Morphology of hepatitis C and hepatitis B virus particles as detected by immunogold electron microscopy

We performed indirect immunogold electron microscopy (EM) for immunological identification and characterization of hepatitis C virus (HCV). To clarify the morphology of HCV, an indirect immunogold EM of two plasma samples from patients with high HCV RNA titers was carried out using antibodies specific for the putative HCV envelope protein (E) 1. Spherical virus particles 55–65 nm in diameter with delicate spike projections were detected in the 1.14–1.16 g/ml fractions after sucrose density gradient centrifugation. Polyclonal and monoclonal antibodies to the putative HCV E1 specifically recognized these particles. In addition, immunogold EM of the samples was also performed to uncover the morphology of HCV core particles. Spherical particles 33–40 nm in diameter (average, 37 nm) were detected in the 1.22- to 1.25-g/ml fractions by conventional EM after sucrose density gradient centrifugation. Immunogold EM using rabbit polyclonal antibody (RR8) specific for the putative HCV core protein and colloidal gold-labeled goat antirabbit IgG showed binding of the gold particles with RR8. Some of the HCV core particles showed icosahedric morphology. Optical rotation technique showed that the HCV core particles exhibit sixfold symmetry and that the length of the regular hexagon side is approximately 20 nm, suggesting that they have an icosahedric structure. Further, the detection limit of the indirect immunogold EM was evaluated in 11 plasma samples from chronic hepatitis B patients with different degrees of hepatitis B virus (HBV) DNA titers using antihepatitis B surface antigen antibody. The study showed that the detection limit of virus using this method is 10⁷ virions/ml.     

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

A mouse model of hepatitis B virus (HBV) infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.     

Unidentified virus-like particles are detected in plasmas with elevated ALT levels: are they significant of etiological agent(s) of non-B,non-C hepatitis?

GB virus C (GBV-C) and hepatitis G virus (HGV) have been proposed as new viruses etiologically implicated in non-B, non-C hepatitis, but the morphology of these particular virus particles is still unknown, and most cases of non-A to E hepatitis do not relate to their infections. We tried to visualize virus-like particles (VLPs) in plasma samples from hepatitis B surface antigen- and antibody to hepatitis C virus (HCV)-negative blood donors with elevated alanine aminotransferase (ALT), and examined the association of the virus-like particles and the genomes of parenterally transmissible GBV-C/HGV. Twenty-three plasma samples, 13 with elevated ALT levels and 10 with normal ALT values, from blood donors without infections of hepatitis B virus (HBV) and HCV, were subjected to a 20%–60% sucrose density gradient centrifugation, and virus-like particles were observed by electron microscopy. GBV-C/HGV RNAs in the plasmas were tested. Virus-like particles were found in the fractions with densities of 1.15–1.16 g/ml from 12 of 13 (92.3%) plasmas with elevated ALT levels and 1 of 10 (10%) normal controls. The ultrastructural morphology of visualized VLPs was pleomorphic in size and appearance; the majority of the VLPs were 50- to 80-nm spherical particles with a 35- to 45-nm inner core and 9- to 12-nm-long surface spikelike projections. Rodlike VLPs 50–70 nm in diameter with a length of 110–160 nm were also observed in the same samples. The incidence of detection of the circulating VLPs was significantly (P < 0.001) related to elevated ALT levels, but GBV-C/HGV RNAs were detected in none of the plasmas containing the virus-like particles. Spherical VLPs are detected in HBV- and HCV-negative plasmas significantly correlated with the elevation of ALT, suggesting that they are implicated in non-B, non-C hepatitis.     

Dane particles and associated DNA-polymerase activity in saliva of chronic hepatitis B carriers

To investigate furhter the problem of salivary transmission of type B hepatitis, salivas free of blood contamination from three HBsAg-positive carriers with chronic active hepatitis were examined by CsCl equilibrium density gradients and electron microscopy (EM). In the CsCl gradient HBsAg of whole salivas distributed in a band centered at 1.19gm/cm3 with a clearly defined shoulder at 1.24 gm/cm3; the HBsAg was found mainly in the mucous component. On EM examination, fractions from the 1.19 gm/cm3 peak contained spherical HBsAg particles of 22 +/- 3 nm diameter, whereas in the 1.24 gm/cm3 shoulder Dane particles 43 nm in diameter with 28 nm cores were found. Specific hepatitis B virus associated DNA-polymerase activity also was found in the 1.24 gm/cm3 shoulder where the Dane particles occurred and was absent from the saliva of healthy controls. When salivas were incubated for three hours at 37 degrees C the total amount of HBsAg diminished and the 1.24 gm/cm3 shoulder disappeared, probably as a result of endogenous degradation of the Dane particles and the free HBsAg. These findings clearly indicate that the hepatitis B viral particle is present in the saliva of chronic HBsAg carriers with active disease and further confirm that saliva is an important vehicle of infection.     

Analysis of intermolecular disulfide bonds and free sulfhydryl groups in hepatitis B surface antigen particles

Summary.  The envelope proteins of hepadnaviruses are highly cross-linked by disulfide bonds in complete virions and 20 nm subviral envelope particles. We have previously shown which of the cysteines in the envelope proteins of the human hepatitis B virus (HBV) are essential for assembly and secretion of 20 nm particles and for the structure of the major antigenic determinants (HBsAg). Now we have analyzed the intermolecular disulfide bonds between S proteins. We have constructed mutants lacking cysteines and have analyzed their capacity for oligomerization in COS-7 cells. We demonstrate that C121 and C147 located in the second hydrophilic region carrying the major antigenic determinants of the HBV S protein participate in intermolecular disulfide bonding. A disulfide bond involving C124 blocks the accessibility of arginine/lysine at position 122, as shown by trypsin digestion of cysteine mutants. Alkylation studies using N-ethyl-maleimide indicate that C76, C90, and/or C221 carry the only free sulfhydryl group(s) present in 20 nm particles secreted from cell lines. Received April 8, 1997 Accepted June 12, 1997     

Ultrastructural and Stereological Study of the Liver in Chronic Mixed HCV + HBV Infection

The stereological values of the structural density of hepatocyte cytoplasmic organelles were similar in chronic mixed HCV + HBV infection irrespective of the form of HBV infection (seropositive or latent). High incidence of HBsAg- and HBeAg-negative forms of HBV infection determines the leading role of PCR diagnosis and studies of liver biopsy specimens with detection of HBcAg structural markers and HBV DNA in native liver tissue in the diagnosis of mixed HCV + HBV infection.     

Effect of multiple freezing and thawing of serum on TT virus and hepatitis B virus DNA positivity

Summary.  This study was done to determine the effect of freezing and thawing of serum on the stability of TTV and HBV DNA levels. Seven TTV DNA positive samples were randomly selected among the sera having HBV DNA with concentrations ranging from 12 pg/ml to 4162 pg/ml and they were frozen and thawed up to eight times and then analyzed for changes on TTV- and HBV DNA levels. TTV DNA positivity and HBV DNA concentrations were tested by using semi-nested PCR and Digene hybrid capture system, respectively. Seven cycles of freezing and thawing did not significantly change HBV DNA concentrations and TTV DNA positivity in any of the samples tested. After eight cycles, only three samples were tested, and all were positive for HBV DNA, but negative for TTV DNA. Our results show that both TTV- and HBV DNA positives continued until the seventh cycle of freezing and thawing in all samples tested. Received July 23, 2001 Accepted November 15, 2001     

Biochemical characterization of Australia antigen. Evidence for defective particles of hepatitis B virus.

Australia antigen exists in the sera of chronic carriers in several particulate forms, one of which may represent the virion of hepatitis B. This report describes the existence of subpopulations of these 43-nm particles, the Dane particles, on the basis of the staining properties of their internal cores and banding characteristics in cesium chloride (CsCl) density gradients. These data suggested that only a minor proportion of Dane particles contained an intact viral genome and represent the standard infectious virus of hepatitis B. The bulk of the Dane particles appeared to be deficient in viral nucleic acid and, as defective interfering particles, may specifically interfere with the growth of standard virus. Such defective interfering particles could thereby play a role in the persistence of HBV infection in man.     

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.     

A new small temperate DNA phage BcP15 isolated from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> DR11

Summary. A Burkholderia cepacia DR11 strain was isolated during the survey of microorganisms from coastal water of deltaic Sunderbans. This strain always released temperate phage BcP15 into culture supernatant. UV irradiation of the strain also induced phage induction. The phage titer was 2.3 × 10⁸. New temperate phage BcP15 has unusual structure. It has a hexagonal head, 65 nm in diameter and a tail 200 nm long, attached with single thick wavy tail fiber (424–705 nm). Phage DNA is double stranded 11.9 kb long. Southern hybridization result indicated that the phage DNA was in lysogenic state into the B. cepacia DR11 genome. SDS-PAGE of phage protein showed two major bands of molecular weight 20 kDa and 40 kDa.     

Detection of HBV infectivity by spot hybridization in HBeAg-negative chronic carriers: HBV DNA in sera from asymptomatic and symptomatic subjects

DNA of hepatitis B virus (HBV DNA) in sera from HBeAg-positive carriers is now the most important and reliable marker of infectivity, but its significance in the progression of chronic hepatitis in anti-HBe carrier status is still under discussion. In this study, viral DNA was tested by a simplified spot hybridization method in sera of 206 HBeAg-negative Italian subjects. In a group of 153 HBsAg carriers, we found that 15.6% of anti-HBe-positive and 10.5% of anti-HBe-negative samples contained viral DNA. No HBV DNA was revealed in 38 HBsAg-negative nor in 15 anti-HBs-positive subjects with different serological markers of HBV. Viral DNA in sera of HBeAg-negative patients with severe chronic liver disease was correlated with increased alaninetransferase activity and IgM anti-HBc. Thus the presence of HBV DNA in these sera not only predicts which subjects are potentially infectious but also indicates chronic progression of hepatitis. Finally, viral DNA extracted from Dane particles of nine anti-HBe-positive sera was characterized by the Southern blot technique. The hybridization pattern shows bands indicating the presence of replicative intermediates.     

Characterization of large surface proteins of hepatitis B virus by antibodies to preS-S encoded amino acids

The major surface protein of HBV, the 226-amino-acid HBsAg, is encoded in the 3' proximal segment of the preS-S gene of 389 codons. To identify gene products from the 5' proximal preS sequence, DNA fragments from the preS region were expressed in Escherichia coli as fusion proteins. Antisera prepared against these fusions were used to screen serum proteins of HBV-infected individuals, and found to react specifically with the two large HBV surface proteins of 39 and 42 kDa. The presence of these proteins could be correlated with acute HBV infection. Analysis by Western blotting using the preS sequence-specific antisera and HBV particles separated into spheres, filaments, and Dane particles confirmed that these proteins were associated with the native virus. Dane particles containing active DNA polymerase could be immune precipitated by the preS-specific antibodies, showing that the preS-coded part of these surface proteins is located on the surface of the virion.     

Hepatitis B virus (HBV) infections in turtles

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.     

Quantitation of hepatitis B virus DNA (HBV DNA) in serum using the spot hybridization technique and scintillation counting

The simple spot hybridization technique to detect HBV DNA in serum was modified to concentrate Dane particles by pelleting. This minimised interference by serum components and allowed larger volumes of sera (up to 500 microliters) to be used in serial dilution on borderline positive samples and increased the efficiency of filtering through a "Hybri.Dot' system. Quantification of HBV DNA by 32P-scintillation Cerenkov counting based on serial standards of cloned HBV DNA processed through the "Hybri.dot' was easy to perform and the assay had good sensitivity (detection limit 2 pg, mode 6.25 pg), precision and accuracy. The pellet-simple spot quantitative assay proved more sensitive than those involving lengthier extraction procedures or direct application of serum to hybridization filters. Scintillation counting curves were linear over a wider range (0-800 pg HBV DNA) than optical densitometry measurements (0-50 pg) of autoradiographs. There was an excellent correlation with DNA polymerase activity (r = 0.948; P less than 0.001) but the assay proved more sensitive since HBV DNA (greater than 45 pg cloned equivalents) was detected in 4 out of 14 DNA polymerase negative sera and in 7 of 7 borderline (DNA polymerase cpm range 116-303) samples. This sensitive, quantitative HBV DNA assay should be of considerable value in studies on infectivity and effectiveness of antiviral therapies.     

Partial characterization of a new virus from ranunculus with a divided RNA genome and circular supercoiled thread-like particles

Summary.  An undescribed virus, here named ranunculus white mottle virus, was isolated in Italy from cultivated ranunculus showing mottle and distortion of leaves. The virus was mechanically transmissible to several herbaceous hosts. In negative stain, the particles appeared as circularised supercoiled threads 3 nm in diameter of different contour lengths; in some conditions the circles collapsed to form linear pseudobranched structures 9 nm in diameter. Immunolabeling of thin sections showed that viral antigen was widely distributed in the cytoplasm of parenchyma cells. The virus was not serologically related to the morphologically similar tenuiviruses, citrus psorosis-ringspot virus and tulip mild mottle mosaic virus. A major 43 kDa protein was present in purified preparations and in infected plant tissue, as also was a minor 28 kDa protein, serologically related to the major one. Nucleic acids extracted from purified particles consisted of at least three RNAs, of approximately 7.5, 1.8 and 1.5 kb, which appeared partly in single- and partly in double-stranded form. Purified preparations, but not viral RNAs, when mechanically inoculated, were infectious. Host range, tissue tropism, particle morphology and coat protein size place the virus closest to citrus psorosis-ringspot and tulip mild mottle mosaic viruses. These three viruses in turn show similarities with the Tenuiviruses and Bunyaviridae. Received May 5, 1997 Accepted July 9, 1997     

Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80–84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radio-labeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited. Received: 9 July 1997 / Accepted: 3 November 1997     

Ultrastructural and physicochemical characterization of the hepatitis C virus recovered from the serum of an agammaglobulinemic patient

Summary.  Hepatitis C virus (HCV) morphology and physicochemical properties remain unclear because HCV usually circulates in a complexed form in association with immunoglobulins. In the present work, we were interested in the characterization of HCV particles derived from the serum of an anti-HCV negative/HCV RNA positive agammaglobulinemic patient suffering from chronic type C hepatitis. Physicochemical properties of the virus particles were determined by serum centrifugation on a 10 60% isopycnic sucrose density gradient. HCV RNA quantified by bDNA was found in a major peak at density 1.13 g/ml and in a minor peak at densities 1.05 1.07 g/ml. By electron microscopy, 45 nm large core-like particles were found at the 1.13 g/ml density while 60 nm large virus-like particles similar to other members of the Flaviviridae family were visualized at the 1.06 1.07 g/ml densities. This confirms some studies reporting the low density of HCV as compared to other members of the Flaviviridae family. Received April 23, 1998 Accepted June 24, 1998     

Antigenicity of mouse hepatitis virus strain 3 subcomponents in C57 strain mice

Summary Morphogenesis of human rotavirus type 2 Wa strain in MA 104 cells was observed. The virus antigen in the cytoplasm was detected by indirect immunofluorescence twelve hours after infection. The cytopathic effect occurred 24 hours after infection when virus particles were detected by EM in the culture fluid as well as in thin sections of the infected cells. Virus particles were observed in the dilated RER, nuclear envelope (perinuclear space), viroplasm, and a lysosome-like body. Three types of virus particles were noted: double-shelled particles 75–85 nm in diameter, single-shelled particles 64–68 nm in diameter and electron-dense nucleoids or cores 32–40 nm in diameter. The outer shell of virus particles was acquired by budding through the membrane of the dilated RER. Tubular structures, similar in diameter to the single-shelled particles, were found in the cytoplasm and nucleus of infected MA 104 cells. Bundles of filaments or the leaflet-like inclusion bodies of membrane-bounded bundles of filaments were found in the cytoplasm and seemed to be associated with virus particles.With 9 Figures     

Non-a, non-b hepatitis: identification of hepatitis-B-like virus particles in serum and liver

Hepatitis B virus-like particles including: small spheres and filaments 15--25 nm in diameter together with a 35--40 nm Dane particle-like virion have been identified in sera of patients with non-A, non-B hepatitis. In a coded serological study, such particles were detected transiently in 3/4 acute, and persistently in 7/8 chronic cases of non-A, non-B hepatitis with non-A, non-B antigenemia. Only 2/12 similar cases without non-A, non-B antigens (Ag) in serum had detectable particles but neither patients with drugs, or type A hepatitis, nor cases of obstructive jaundice. The particles did not express hepatitis B surface (HBs) or non-A, non-B Ag at their surface but were associated, in three patients, with significant endogenous DNA polymerase activity. Furthermore, particles similar to hepatitis B cores (BHc) and also associated with DNA polymerase activity were demonstrated by sucrose gradient ultracentrifugation of a liver homogenate obtained from a patient who had died of non-A, non-B hepatitis. The non-A, non-B hepatitis virion described here appears, therefore, as a hepatitis B-like virus. The exact kinship between these two agents is currently being investigated.     

Hepatitis B and herpes viral components in the cerebrospinal fluid of chronic schizophrenic and senile demented patients

Cerebrospinal fluids (CSF) or sera or both from 57 chronic schizophrenics and 18 senile demented patients were examined by various tests for HBsAg. both CSF and serum were positive in 2 schizophrenics while in six HBsAg was detected in the CSF only. Elevated values of circulating immune complexes were found in positive patients. Most CSF positive for HBsAg also contained neutralizing antibodies to Herpesvirus hominis type 1. Ultramicroscopic structures similar to hepatitis B virus (HBV) components and herpesviral particles were visualized in the CSF of one patient on electron microscope (EM) grids coated with anti-human IgG serum. Most HBsAg positive patients appeared to be liver-symptoms-free carriers. In the CSF of a 79 years old senile demented man HBsAg was proved serologically. Several herpesviral particles complexed with globular material and spherical structures for 15 to 25 nm in diameter were visualized in the same CSF on EM grids coated with anti-human IgG serum. The findings support the importance of herpesviruses in mental illness. Penetration of HBsAg through the blood-brian barrier might be involved as an iatrogenic factor in the course of late psychoses.