates among immunocompromised patients.

Introduction:Herpes simplex virus (HSV) Type 2 primarily causes genital herpes, while HSV Type 1 is responsible for oral and facial lesions. The objective of this study was to isolate and characterize HSV from herpetic lesions among human immunodeficiency virus (HIV) infected patients and to evaluate their acyclovir susceptibility pattern.Materials and Methods:Blister fluid and swabs from ulcers were collected from patients with clinical diagnosis of HSV infection among patients attending the HIV clinic of two tertiary care centers – Medical College, Kolkata, and School of Tropical Medicine, Kolkata. These samples were cultured in the Vero cell line. Growth of virus was noted by observing the characteristic cytopathic effect of HSV, which was further confirmed by immunofluorescence and polymerase chain reaction (PCR). These isolates were then subjected to the Vero cells with serial dilutions of acyclovir for determining the susceptibility pattern.Results:Among the 52 samples received, 8 (15.38%) showed growth of HSV. After confirmation by immunofluorescence and PCR, all seven isolates from genital samples were identified as HSV-2 and the lone isolate from oral lesion was confirmed as HSV 1. Out of the eight isolates, 25% showed resistance to acyclovir. The overall isolation rate was more from genital blister than genital ulcer which was 46.15% and 2.86%, respectively.Conclusion:HSV was isolated in 15.38% of cases of clinical herpes. There was a higher isolation rate of virus from blister fluid as compared to ulcer scrapings. Acyclovir resistance in 25% of cases is alarmingly high.

Inhibition of Herpes Simplex Virus by Polyamines

Herpes simplex virus (HSV) establishes latent infection in humans with periodic reactivation. Acyclovir, valacyclovir and foscarnet are in medical use today against HSV type-1 (HSV-1) and type-2 (HSV-2), inhibiting the DNA synthesis of the viruses. Additional drugs that will affect the growth of these viruses by other mechanisms and also decrease the frequency of appearance of drug-resistant mutants are required. Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination. Polycations of dextran, pullulan and arabinogalactan were grafted with oligoamines of 2-4 amino groups forming Schiff-base imine-based conjugates followed by reduction with borohydride to obtain the stable amine-based conjugate. Evaluation of toxicity to BS-C-1 cells and antiviral activity against HSV-1 and HSV-2 of the different compounds was performed in vitro by a semiquantitative assay. A quantitative study with a selected compound followed. Structure-activity relationship studies showed that the nature of the grafted oligoamine of the polycation plays an essential role in the antiviral activity against HSV-1 and HSV-2. Dextran-propan-1,3-diamine (DPD) was found to be the most potent of all the compounds examined. DPD did not decrease the infectivity of HSV upon direct exposure to the virions. The growth of HSV was significantly inhibited when DPD was added to the host cells 1 h prior to infection, thus preventing the adsorption and penetration of the virus into the cells. Our in vitro data warrant clinical investigation. DPD could have an advantage as a topical application in combination therapy of HSV lesions.

Immunological Aspects of Herpetic Stromal Keratitis

Herpetic stromal keratitis (HSK) is an immune reaction related to herpes simplex virus (HSV) corneal infection, and has many important immunological aspects. CD4+ T lymphocytes, especially Th1 cells, are the principal mediators for HSK. In addition, neutrophils and antigen-presenting cells play vital roles in HSK. CD8+ T lymphocytes, B cells, and natural killer cells all participate in the pathogenesis of HSK under certain circumstances. Many molecules are involved in the pathogenesis of HSK. Th1 cytokines such as interleukin 2 (IL-2), IL-12 and interferon γ, and inflammatory cytokines such as IL-1α and IL-6 are especially important ones. Among various chemokines that take part in HSK, MIP-1α is one of the most important aggravating factors. Vaccination therapy against HSK has been developed; glycoprotein D is a particularly promising candidate. However, the possibility of HSK exacerbation due to vaccination is the final problem to be solved before vaccination can be clinically applied to HSK. Molecular mimicry theory and bystander activation theory are the two new autoimmune theories that have been advocated. Since genuine autoimmune HSK without HSV growth can hardly be the case in clinical practice, some part of these new theories remains controversial. In the future, better understanding of the pathogenesis of HSK is essential to resolve the paradox between suppressing the immune reaction to avoid corneal scarring and preventing viral proliferation.

Inhibition of herpes simplex virus replication by a 2-amino thiazole via interactions with the helicase component of the UL5-UL8-UL52 complex.

With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC50 (concentration of compound that yields 50% inhibition) of 5 microM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC50 = 20 microM). T157602 inhibited HSV growth in a one-step viral growth assay (IC90 = 3 microM), and plaque formation was completely prevented at concentrations of 25 to 50 microM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 microM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.

A Bismuth Salt Inhibits Herpes Simplex Virus

Bismuth has been shown not only to heal peptic ulcers but also to reduce recurrence. Helicobacter pylori (H pylori) plays an important role in the pathogenesis of peptic ulcers, and since bismuth has a bactericidal effect on this organism, the beneficial effect of bismuth on peptic ulcer disease has been attributed to the effect on H pylori . However, not all pathophysiological changes found in patients with duodenal ulcer disease can be explained by H pylori infection, and bismuth has only a weak bactericidal effect on H pylori . Infection with Herpes simplex virus (HSV) in autonomic ganglion cells has also been suggested as an etiological factor of peptic ulcer. In the present study we accordingly examined the effect of bismuth on the growth of HSV and compared it with that on H pylori . Ammonium bismuth citrate trihydrate (ABC) inhibited completely the growth of H pylori at the concentration of 32 mg/l and HSV-1 and HSV-2 at the concentrations of 2000 mg/l and 1000 mg/l, respectively. Since bismuth after oral ingestion is concentrated or even precipitated at the base of peptic ulcers, an effective concentration of bismuth with regard to HSV may also be obtained. Keywords : bismuth, duodenal ulcer disease, Herpes simplex virus, Helicobacter pylori , pathogenesis.

A Bismuth Salt InhibitsHerpes SimplexVirus

Bismuth has been shown not only to heal peptic ulcers but also to reduce recurrence. Helicobacter pylori (H pylori) plays an important role in the pathogenesis of peptic ulcers, and since bismuth has a bactericidal effect on this organism, the beneficial effect of bismuth on peptic ulcer disease has been attributed to the effect on H pylori. However, not all pathophysiological changes found in patients with duodenal ulcer disease can be explained by H pylori infection, and bismuth has only a weak bactericidal effect on H pylori. Infection with Herpes simplex virus (HSV) in autonomic ganglion cells has also been suggested as an etiological factor of peptic ulcer. In the present study we accordingly examined the effect of bismuth on the growth of HSV and compared it with that on H pylori. Ammonium bismuth citrate trihydrate (ABC) inhibited completely the growth of H pylori at the concentration of 32 mg/l and HSV-1 and HSV-2 at the concentrations of 2000 mg/l and 1000 mg/l, respectively. Since bismuth after oral ingestion is concentrated or even precipitated at the base of peptic ulcers, an effective concentration of bismuth with regard to HSV may also be obtained.

Potent Inhibition of Herpes Simplex Virus by MDL 101028, a Novel Biphenyl Disulfonic Acid Urea Co-Polymer

A novel low-molecular-weight sulfonic acid polymer, MDL 101028, was investigated for antiviral activity against herpes simplex virus (HSV) type 1 and type 2 in human embryonic diploid fibroblasts (MRC-5) and in a line of monkey kidney cells (Vero). Potent antiviral activities were obtained when treatment was restricted to the adsorption phase of virus propagation as measured by plaque reduction assay, and this was confirmed by experiments at high multiplicity of infection. Inhibition of virus binding to host cells by MDL 101028 was compared to that of heparin, a known antagonist of virus attachment. Both compounds showed dose-dependent inhibition, with IC50 values of 1.1 and 0.43 μg ml−1, respectively. The effect on cell-to-cell fusion was investigated using a syncytial-positive (syn+) phenotypic mutant of HSV-1 (strain 17i). MDL 101028 (3-1 μm) dramatically halted the spread of syncytial lesions; this effect was accompanied by the destruction of the syncytium and regrowth into the lesion by uninfected Vero cells. In contrast, heparin (10 μg ml−1) only partially reduced the spread of syncytia. Protection was also observed with MDL 29797 (100 μg ml−1), an antagonist of glycoprotein maturation. Unexpectedly, only a marginal effect was observed with acyclovir (10 μg ml−1), a potent inhibitor of viral DNA synthesis and HSV growth. As an extension to the current therapy for herpes simplex infection, acyclovir, the potent anti-fusion activity of MDL 101028 may have a clinical use in tissues or organs in which cell-to-cell fusion contributes to the pathology.

The effect of rolling and orbital motion on herpesvirus replication in tube cultures and shell vials

Background: Isolation of viruses from clinical specimens remains a viable diagnostic manoeuvre but positive isolation rates may be low and time to observe cytopathic effects (CPE) may be longer than preferred for optimal management. Both shell vial (SV) centrifugation and rolling of conventional tube cultures (TC) have been used to enhance the isolation process Objectives: To determine the effect of rolling and orbital motion on the replication of herpes simplex virus (HSV) and cytomegalovirus (CMV) TC and SV. Study design: TC were inoculated with HSV or CMV at varying dilutions and subjected to rolling or incubated stationary. Samples were selected to determine the impact of motion on viral yield, time to CPE according to initial multiplicity of infection (MOI) and stage of the infection process. Similar studies were performed with SV centrifuged with virus followed by stationary incubation or not centrifuged but incubated on an orbital shaker. Results: Rolling HSV-infected TC 383 rotations per minute (RPM) (30.2 × g) for 4 days enhanced viral yields by 53-fold over TC without motion. The optimal RPM response for HSV replication occurred at 96 RPM (1.9 × g) where an 89-fold increase in viral yield was detected, P<0.01. One-step growth studies at 0,2 and 96 RPM demonstrated enhancement of HSV replication at 2 and 96 RPM. TC infected with HSV at low MOI and rolled at 96 RPM had more CPE-positive cultures after 1–3 days than controls. Late in the infection process, no differences in CPE-positive were detected between rolled and non-rolled TC. Studies with CMV and rolling at 96 RPM resulted in more positive TC and greater CPE. SV that were not centrifuged but incubated for 16 h on an orbital shaker had significantly more HSV foci than those handled by a conventional SV method. Conclusion: Our studies indicate that orbital motion and motion yielding a force near 2 × g enhances the isolation and growth of HSV and CMV in TC and SV.

Enhanced inhibition of herpes simplex virus type 1 growth in human corneal fibroblasts by combinations of interferon-alpha and -gamma.

Trials testing the topical treatment of herpes simplex virus (HSV) ocular infections with single interferons (IFN) have provided mixed results. To determine if combination therapy with IFN may be more effective, the ability of combinations of IFN-alpha and IFN-gamma to inhibit HSV growth in human corneal fibroblasts (HCF) was assessed. Virus yields were reduced 282-fold and 37-fold, respectively, in HCF treated with either IFN-alpha or IFN-gamma (10(3) units/mL each). In cells treated with a combination of IFN-alpha and IFN-gamma (10(3) units/mL each), an average reduction of 5.1 x 10(5)-fold in the yield of infectious virus was achieved. Combinations of IFN-alpha and IFN-gamma considerably enhanced the antiviral effect in HCF, suggesting that combination treatment may be efficacious against ocular HSV infections; these findings provide a possible explanation at the cellular level for the poor results achieved in previous clinical trials.

Protective effects of immunoactive peptide, FK565 against systemic and local infections with herpes simplex virus and murine cytomegalovirus and respiratory tract infection with influenza virus in mice.

The protective effects of FK565 against systemic infections with herpes simplex virus (HSV) and murine cytomegalovirus (MCMV), respiratory tract infection with influenza virus and zosteriform rash with HSV investigated in mice. FK565 showed excellent protective activities against systemic infections with both acyclovir (ACV)-sensitive and -resistant HSV at intravenous and subcutaneous doses of 0.1 and 1 mg/kg and oral dose of 1 mg/kg. FK565 showed superior protective activities at subcutaneous doses of 0.01 and 0.1 mg/kg compared to ACV at subcutaneous dose of 15 mg/kg against MCMV infection. In respiratory tract infection with influenza virus, FK565 showed potent protective effects at intravenous, subcutaneous and oral doses of 0.001 to 1 mg/kg. FK565 markedly inhibited zosteriform spread of HSV on the flank skin at an intravenous dose of 0.1 mg/kg and the mice given FK565 survived longer than the control mice. The peritoneal exudate cells from FK565-treated mice suppressed the growth of HSV in mouse embryo fibroblast more strongly than those from the control mice, although FK565 had no direct antiviral activity against HSV. These findings suggest that FK565 enhanced the host defense ability against viral infections by nonspecific activation of macrophages.

In vitro antiviral activity of polyoxotungstate (PM-19) and other polyoxometalates against herpes simplex virus

Polyoxotungstates with Keggin-type structure were found to demonstrate marked antiherpetic activity. K 7[Ti 2W 10PO 40]·6H 2O (PM-19) caused a decrease in plaque formation by several strains of herpes simplex virus (HSV) type 1, including acyclovir-resistant (thymidine kinase-negative) strains, at concentrations which were not toxic to the host cells. The 50% plaque-inhibiting concentration (EC 50) for the different strains was between 20 and 50 μg/ml. Single-cycle HSV growth was also inhibited by PM-19. PM-19 inhibited viral DNA synthesis in HSV-infected cells at a concentration of 5 μg/ml but did not exhibit a virucidal effect, and pretreatment of the host cells with PM-19 did not provide resistance to herpes infection. Yet, virus adsorption to the cells was markedly affected at PM-19 concentrations higher than 25 μg/ml. PM-19 was also effective against human cytomegalovirus, but not against adenoviruses and varicella-zoster virus, although it did delay the development of the cytopathic effect of these viruses.

Nucleotide sequences responsible for the inability of a herpes simplex virus type 2 strain to grow in human lymphocytes are identical to those responsible for its inability to grow in mouse tissues following ocular infection

A study was undertaken to determine whether genes associated with herpes simplex virus (HSV) neuroinvasiveness in mice influence the growth of HSV in man, the virus's natural host. HSV-2 (186), a nonneuroinvasive HSV strain, was found to replicate poorly (<3-fold) in cultures of phytohemagglutinin (PHA) stimulated human peripheral blood mononuclear cells (PBMC). In contrast, seven other HSV strains all multiplied 40- to 100-fold. The paucity of HSV2(186) growth in PBMC was not due to a failure of this strain to grow in primary human cells because high titers (10 8 PFU/ml) were obtained following infection of human foreskin fibroblasts. The genetic basis for the deficient growth was analyzed by marker rescue experiments. Recombinant HSV-2 strains were generated in marker rescue experiments utilizing HSV-2(186) DNA and plasmids containing a cloned DNA polymerase gene isolated from a neuroinvasive HSV strain possessing the capacity to replicate in human PBMC. Progeny which rescued DNA from the cloned HSV DNA polymerase gene replicated 40- to 100-fold in PHA-stimulated PBMC. Moreover, unlike the HSV-2(186) parent, HSV-2(186) isolates possessing rescued DNA grew well in the eye, trigeminal ganglion, and brain of mice and induced fatal encephalitis. The results indicate that nucleotide sequences responsible for increasing the capacity of HSV-2(186) to grow in PBMC of man are identical to those responsible for increasing the capacity of this strain to grow in mouse tissues and to spread from the eye to the brain.

Herpes simplex virus and intraoral ulcers in immunocompromised patients with haematologic malignancies

Possible factors predisposing to the development of intraoral ulcers in immunocompromised patients with haematologic malignancies were investigated. Among 46 patients undergoing antineoplastic treatment, 18 developed an intraoral ulcer during the prospective study. Patients with or without ulcers were comparable with respect to underlying disease, presence of teeth, the qualitative composition of the aerobic and facultatively anaerobic oral microflora, herpes simplex virus (HSV) titer positivity, a past history of herpes labialis, leukocyte and thrombocyte counts, and duration of fever. Viral cultures from saliva revealed an association between the presence of HSV in saliva and the presence of intraoral ulcers. HSV was not isolated from the saliva of any patient without ulcers. Viral cultures from the ulcers revealed growth of HSV in 11 (61%) of the 18 patients with ulcers. HSV-positive ulcers were located more often on the alveolar process than elsewhere in the oral cavity. Ulcers on the lateral borders of the tongue and on the buccal mucosa were found only in dentate patients. Development of intraoral ulcers was not associated with an increase of the HSV titer in serum in any patient. It is concluded that some intraoral ulcers, especially when located on the alveolar process, are associated with and probably caused by HSV, while mechanical trauma during mastication seems likely to be involved in the pathogenesis of ulcers elsewhere in the oral cavity.

Effect of Nα-acetylmuramyl- l-alanyl- d-isoglutaminyl- Nϵ-stearoyl- l-lysine on resistance to herpes simplex virus type-1 infection in cyclophosphamide-treated mice

The restoration of resistance by N α -acetylmuramyl- l-alanyl- d-isoglutaminyl-N ϵ-stearoyl- l-lysine [MDP-Lys(L18)] on herpes simplex virus (HSV) type-1 infection was examined in cyclophosphamide (CY)-treated mice. MDP-Lys(L18) was shown to restore the resistance to HSV infection in CY-treated mice when it was injected either subcutaneously, intravenously, or intraperitoneally before infection. Treatment with MDP-Lys(L18) in CY-treated mice restored impaired activity for inhibiting HSV growth in the liver.

Successful Treatment with Foscarnet of an Acyclovir-Resistant Mucocutaneous Infection with Herpes Simplex Virus in a Patient with Acquired Immunodeficiency Syndrome

THE most widely used drug therapy for herpes simplex virus (HSV) is the nucleoside analogue acyclovir.1 2 3 To inhibit the growth of HSV, acyclovir must first be phosphorylated to the monophosphate...

Effect of smokeless tobacco on the replication of herpes simplex virus in vitro and on production of viral lesions in hamster cheek pouch

Previous experiments have shown that combination of herpes simplex virus (HSV) infection and simulated snuff-dipping in hamster buccal pouches enhances the development of micro-invasive squamous cell carcinoma in cheek pouch epithelium. The effect has now been determined of waterextractable components of snuff (snuff-extract) on the growth and the cell-lysing activity of HSV. Various dilutions of snuff-extract in tissue culture medium significantly inhibited the growth of HSV in Vero cell monolayers by inhibiting the viral DNA replication. Moreover, HSV was inactivated and its cell-lysing activity lost when it was incubated with snuff-extract in cell-free condition. Snuff also had a similar anti-herpetic effect in vivo: HSV infection of pouch tissues followed by simulated snuff-dipping resulted in significant inhibition of viral growth. Thus snuff interferes with the DNA synthesis and cytolytic activity of HSV in vitro and in vivo, and this in turn, may increase its oncogenic capacity.

Herpes simplex virus type 1 and neuronal cells--a special cell-virus interaction.

Rat brain glioma cells were semipermissive for herpes simplex virus (HSV) replication, because the growth of HSV was multiplicity-dependent in these cells. By using this property, we successfully isolated 'survivor' glioma cells following HSV infection at low multiplicity and without using any special treatment (such as UV irradiation) either of the cells or of the virus. Under the same conditions there were no survivor BHK or 3T3 cells, which suggests the uniqueness of the glioma cell-HSV interaction. The survivor cells ceased to produce infectious virus after two subcultures, but were highly resistant to superinfection for at least 20 subcultures. Parental cells were significantly more permissive for homologous virus growth than survivor cells. Interferon was apparently not induced in the survivor cells, because they were as susceptible as the parental cells to infection with vesicular stomatitis virus. The survivor cells produced HSV-specific antigens and contained HSV-specific DNA.

Interaction of Herpes simplex virus with human cell lines at various stages of lymphoid differentiation.

The growth of herpes simplex virus (HSV) type 1 in human lymphoid cell lines, arrested at various stages of differentiation, was studied. The synthesis of viral DNA and antigens and the production of infectious virus were followed in null, B and T leukemia-lymphoma cell lines. It was found that while "nondifferentiated" null cells and cells differentiating along the B pathway, even at very early stages of differentiation, supported HSV growth, cell lines of T origin at all stages of differentiation were generally nonpermissive. The failure of T cells to support the growth of HSV did not result from inefficient adsorption to the host cells. However, viral DNA synthesis was not detected, and a significant reduction in the ability of synthesize HSV antigens was observed. It is suggested that the block in the growth cycle of the virus occurs at a stage after adsorption of the virion to the cells, but prior to synthesis of its DNA and proteins.

Effect of prostaglandins and cyclic adenosine 3',5'-monophosphate modulators on herpes simplex virus growth and interferon response in human cells

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.

Growth of Type 2 Herpes Simplex Virus in Newborn and Adult Mononuclear Leukocytes

Growth of type 2 herpes simplex virus (HSV) in newborn and adult human mononuclear leukocytes (MNL) was compared. Phytohemagglutinin stimulation of cultures for 3 days yielded comparable peak titers in newborn (10(5.3) PFU) and adult (10(5.1) PFU) MNL. Unexpectedly, 3-day cultures of unstimulated newborn MNL also substantially replicated HSV (10(4.7) PFU), whereas similarly treated unstimulated adult cells did not. Growth of HSV in freshly isolated human MNL was next investigated. MNL from 4 mothers and 6 nonpregnant adults showed no evidence of virus growth; however, leukocytes from 11 of 24 newborns (46%) supported replication. Newborn MNL manifested an increased ability to replicate HSV within 1 day of culture, whereas comparable growth in adult MNL was not achieved until the 4th day of culture. The significance of the above observations as it relates to visceral dissemination of HSV in the neonate is discussed.