Inactivation of Herpes Simplex Virus by Concanavalin A

The infectivity of herpes simplex virus type 1 (HSV-1) was inactivated after treatment with either concanavalin A (ConA) or periodate. Phytohemagglutinin, wheat germ agglutinin, pokeweed mitogen, and neuraminidase failed to inactivate the virus. The effect of ConA could be specifically inhibited or reversed by the addition of α-methyl-d-glucoside or α-methyl-d-mannoside. Evidence was obtained that HSV-1 inactivated by ConA could adsorb to host cells. Viral aggregation was not a major mechanism in the inactivation of HSV-1 by ConA. Under the experimental conditions employed, inactivation of HSV-1 was faster by ConA than by antiserum and less temperature dependent. A ConA-resistant fraction was detected which appeared to adsorb less quickly than untreated virus, and penetration of ConA-resistant fraction was strikingly slow. The presence of aggregates in the virus preparation did not appear to account for the ConA-resistant fraction. Inactivation of viral infectivity by ConA was obtained only with enveloped viruses, since HSV-1, HSV-2, pseudorabies, and vesicular stomatitis virus were inactivated and vaccinia and echovirus type 6 were not.     

Colorado tick fever virus in cell culture. II. Physical and chemical properties

Trent, Dennis W. (University of Oklahoma School of Medicine, Oklahoma City), and L. Vernon Scott. Colorado tick fever virus in cell culture. II. Physical and chemical properties. J. Bacteriol. 91:1282-1288. 1966.-Heat-inactivation kinetics for Colorado tick fever (CTF) virus grown in L cells indicated that more than one rate constant was involved for inactivation at each exposure temperature. An Arrhenius plot of the data indicated the inactivation rate constants to be dependent on the absolute temperature. The energy of activation, for thermal inactivation of the virus, was 17,289 calories per mole, with the Q(10) being 2.6. The optimal pH range for maintenance of CTF viral infectivity was determined to be 7.5 to 7.8. The infectivity of CTF virus was stable to freezing and thawing in diluents which contained: 50% calf serum, 20% glucose, 20% glycerol, 10% bovine serum albumin, 20 mm glutamine, and 2% gelatin. CTF virus replication was insensitive to inhibition by 5-fluoro-2'-deoxyuridine and 5-bromo-2'-deoxyuridine, whereas herpes simplex virus was markedly inhibited, as reported by others. Actinomycin D inhibited CTF virus replication when cells were pretreated for 24 and 12 hr prior to infection, but not when the inhibitor was added at the time of infection. The nucleic acid of CTF virus appears to be of the ribose type.     

Oncogenic Transformation of Hamster Cells after Exposure to Herpes Simplex Virus Type 2

THE possibility of a relationship between herpes simplex viruses (HSV) and human cancer has been suggested^1–4 chiefly on the basis of studies of the epidemiology of cervical cancer, but so far it has not been possible to demonstrate that human herpes viruses can induce primary transformation of normal cells. Injection of herpes simplex virus type 1 (ref. 5) or type 2 (ref. 6) into Syrian hamsters rarely leads to the production of a tumour and it has been difficult to demonstrate herpes viral antigens in tumour cells. Human herpes simplex viruses grown in vitro are characterized by the rapidity with which the infected cell is destroyed, so that cell transformation is impossible, but this effect can be mitigated by inactivation of the herpes virus by ultraviolet irradiation. Indeed, this procedure may have the additional advantage that viral infectivity is removed more quickly than the viral transforming potential⁷.     

Interaction of infectious viral particles with a quaternary ammonium chlorid (QAC) surface

The antiviral activity of a surface-bonded quaternary ammonium chloride (QAC) was examined in this study. The mechanism of inactivation was elucidated by a combination of infectivity assay, radioactive labeling assay, and sedimentation analysis. Although the virions are still infectious when attached onto the chemically modified surface, we found these viruses are inactivated if they are eluted from the surface. The inactivation is caused by the disruption of the viral envelope with subsequent release of the nucleocapsid. No evidence indicates the released nucleocapsid is further disrupted. An enveloped virus shows a much higher affinity for the QAC-treated surface than a nonenveloped one due to hydrophobic interaction. The QAC-treated beads can effectively remove the enveloped viruses at low protein concentrations. The titer of herpes simplex virus was reduced by a factor of nearly 5 logarithm units in a 0.5 wt % bovine serum albumin solution with less that 10% protein loss. However, the presence of proteins in the solution reduced both the rate and capacity of this nonspecific adsorption-inactivation process. As a consequence, the removal efficiency is relatively poor in solutions with high protein content.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Cell fusion induced by herpes simplex is inhibited by hen egg-white lysozyme

The formation of syncytia in cell monolayers infected with a macroplaque strain (MP) of herpes simplex virus was found to be inhibited by hen egg-white lysozyme. Inhibition was roughly proportional to the enzyme concentration. The virus titres in supernatant fluids of lysozyme-treated cultures were also reduced compared with untreated cultures. Control experiments excluded the possibility that lysozyme altered the virus viability and infectivity or impaired cell growth. Since lysozyme is a cationic protein, further experiments were performed in order to discover whether its antisyncytiogenic effect depended on its enzymatic activity or on its positive charge. Inhibition of the MP-induced polycaryocytosis was found to be caused by heat-inactivated lysozyme and three chemically-modified lysozymes with a higher positive charge (one retaining and two lacking enzymatic activity).     

Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.     

Isolation of a protein kinase induced by herpes simplex virus type 1

We have isolated a new cyclic AMP-independent protein kinase activity induced in HeLa cells by infection with herpes simplex virus type 1. Induction of the enzyme does not occur in cells treated with cycloheximide at the time of infection, or in cells infected with UV-inactivated herpes simplex virus type 1. The amount of enzyme induced in infected cells is dependent upon the multiplicity of infection. An enzyme with identical properties to the appearing in infected HeLa cells is also induced by herpes simplex virus type 1 in BHK cells.     

In vitro activity evaluation of Parkia pendula seed lectin against human cytomegalovirus and herpes virus 6.

Human cytomegalovirus (HCMV) in vitro infectivity was inhibited by Parkia pendula seed lectin (PpeL) in contrast to human herpes virus 6 (HHV-6) which was not affected. The antiviral activity was detected for HCMV in human embryo lung (HEL) cells using a microtechnique in culture plates. The assay showed a reduction of cellular infectivity from approximately 95%, at a concentration of 150microg/mL with minimal cytotoxicity (25%). Also, a reduction of 75% was observed in HEL cells at a concentration of 75microg/mL without toxic effect. The reduction on infectivity was observed even after virus pre-adsorption to cells suggesting that this action should occur after virus penetration, in the intracellular replication phase. MT4 lymphocytes and cord blood mononuclear cells (CBMC) were used to evaluate the lectin effect on HHV-6 following the same technique. Lectin concentrations with few or no toxic effects on lymphocytes did not show inhibitory action of HHV-6 cytopathic effect. The results obtained with PpeL demonstrate that it may have an impact in the design of pharmacological strategies to infection of cytomegalovirus.     

The effect of the UL42 protein on the DNA polymerase activity of the catalytic subunit of the DNA polymerase encoded by herpes simplex virus type 1.

The effect that the UL42 protein of herpes simplex virus type 1 has on the DNA polymerase activity of the DNA polymerase catalytic subunit (Pol) of the same virus has been investigated. The observed effects are critically dependent on the salt used and its concentration, such that the UL42 protein may inhibit, have little or no effect on, or activate the Pol activity, depending on the condition used. The observed effects are due to the values for Km(app) for activated DNA and Vmaxapp for Pol and the Pol-UL42 protein complex differently varying with salt concentration.     

Induction of uracil-DNA glycosylase and dUTP nucleotidohydrolase activity in herpes simplex virus-infected human cells

HeLa BU cells infected with either the type 1 or the type 2 forms of herpes simplex virus show an increase in the activities of uracil-DNA glycosylase and dUTP nucleotidohydrolase. Under optimal conditions, uracil-DNA glycosylase activity increases approximately 40-fold in HSV type 2-infected cells. In herpes simplex virus (HSV) type 1-infected cells, uracil-DNA glycosylase activity increases only 6-fold. At a KCl concentration of 100 mM, uracil-DNA glycosylase derived from HSV type 2-infected cells is activated 2-fold, while the glycosylase extracted from mock infected HeLa BU cells is inhibited almost 90% at 100 mM KCl. dUTP nucleotidohydrolase activity increases 4-fold and 3-fold, respectively, in HSV type 1- and HSV type 2-infected HeLa BU cells. Nondenaturing polyacrylamide gel electrophoresis of extracts derived from the type 1- and type 2-infected cells indicates distinct electrophoretic mobilities from the host cell enzyme. dUTP nucleotidohydrolase RF values for the mock infected cells, HSV type 1, and HSV type 2 are 0.5, 0.25, and 0.33, respectively. Serum from rabbits immunized against cells infected with herpes simplex virus type 1 or type 2 specifically neutralizes the dUTPase and uracil-DNA glycosylase activities extracted from herpes simplex virus-infected cells. This serum does not neutralize dUTPase or uracil-DNA glycosylase activity derived from mock infected cells.     

Inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase

Genital herpes simplex virus type 2 (HSV-2) is a significant clinical problem. Infection in pregnancy may result in disseminated infection of the newborn with encephalitis. We analyzed the antiviral effects induced by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in cervix carcinoma cells (HeLa) and astrocytoma cells (86HG39). We found that replication of HSV-2 in HeLa cells and in 86HG39 cells is inhibited after stimulation of the cells by IFN-gamma and TNF-alpha. The antiviral effect of IFN-gamma is enhanced in the presence of TNF-alpha, while stimulation by TNF-alpha alone did not induce antiviral activity. We found that IFN-gamma induces a strong activation of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and in addition, that the IFN-gamma-induced IDO activity was enhanced in the presence of TNF-alpha. Furthermore, we found that the induction of IDO activity is responsible for the inhibition of herpes simplex virus replication, since the presence of excess amounts of l-tryptophan abrogates the antiviral effect induced by IFN-gamma and the combination of IFN-gamma and TNF-alpha. We therefore conclude that the antiviral effect against HSV-2 mediated by type II interferon and TNF-alpha are dependent on IDO activation.     

Neutralizing antibodies specific for glycoprotein H of herpes simplex virus permit viral attachment to cells but prevent penetration.

Monoclonal antibodies specific for gH of herpes simplex virus were shown previously to neutralize viral infectivity. Results presented here demonstrate that these antibodies (at least three of them) block viral penetration without inhibiting adsorption of virus to cells. Penetration of herpes simplex virus is by fusion of the virion envelope with the plasma membrane of a susceptible cell. Electron microscopy of thin sections of cells exposed to virus revealed that neutralized virus bound to the cell surface but did not fuse with the plasma membrane. Quantitation of virus adsorption by measuring the binding of purified radiolabeled virus to cells revealed that the anti-gH antibodies had little or no effect on adsorption. Monitoring cell and viral protein synthesis after exposure of cells to infectious and neutralized virus gave results consistent with the electron microscopic finding that the anti-gH antibodies blocked viral penetration. On the basis of the results presented here and other information published elsewhere, it is suggested that gH is one of three glycoproteins essential for penetration of herpes simplex virus into cells.     

The inhibitory effect of essential oils on herpes simplex virus type-1 replication in vitro

The antiviral effect of 12 essential oils on herpes simplex virus type-1 (HSV-1) replication was examined in vitro. The replication ability of HSV-1 was suppressed by incubation of HSV-1 with 1% essential oils at 4 C for 24 hr. Especially, lemongrass completely inhibited the viral replication even at a concentration of 0.1%, and its antiviral activity was dependent on the concentrations of the essential oil. When Vero cells were treated with the essential oil before or after viral adsorption, no antiviral activity was found, which suggests that the antiviral activity of essential oils including lemongrass may be due to the direct interaction with virions.     

In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein.

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.     

Anti-herpesviral property and mode of action of a polysaccharide from brown seaweed (<Emphasis Type="Italic">Hydroclathrus clathratus</Emphasis>)

A sulfated polysaccharide, designated HC-b1, was isolated from the brown seaweed Hydroclathrus clathratus. It was found to be a strong inhibitor of herpes simplex virus type 1 (HSV-1), including acyclovir-resistant strain and clinical strain. HC-b1 inhibited the plaque formation of HSV-1 in a dose-dependent manner. It could protect Vero cells from infection by HSV-1 if the cells were incubated with HC-b1 before exposure to the virus. It also had inactivating effect against HSV-1 since the pretreatment of the virus with HC-b1 caused significant reduction of viral infectivity. Time of addition studies demonstrated that HC-b1 exerted its antiviral action at the early stage of virus replication cycle. The presence of HC-b1 could not effectively inhibit the replication of HSV-1 about 45 min after the penetration period started. The antiviral action of HC-b1 appeared to inhibit the attachment of herpes simplex virus on host cell membrane through interfering with the processes of adsorption and penetration.