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T I Michalak

Publications and source records attributed to T I Michalak.

At least 19 recordsLinked to original sources
PubMed2005

Comparison of biological activity of recombinant woodchuck interferon gamma and tumor necrosis factor alpha produced in baculovirus and Escherichia coli expression systems.

The full-length cDNAs of recombinant woodchuck interferon gamma (rwIFN gamma) and woodchuck tumor necrosis factor alpha (rwTNF alpha) were cloned into baculovirus transfer vectors and expressed in insect Sf9 cells. The recombinant proteins secreted by the insect cells, bac-rwIFN gamma and bac-rwTNF alpha, were found to be functionally competent. Their biological activities were compared to those of rwIFN gamma and rwTNF alpha produced in the Escherichia coli (E. coli) expression system. The bac-rwIFN gamma demonstrated a 4.5-fold greater protective activity against encephalomyocarditis virus-induced cytolysis of woodchuck hepatocytes and that of class I MHC antigen presentation on the hepatocytes than rwIFN gamma derived from E. coli. The bac-rwTNF alpha was cytotoxic towards murine fibroblasts and able to upregulate class I MHC antigen display and these effects were about 18-fold greater than those triggered by rwTNF alpha from E. coli at a comparable protein level. In addition, the antiviral activity of bac-rwIFN gamma was inhibited by anti-wIFN gamma antibodies and the cytotoxicity of bac-rwTNF alpha neutralized by cross-reactive antibodies to murine TNF alpha. The study showed that the expression of rwIFN gamma and rwTNF alpha in the baculovirus system generated biologically active cytokines whose potency was considerably greater than those produced in E. coli.

Animals↗
PubMed2001

Augmented hepatic interferon gamma expression and T-cell influx characterize acute hepatitis progressing to recovery and residual lifelong virus persistence in experimental adult woodchuck hepatitis virus infection.

Woodchucks infected with woodchuck hepatitis virus (WHV) have profiles of liver disease and age-dependent rates of progression to chronic hepatitis (CH) comparable with those seen in human hepatitis B. The mechanism of recovery from acute hepadnaviral infection or its evolution to chronicity remains unknown, although the liver immune responses are expected to play an important role. To determine the dynamics of intrahepatic cytokine expression and T-cell involvement, and to assess their value in predicting the outcome of acute hepatitis (AH) in the adult onset of WHV infection, we evaluated liver transcription of interferon gamma (IFN-gamma); tumor necrosis factor alpha (TNF-alpha); interleukins (IL)-2, -4, and -6; and the T-cell influx in relation to disease histologic severity and virus load in serial liver biopsies collected during the life span of experimentally infected woodchucks. Our results show that recovery from viral AH in adulthood is preceded by a significantly greater hepatic expression of IFN-gamma and CD3, an increased TNF-alpha transcription, lower hepatic WHV load, and a greater degree of liver inflammation than those in acute infection with CH outcome. Furthermore, we have learned that the elevated IFN-gamma, TNF-alpha, and CD3 expression in the liver endures for years not only in CH, but also, although to a lesser extent, in apparently completely resolved infection. This is consistent with our previous findings that residual WHV replication and remnant liver inflammation continue for life after recovery from AH. This study indicates that antiviral cytokines, in particular IFN-gamma, may play a central role in the long-term control of occult hepadnavirus persistence in the liver.

Acute Disease↗
PubMed2001

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus.

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.

Animals↗
PubMed2000

Occult persistence and lymphotropism of hepadnaviral infection: insights from the woodchuck viral hepatitis model.

Hepatitis B virus (HBV) is a major human pathogen that causes chronic infection and life-threatening liver diseases in millions of individuals. While pathological and epidemiological consequences of clinically evident HBV infections are well recognized, there is no similar knowledge on an asymptomatic, silently progressing virus persistence. Contrary to previous opinion, current evidence indicates that a serologically undetectable (occult) HBV carriage is a common outcome of recovery from symptomatic illness and that scanty amounts of the virus are carried by apparently healthy individuals for years after resolution of hepatitis B despite the presence of presumably protective antiviral antibodies. Recent studies on this silent form of hepadnavirus carriage in an experimental woodchuck hepatitis virus (WHV) infection, which is considered to be the closest natural model of HBV disease, revealed that the life-long occult persistence of traces of pathogenic virus is an invariable consequence of recovery after hepadnaviral invasion and that this state always co-exists with a steady low-rate virus replication in both the liver and the lymphatic system. Importantly, this serologically concealed infection can be accompanied by development of hepatocellular carcinoma in convalescent animals and is transmittable from mothers to offspring as an asymptomatic, indefinitely long infection which involves the lymphatic system but not always the liver. This review focuses on the features of hepadnavirus occult persistence and its lymphotropism, and on what is currently understood about the contribution of the lymphatic system in maintaining hepadnavirus carriage based on insights provided by analysis of the woodchuck-WHV experimental system.

Animals↗
PubMed2000

Posttranscriptional inhibition of class I major histocompatibility complex presentation on hepatocytes and lymphoid cells in chronic woodchuck hepatitis virus infection.

Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, beta(2)-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence.

Acute Disease↗
PubMed1999

Occult lifelong persistence of infectious hepadnavirus and residual liver inflammation in woodchucks convalescent from acute viral hepatitis.

Traces of hepatitis B virus (HBV) genome can persist for years following recovery from hepatitis B. To determine overall duration, molecular characteristics, and pathological implications of this serologically undetectable form of hepadnaviral carriage, we have analyzed the expression of transcriptionally active virus genomes, their infectivity, and examined liver alterations during the natural lifespan of woodchucks convalescent from acute infection with HBV- related woodchuck hepatitis virus (WHV). In this study, we document lifelong persistence of scanty amounts of replicating virus both in the liver and lymphatic system after spontaneous resolution of an episode of experimental hepadnaviral hepatitis. Antibodies to virus nucleocapsid (core) were found to be the most reliable immunovirological marker coexisting with occult infection. In the majority of convalescent woodchucks, serial liver biopsies showed protracted minimal to mild necroinflammation with periods of normal morphology; however, hepatocellular carcinoma (HCC) ultimately developed in 2 of 9 animals studied. Inocula derived from lymphoid cells of convalescent animals induced classical acute hepatitis in virus-naive woodchucks that progressed to chronic hepatitis and HCC in 1 of the animals, demonstrating infectivity and pathogenic competence of the carried virus. Our results reveal that low levels of infectious WHV and residual hepatic inflammation usually continue for life after resolution of hepatitis and that this recovery does not avert HCC development. They also demonstrate that, in addition to the liver, the lymphatic system is the site of the occult lifelong maintenance of replicating hepadnavirus.

Acute Disease↗
PubMed1999

Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis.

Acute Disease↗
PubMed1999

Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis.

Mother-to-child transmission is an important route for hepatitis B virus (HBV) dissemination. It has been established that HBV traces persist for years after complete clinical recovery from hepatitis B. Similarly, resolution of hepatitis caused by HBV-related woodchuck hepatitis virus (WHV) is followed by occult lifelong carriage of pathogenic virus. In this study, we documented that WHV persisting after termination of acute hepatitis is transmittable to newborns as an asymptomatic long-term infection. All 11 offspring from 4 dams studied carried transcriptionally active WHV genomes for 3.5 years after birth without immunovirological markers of infection. WHV genomes and mRNA were detected both in the liver and lymphoid tissue in the majority of offspring; WHV covalently closed circular DNA was detected in some samples. In 4 offspring, however, the virus was restricted to the lymphatic system. In the circulation, WHV DNA-reactive particles were DNase resistant and of comparable size and density to complete virions. Importantly, the virus in offspring with or without hepatic WHV DNA expression was infectious to WHV-naive woodchucks. Finally, offspring challenged with WHV were not protected against reinfection. These findings show that mothers with occult hepadnaviral carriage transmit pathogenic virus to their offspring, inducing a persistent infection invariably within the lymphatic system but not always in the liver.

Animals↗
PubMed1998

Complement-mediated cytotoxicity and inhibition of ligand binding to hepatocytes by woodchuck hepatitis virus-induced autoantibodies to asialoglycoprotein receptor.

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% +/- 0.3% to 33.6% +/- 3.6% (mean +/- SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.

Animals↗
PubMed1997

Virus-induced anti-asialoglycoprotein receptor autoimmunity in experimental hepadnaviral hepatitis.

The relationship between hepatitis virus invasion and emergence of liver-specific autoantibodies against asialoglycoprotein receptor (anti-ASGPR) and the occurrence patterns, prognostic value, and specificity of these autoantibodies toward polypeptides of host ASGPR were investigated in experimental viral hepatitis in the woodchuck system. Sequential sera (n = 231) obtained before and after inoculation with woodchuck hepatitis virus (WHV) from animals which resolved acute infection (n = 7) or developed chronic hepatitis (n = 6) were tested for anti-ASGPR using radio and enzyme-immunodetection assays. In addition, the outcome of WHV hepatitis was analyzed in 30 other woodchucks whose preinoculation sera were tested for anti-ASGPR. The receptor subunit specificity of virus-induced anti-ASGPR was determined by Western blotting and compared with that of anti-ASGPR raised in woodchucks challenged with a heterologous (rabbit) receptor. The results revealed that WHV infection triggered anti-ASGPR in all except one of the initially autoantibody nonreactive animals (eight of nine; 89.9%). Once induced, anti-ASGPR were detectable throughout the entire follow-up independent of histological severity of liver damage or the outcome of hepatitis. In healthy WHV-naive woodchucks, anti-ASGPR occurred at low titers in approximately one third of the animals. Importantly, woodchucks reactive for anti-ASGPR before WHV inoculation developed chronic hepatitis with a significantly greater frequency (55.5%) than those autoantibody negative (15.6%; P < .05). In contrast to anti-ASGPR elicited by immunization with a heterologous receptor, which initially recognized only the ASGPR 40-kd polypeptide, anti-ASGPR emerging after virus invasion reacted with both the ASGPR 40- and 47-kd subunits from the moment of their appearance. This study provides the first direct evidence that hepatitis virus in the natural host triggers autoantibodies against a unique hepatocyte antigen and shows that anti-ASGPR autoimmunity existing before virus infection is associated with a high rate of progression to chronic disease in experimental hepadnaviral hepatitis.

Animals↗
PubMed1996

Protease-activated lymphoid cell and hepatocyte recognition site in the preS1 domain of the large woodchuck hepatitis virus envelope protein.

A site capable of strictly host- and cell type-specific recognition was identified in the preS1 domain of woodchuck hepatitis virus (WHV) through the use of antipeptide antisera generated against the extreme N-terminal fragment of the large virus envelope protein. The crucial determinant of this binding site was mapped to amino acids 10-13. Although a synthetic analogue of the site was highly immunogenic, natural WHV envelope did not display the site activity unless it was modified by proteolysis or acidic pH treatment, indicating an internal location of the determinant in viral envelope. Synthetic peptides encompassing the sequence of this site bound woodchuck lymphoid cells and hepatocytes in a species-restricted manner which followed characteristics of a specific ligand-receptor interaction, although their ability to interact with lymphoid cells was considerably greater than that for hepatocytes. In WHV-infected animals, a natural antibody to the identified cryptic cell-binding site arose independently of that directed against epitopes of unmodified virus envelope and its appearance constituted the earliest immunovirological indicator of virus invasion. Our results demonstrated that the preS1 domain of the large WHV envelope protein is endowed with the species- and cell type-specific recognition site which is protected against antibody surveillance by the natural tertiary structure of the protein and we suggest that proteolytic cleavage is required to induce the binding activity.

Amino Acid Sequence↗
PubMed1995

Detection of hepatitis B and woodchuck hepatitis viral DNA in plasma and mononuclear cells from heparinized blood by the polymerase chain reaction.

Amplification by the polymerase chain reaction (PCR) of hepatitis B virus (HBV) DNA extracted from parallel samples of serum and heparinized plasma gave contradictory results, indicating that heparin inhibits virus detection. Similarly, analysis of PCR products of woodchuck hepatitis virus (WHV) DNA showed that heparinization of blood abolished WHV DNA amplification, while anticoagulation with sodium EDTA or acid citrate dextrose did not. Amplification of recombinant WHV and HBV DNA in the presence of increasing concentrations of sodium heparin progressively inhibited and finally abolished virus genome detection. The inhibitory effect of heparin was reversed by treatment of either plasma or isolated DNA with heparinase (5 U/reaction, 1 h at 28 degrees C) prior to PCR. In contrast, heparin did not influence the detection of hepadnavirus in peripheral blood mononuclear cells (PBMC), even after prolonged incubation of the cells with heparin in culture. These findings confirm that heparin exerts a dramatic inhibitory effect on hepadnaviral DNA detection by PCR and they demonstrate that this effect can be reversed by heparinase. The findings also show that extensively washed PBMC derived from heparinized blood can be a reliable source of nucleic acids for amplification of hepadnavirus genome. These results imply that previous data should be reassessed if samples of heparinized plasma were found hepadnavirus DNA nonreactive by PCR or when these samples were used as a starting material for PCR quantitation of viral genome.

Animals↗
PubMed1995

Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology.

Adult↗
PubMed1995

Identification of woodchuck class I MHC antigens using monoclonal antibodies.

Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43- and 39-kDa were identified in the cell surface membranes of normal woodchucks using a newly developed antiwoodchuck class I monoclonal antibody (mAb) B1b.B9 and immunoblotting. B1b.B9 was generated by immunizing mice with viable woodchuck peripheral blood mononuclear cells and was selected for anti-class I MHC reactivity using a cellular enzyme-linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43-kDa, whereas splenocyte plasma membranes showed intense expression of a 43-kDa species, as well as the presence of a 39-kDa protein. The 39- and 43-kDa proteins were extracted with Triton X-114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.

Animals↗
PubMed1994

Autoreactive IgG elicited in mice by the non-dominant but pathogenic thyroglobulin peptide (2495-2511): implications for thyroid autoimmunity.

We have previously shown that mice challenged with the rat thyroglobulin (Tg) peptide TgP1 (corresponding to aa 2495-2511 of human Tg) develop experimental autoimmune thyroiditis (EAT) and produce IgG antibodies that cross-react with Tg from various species. It was not clear, however, whether such antibodies were TgP1-specific or were induced secondarily--i.e. by autologous Tg released from the destroyed gland--and therefore directed to determinants other than TgP1. In this study we describe that, 5 weeks after priming with TgP1, the binding of serum IgG on native Tg is completely inhibited by free peptide, suggesting lack of recognition of other determinants on mouse Tg (mTg). In addition, TgP1-induced but not mTg-induced IgG bound better to heat-denatured than intact mTg, a result compatible with the recognition of a linear epitope by the peptide-induced antibodies. Comparison of the IgG subclass distribution among mTg-induced versus TgP1-induced IgG did not reveal qualitative differences, since all subclasses were represented in the order IgG1 > IgG2b > IgG2a > IgG3. Finally, TgP1-specific IgG reacted strongly with the follicular colloid in sections of normal thyroids, indicating the potential to bind to native Tg in vivo. These data: (i) highlight TgP1 as the only, so far, Tg sequence known to generate both EAT and Tg-reactive IgG in mice; and (ii) do not provide evidence for an amplification of the Tg-specific IgG response through the involvement of endogenous autoantigen in EAT.

Amino Acid Sequence↗
PubMed1994

Identification and characterization of the cell surface 70-kilodalton sialoglycoprotein(s) as a candidate receptor for encephalomyocarditis virus on human nucleated cells.

The attachment of encephalomyocarditis (EMC) virus to human nucleated cells susceptible to virus infection was examined with HeLa and K562 cell lines. Both cell types showed specific virus binding competitively blocked by unlabeled virions. The number of binding sites for EMC virus on HeLa and K562 cells were approximately 1.6 x 10(5) and 3.5 x 10(5) per cell, respectively, and dissociation binding constants were 1.1 and 2.7 nM, respectively. Treatment of cells with cycloheximide after pretreatment with trypsin eliminated EMC virus attachment, suggesting that the virus-binding moiety is proteinaceous in nature. Digestion of cells, cell membranes, and sodium deoxycholate-solubilized cell membranes with proteases or neuraminidases or treatment of cells with lectins demonstrated that the EMC virus-cell interaction is mediated by a sialoglycoprotein. Proteins with a molecular mass of 70 kDa were isolated from detergent-solubilized cell membranes of both HeLa and K562 cells by EMC virus affinity chromatography. The purified proteins, as well as their 70-kDa-molecular-mass equivalents detected in intact surface membranes of HeLa and K562 cells, specifically bound EMC virus in a virus overlay protein blot assay, whereas membranes from nonpermissive K562 D clone cells did not. Western immunoblot analysis with glycophorin A-specific antibody confirmed that the identified 70-kDa binding site on K562 cells is not glycophorin A, which is the EMC virus receptor molecule on virus-nonpermissive human erythrocytes (HeLa cells do not express glycophorin A). These results indicate that EMC virus attachment to permissive human cells is mediated by a cell surface sialoglycoprotein(s) with a molecular mass of 70 kDa.

Blotting, Western↗
PubMed1994

Hepatitis B virus persistence after recovery from acute viral hepatitis.

Contrary to current opinion, the disappearance of hepatitis B surface antigen (HBsAg) from the serum, the development of anti-HBs antibodies, and normalization of liver function may not reflect complete virological recovery from acute hepatitis B virus (HBV) infection. By using the polymerase chain reaction (PCR), in the current study we demonstrate long-term persistence of HBV DNA in the serum and peripheral blood mononuclear cells (PBMC) of four patients for up to 70 mo after complete clinical, biochemical, and serological recovery from acute viral hepatitis. Serum HBV DNA reactivity co-sedimented with HBsAg in sucrose gradients, and it displayed the size and density characteristics of naked core particles and intact HBV virions, presumably contained within circulating immune complexes in these anti-HBs antibody-positive sera. HBV DNA was also present in PBMC in late convalescent samples from all four patients, and HBV RNA was detected in late convalescent phase PBMC in two of these patients. These results suggest that HBV DNA, and possibly HBV virions, can be present in the serum, and that the viral genome can persist in a transcriptionally active form in PBMC for > 5 yr after complete clinical and serological recovery from acute viral hepatitis.

Adult↗