Variants Of Herpes Simplex Virus Research Articles

In vitro isolation of variants of herpes simplex virus attenuated with altered thymidine kinase and DNA polymerase genes using carrageenans as selection agents

Natural polysaccharides known as carrageenans are potent and selective inhibitors of herpes simplex virus through blocking the interaction of the virus with the cellular receptor. Passaging the virus in vitro in the presence of carrageenans types k and ι enabled us to generate variants of herpes simplex type 2. At passage 22, four clones were selected for further characterization: ι22-9, ι22-12, k22-12 and k22-13. Variants showed a syncytial phenotype, grew at similar titers when compared to parental virus and exhibited moderate resistance to carrageenans. These were found to have a mutation in the thymidine kinase gene in the case of k22-13 (aa 149 Val to Ala) and in the DNA pol gene in the case of ι22-12 (aa 789 Met to Thr). In variant k22-12, three substitutions in the DNA pol gene were identified. Variants were less virulent than parental strain when tested intravaginally or intranasally in mice. Attenuation correlated with higher levels of IL-6 and TNF-α in animals inoculated with the variants. Selective pressure on the external glycoproteins of the virus may generate viruses with alterations in genes unrelated to the target of action of the selection agent. Study of this type of variation might help us to understand the basis of persistent viral strategies, which in turn may play a role in the development of virus-host symbioses.

The pathogenicity of drug-resistant variants of herpes simplex virus

The pathogenicity of drug-resistant variants of herpes simplex virus

Colonization of adrenal glands and ovaries of mice by variants of HSV 1 and 2

The herpes simplex virus (HSV)-infected mouse model was used to correlate histopathological lesions in adrenal glands and ovaries with the localisation of viral nucleic acids and viral antigens, employing in situ hybridization and immunohistochemistry. In the adrenals, the lesions were mainly restricted to the zona fasciculata and the zona reticularis, sometimes extending to the medulla. In the ovaries, lesions were detected in follicles and in the stroma. During the course of infection, HSV nucleic acids could be detected earlier than HSV proteins. Next to the center of necrotic foci mainly HSV proteins were detected, whereas peripheral cells were found to contain viral nucleic acids. In situ hybridization revealed no proof of HSV latency in either organ. Among HSV-1 and HSV-2 strains of different neurovirulence, only HSV-2 variant ER- failed to replicate in adrenal glands and ovaries, whereas the neuroinvasive variant ER+ showed the same patterns as the HSV-1 strains used.

Characterization and sequence analyses of antibody-selected antigenic variants of herpes simplex virus show a conformationally complex epitope on glycoprotein H

Thirteen antigenic variants of herpes simplex virus which were resistant to neutralization by monoclonal antibody 52S or LP11 were isolated and characterized. The antibodies in the absence of complement potently neutralize infectivity of wild-type virus as well as inhibit the transfer of virus from infected to uninfected cells ("plaque inhibition") and decrease virus-induced cell fusion by syncytial strains. The first variant isolated arose in vivo. Of 66 type 1 isolates analyzed from typing studies of 100 clinical isolates, one was identified as resistant to neutralization by LP11 antibody. The glycoprotein H (gH) sequence was derived and compared with those of wild-type and syncytial laboratory strains SC16, strain 17, and HFEM. The sequences were highly conserved in contrast to the diversity observed between gH sequences from herpesviruses of different subgroups. Only four coding changes were present in any of the comparisons, and only one unique coding change was observed between the laboratory strains and the clinical isolate (Asp-168 to Gly). These sequences were compared with those of antigenic variants selected by antibody in tissue culture. Twelve variants were independently selected with antibody LP11 or 52S from parent strain SC16 or HFEM. For each variant, the gH nucleotide sequence was derived and a point mutation was identified giving rise to a single amino acid substitution. The LP11-resistant viruses encoded gH sequences with amino acid substitutions at sites distributed over one-half of the gH external domain, Glu-86, Asp-168, or Arg-329, while the 52S-resistant mutant viruses had substitutions at adjacent positions Ser-536 and Ala-537. One LP11 mutant virus had a point mutation in the gH gene that was identical to that of the clinical isolate, giving rise to a substitution of Asp-168 with Gly. Both LP11 and 52S appeared to recognize distinct gH epitopes as mutant virus resistant to neutralization and immunoprecipitation with LP11 remained sensitive to 52S and the converse was shown for the 52S-resistant mutant virus. This is consistent with previous studies which showed that while the 52S epitope could be formed in the absence of other virus products, virus gene expression was required for stable presentation of the LP11 epitope, and for transport of gH to the cell surface (Gompels and Minson, J. Virol. 63:4744-4755, 1989). All mutant viruses produced numbers of infectious particles that were similar to those produced by the wild-type virus, with the exception of one variant which produced lower yields.(ABSTRACT TRUNCATED AT 400 WORDS)

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.

Molecular epidemiology of herpes simplex virus and geographic localization of a variant of herpes simplex virus type 1

An analysis of nucleotide sequence of herpes simplex virus (HSV) DNA by restriction endonuclease digestion is being applied to epidemiologic studies. We examined basic characteristics of the DNA fingerprinting analyses, and observed that no two Japanese epidemiologically unrelated isolates yielded identical patterns of DNA fragments. This paper reports a comparison of restriction enzyme cleavage profiles of 224 isolates of HSV type 1 (HSV-1) from five regions in Japan. A variant with a characteristic Bfl II cleavage pattern was found. The variant was isolated at a much higher rate in the western regions than in the eastern regions of Japan. These results seem to suggest that there are some geographic differences among HSV isolates in Japan where migration of the people was very strictly controlled for a long time until a century ago. Thus, HSV variants may have occurred and accumulated in human populations, persisting through intimate personal contacts with the virus carriers.

Susceptibility to other antiherpes drugs of pathogenic variants of herpes simplex virus selected for resistance to acyclovir

Cross-resistance data for a group of nine acyclovir-resistant variants of herpes simplex virus type 1 are reported. These mutants, which express either altered thymidine kinase (TK) or DNA polymerase, were all derived from the same wild-type (wt) strain after exposure to acyclovir in tissue culture. Furthermore, all variants have pathogenic properties similar to the wt parental strain as assessed using mouse model systems (G. Darby, H.J. Field, and S.A. Salisbury, Nature (London) 289:81-83, 1981; B.A. Larder and G. Darby, Virology 146:262-271, 1985). Two groups of antiherpes compounds were used: those requiring activation by TK and those whose action is independent of that enzyme. The TK substrate-specificity mutants were generally resistant to the TK-activated drugs but showed wt susceptibility to phosphonoacetic acid, 9-beta-D-arabinofuranosyladenine, and aphidicolin. The DNA polymerase mutants were relatively susceptible to most TK-activated drugs, although two were resistant to 5-(trifluoromethyl)-2'-deoxyuridine. The polymerase mutants showed a more complex pattern of susceptibility, however, to those compounds whose mode of action is independent of TK. In general, these variants showed similar responses to phosphonoacetic acid, phosphonoformate, and 9-beta-D-arabinofuranosyladenine, a particular variant being either resistant, susceptible, or hypertensive to all three. The response of each variant to aphidicolin, however, appeared to be the inverse of its response to the other three drugs. The cross-resistance patterns are discussed, and their implications for combined or successive therapies are considered.

Characterization of abnormal thymidine kinases induced by drug-resistant strains of herpes simplex virus type 1.

Two TK+ acyclovir-resistant variants of herpes simplex virus (HSV) (S1 and Tr7) and one TK+ BVdU-resistant variant (B3) induce abnormal thymidine kinases with impaired ability to phosphorylate the drugs used in their isolation. These enzymes have been purified and their properties compared with those of the wild-type (wt) parent, SC16. The enzyme induced by S1 differed markedly from the other three in both its responses to salt and to pH. B3 TK recognized the enzyme's natural substrates, thymidine, deoxycytidine, dTMP and ATP as well as the wt enzyme. In contrast, Tr7 and S1 TKs failed to bind deoxycytidine and bind thymidine less well than wt. Tr7 and S1 TKs had affinities for dTMP similar to those of B3 and the wt enzymes. ATP binding to wt, Tr7 and B3 enzymes was similar but this substrate bound only weakly to S1 TK. Each mutant displayed a characteristically distinct pattern of affinities for a range of nucleoside analogue substrates, suggesting that they will show some cross-resistance to drugs which have a similar mechanism of action to acyclovir and BVdU.

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.

Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus. Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase.

The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.

Management of herpes simplex infections of the skin

MANY REMEDIES, folk, over-the-counter, and prescribed, are recommended for treatment of cold sores and fever blisters. This immediately suggests two possibilities: either everything is effective in the management of the troublesome skin lesions of herpes simplex infection, or no treatment is really very effective. A brief review of the course and pathogenesis of herpetic infections will explain why both of these possibilities may be true and will give insight into the problems confronting both patient and physician. There are two variants of herpes simplex virus that affect man—type 1, which is most commonly isolated from facial lesions, and type 2, which normally causes herpes infections of the genitalia. Other than the differences in location and the earlier appearance of clinical and serological evidence of infection with herpesvirus type 1, the course of the disease caused by the two variants is quite similar. Most persons contract infection with herpesvirus type 1

Management of Herpes Simplex Infections of the Skin

MANY REMEDIES, folk, over-the-counter, and prescribed, are recommended for treatment of cold sores and fever blisters. This immediately suggests two possibilities: either everything is effective in the management of the troublesome skin lesions of herpes simplex infection, or no treatment is really very effective. A brief review of the course and pathogenesis of herpetic infections will explain why both of these possibilities may be true and will give insight into the problems confronting both patient and physician. There are two variants of herpes simplex virus that affect man—type 1, which is most commonly isolated from facial lesions, and type 2, which normally causes herpes infections of the genitalia. Other than the differences in location and the earlier appearance of clinical and serological evidence of infection with herpesvirus type 1, the course of the disease caused by the two variants is quite similar. Most persons contract infection with herpesvirus type 1

Low virulence and immunogenicity in mice and in rabbits of variants ofHerpes simplex virus resistant to 5-iodo-2-deoxyuridine

Par passage en serie sur des cellules en culture et en presence de 5-iodo-2-desoxyuridine (IUdR) on a obtenu une souche de virus herpetique resistant a l'IUdR. Cette souche est tres peu virulente chez la souris et le lapin, mais est capable de proteger ces animaux contre le virus pathogene d'origine.

Studies on increased turnover of brain mono-amines induced by experimental herpes simplex infection.

Intracerebral infection of mice with different herpes simplex virus (HSV) strains caused raised dopamine and 5‐hydroxytryptamine turnover, reflected in increased brain concentrations of the acid metabolites of the amines i. e. homovanillic acid (HVA) and 5‐hydroxyindole‐acetic acid (5‐HIAA). Inhibitors of virus multiplication (actinomycin D, mitomycin C and iododeoxyuridine) and different variants of HSV were used to study whether the rise in mono‐amine metabolism was an event dependent upon the direct action of the virus or viral metabolites on pathways of mono‐amine synthesis. The results obtained were not compatible with such a hypothesis. The histopathological study of the distribution of the neuronal lesions indicated that the lower brain stem from which most of the monoaminergic neurons originate was hardly affected at all. The degree of severity of the neuronal lesions in relation to the HVA and 5‐HIAA concentrations found in infected brains was studied for 13 different HSV variants. The more extensive and frequent the lesions, the higher were the levels of the acids encountered. It was assumed that impaired functions of postsynaptic cells or altered receptor‐functions possibly affecting regulatory feed‐back mechanisms could be responsible for the increased mono‐amine turnover.

Heterogeneity in the Ability of Rabbit 7 S and 19 S Neutralizing Antibodies to Differentiate Between Intratypic Antigenic Variants of Herpes Simplex Virus

Summary The ability of early (7 day) and late (7 week) rabbit 7 S and 19 S antibodies to differentiate between two intratypic herpes simplex virus (HSV) antigenic variants was studied by carrying out neutralization kinetic tests. The early and late 19 S antibodies could differentiate between these HSV variants in the presence of complement (C′), and the late 19 S antibody could also differentiate between these variants in the absence of C′. However, neither the early nor the late 7 S antibodies could differentiate between these HSV variants in the presence of C′, but the late 7 S antibody could differentiate between these variants in the absence of C′. The ability of the late 7 S antibody to differentiate between these HSV variants in the absence of C′ was destroyed by treatment of the antibody with 2-mercaptoethanol (2-ME), although 2-ME treatment did not reduce the neutralization titers of the late 7 S antibody.

Cumulative Changes in the Antigenic Properties of Herpes Simplex Virus from Persistently Infected Cell Cultures

Summary Reciprocal neutralization kinetic studies were carried out between the virus isolated at different times from persistent herpes simplex virus (HSV) infected cell cultures and their respective antisera. The results indicated that the virus acquired new antigenic determinants with time, while still retaining the determinants of the antecedent virus. The serologic relationship between the HSV variants isolated from these persistent infections was similar to that observed by others between HSV and herpes B virus, but not previously noted with strains of HSV.

VARIANTS OF HERPES SIMPLEX VIRUS: ISOLATION, CHARACTERIZATION, AND FACTORS INFLUENCING PLAQUE FORMATION.

Rapp, Fred (Baylor University College of Medicine, Houston, Tex.). Variants of herpes simplex virus: isolation, characterization, and factors influencing plaque formation. J. Bacteriol. 86:985-991. 1963.-Variants of herpes simplex virus were isolated which differed in plaque size and in virulence for rabbits and mice. Keratitis on the rabbit cornea and generalized disease were associated with the large, but not the small, plaque variants. The large plaque variants were about as neurovirulent as was the parent strain for weanling mice, but the majority of the small plaque variants were only 1% as virulent for mice as was the parent virus. Agar inhibited the formation of plaques by the parent strain and all variants tested; development of plaques was retarded and the number of plaques was approximately 60% less than those developing under an overlay in which methyl cellulose had been substituted for the agar. Rabbit kidney cells and human embryonic lung fibroblasts were more sensitive than mouse embryo fibrolasts to infection with all variants tested.

Further studies of variants of herpes simplex virus that produce syncytia or pocklike lesions in cell cultures.

Journal Article FURTHER STUDIES OF VARIANTS OF HERPES SIMPLEX VIRUS THAT PRODUCE SYNCYTIA OR POCKLIKE LESIONS IN CELL CULTURES Get access M.DAVID HOGGAN, M.DAVID HOGGAN Search for other works by this author on: Oxford Academic PubMed Google Scholar BERNARD ROIZMAN, BERNARD ROIZMAN Search for other works by this author on: Oxford Academic PubMed Google Scholar PHILIP R. ROANE, JR PHILIP R. ROANE, JR Search for other works by this author on: Oxford Academic PubMed Google Scholar American Journal of Epidemiology, Volume 73, Issue 1, January 1961, Pages 114–122, https://doi.org/10.1093/oxfordjournals.aje.a120162 Published: 01 January 1961 Article history Received: 13 October 1960 Published: 01 January 1961