Herpes simplex virus: dry mass.

Dry mass of herpes simplex virus particles was measured by quantitative electron microscopy after isolation by surface spreading and critical-point drying of infected cells. The core weighed about 2 x 10(-16) gram, the empty naked capsid 5 x 10(-16) gram, the full naked capsid 7 x 10(-16) gram, and the enveloped nucleocapsid 13 x 10(-16) gram.

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses.

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.

Genetic engineering of novel genomes of large DNA viruses.

Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.

Examination of oral cancer tissue for the presence of the proteins ICP 4, ICP 5, ICP 6, ICP 8, and gB of herpes simplex virus type 1.

Although patients with oral cancer have increased levels of antibody to herpes simplex virus type 1, the origin of the antigenic stimulation remains unknown. We have therefore looked for proteins of herpes simplex in oral squamous cell carcinomas by staining frozen sections with monoclonal antibodies to the proteins ICP 4, ICP 5, ICP 6, ICP 8, and gB. No staining was seen of the tumor cells of any of 11 oral cancer cases or of the epithelium of 29 other oral lesions, which included cases of leukoplakia, lichen planus, and aphthous ulcers. Frequent staining of mast cells was seen in the connective tissue associated with oral cancer when ascitic fluid was used as the source of monoclonal antibody, but such staining was not seen when the precipitated IgG fraction was used.

Analysis of chromosomes, nucleic acids, and polypeptides in hamster cells transformed by herpes simplex virus type 2.

Syrian hamster embryo fibroblasts were oncogenically transformed by UV-inactivated Herpes simplex type 2. Eighteen clones were isolated shortly after transformation occurred. Two clones and their tumor derivatives were studied using several techniques. The karyotype analysis revealed different chromosome patterns in the two clones and a tendency toward hypodiploidy in the tumor derivatives. All of these cell lines were shown by molecular hybridization to contain 40% of the HSV-genome in several copies. The viral DNA sequence complexity was retained in the tumor derivatives, but a decrease in the copy number was observed. Viral RNA's were detected by in situ hybridization in all the lines that were tested. Viral antigens could be observed in these transformed cells by immunofluorescence. Finally, polypeptide analysis showed three differences between normal and transformed cells.

Functional regions and structural features of the gB glycoprotein of herpes simplex virus type 1. An analysis of linker insertion mutants.

Glycoprotein B (gB) of Herpes simplex virus type 1 (HSV-1) plays an essential role in viral entry. A set of more than 100 HpaI (GTTAAC) linker insertion mutations and their derivatives were isolated in plasmids specifying the gB coding and flanking sequences. Mutations including addition, deletion and nonsense mutations at 34 independent sites were identified by DNA sequence analysis of 48 plasmids. A map was constructed for the ability of addition mutants to complement a gB-null virus. The expression of gB activity for some plasmids was temperature-dependent. Many complementation-negative plasmids inhibited the complementation activity of a plasmid specifying wild-type gB, suggesting an interaction between active and inactive molecules to form oligomers. The interaction was localized to 328 of the total of 904 amino acids comprising gB. Partial Endo H digestion of nonsense polypeptides revealed that five of the six potential N-linked oligosaccharide sites are glycosylated; the most C-terminal site appears not to be glycosylated. A number of mutations, including some on the cytoplasmic side, were identified that blocked processing, transport and secretion. Addition mutations that blocked processing of membrane polypeptides also blocked processing and secretion when combined into a nonsense mutant that by itself was processed and secreted. The previously predicted membrane spanning domain and the membrane orientation of the N-terminal portion of gB were confirmed.

Herpes simplex encephalitis: analysis of a cluster of cases by restriction endonuclease mapping of virus isolates.

In December 1979, there were three deaths from culture-proven herpes encephalitis in 3 weeks in the New Haven area, and a nurse caring for one of these patients developed a herpetic lesion on her nose. The three brain isolates, the isolate from the nurse, and several epidemiologically unrelated strains were analyzed by restriction endonuclease mapping. All were determined to be distinct strains of herpes simplex virus. The possibility that a single strain of virus caused this cluster of cases was therefore examined directly and disproved.

Immunoelectron microscopic localization of herpes simplex virus antigens in infected cells using the unlabeled antibody-enzyme method.

Subcellular localization of viral antigens was demonstrated during viral morphogenesis using herpes simplex virus type 1 (HSV-1) infected monolayers of rabbit cornea cells. The localization was done by immunoelectron microscopy employing the peroxidase-antiperoxidase (PAP) immunocytochemical technique and the postembedding staining method. The localization of viral antigens was followed at time intervals during infection from 2 to 19 hr. After exposure of sections to either polyspecific antibodies against total HSV-1 antigens or monospecific antibodies against HSV-1 antigen No. 8, specific immunological reaction products were identified both in the cytoplasm and nucleus after 2 hr. The distribution and quantity of reaction products varied in the infected cells during the viral morphogenesis. The present results on the subcellular distribution of the HSV-1 antigens are related to current biochemical findings.

Differential feulgen-deoxyribonucleic acid hydrolysis patterns of Herpes simplex virus type 1 and type 2 infected cells.

Infection of human embryonic lung cells with herpes simplex virus type 1 (HSV-1) and herpes simplex type 1 (HSV-2) resulted in: (a) qualitative (nuclear cytopathologic) alterations and quantitative (nuclear area) differences in infected compared to control nuclei; (b) increased Feulgen-deoxyribonucleic acid (F-DNA) amounts in infected cells, probably due to viral DNA; (c) higher F-DNA levels in HSV-2 infected cells; and (d) increased rates of F-DNA hydrolysis in viral-infected as compared to uninfected nuclei.

Cytochemical analysis of DNA organization during encapsidation in herpes simplex virus.

The organization of intranuclear Herpes simplex virus DNA in rabbit fibroblast cells infected for 7 hr with HSV type 1 was examined before and during encapsidation by electron microscopic cytochemistry. Most non-encapsidated viral deoxyribonucleoprotein fibers exhibited a non-nucleosomal configuration. Empty capsids within the virus-specific regions of infected nuclei were wrapped with portions of the viral genome which adhered tightly to their surfaces even under conditions that loosened and spread apart other nucleoprotein fibers. During encapsidation, the internal surface of the capsid shell also appeared to bind a part of the viral genome, specifically the outer cage portion, which is detectable in methanol-dehydrated cells. Variations in the amount of DNA within the capsids indicated that the insertion of HSV genome into the capsid is a progressive process. The cage and core cylinder portions of the viral nucleoid appear to form and develop simultaneously. We propose that there may be binding sites on both the external and internal surfaces of the capsid shells which might play a role in the encapsidation process.

Intracellular maturation and sorting of two herpes simplex virus type 1 glycoproteins. Immunogold staining of ultrathin cryosections.

Simultaneous immunocytochemical triple staining of ultrathin cryosections of herpes simplex virus type 1-infected cells was carried out using monoclonal antibodies specific for glycoprotein C, glycoprotein D and alpha + beta tubulin. The viral glycoproteins were identified in the cytoplasm, in the Golgi sacs, on the plasma membrane and on the surface of intra- and extracellular virus particles, but not on the nuclear membrane. The glycoproteins identified in the cytoplasm outside the Golgi region were not always confined to the membranes of vesicles, but were often located in close proximity to the tubulin-labelled structures. The glycoproteins C and D were usually codistributed in the cytoplasm, and both accumulated in the Golgi sacs in the same membrane domains. As the glycoproteins occur in close proximity to the microtubular structures, we speculate that these might be directly involved in the intracellular transport of viral glycoproteins.

Effect of interferon treatment on expression of gC and gE glycoproteins in herpes simplex virus-infected cells.

The effect of interferon treatment on the herpes simplex virus type 1 (HSV-1)-specific glycoproteins gC and gE in homologous and heterologous cells has been investigated. In human embryonic fibroblastic cells, human leukocyte interferon inhibited virus multiplication and expression of the HSV-1-specific glycoproteins gC and gE on the cell surface in a dose-dependent manner. In heterologous baby hamster kidney cells, the human interferon had no effect on virus multiplication. However, the surface expression of the HSV-1-specific glycoproteins was reduced, as shown by erythrocyte rosette formation, by attachment of monodisperse polystyrene particles coated with antibodies and by immunogold scanning electron microscopy.

Infections due to the human herpesviruses in southern India: a seroepidemiological survey.

We studied the prevalence of IgG and IgM antibodies to the human herpesviruses in a hospital-based population of 181 individuals aged 0 to 25 years, who were resident in Vellore, south India or surrounding rural areas. Antibodies to the Epstein-Barr virus (EBV) viral capsid antigen were determined by indirect immunofluorescence, while antibodies to the remaining herpesviruses were determined by enzyme-linked immunosorbent assay. The age-specific prevalence rates of IgG antibodies to EBV and cytomegalovirus (CMV) rose rapidly after birth to reach a value of over 90% by the fourth year of life. High age-specific IgM prevalence rates and geometric mean titres (GMT) of IgG antibody in children 6 months to 2 years of age, and the early median age of virus infection (1.4 years for EBV and less than 1 year for CMV) indicate that primary infection with these viruses occurs early in life. In contrast, age-specific prevalence rates of IgG antibodies to varicella-zoster virus (VZV) and herpes simplex virus (HSV) rose gradually after birth to attain maximal values of only 72% (VZV) and 83% (HSV) in the 15-25 year age group, and the median ages of infection were delayed (12.25 years for VZV and 8.2 years for HSV). The age-specific IgG prevalence rates of VZV and HSV, and of EBV and HSV showed statistically significant positive correlations, suggesting that common epidemiological factors may underlie the pattern of infections due to these groups of viruses.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of herpes simplex virus (HSV) type-1 IgG and IgM antibodies by enzyme-linked immunosorbent assay (ELISA).

A sensitive enzyme linked immunosorbent assay (ELISA) is described for detection of herpes simplex virus (HSV) type 1, IgG and IgM antibodies. The antigen consisted of a crude extract of HSV infected human foreskin fibroblast cells. Specific horseradish peroxidase conjugated antisera were used to detect total immunoglobulin, IgG and IgM antibodies bound to viral antigen. The substrate was a solution of 5-aminosalicylic acid and hydrogen peroxide, which yielded a readily visible endpoint. Results obtained by the ELISA method were compared with the micro-complement fixation (CF) method on 36 sera. ELISA was shown to be at least 10-20 fold more sensitive than CF, with a correlation coefficient of 0.752 (p less than .001). HSV antibodies in these sera were mainly IgG, although IgM antibodies could also be detected by ELISA. HSV antibodies were not found in 16 cerebrospinal fluids from patients without HSV encephalitis (HSVE). ELISA appears to be a rapid, sensitive, and specific method for demonstration of IgG and IgM HSV antibodies. It may have possible application in the diagnosis of HSVE.

Variations in herpes simplex virus isolated from human ganglia and a study of clonal variation in HSV-1.

Growth of human sensory ganglia in culture has led to the reactivation of herpes simplex virus from over 50% of cases studied. Infected cell polypeptide and restriction enzyme analysis has led to the conclusion that each individual has one unique latent strain of HSV-1 that can be present in more than one ganglion in the body. Analysis of 91 isolates has shown that the long region of the genome is variable in terms of DNA restriction sites, DNA sequences, and in coding for the majority of variable polypeptides. The short region is stable with only polypeptides Vmw 21 and 22, restriction sites HindIII-(M-N) and BglII-(G-H) and the DNA sequence BamHI-1' having been found to vary. The insertion and deletion of small DNA sequences at specific locations allows individual reactivation events to be distinguished. Detection of information able to complement and produce ts+ virus on superinfection of otherwise negative ganglia with ts mutants, has led to the conclusion that ganglion cells may harbor herpes virus-related information that is only detectable by use of such genetic probes.

Detection of herpes simplex virus in clinical specimens by DNA hybridization.

An assay to detect herpes simplex virus (HSV) DNA in clinical specimens has been developed. It utilizes nucleic acid hybridization with a 32P-labeled DNA probe prepared from a fragment of HSV DNA cloned in a plasmid vector. This assay can detect 5 X 10(4) plaque-forming units of cell-free HSV and as few as four virus-infected cells. The assay has a sensitivity of 78% and a specificity of 100% compared to virus culture for the detection of HSV in swab specimens from genital lesions. No hybridization is observed with uninfected, varicella-zoster virus infected, or cytomegalovirus infected cells, and specimens from herpes zoster lesions are uniformly negative. While hybridization with a 32P-labeled probe is not optimally suited for routine diagnostic use, this report establishes the feasibility of using nucleic acid hybridization to detect HSV in clinical specimens.

Detection of herpes simplex virus by biotinylated DNA probes.

Clinical specimens from 159 patients suspected with herpes simplex virus (HSV) were examined by monoclonal antibody immunofluorescence (IF) and by a commercial biotinylated DNA probe kit following cell culture isolation. Herpes simplex virus was isolated from 57 samples. All cultures were positive by IF when the cytopathic effect (CPE) was less than 1+ but only 49 (86%) yielded positive reaction with the DNA probe when CPE was at least 1+. A total of 54 clinical specimens was also examined directly by immunoperoxidase histopathology (IHP), IF, and DNA hybridization. Of these, 16 were positive by IHP, 15 by IF, and only five by DNA probe. The DNA probe kit was found to be reasonably sensitive only after cell culture isolation of HSV. Compared to the IF procedure, the DNA probe kit was found to be costly, labor intensive, and time consuming.

The use of lectin affinity columns for selection of precursor or fully glycosylated forms of glycoprotein gD1 of herpes simplex virus type 1.

Several lectins were examined for their ability to bind to the glycoprotein gD1 polypeptide from Vero cells infected by herpes simplex virus type 1 (HVS-1), strain KOS. At least four distinct forms of gD1 (1, 2, 3 and 4), ranging in size from 59K to 52K, were resolved by SDS-10% polyacrylamide gel electrophoresis. Wheat germ agglutinin (WGA) did not bind to any of these forms, suggesting that if any sialic residues are present in the carbohydrate moieties of gD1, they are not available for binding to WGA. The entire population of forms 1 and 2 (approximately 59K) bound to castor bean-120 (CB-120) lectin affinity columns, suggesting the presence of terminal galactose residues on the mature and more fully glycosylated carbohydrate moieties of gD1. The forms 3 and 4, representing precursor gD1 molecules, did not bind. The majority of forms 2 and 4, and a portion of form 3 bound to lentil lectin, suggesting the presence of fucose and alpha-linked mannosyl residues on these molecules. A gD1-specific, high molecular weight species (120-125K) was detected in the lentil lectin-binding fraction but not in the fraction bound to CB-120 lectin or in the original infected-cell extract. The results indicated that lectin affinity chromatography, using lentil and CB-120 lectins, is useful as an initial step for the selection and purification of the individual glycosylated forms of gD1.

A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins.

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosylated processive intermediates bound to lentil lectin indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosylated, no gD and only a portion of pgD bound to lentil lectin when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1 and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.

Trifluorothymidine: potential non-invasive diagnosis of herpes simplex infection using 19F nuclear magnetic resonance in a murine hepatitis model.

Trifluorothymidine (TFT) is known to be concentrated in herpes simplex virus (HSV) infected cells in vitro in the form of phosphorylated derivatives. We studied a murine hepatitis model of HSV infection to determine whether this in vitro observation would also be demonstrable in vivo. Following i.v. injection of 100 or 160 mg/kg TFT, TFT was found in significantly higher concentrations in the livers of HSV-2 infected mice than in the livers of uninfected mice, mice infected with murine hepatitis virus (MHV-A59) or mice with hepatitis from carbon tetrachloride treatment. Neither altered renal function, nor altered pharmacokinetics could account for this difference. 19F Nuclear Magnetic Resonance spectroscopy readily detected the 19F from TFT in both liver extracts and whole livers, particularly at higher tissue levels, i.e. greater than 50 micrograms/g tissue. If further studies with living animals support these preliminary observations, clinical application could be pursued.