Evidence for incomplete replication of a penguin poxvirus in cells of mammalian origin.

The recent discovery of a novel poxvirus [penguin-pox virus (PPV)] from Jackass penguins offers the potential of a unique candidate vaccine vector for use in mammals. Infectivity studies were therefore undertaken using a number of mammalian cell lines and chick embryo fibroblasts (CEF). It was shown that the simian CV-1 cell line was able to support replication of the PPV DNA, but no infectious progeny virus could be recovered from the infected cells. Electron microscopy was used to establish the extent of virus morphogenesis in CV-1 cells as compared to that in both chorio-allantoic membranes (CAMs) of hens' eggs and CEF cells. It appears that CV-1 cells are able to support partial maturation of PPV, but that morphogenesis does not proceed to the stage of mature infectious particles. Vaccinia virus promoters were successful in achieving transient gene expression in PPV-infected cells.

Direct immunogold labeling of herpesvirus suspensions.

Gold particles in colloidal suspension are particularly well suited as markers for immune electron microscopy. Their extreme electron opacity ensures that they are detected with accuracy even at particle sizes of less than 3 nm. Gold spheres can be made easily and inexpensively by reduction of gold chloride with mild acid and heat (1), and particles can be prepared in a variety of sizes by varying the nature of the reducing agents (2).

DNA single strand conformation polymorphism identifies five defined strains of hepatitis B virus (HBV) during an outbreak of HBV infection in an oncology unit.

An outbreak of hepatitis B virus (HBV) infection in a children's oncology unit was identified in which 61 children were shown to have been infected, 59 of them asymptomatically. In order to establish whether intra-unit cross infection had occurred, we used the single strand conformational polymorphism (SSCP) technique to analyse viral isolates from 57 of the infected children and 40 unrelated controls. HBV-specific primers were designed to amplify a 189 bp fragment of DNA encompassing part of the hypervariable pre-S1 region of the HBV genome. Denatured PCR products were compared after electrophoresis through polyacrylamide gels and staining with silver. By SSCP analysis, the unrelated infections each yielded a unique electrophoretic banding pattern, indicative of a variety of distinct virus strains. In contrast, most of the oncology patients had been infected with one of only five different strains. Three major groups comprising 19, 16, and 9 patients, respectively, and two minor groups of 5 and 3 patients were identified. Results indicate the occurrence of multiple episodes of cross infection, and demonstrate the sensitivity and value of SSCP as a technique to establish common sources of infection.

Intra-nuclear localization of two envelope proteins, gB and gD, of herpes simplex virus.

The envelopes of herpes simplex virus (HSV) particles are acquired from the inner nuclear membrane (INM) of the infected cell and virus-coded glycoproteins are present in the envelope of mature virions. Our ultrastructural study examined the process of virus envelopment and the targeting of two major viral glycoproteins, gB and gD, to the INM in HSV-infected human embryonic fibroblasts. It was shown that envelopment and transport of virus particles from the nucleus is facilitated by the formation of a dynamic tubulo-reticulum arising from the INM. Capsids were assembled in the nucleus and collected within INM tubules which protruded into the perinuclear space and thence into the cisternae of the endoplasmic reticulum (ER). Envelopment occurred by constriction and fusion of the tubular channel walls, releasing enveloped virions into the ER. Transport to the cell surface took place in membrane-bound compartments and probably followed the normal secretory pathway through the Golgi apparatus. Immunogold probes, tagged with specific monoclonal antibodies, were used to localize gB and gD during the process of virus maturation. Cytoplasmic membranes were not labelled, but probes bound inside the nucleus, mainly at sites of virus assembly. Labelling occurred on the nucleoplasmic side of the INM which surrounded capsids in the process of envelopment, but not on the outside of that membrane, although characteristic gB glycoprotein spikes were labelled on the envelopes of extracellular virus particles and on virions in trans-Golgi transport vesicles just prior to their release from the infected cell. gB was not detected on the surface of enveloped virions in the perinuclear space, or the cisternae of the ER or cis-Golgi, which suggests that the specific epitope was masked during that stage of intracellular processing. gD probes bound to virion envelopes and also to the tegument region of some particles found in both perinuclear and extracellular sites. We postulate the precursor core proteins for both gB and gD are transported first to the nucleus, and then, together with maturing capsids, are targeted to the INM, and later inserted into viral envelopes at the site of budding. Post-translational glycosylation of envelope proteins could occur as virus particles exit the nucleus and travel through the ER and Golgi compartments.

Immunogenicity of influenza and HSV-1 mixed antigen ISCOMs in mice.

Immunostimulating complexes (ISCOMs) were prepared with mixtures of antigens from influenza A virus (A/PR/8/34 or A/Sichuan/2/87) and herpes simplex virus type 1 (HSV-1), and were characterised by enzyme linked immunosorbent assay (ELISA) and electron microscopy using double-labelling immunogold techniques employing monoclonal antibodies to influenza or HSV-1 glycoproteins. The immunogenicity of the mixed antigen ISCOMs was evaluated in mice, following administration by the subcutaneous route, by measuring the total and subclass IgG antibody responses. Protection of these animals against challenge with live influenza A/Sichuan virus or live HSV-1, was compared with that induced by immunization with aqueous mixed antigen preparations. It was found that relatively high humoral responses to both influenza and HSV antigens, and increased levels of protection to both influenza and HSV viruses were elicited in mice receiving the mixed antigen ISCOM preparation compared to those observed in animals receiving the mixed aqueous subunit preparation. The findings also indicate that antigens from more than one virus can be used in an ISCOM formulation to produce immunity and protection.

An Fc receptor for human immunoglobulin G is located within the tegument of human cytomegalovirus.

Immunogold electron microscopy has demonstrated that human immunoglobulin G (IgG) can bind to the tegument of human cytomegalovirus virions by the Fc portion of the molecule. This binding was inhibited by preincubation of the Fc probes with protein A. Treatment of AD169 virions with Triton X-100 allowed release of the Fc-binding proteins, which were precipitated and characterized by polyacrylamide gel electrophoresis (PAGE). Polypeptides of approximately 69 and 33 kDa were recovered and shown by immunoblotting to retain their capacity to bind Fc-gold after separation under both reducing and nonreducing conditions. The combined results of blocking experiments, PAGE of precipitates, and Western blots (immunoblots) indicate that the tegument proteins which bind IgG-Fc are identical to those which bind beta 2 microglobulin.

Beta 2 microglobulin binds to the tegument of cytomegalovirus: an immunogold study.

Previous reports have provided evidence for the ability of human cytomegalovirus (HCMV) to bind the host protein beta 2 microglobulin (beta 2m) from body fluids or culture medium, and thus enhance infectivity of the virus, both by evasion of immune neutralization and the capacity to employ the bound beta 2 m for attachment to the host cell. Immunocytochemical techniques and negative stain electron microscopy were used to identify the ultrastructural components of HCMV involved in its interaction with beta 2m. Probes comprising colloidal gold coupled to beta 2m were seen to bind not to the envelope as previously suspected, but to material closely surrounding the nucleocapsids. It is postulated that the tegument proteins of HCMV, via their capacity to bind beta 2m, play an important role in the preservation of infectivity of disrupted virions by enabling unenveloped capsids to bind to cells and gain entry by a pathway other than that normally taken by intact virions.

Morphology and distribution of gp52 on extracellular human cytomegalovirus (HCMV) supports biochemical evidence that it represents the HCMV glycoprotein B.

Glycoprotein gp52 exists within the mature human cytomegalovirus (HCMV) envelope in heterodimeric, disulphide-linked complexes with glycoproteins gp95 and gp130. Biochemical studies involving immunoprecipitations and Western blots have demonstrated that gp52 is the glycoprotein B (gB) homologue of HCMV but that gp95 and gp130 are probably separate gene products. The distribution of this putative gB on extracellular HCMV particles was revealed by high resolution electron microscopy of preparations labelled with a monoclonal antibody, F5, directly coupled to colloidal gold. F5-gold probes, specific for HCMV gp52, bind to the distal end of 12 nm long, slender spikes projecting from virion and dense body envelopes. Labelled spikes were most often present in closely packed, homogeneous clusters and were frequently present on envelope protrusions. The degree of labelling on individual HCMV particles was highly variable. Both the morphology and distribution of HCMV gp52 show strong similarity with that previously reported for the gB of herpes simplex virus. Other morphologically distinct spikes occur in the HCMV envelope but these were not recognized by F5-gold probes.

The morphology of human immunodeficiency virus particles by negative staining electron microscopy.

Negative staining electron microscopy was used to examine culture fluids from the H9/HTLV-III cell line after concentration by centrifugation. Characteristic retrovirus-like particles bearing distinctive envelope projections were seen. The virion envelope was frequently extended in the form of a bleb or a tail. These particles were morphologically virtually indistinguishable from similar preparations of Friend murine leukaemia virus. H9/HTLV-III culture fluids contained, in addition, numerous comet-shaped particles with a dense head and flared tail. These particles were clumped by the addition of anti-HTLV-III-positive serum suggesting that they may represent intermediate forms of the virus.

Herpes simplex virus glycoproteins associated with different morphological entities projecting from the virion envelope.

Spikes of different kinds, distinct in size and appearance were detected on the surfaces of herpes simplex virions by electron microscopy of negatively stained preparations. Use of monoclonal antibodies coupled to colloidal gold permitted identification of viral glycoproteins present in different structures projecting from the virion envelope. Antibodies specific for the glycoprotein designated gB bound to the most prominent spikes, which were about 14 nm long and, in side view, had a flattened T-shaped top. Antibodies specific for gC bound to structures that, in some instances, appeared to extend as much as 24 nm from the surface of the envelope and were too thin to resolve. Antibodies specific for gD bound to structures that extended as much as 8 to 10 nm from the surface of the envelope. The gB spikes were invariably clustered, usually in protrusions of the envelope varying from small bulbous distentions to long tail-like projections. The gC components were randomly distributed and widely spaced and the gD components were irregularly clustered in patterns distinct from those of the gB spikes. These three glycoproteins therefore form structures that are different in size, morphology and distribution in the envelope.

Hepatitis B virus associated membranous glomerulonephritis.

The incidence of persistent hepatitis B surface (HBs) antigenaemia was studied in 114 nephrotic children with glomerulonephritis. Twenty five (24 boys) of 28 cases of membranous glomerulonephritis were HBs antigen (HBsAg) carriers. Only 9 of the remaining 86 patients with nephropathies other than membranous glomerulonephritis were HBsAg positive. HBsAg immune complexes were seen in the sera by electron microscopy. On radioimmunoassay both HBsAg and antibody (anti-HBs), and HBeAg and antibody (anti-HBe) were often detected concurrently, HBsAg was not shown in the glomerular capillary wall. HBs antigenaemia persisted in 80% of patients after recovery from glomerulonephritis but remission of the proteinuria correlated well, although not fully, with seroconversion to anti-HBe. The natural history of hepatitis B virus (HBV) associated glomerulonephritis in childhood is one of slow recovery. A few patients are left with mild asymptomatic proteinuria but progressive renal failure is rare. The 14% incidence of membranous glomerulonephritis in nephrotic children in this area is much higher than that found by the international study of kidney disease in children in well developed countries and is probably related to a high HBV carrier rate. A search for HBV markers should be included in the investigation of persistent glomerulonephritis, particularly in countries with a high prevalence of HBV carriers.

Antigenic cross-reactions between woodchuck hepatitis virus and human hepatitis B virus shown by immune electron microscopy.

Using immune electron microscopy (IEM), low-level cross-reactions could be demonstrated between the surface antigens of hepatitis B and woodchuck hepatitis. However, immune complex formation was greatly enhanced by pre-exposure of the antigens to 0.5% deoxycholate. Cross-reaction between the core antigens and e antigens of both viruses was also confirmed by IEM as well as radioimmunoassay. It appears that the woodchuck sera used in this study may well contain an anti-immunoglobulin akin to rheumatoid factor.

An electron microscopic demonstration of immune complexes of hepatitis B e-antigen using colloid gold as a marker.

Immune complexes of the hepatitis B e-antigen (HBeAg) could be labeled and thus visually identified in the electron microscope by using antibody to HBeAg (anti-HBe) tagged with colloidal gold particles. Circulating immune complexes of HBeAg were detected in sera from patients with acute hepatitis B infections as well as from asymptomatic carriers of hepatitis B surface antigens (HBsAg). Sera positive for rheumatoid factor frequently contained mixed aggregates in which immune complexes of HBsAg were closely bound to immune complexes of HBeAg.

A new phenomenon (SMOG) associated with solid phase immune electron microscopy.

Solid phase immune electron microscopy (SPIEM) has been used to study the interaction of virus and antibody both quickly and with economy of reagents. Unexpectedly, the present study shows that when virus-coated grids are floated on drops of specific antibody the virus particles migrate to form complexes. This has been termed specific migration on the grid (SMOG) and it is suggested that it can be used both to assess the properties of an antiserum and to make virus more readily detectable. The mechanism by which SMOG occurs is not understood but various possibilities are discussed.

The role of rheumatoid factor in an electron microscope study of hepatitis B antigens.

An immune electron microscopic (IEM) study of the hepatitis B e antigen (HBeAg) system has shown that rheumatoid factor (RE) can be an important complicating factor when examining preparations containing multiple antigenic specificities. For example, in the presence of RF, mixed immune complexes were produced that suggested HBeAg might be antigenically related to either Dane particles or cores. When RF was removed, however, the putative HBeAg--anti-Hbe complexes showed no relationship with any other hepatitis B component. It has been shown that RF can have a positive practical application in IEM by using it to link preformed marker complexes to immune complexes which do not contain morphologically recognizable antigen.

Morphological irregularities in Dane particle cores.

Electron microscopy of hepatitis B antigen has revealed Dane particles with abnormal morphology. These aberrant Dane particles contain incomplete cores. They were seen in large numbers in the serum of an HBsAg-carrying renal dialysis patient and were less numerous but invariably present in other Dane particle-containing sera --12 from asymptomatic carriers, 2 chronic hepatitis patients and 1 patient with acute hepatitis. All 16 sera were shown to contain HBeAg.

Observations on the morphology of two rotaviruses.

The negative staining technique was used to study the morphology of two rotaviruses, the epizootic diarrizootic diarrhoea of infant mice (EDIM) and the simian virus SA II. It is proposed that the inner capsid of the virion has icosahedral symmetry and consists of 180 morphological units arranged in an open lattice formation with the 12 spaces at the apices being surrounded by 5 capsomeres and the other 80 spaces each surrounded by six capsomeres. The outer capsid of the virion consists of a honeycomb-like lattice which corresponds to the lattice arrangement of the inner capsid.

Hepatitis B antigen: IgG components shown by immune electron microscopy.

Immune complexes could be formed with hepatitis B Ag and anti-IgG serum after treatment of the antigen with detergent, CsCl or glycerol, but not before. It is suggested that an antigenic determinant specifically reacting with anti-IgG forms an integral part of hepatitis B Ag. This determinant is ordinarily obscured in the undamaged antigen.

The problem of the demonstration of hepatitis B antigen in faeces and bile.

Attempts to detect hepatitis B antigen in faeces and bile should take into account the degradation and disappearance of the surface antigens in an environment containing proteolytic enzymes in the presence of bile salts as in the intestinal lumen. The cores of the Dane particles are much more stable than the surface antigens and these may best be identified by immune electron microscopy using core antibody.