Occult herpesvirus folliculitis clinically simulating pseudolymphoma.

Two cases of cutaneous herpesvirus infection are described that clinically masqueraded as pseudolymphoma. Light microscopy demonstrated typical viral changes involving pilosebaceous complexes with sparing of the surface epithelium. Dermal changes consisted of a dense perivascular and perifollicular inflammatory infiltrate. Multinucleated lymphoid cells were found in the dermis in one case and viral inclusions in fibroblasts were present in the other case. Immunoperoxidase stains with antisera to herpes simplex virus types I and II were positive in one case and negative in the other case. Ultrastructural examination demonstrated viral particles consistent with herpesvirus in both cases. Recognition of typical histologicl features of herpesvirus folliculitis will lead to an accurate diagnosis in these types of clinically unsuspected cases.

Properties of the human herpesvirus 6 strain Z29 genome: G + C content, length, and presence of variable-length directly repeated terminal sequence elements.

We have studied the structure of the human herpesvirus 6 (HHV-6) genome. The density of genomic DNA is approximately 1.702 g/cm3 as determined by isopycnic density gradient centrifugation, from which a mean G + C content of 43% was calculated. The genomic termini were examined by exonuclease digestion and DNA/DNA hybridization; relative molarities of restriction fragments were determined by quantitative densitometry. The results indicate that the HHV-6(Z29) genome has two unique termini and consists of a long unique segment bounded by a directly repeated sequence element found in one copy at each end of the genome. We estimated the length of the genome by pulsed-field gel electrophoresis and by summation of restriction endonuclease fragment lengths. We observed two forms of HHV-6(Z29) DNA of approximately 162 and 168 kb in length. The length heterogeneity was localized within the terminal repeat element, each copy of which is approximately 10.1 kb in length in the shorter form of the genome and 13.2 kb in length in the longer form of the genome.

Characterization of envelope proteins of alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 is a gammaherpesvirus which causes malignant catarrhal fever, an acute lymphoproliferative disorder of cattle and other susceptible Bovidae, which is almost invariably fatal. A preliminary analysis of proteins induced by the virus indicated that as many as six glycoproteins and one nonglycosylated molecule might be present in the virus envelope. Monoclonal antibodies selected for recognition of virion envelope proteins included two that recognized a complex of infected cell proteins, designated the gp115 complex, and neutralized virus infectivity in the absence of complement. The gp115 complex consisted of five glycoproteins of 115, 110, 105, 78, and 48 kilodaltons (kDa), and all except the 48-kDa species reacted with antibody in Western blots (immunoblots). Pulse-chase experiments analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions suggested that the 110-kDa protein was the precursor molecule which was processed by addition of sugars to 115 kDa. The 115-kDa protein was cleaved to form a disulfide-linked heterodimer of 78 and 48 kDa, which was the mature form of the molecule incorporated into the virion envelope. The glycoprotein contained N-linked sugars, but little or no O-linked sugar was present. The relative abundance of the mature protein and its ability to induce neutralizing antibodies suggest that it will prove useful to studies aimed at elucidating the biology and pathogenesis of alcelaphine herpesvirus 1.

[Analysis of B-lymphotropic oncogenic herpesvirus of Macaca arctoides]. / Analiz B-limfotropnogo onkogennogo virusa gerpesa makakov burykh.

Some physico-chemical and molecular-biological parameters of B-lymphotropic herpes virus of Macaca arctoides (HVMA) have been analyzed. The buoyant density of virions and nucleocapsids in a gradient of the percoll density is 1.10 and 1.14 g/ml, respectively, while that of DNA in CsCl gradient is 1.717 g/ml, which is typical for herpes viruses of primates. At the same time the homology of DNA HVMA with DNA EBV and DNA HVP is 30-45%. Differences in restrictions sites between DNA HVMA, DNA HVP and DNA EBV are determined. The intraspecific heterogeneity of HVMA strains has been detected according to restriction sites of DNA. The HVMA genome size is about 170 kb. The DNA HVMA is colinear to the EBV genome, which is also typical of B-lymphotropic herpes viruses of primates.

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.

Nucleocapsid mass and capsomer protein stoichiometry in equine herpesvirus 1: scanning transmission electron microscopic study.

The Brookhaven scanning transmission electron microscope was employed to measure the masses of two nucleocapsid species (of light and intermediate densities) of equine herpesvirus 1. These were found to be 196.7 +/- 9.2 and 229.0 +/- 9.5 megadaltons (MDa), respectively. Biochemical assays showed that neither nucleocapsid contained any significant amount of DNA (less than 0.2% [wt/wt]). Taking into account data on protein composition, we conclude that the difference between their masses is essentially contributed by viral protein 22 (46 kDa), which is an integral component of the maturable intermediate nucleocapsid but not of the abortive light nucleocapsid. In view of earlier ultrastructural information on capsomer symmetry, our mass determinations are consistent only with the 150 hexavalent capsomers being hexamers of the 148-kDa major capsid protein.

Identification of proteins specific for human herpesvirus 6-infected human T cells.

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [35S]methionine- and [3H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [35S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-Mr polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k.

Virion and nonstructural polypeptides of human herpesvirus-6.

Human herpesvirus-6 (HHV-6) was purified from HHV-6-infected mononuclear cells from cord blood or infected culture medium. Virion was found to have at least 29 polypeptides ranging from 280 to 30 kDa. Six polypeptides were identified in the envelope fraction. Twenty-five viral polypeptides were also identified in HHV-6 infected cells by immunoprecipitation with immune serum.

Identification of the gB homologues of equine herpesvirus types 1 and 4 as disulphide-linked heterodimers and their characterization using monoclonal antibodies.

Equine herpesvirus types 1 and 4 (EHV-1 and EHV-4) labelled with [14C]glucosamine were purified from infected cell culture medium and profiles of their structural proteins were obtained that enabled identification of the major glycoproteins. Nine glycosylated polypeptides were identified for each virus. Preparations of the purified viruses each contained a glycoprotein which was linked by disulphide bonds, as determined by diagonal gel electrophoresis under reducing/non-reducing conditions. High Mr forms of this glycoprotein were detected for EHV-1 when the sample was not heated. The EHV-1 protein consisted of three polypeptides of Mr 108K, 76K and 58K and the EHV-4 protein consisted of three polypeptides of Mr 112K, 74K and 61K. Western blotting and immunoprecipitation with monoclonal antibodies confirmed that the EHV-1 gB homologue migrates with an apparent Mr of 108K (140K under non-reducing conditions) but is cleaved to give glycoproteins of 76K and 58K which are held together by disulphide bonds. The EHV-4 gB homologue consists of a 112K glycoprotein which is cleaved to give glycoproteins of 74K and 61K which are also linked by disulphide bonds.

Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer).

Eleven virus isolations were made from the blood of 45 free living healthy African buffaloes by long term cocultivation of their leucocytes with bovine thymus or spleen cells. The isolates were indistinguishable from each other or from herpesviruses isolated from a severely ill buffalo calf and from a dead buffalo. These viruses possessed the characteristics of the bovine herpesvirus-3 (BHV-3) group and were indistinguishable by serology and restriction endonuclease analysis from the BHV-3 type strains Movar 33/63 and DN599. There was a 93.6 per cent prevalence of indirect immunofluorescent antibody to BHV-3 in the sera of 94 buffaloes in the sample population. No clinical signs or viraemia were detected in five cattle inoculated with 10(8.7) log10 TCID50 of the isolate from the sick buffalo calf. Two of three cattle hyperimmunised with this virus resisted challenge with malignant catarrhal fever herpesvirus, which proved fatal for the other immunised animal and for three control cattle.

Utilization of human hematopoietic cell lines for the propagation and characterization of HBLV (human herpesvirus 6).

In 1986, we reported the discovery and isolation of a novel human herpesvirus (HBLV) from AIDS and other lymphoproliferative disorders. Because HBLV is distinct from other members of the herpesvirus family and can infect B- and T-lymphocytes and other human cells (megakaryocytes and glioblastoma cells), we suggested human herpesvirus-6 (HHV-6) as the taxonomic designation for this virus. In cultures from patients' peripheral blood, the evidence of HBLV can be recognized from the appearance of short-lived giant cells (2-10%), which are large, refractile, and are often mono- and binucleated. As these cells degenerate, extracellular virus particles are found in the culture medium. HBLV can infect fresh mononuclear cells, established B- and T-lymphoblastoid cell lines, megakaryocytes and glioblastoma cell lines. HBLV infection can be detected by: a. morphological changes; b. indirect immunofluorescence assay, in situ hybridization, southern blot analysis, polymerase chain reaction amplification; and c. electron microscopy. Because of its wide cell tropism, HBLV DNA sequences have been detected in B-cell lymphomas and short term cultured cells from Sjogren's patients. Expression of HBLV RNA was also detected in sarcoidosis. The etiological role of HBLV in human tumors is unclear. While in vitro data may not necessarily apply to in vivo conditions, the infection of various cell lines from tumors and fresh mononuclear cells suggests HBLV involvement in a variety of diseases.

Further biological, serological and biochemical characterization of North American, European and Southeast Asian strains of bovine herpesvirus 1 compared with other alphaherpesvirinae members.

Hemagglutination activity, structural protein profiles and neutralization assays were used in a comparative study of bovine herpesvirus 1 strains from the U.S.A., Canada, Great Britain, Denmark and Malaysia with equine, feline and human herpesviruses in order to further characterize the bovine herpesvirus 1 hemagglutinin. Bovine herpesvirus 1 strains of different geographical origins all showed hemagglutinating activity for mouse erythrocytes; furthermore, feline herpesvirus 1 was also shown to hemagglutinate mouse erythrocytes. Analyses of partly purified viruses showed that a distinctive and specific polypeptides profile is associated with each species of herpesviruses used in our study; strains of bovine herpesvirus 1 from North America, Europe and Southeast Asia however, presented a remarkable similarity as to their electrophoretic protein patterns. A protein similar to the 97-kDa bovine viral hemagglutinin was not identified with the hemagglutinating feline herpesvirus. An important neutralization epitope on the bovine viral hemagglutinin was also not found on feline, equine and human herpesviruses but was identified on all bovine strains tested from North America, Europe and Southeast Asia stressing the importance of the bovine hemagglutinin for eventual prophylactic purposes.

DNA-binding proteins induced by the cottontail rabbit herpesvirus CTHV.

Several new polypeptides were detected in cells infected with CTHV, a cottontail rabbit herpesvirus. All of them (Mr 150K, 110K, 93K, 83K, 75K and 35K) accumulated in the nucleus during the infectious cycle, and all except the 150K species bound to DNA-cellulose affinity columns in low-salt buffers. Polyclonal antisera prepared against the 35K DNA-binding protein also recognized the 75K species. Although the 75K protein could be detected earlier in infection than the 35K protein, late in the infectious cycle the latter increased to an abundance approaching that of cellular histones. Treatment of partially purified virions with a non-ionic detergent indicated that the 35K protein, but not the 75K protein, is a component of capsid/tegument structures. The anti-35K/75K serum did not cross-react with herpesvirus sylvilagus virion proteins, which, in an electrophoretic comparison, exhibited both similarities to and differences from the virion proteins of CTHV. Labelling of CTHV-infected cells with [32P]orthophosphate revealed the presence of phosphoproteins electrophoretically comigrating with the 93K, 83K, 75K and 35K proteins.

Characterization of equine herpesvirus type 1 immediate early proteins.

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.

Comparative polypeptide analysis of human, murine and strigis herpesviruses with murine cytomegalovirus by polyacrylamide gel electrophoresis.

The polypeptide composition of five purified murine herpesvirus (MHV) strains grown in a stable line of rabbit embryo fibroblasts (REF) was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and compared with herpes simplex virus type 1 (HSV-1). About 24 structural polypeptides of molecular mass ranging from 275,000 to 25,000 were identified in MHV and HSV-1. The polypeptide profiles of MHV and HSV-1, showed a close similarity. The polypeptides of MHV were further compared with those of HSV-1, HSV-2, herpes virus strigis (HVS) and murine cytomegalovirus (MCMV). Differences were found between herpesviruses of different origin and MCMV. SDS-PAGE analysis of the six strains of MCMV labelled with 14C-amino acid hydrolysate also revealed differences in electrophor eticprofiles of MHV and MCMV proteins, what was confirmed by densitometric scanning of HSV-1, MHV and MCMV.

A comparison of the acid-soluble polypeptides of five herpesviruses.

The polypeptides soluble in 0.25 M-HCl were extracted from the nuclei of BHK cells infected with herpes simplex virus type 1 or type 2 and separated by SDS-PAGE. Seventeen polypeptides were detectable in each extract of which 10 type 1 and nine type 2 polypeptides were reproducibly effectively extracted. In cells infected with bovine mammillitis virus, pseudorabies virus or equine herpesvirus type 1, at least 12, 13 and eight polypeptides respectively were acid-soluble. In addition to histones, three other cellular polypeptides were present in sizeable quantities in the acid extracts and could obscure other acid-soluble viral polypeptides. Possible relationships between some polypeptides of the five herpesviruses are discussed.

Antigenic and biochemical analysis of gB of herpes simplex virus type 1 and type 2 and of cross-reacting glycoproteins induced by bovine mammillitis virus and equine herpesvirus type 1.

An antiserum was produced to the oligomeric form of glycoprotein B (gB) induced by herpes simplex virus type 1 (HSV-1) strain 17. This antiserum gave a single common precipitin line in agar gel immunodiffusion with HSV-1, HSV-2, bovine mammillitis virus (BMV) and equine herpesvirus type 1 (EHV-1). It also neutralized HSV-1, HSV-2 and BMV but not EHV-1. Absorption of the antiserum with excess HSV-2 or BMV antigen resulted in an HSV-1-specific neutralizing antiserum. In immunoprecipitation, two proteins, gB and pgB, were precipitated from HSV-1- and HSV-2-infected cells and at least three from BMV- and EHV-1-infected cells. Glycoprotein B and pgB of three HSV-1 and three HSV-2 strains and the corresponding antigenically related glycoproteins of BMV- and EHV-1-infected cells were labelled with 125I, digested with trypsin and the resulting peptides separated by two-dimensional thin-layer chromatography or high-pressure liquid chromatography. The resulting profiles were found to be almost identical, suggesting considerable structural conservation of the peptide backbone of the antigenically related glycoproteins of these four viruses.

Gel electrophoretic analysis of polypeptides from nucleocapsids of Marek's disease virus strains and herpesvirus of turkey.

The polypeptides of nucleocapsids of Marek's disease virus (MDV) strains with different biological properties and of antigenically related herpesvirus of turkey (HVT) strains were analysed by one- and two-dimensional (1D and 2D, respectively) gel electrophoresis. Based on small differences in migration behaviour (size and charge) of a number of corresponding nucleocapsid polypeptides, the virus strains could be differentiated into three groups. The polypeptide pattern of group I, comprising the virulent MDV-strain K and the attenuated strains, HPRS-16/att and CVI988 37th passage, was composed of four major polypeptides (i.e. 140K, 50K, 40K and 33K daltons) and at least four minor polypeptides. The pattern of group II, comprising the naturally occurring non-oncogenic MDV-strains SB-1 and HPRS-24, contained one additional major polypeptide of 39K daltons. The nucleocapsid-specific 2D polypeptide patterns of the HVT strains HVT-Fc 126 and PB-THV1, comprising group III, were distinguishable from each other on the basis of a small difference in size of one major 50K polypeptide. Results were further substantiated by coelectrophoresis experiments.

Immunogenic proteins of feline rhinotracheitis virus.

Feline rhinotracheitis virus is an upper-respiratory-tract pathogen of cats. It may also cause generalized infections or abortions. Antigens present in [35S]methionine- or [14C]glucosamine-labeled purified virions, in Nonident P-40 (NP-40) extracts of a mixture of virions and infected cells, and in virion-free cell culture medium, along with mock-infected Crandell -Rees feline kidney cell controls, were analyzed by direct sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or by SDS-PAGE preceded by Staphylococcus aureus protein A immunoprecipitation. The direct SDS-PAGE analysis revealed at least 17 virus-specific peptides with molecular weights ranging from less than 200,000 ( 200K ) to more than 30K . Three of these peptides were glycosylated and had molecular weights of 105K , 68K , and 60K. Immunoprecipitates of purified virions and NP-40 extracts contained three major glycoproteins with the same estimated molecular weights as those found by the direct analysis. A prominent 105K glycoprotein was present in virion-free cell culture medium immunoprecipitates. In addition, a number of nonglycosylated feline rhinotracheitis virus-specific polypeptides (eight in virions, three in NP-40 extracts, and nine in virion-free cell culture medium), ranging in molecular weight from 145K to 32K, were present in the various immunoprecipitates.