Characterization of the major glycoproteins of equine herpesviruses 4 and 1 and asinine herpesvirus 3 using monoclonal antibodies.

A panel of 14 monoclonal antibodies (MAbs) was used to characterize the high abundance glycoproteins of equine herpesviruses 4 (EHV-4) and 1 (EHV-1), and asinine herpesvirus 3 (AHV-3). The specificities of the MAbs, which had been determined previously for strains of EHV-4 and -1 from the U.S.A., in general were confirmed by ELISA for Australian strains of these viruses. Of the 14 MAbs seven were EHV-4 and -1 type-common and cross-reacted with AHV-3. Of the five MAbs that were EHV-1 type-specific, four cross-reacted with AHV-3, whereas neither of the EHV-4 type-specific MAbs reacted with AHV-3, providing further evidence for a closer evolutionary relationship between EHV-1 and AHV-3 than that between either of these viruses and EHV-4. By Western blot and immunoprecipitation analyses, the identity of the six major glycoproteins, gp2, gp10, gp13, gp14, gp18 and gp21/22a, of an Australian EHV-1 isolate was verified, and it was shown that AHV-3 had cross-reactive glycoproteins of very similar Mr to those of EHV-1; five homologous glycoproteins of EHV-4 were also identified. It was determined that the EHV-4 gp13 homologue had a much reduced Mr (67K) when the virus was grown in a continuous cell line than when grown in equine foetal kidney cells (95K). It is suggested that altered glycosylation by the cell line is responsible for this change in Mr. Those glycoproteins acting as major immunogens in the naturally infected host, at least in their ability to elicit antibody, were identified. It was found that gp2, gp13, gp14, gp18 and a glycoprotein at 120K (EHV-1) or 116K (EHV-4) were all important immunogens in mares following EHV-1-induced abortion, and in a specific pathogen-free foal experimentally infected with EHV-1 and later cross-challenged with EHV-4. Gp2, gp14 and gp18 were the major immunogens in the donkey in response to AHV-3 infection. The type specificity associated with these glycoproteins was also examined and it was found that although most if not all contain type-specific epitopes, gp2 and a glycoprotein at 120K, and to a lesser extent gp13 and gp18, were significantly type-specific in the serum from a mare following natural EHV-1 infection and abortion.

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.

Comparison of proteins of simian herpesvirus aotus type 2 and bovine herpesvirus type 4.

Genomes of herpesvirus aotus type 2 (HVA-2) and bovine herpesvirus type 4 (BHV-4) have previously been shown to be closely similar. Moreover, preliminary serological data indicated that HVA-2 is antigenically related to BHV-4. To extend this study, structural components of four BHV-4 strains and HVA-2 were compared by SDS-PAGE, radioimmunoprecipitation and Western blotting. The overall pattern of structural proteins was the same for HVA-2 and BHV-4 but variations were observed in electrophoretic profiles of glycoproteins, mainly of the two major ones (gp6/gp10/gp17 and gp11/VP24). Variations between HVA-2 and BHV-4 glycoproteins were greater than those observed among BHV-4 strains.

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.

Viremia and neutralizing antibody response in infants with exanthem subitum.

Mononuclear cell-associated viremia caused by human herpesvirus type 6 was detected in 39 (66%) of 59 blood samples from 38 children with exanthem subitum between day 0 and day 7 of the disease. The rate of virus isolation from mononuclear cells was 100% (26/26) on days 0 to 2 (just before appearance of skin rash), 82% (9/11) on day 3, 20% (2/10) on day 4, 7% (2/12) on days 5 to 7, and 0% (0/37) on day 8 and thereafter. The cell-free virus was detected in blood in 10 (21%) of 47 blood samples during the same period. The antibody activity to the virus, evaluated by a newly developed neutralization assay, was first detected on day 3 of the disease with a positive rate of 18% (2/11). It became 60% (6/10) on day 4, 75% (9/12) on days 5 to 7, and 100% on day 8 and thereafter. Thus the disappearance of the virus from blood was associated with the induction of specific immunity to the virus.

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.

Application of cloned fragments of equine herpesvirus type-1 DNA for detection of virus-specific DNA in equine tissues.

Tissue specimens obtained from equine herpesvirus-1 (EHV-1), subtype 1-infected aborted foetuses were analysed for the presence of virus DNA by means of Southern blot and dot blot hybridisations. The specificity of the methods was confirmed although the sensitivity was inferior to classical techniques such as virus isolation. However, the possibility of detecting the state of the virus DNA and the ability to distinguish between subtypes were important features, and the dot blot method was shown to have potential for a rapid diagnostic test. This report demonstrates some potential practical applications of hybridisation methods for studying the pathogenesis and epidemiology of EHV-1 but also reveals limitations of the techniques.

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.

Vertical transmission of channel catfish virus.

Channel catfish broodfish were examined nondestructively for the presence of channel catfish virus (CCV), using a nucleic acid-probing technique. A dot blot assay was tested and shown to be accurate and rapid in the diagnosis of CCV. Two CCV-positive fish were successfully mated, and fertilized eggs were collected. Offspring that hatched were tested and were shown to have CCV. This result indicated vertical transmission of CCV, a herpesvirus.

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.

Comparison of a cheetah herpesvirus isolate to feline herpesvirus type 1.

A cytopathogenic virus with size and structural characteristics of a Herpesviridae was isolated from a cheetah with severe ulcerative dermatitis. Restriction endonuclease analysis and cross-hybridization studies revealed that the isolate was related to feline herpesvirus type 1 (FHV-1). Antigenic comparison studies using anti-FHV-1 serum demonstrated the presence of common antigens in the FHV-1 and the isolate from the cheetah.