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The hepatitis C virus (HCV) interferon-alpha (IFN-alpha) sensitivity-determining region (ISDR) has been shown to suppress double-stranded RNA-dependent protein kinase (PKR) activity in vitro in a yeast PKR expression system. Since variability of ISDR was shown to correlate with nonresponsiveness to IFN-alpha therapy in chronically HCV-infected patients, it has been suggested that prototype ISDR might be a viral inhibitor of cellular PKR. The present study evaluates the biological significance of ISDR variability in situ, relating it to PKR-mediated cellular antiviral responses within the liver. ISDR variability was determined in patients chronically infected with HCV genotypes 1a, 1b, and 3a by direct sequencing using liver-derived RNA preparations as starting material. As surrogate parameters for PKR-mediated cellular responses, hepatic endogenous IFN-alpha gene expression as well as MxA expression were analysed by a competitive, quantitative reverse transcription-polymerase chain reaction technique. Irrespectively of intra- or intergenotypic ISDR amino acid substitutions, ISDR variability was found not to correlate with endogenous hepatic IFN-alpha or with hepatic MxA gene expression. The data suggest that at least two prominent PKR-mediated cellular responses might be largely unaffected by HCV ISDR variability.

Chronic hepatitis C virus (HCV) infection in humans is treated at present with interferon (IFN)-α. Because the proportion of patients responding to therapy with sustained or even just with transient elimination of viral RNA is low, several potential prognostic parameters have been evaluated to predict the outcome of the therapy. The present study aimed to prove the validity of a predictive parameter described previously for initial virological response, namely the ratio of serum γ-glutamyltransferase/alanine transaminase (γ-GT/ALT) activity in connection with virus genotypes 1a, 1b, and 3a, prospectively and to compare the predictive value of these combined parameters with amino acid variability within the interferon sensitivity determining region (ISDR). The prospective analysis confirmed previous data on the predictive value of the serum γ-GT/ALT ratio. Concerning ISDR variability, the majority of ISDR sequences obtained from the mostly nonresponding type 1b-infected individuals (23/28) resembled nonmutant types (27/28). Isolates from type 3a-infected patients responding to therapy in the majority of cases (13/20) exclusively resembled nonmutant types when compared with databank type 3a sequences, but were mutant when compared with the prototype sequence HCV-J. However, the initial virological responsiveness among both type 1b- and type 3a-infected patients did not correlate to ISDR variability. In contrast, virological responsiveness was closely related to serum γ-GT/ALT ratio. The data are not necessarily contrary to the concept that the number of amino acid exchanges within the ISDR compared with the prototype HCV-J sequence is related to some extent to IFN-α sensitivity. The ratio of serum γ-GT/ALT in combination with HCV genotype, however, was found to be a more reliable and stringent predictive parameter. J. Med. Virol. 58:227–234, 1999. © 1999 Wiley-Liss, Inc.

We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector. The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions. The R-rich 30-amino-acid C-terminal domain was not analyzed. A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen. A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped. Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B. Bottcher, S. A. Wynne, and R. A. Crowther, Nature 386:88–91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop). Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein. Of the 110 mutations, 38 allowed capsid formation in Escherichia coli . They mapped exclusively to nonhelical regions of the proposed fold. The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.

Hepatitis C virus (HCV) binds to different human cell lines in vitro. However, the efficiency of adsorption is very low due mainly to a relatively small fraction of the virus being able to bind to these cells. Free low density lipoprotein (LDL > 200 μg/ml) is able to block the attachment of HCV to human fibroblasts in vitro completely. COS-7 cells being primarily not able to bind HCV were transfected with a vector containing the entire coding sequence of the human LDL-receptor (LDLR). HCV was now bound to these cells. We propose that HCV and LDL are competitive for the cellular LDLR and that LDL in sera of patients may regulate the binding of HCV to this target. J. Med. Virol. 57:223–229, 1999. © 1999 Wiley-Liss, Inc.

Quantitative detection of hepatitis B virus (HBV) in serum or plasma is of significance for monitoring of therapy and establishment of the prognosis of the disease, as well as for infectivity assessment and quality control of the diagnosis. Unfortunately, various commercially available test kits for HBV DNA yielded conflicting quantitative results, with differences of up to a factor of 120. The Eurohep Pathobiology Group has established two reference samples of plasma from HBV carriers and determined as accurately as possible the number of HBV DNA molecules in these samples. Plasma donations from two single highly viremic carriers of HBV genotype A (HBV surface antigen subtype adw2 ) and genotype D ( ayw2/3 ), respectively, were collected, and coded dilutions of these samples were analyzed by members of the Eurohep Pathobiology Group. Quantitative results from the seven laboratories reporting consistent results were initially divergent. Limiting dilution and nested PCR assays suffered from incomplete DNA extraction. Hybridization assays used inaccurately quantitated cloned DNA as a reference. Two hybridization assays could not be calibrated directly with cloned HBV DNA, because virion-derived DNA reacted much less efficiently. After identification and elimination of these problems, limiting-dilution assays from three laboratories and hybridization assays from two producers generated consistent and concordant results: 2.7 × 10 9 HBV DNA molecules/ml (range, 2.1 × 10 9 to 3.4 × 10 9 HBV DNA molecules/ml) in the plasma from the carrier of genotype A and 2.6 × 10 9 HBV DNA molecules/ml (range, 2.1 × 10 9 to 3.0 × 10 9 HBV DNA molecules/ml in the plasma from the carrier of genotype D. The two Eurohep reference plasma samples have already been used for the standardization of test kits and in quality control trials, and the plasma from the carrier of genotype A will probably be the basis of a World Health Organization reference sample.

To determine whether Lyme disease neuropathogenesis may result from infection by a particular segment of the locally extant population of spirochetes, genetic markers of spirochetes found in cerebrospinal fluid (CSF) of 12 pediatric patients were compared with those in spirochetes from 40 vector ticks sampled in the vicinity of their homes. The primary structure of the outer surface protein A served as the marker of variation; a fragment of the corresponding gene was amplified by nested polymerase chain reaction and the products sequenced. Tick-derived variants clustered in seven distinct categories, of which four were present in CSF. One of the CSF variants differed from any found in ticks. Coinfection by different spirochete variants was infrequent in ticks and absent in human samples. Spirochetal neuropathology in children in our study site does not correlate with a particular segment of the tickborne pathogens present in nature.

Cellular immune responses to the HBc or HBe antigen of hepatitis B viruses contribute to the pathogenesis of hepatitis B and to the elimination of the virus. Insufficient cytotoxic immune reactions against the core antigen may be one major reason for viral persistence in the absence of severe clinical symptoms. We attempted to stop viral persistence in the animal model of congenitally infected ducks by injection of recombinant DHBc particles, together with the strong immunostimulator Freund's adjuvant. However, the duck HBc antigen (DHBcAg)-treated ducks did not develop detectable liver disease, nor was the virus persistence affected. The congenitally infected ducks did not contain antibodies against DHBcAg before injection despite continuous production of duck hepatitis B virus, and developed only a weak transient antibody response after injection of recombinant DHBcAg together with Freund's adjuvant. Noninfected ducks developed, in contrast, a strong antibody response to the injected DHBcAg. We conclude that congenitally infected ducks are immunotolerant to DHBcAg and cannot be cured by immunotherapy with exogenous recombinant DHBcAg.

Antibodies directed against the autoantigen p26 were detected in sera from 32 patients with acute Epstein-Barr virus (EBV) infection and clinical symptoms of infectious mononucleosis. P26 has now been identified as the enzyme manganese superoxide dismutase (MnSOD) by comparison of the NH2-terminal amino acid sequence. Antibodies against MnSOD belong to the immunoglobulin class M. They are not detectable in sera of patients with other herpesvirus infections. In the 32 patients investigated, the rise and fall of the autoantibodies coincides with the clinical symptoms. In vitro, the autoantibodies were shown to inhibit the dismutation of superoxide radicals by blocking MnSOD. As presented in the discussion this effect may contribute to the pathogenesis of acute EBV infection.

The hepatitis B virion is a spherical double-shelled particle carrying three surface proteins (large [L], middle [M], and small [S]) in its envelope. All three proteins are translated from a single open reading frame by means of three different in-frame start codons from unspliced mRNAs. This organization defines three protein domains (pre-S1, pre-S2, and S). All three domains together form the L protein, whereas the M protein consists of domains pre-S2 plus S. The L and S proteins are both necessary for virion production, whereas the M protein is dispensable, suggesting an important function of the pre-S1 domain in virion morphogenesis. To investigate this point, we created a series of N-terminal-truncated L mutants and tested their ability to substitute for the wild-type L protein in virion formation. We found that the constructs fell into two classes, (i) N-terminal deletion mutants lacking up to 102 of the 119 amino acids of the pre-S1 domain still allowed virion maturation, showing that the N-terminal 5/6 of the pre-S1 sequence is dispensable for this process. (ii) Mutants lacking 110 or more N-terminal amino acids were unable to substitute for the L protein in virion assembly, although they were stably expressed and secreted as components of subviral 20-nm hepatitis B surface antigen particles. This suggests that a short C-terminal region of pre-S1 is important for virion formation. Like the wild-type L protein, the mutants of the first class were not glycosylated in their pre-S2 domains; however, this site was used for glycosylation in mutants of the second class, similar to that in the M protein. These findings can be related to a model for the function of the L protein in virion maturation.

Hepatitis C virus (HCV)-RNA in sera of patients with viral hepatitis C is supposed to be included, at least partially, into HCV particles. We found that the density of HCV-RNA-carrying material was variable, as determined by sucrose gradient density centrifugation (1.03-1.20 g/cm3). In some of the sera examined HCV-RNA was restricted to low densities between 1.03 and 1.08 g/cm3. In other sera additional densities of HCV-RNA were found distributed over the whole gradient with peaks at 1.12 and 1.17 and at 1.19-1.20 g/cm3. HCV-RNA banding at low densities could be completely co-precipitated with anti-beta lipoprotein, whereas HCV-RNA fractions of higher densities were only partially precipitated or not at all. In 8 of 20 sera directly examined, HCV-RNA could be completely and in 9 sera only partially co-precipitated by anti-beta lipoprotein. In 3 sera no significant precipitation could be observed.

On the basis of published sequence data the preS1 attachment region of hepatitis B virus (HBV) appears to be highly variable. Using a novel method for rapid DNA sequencing by the polymerase chain reaction we screened 34 HBV DNA-positive sera for mutations in a variable part of the preS1 region of the HBV genome. The sequence data were used to analyse potential chains of infection, and strongly supported the expected routes of HBV transmission among patient groups. Furthermore, sequence comparisons permitted sub-genotyping of the viruses. In the 22 cases of subtype adw, we found a very low number of point mutations. This shows that the attachment site of HBV is more highly conserved than that of other blood-transmissible viruses such as human immunodeficiency virus or hepatitis C virus.

Open reading frames of human papillomaviruses were expressed in Escherichia coli as β-galactosidase fusion proteins. These bacterially derived papillomaviral gene products were used to examine sera from 67 women (63 healthy subjects, 4 patients with genital carcinoma) for antibodies to papillomavirus type-16 antigens (El, E2, E4, E5, E6, E7, L1, L2) and the L2 proteins of HPV-6b and HPV-18 by Western-blot analysis. The serologic data were compared with cytological findings classified according to Papanicolaou and with nucleic acid hybridization data from cervical smears of the same individuals. Twenty-three of the normal individuals showed antibodies exclusively directed against L2 gene products; whereas in the sera from the four genital cancer patients, antibodies to the early gene products E4 and/or E7 could be detected. In one case these antibodies were found to be combined with antibodies to L2 of HPV-16 and −18 and in another case with those to El and E2 of HPV-16. In none of the sera examined could antibodies to L1, E5 or E6 be identified. Three of the antibody positive normal women were found to be also positive for HPV-16/18 DNA, while all of the 40 seronegative women were HPV-16/18 DNA negative. These data indicate that serology may be a valuable means to study the epidemiology of genital human papillomavirus infection.

We have developed a modified ELISA for the detection of anti-Borrelia burgdorferi (Bb) antibodies based on a peroxidase enzyme labelled antigen (ELAT). Microtiter plates were coated with antigen of Bb. The immunoglobulins of the serum samples were bound to the antigen and specific antibodies were detected by an enzyme labelled antigen. The test principle facilitates the recognition of specific antibodies in different collectives of human and animal sera. We performed epidemiological studies with the ELAT on 231 sera from mothers in maternity wards (9.5% positive), 219 patient sera sent to the Bb routine diagnostics (15% positive) and 230 sera from forestry workers (21.3% positive). We further investigated sera from red deer from South Lower Saxony which remained 55% Bb-antibody positive; deer were 37% and fallow deer were 29% positive.

The diagnostical significance of the large hepatitis B surface protein with its preS1 attachment site and of anti-preS antibodies are not yet well known. We investigated the epitope of the preS1 attachment site to see whether it is a marker of viremia and whether antibodies against it occur in convalescents and vaccinees. For comparison, sera were also tested for the presence and relative amount of a preS2 epitope. The epitopes were detected by binding to specific monoclonal antibodies (mAb MA18/7 for the preS1 epitope and mAb Q19/10 for the preS2 epitope) at the solid phase of a sandwich enzyme-linked immunosorbent assay. Antibody against the preS1 epitope was detected by inhibition of binding to mAb MA18/7. This mAb inhibits attachment of preS1 antigen to hepatocytes and reacts with a subtype-independent sequential epitope at the surface of hepatitis B virus between amino acid 29-36. This preS1 epitope occurs in most hepatitis B surface antigen (HBsAg) carriers, irrespective of viremia. Free preS2 epitope Q19/10 is present in samples with more than 8 micrograms/ml total HBsAg and it is masked in sera with less HBsAg. Antibodies which compete with mAb MA18/7 for its viral preS1 epitope occur in one third of HBsAg carriers who were negative for hepatitis B e antigen. It also occurs in one third of convalescents and in most good responders to plasma-derived vaccines.

During the course of acute Epstein-Barr virus (EBV) infection, there is a rise of oxygen radical production. As a consequence, the production of the oxygen radical scavenger manganese superoxide dismutase (MnSOD) is increased. Patients with acute EBV infections regularly develop autoantibodies against MnSOD that are able to inhibit the enzyme activity in vitro. To elucidate the origin of the autoantibodies, the epitopes on MnSOD were determined. The entire sequence of MnSOD was synthesized as overlapping pentadecapeptides, which were scanned for their reactivity with sera of patients with acute EBV infections. Sera as well as affinity-purified anti-MnSOD antibodies reacted with the peptides p(no15) (amino acids 47-61) and p(no30) (amino acids 122-136) lying in crucial parts of the MnSOD tetramer. The two main epitopes p(no15) and p(no30) showed sequence homologies with EBV-encoded proteins. Reactivity of affinity-purified antibodies with a peptide of the homologous BGLF4 points to a molecular mimicry causing the occurrence of anti-MnSOD antibodies. Anti-MnSOD antibodies were able to block the protective effects of MnSOD in a model for oxidative damage produced by xanthine/xanthine oxidase in EAhy926 endothelial cells. Thus, these autoantibodies may contribute in vivo to the clinical symptoms by accumulation of toxic oxygen radicals.

In most sera of hepatitis C virus (HCV)-infected patients beta-lipoproteins are bound to HCV RNA-carrying material, most often simultaneously with immunoglobulins (IgG, IgM) and sometimes additionally with high-density lipoproteins, forming complexes of low density (1.04-1.06 g/ml). To separate HCV particles from bound material, we tried to destroy the lipoprotein enzymatically by incubating HCV-positive human sera with lipoprotein lipase derived from Pseudomonas spp. (LPL-Ps). After this treatment, titers of HCV RNA in human sera (determined by a simple semiquantitative reverse transcription-PCR assay) were strongly reduced, regardless of whether primers of the NTR or NS5 region were used. Inactivation of HCV RNA could be inhibited by the addition of RNAguard to the serum-enzyme mixture. The lytic effect of the LPL-Ps preparation could be inhibited by tetrahydrolipstatin. Hence LPL-Ps seems to disrupt the HCV particle structure, including the putative core of the virus, and makes HCV RNA sensitive for RNase present in the reaction mixture. HBV was not destroyed by LPL-Ps. Porcine pancreatic lipase had no effect on HCV. The implications of these observations for the structure and biology of HCV and for its stability and inactivation in human sera are discussed.

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.

Infection with hepatitis C virus (HCV) is still a serious problem in hemodialysis patients, despite screening of blood products for anti-HCV antibodies. The prevalence of HCV in HD patients is between 15% and 30% in Germany. We report the molecular epidemiology of an HCV outbreak in a hemodialysis unit in 1997 is determined. HCV hypervariable region 1 (HVR1) was amplified from serum samples of 19 patients by polymerase chain reaction (PCR) and sequenced directly. In addition, HCV isolates from 3 of these 19 patients were cloned and sequenced. 14 newly infected patients and two patients, who had been infected for several years had very closely related HCV isolates. Unrelated HCV isolates as well as sequences obtained from an HCV outbreak in a plasmapheresis center were found in different, distantly related branches. These findings provide strong evidence for nosocomial transmission of the virus, despite following strict general hygiene precautions. The production of anti-HCV antibody was delayed significantly or seroconversion did not occur at all during the period of observation in 8 out of 14 newly infected HCV RNA positive patients. Close-meshed reverse transcription-polymerase chain reaction (RT-PCR) analyses on apparently non infected patients within hemodialysis units and upon admission of new patients is strongly recommended for the early detection and prevention of outbreaks of HCV.

Objective To determine if end-stage dilated cardiomyopathy (DCM) is associated with the presence of Lyme disease causing spirochete Borrelia burgdorferi in the myocardium, we used nested polymerase chain reaction to detect B burgdorferi DNA in myocardial samples from explanted hearts of patients with end-stage DCM. Patients originated from endemic areas for Lyme disease (Bavaria, Lower Saxony, Germany). Methods and Results This was a retrospective study. Polymerase chain reaction was used to detect the specific B burgdorferi recombinant outer surface protein A (OspA) gene in myocardial tissue from 68 patients with end-stage DCM who had undergone heart transplantation. The clinical history of Lyme disease, the presence of Borrelia burgdorferi OspA, and antibodies against OspA in myocardial tissue and serum were investigated. B burgdorferi DNA was not detected in any of the 68 human hearts. Immunoglobulin G antibodies against specific B burgdorferi antigens were observed in 3 (12.5%) of 24 patients. In contrast, 4 hearts from rats experimentally infected with B burgdorferi were all positive for OspA DNA as measured by polymerase chain reaction. Conclusion Our data show that cardiac myocytes of hearts obtained from subjects with end-stage DCM did not contain B burgdorferi DNA as investigated by polymerase chain reaction. However, B burgdorferi shows a high affinity for myocardial tissue as shown by the animal studies, indicating that myocardial infections are nevertheless possible. (Am Heart J 1999;138:269-72.)

Hepatitis B virus DNA was extracted from serial serum samples of a hepatitis B surface antigen-negative patient with antibodies to the core protein as the only marker of an infection with hepatitis B virus. This patient showed no symptoms of hepatic injury. Sequencing of the amplified viral DNA demonstrated multiple amino acid changes clustering in surface-exposed regions of the surface protein. Synthesis and association of the middle (M) and small (S) surface proteins could be shown in vitro. The variant surface antigens were recognized neither by monoclonal antibodies to the surface antigen nor by the vaccinee’s sera. Consequences for hepatitis B surface antigen testing and vaccine development are discussed.

The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3'-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as is common in microbiological laboratories.

Having returned from a holiday in Southeast Europe, a 30-year-old German woman developed acute hepatitis. Hepatitis A virus (HAV) infection was diagnosed serologically. During the course of the infection, hemolysis was found. IgM antibodies against triosephosphate isomerase (IgM anti-TPI) were detected in the patient's serum from the acute phase of the HAV infection. Affinity purified IgM anti-TPI from the serum reduced the enzyme activity in vitro and caused an increased51 Cr release from erythrocytes. IgM anti-TPI is assumed to be one of the causative agents of hemolysis in HAV infection.

Heterogeneities in the density of hepatitis C virus RNA-carrying material (HCV-RNA-CM) found in human sera (1.03-1.20 g/cm3) are attributed to the binding of low-density lipoproteins and/or of IgG. In some sera HCV-RNA-CM seems to be nearly totally bound to beta-lipoproteins and cannot be precipitated by anti-IgG (gamma); in others more than 95% of HCV-RNA-CM is bound to IgG and cannot be precipitated by anti-beta-lipoprotein. Furthermore, there are sera from which HCV-RNA-CM can be completely be precipitated by either anti-beta-lipoprotein or anti-IgG (gamma), pointing to a binding of the two serum proteins to the same HCV-RNA-CM. There are other sera from which HCV-RNA-CM can be partially precipitated by the one or the other antiserum, leaving behind fractions, which are bound to beta-lipoprotein or to IgG. HCV-RNA-CM cannot be precipitated from some sera either by anti-beta-lipoprotein or by anti-IgG (gamma).

The prevalence and time course of the occurrence of antibodies to the hepatitis B virus polymerase (anti-HBpol) were investigated in acutely and in chronically HBV-infected individuals by using recombinant HBpol protein for Western blot analysis. One group consisted of 19 patients who were acutely infected and recovered completely. Five of these patients (26%, 69 serum samples examined) exhibited anti-HBpol. Among those anti-HBpol positive patients, recovery from the disease was combined with a complete loss of this antibody. In contrast, in a second group of 15 individuals who developed chronic hepatitis B, 13 (87%, 102 serum samples examined) had anti-HBpol during the acute phase of the disease. The difference between the anti-HBpol prevalence rates of the two patient groups is statistically significant (Exact Fisher test, P < .002), implying that the occurrence of anti-HBpol may be indicative of a potential chronic course of hepatitis B. Remarkably, anti-HBpol was found in one case of a clinically suspected hepatitis B in which no other serological HBV parameters were found. This serum sample was positive in HBV PCR, supporting a possible diagnostic value of anti-HBpol. © 1993 Wiley-Liss, Inc.

In half of the sera from patients with acute bacterial infections and 15% of the sera from patients with acute viral infections glycoproteins were found that form a scalariform pattern in the cationic range upon isoelectric focusing. The cationic glycoproteins appeared with the clinical illness. After subsidence of the symptoms they disappeared within 4 to 6 weeks. The proteins were identified as immunoglobulin G (IgG) by determination of the NH2-terminal amino acid sequence. Remarkably, these IgG only contained light chains of the kappa type with high proportions of carbohydrates. Both, N-glycosidic- and O-glycosidic-bound glycans were present. The glycosylated light chains may render the cationic IgG multireactive. Thus, it may be part of an early nonspecific immune defense mechanism.

During the assembly of the nucleocapsid of the hepatitis B virus a protein kinase, probably of cellular origin, is encapsidated. This enzyme phosphorylates serine residue(s) localized within the lumen of the particle. By using purified, liver-derived core particles, we characterized the protein kinase activity in the presence of different ions and inhibitors. Controls were performed with cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) and recombinant core particles. We showed that the endogenous protein kinase of the core particles was not inhibited by H89, a specific inhibitor of PKA. Staurosporine, a selective inhibitor of PKC inhibited the endogenous kinase activity only within the first minutes of the reaction. In contrast, quercetine, a selective inhibitor of the protein kinase M (PKM) did not inhibit during the first minutes but inhibited efficiently during later phases of incubation. PKM represents an enzymatically active proteolytic fragment of PKC. These results suggest that PKC is encapsidated into human core particles and is converted to PKM during the in vitro reaction. This conclusion implies the association of a protease activity localized with the HBV nucleocapsid inside liver-derived core particles.

The hepatitis B virus, although containing a DNA genome, replicates by reverse transcription of an RNA pregenome. The viral Pol gene encodes the reverse transcriptase which catalyzes viral DNA synthesis. To study the interaction of this protein with HBV RNA, the entire Pol gene product was expressed except its eight amino-terminal codons in Escherichia coli as fusion protein with β-galactosidase. In the absence of competing nucleic acids full-length expression products were able to nonspecifically bind in vitro synthesized HBV RNAs of different polarity and length. However, if competed with an excess of unspecific RNA, only those HBV RNAs were bound which contained besides the direct repeats 1 and 2 nucleotide sequences downstream of direct repeat 1. The corresponding binding site was found to be located within the adjacent 134 nucleotides downstream of DR1. We conclude from our data that this region which is in part homologous to the U5 region of retroviral genomes may be important for the binding of the HBV Pol gene product to the viral pregenome.

In the course of infectious mononucleosis, IgM antibodies are formed against 2 proteins present in nucleated and non-nucleated vertebrate cells. Antibodies were found in sera of all patients suffering from acute Epstein-Barr virus infection. In 40% of the cases these antibodies are monoclonal. Persons with former Epstein-Barr virus infection were negative. The antibodies against the 2 proteins were first detected in Raji cells with an IgM-specific immunofluorescence test. The proteins were demonstrated in extracts of different cells and tissues by immunoblot technique. The molecular weight of the proteins measured in SDS polyacrylamide gel electrophoresis was 26 kd and 29 kd, respectively. Their presence in the cells does not depend on the presence of the Epstein-Barr virus genome. The relevance of the new findings concerning diagnostics as well as pathogenetic aspects remains to be established.

Specific recognition of antigens by cytolytic T lymphocytes sensitized to vaccinia virus was tested by monolayer adsorption. Adsorption was possible only on monolayers also expressing syngenic H-2 as viral antigens present during the sensitization phase. Vaccinia-virus-infected target cells were subjected to papain and neuraminidase treatment. H-2 antigenic determinants could be removed by papain treatment. Due to virus-specific inhibition of host-cell protein synthesis, reexpression of H-2 antigenic determinants did not take place, but viral surface antigens were resynthesized. Susceptibility of target cells to T-cell-mediated lysis was decreased after papain treatment. Substrains of vaccinia virus were used in order to define the minimal changes induced by vaccinia virus necessary for T-cell sensitization in vivo and target-cell lysis in vitro. When the immune response to a conditioanl lethal mutant strain of vaccinia virus was investigated, it could be demonstrated that expression of early surface antigens is sufficient for induction of the cellular immune reactions. These data were confirmed by inhibition studies with virus-specific antisera.

IgM antibody to hepatitis B core antigen (anti-HBc) was determined by a reverse enzyme immunoassay. In all of 58 patients with transient hepatitis B surface antigen (HBsAg)-positive acute hepatitis, IgM anti-HBc was detected in high titer. In 79.3%, IgM anti-HBc disappeared within two years, but in the remaining 12, it was still detectable. In 20 of 21 patients who developed chronic hepatitis, IgM anti-HBc was present after two years. High titers of IgM anti-HBc were found in 13 patients with histologically confirmed hepatitis B in whom HBsAg could not be detected in the initial serum samples; 11 of them later developed antibody to HBsAg and/or e antigen. Furthermore, IgM anti-HGc was detected in eight (5.6%) of 142 HBsAg-negative blood donors with elevated levels of serum transaminases and in 11 (0.5%) of 2,400 HBsAg-negative blood donors with normal levels of serum transaminases. Thus, IgM anti-HBc might be a better indicator of hepatitis B than HBsAg and help differentiage acute from chronic infection in HBsAg-positive patients.

The frequency of hepatitis B was examined using three serological parameters: HBsAg, antiHBs and antiHBc. All three substances were detected qualitatively by means of sensitive radioimmunological techniques. Of 1216 patients with acute hepatitis, 55% were HBsAg and antiHBc positive on admission to hospital. A further 17.8% had no HBsAg but were antiHBc positive and also partly antiHBs positive. These cases can be divided into 2 groups: In one group, in 8.6% of patients had a high antiHBc concentration during the acute phase. Similar antiHBc concentrations were seldom found (0.04%) in HBsAg negative blood donors. AntiHBs in the patients was at first mainly negative and then appeared during reconvalescence. These cases were considered to be acute hepatitis type B, although HBsAg was absent. In the second group, comprising 9.2% of the patients, antiHBc was present in low concentrations and in the majority of cases antiHBs had been present from the beginning. The same antibody constellation was found in 3.6% of 2341 blood donors. In the group of patients it is supposed that the acute hepatitis present is not of type B and has a different aetiology. The low concentration of antibody is interpreted to be a sign of an earlier HBV-infection.

A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The method's detection limit was 10(5) genome equivalents or 0.3 pg DNA per ml. Titers of 5 X 10(7) to 5 X 10(8) genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10(5)-5 X 10(7)) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (greater than 5 X 10(7)), moderate (10(5) -5 X 10(7)) and low (less than 10(5)) infectivity.

The development of cytopathogenic changes in chicken embryo fibroblasts infected with the herpesvirus of the turkey, strain PB-THVI, and the release of virus particles into the supernatant of infected cultures is accelerated at temperatures higher than 37 degrees C and is fastest at 41 degrees C, the normal body temperature of chickens. The growth rate of HVT in CEF cultures was followed by determination of the number of virus genome equivalents within infected cells at various time intervals p.i. A temperature-dependent increase in the amount of virus DNA per infected cell could be detected, which is highest at 41 degrees C. At all temperatures tested (34, 36, 41 and 43 degrees C) the number of virus genome equivalents per infected cell ultimately reaches the same level. In the course of infection, virus DNA in CEF cultures at 37 and 41 degrees C becomes associated with the cellular DNA, as determined by neutral CsCl gradients of the total cellular DNA and hybridization of each fraction with 32P-labelled virus-specific complementary RNA. Association of virus to cellular DNA occurs earlier at 41 than at 37 degrees C. However, the same proportion (45%) of the total virus DNA appears ultimately to be associated with cellular DNA at both temperatures. A temperature-shift of CEF cultures infected with PB-THVI from 41 to 37 degrees C 24 h p.i. resulted in the same replication kinetics of virus DNA as was found at 41 degrees C.

Vaccinia virus specific cytotoxicity against infected target cells was observed in vitro. Spleen lymphocytes from normal and immunized mice of the inbred strains C3H and DBA/2 were incubated with vaccinia virus-infected and non-infected 51 Cr-labeled mastocytoma P-815-X2 cells and L-929 fibroblasts, which were used as targets. Cytotoxic lymphocytes could be isolated from the mice as early as 2 days after infection with vaccinia virus. The highest cytotoxic effect was obtained with lymphocytes taken 6 days after infection. The degree of lysis was correlated with the ratio of immune lymphocytes to target cells. Specific blocking of target cell lysis resulted after addition of anti-vaccinia antibody from different sources. The effector cells could be characterized as T cells by elimination of macrophages and B cells. Target cell killing was only possible in a syngeneic system; allogeneic infected target cells were not lysed significantly.

Hepatitis B surface Antigen (HBsAg) wurde aus menschlichem Plasma durch Gelchromatographie, isopkynische Zentrifugation und zonale Sedimentation weitgehend von Plasmaproteinen und Hepatitis B Viruspartikeln (HBV) mit 60%iger AktivitAtserhaltung gereinigt. Eine Behandlung mit Formaldehydkonzentrationen bis 0,1% zur Inaktivierung moglicher RestinfektiositAt im gereinigten HBsAg reduziert die AntigenitAt in vitro und die ImmunogenitAt im Meerschweinchen nur gering. Adsorption an Aluminiumhydroxyd ergibt etwa 16 mal hohere Werte an Antikorpern gegen HBsAg (anti-HBs) als die Verabreichung von gelostem HBsAg. Nach zwei Injektionen von 0,2Μg Formaldehyd-behandeltem HBsAg mit Aluminiumhydroxyd wurde in Meerschweinchen ein mittlerer anti-HBs-Titer von 4 internationalen Einheiten/ml (Normalwert bei menschlichen Rekonvaleszenten etwa 0,1) gefunden, der ohne weitere Injektionen fur ein Jahr stabil blieb. Eine passive Verabreichung von einem zwolffachen Aquivalent an Meerschweinchen-anti-HBs kurz vor der ersten Injektion von HBsAg beeintrAchtigte die aktive Bildung von anti-HBs nach einer zweiten Injektion nicht. Zur Verringerung eines nicht vollig auszuschlie\enden Infektionsrisikos und zur Vermittlung eines raschen Schutzes konnte bei zukunftigen Versuchen mit einer HBsAg-Vaccine gleichzeitig Hepatitis B Immunoglobulin verabreicht werden. Um eine moglichst vollstAndige Freiheit von HBV in der Vaccine zu gewAhrleisten, sollte neben einer effizienten Reinigung und Inaktivierung als Ausgangsmaterial anti-HBe-positives Plasma verwendet werden.

Seventy-four laboratories tested identical panels of 50 coded samples containing 0·02 to 16·000 ng HB S Ag protein per ml serum. The median value and the variance of detection limits for HB S Ag in the different laboratories were determined for following methods: Agargeldifussion, counterimmunoelectrophoresis, complement fixation, reverse passive hemagglutination and solid phase radioimmunoassay. The significance of the tests for the screening of blood donors and hepatitis patients is discussed. For the control of the sensitivity and accuracy of HB S Ag determination it is proposed to use HB S Ag reference samples with defined contents of antigen specific protein.

Vacciniavirusinduzierte Zelloberflachenveranderungen konnen durch den zytoziden Effekt virusspezifischer Antikorper auf infizierte Zellen sowie durch die Agglutination virusinfizierter Zellen durch Concanavalin A demonstriert werden. Diese Veranderungen treten zeitlich korreliert mit einer gesteigerten Immunogenitat der Wirtszelle 2–3 Stunden nach Infektionsbeginn bereits vor der Produktion reifer infektioser Viruspartikeln auf. Eine gesteigerte Antikorperproduktion gegen Wirtszellantigene wird auch nach Immunisierung mit isolierten Plasmamembranen vacciniavirusinfizierter Zellen erreicht, wahrend Zellkernfraktionen diesen Effekt nicht auslosen. Bei den zytotoxischen Antikorpern, die zu den 7 S-Immunglobulinen gehoren, handelt es sich — wie durch Absorption mit BHK-Zellmembranen gezeigt werden kann — um spezifisch gegen BHK-Zellen gerichtete Antikorper.

Nach aktiver Immunisierung mit virusinfizierten Lymphoblasten bilden Meerschweinchen vermehrt Antikorper gegen Antigene nicht infizierter Lymphocyten. Dieser immunologische Adjuvanseffekt wurde nach Infektion von menschlichen und Mauselymphoblasten mit Herpes simplex-Virus, VSV und Vacciniavirus beobachtet. Die vermehrt gebildeten Antikorper wurden im Mikrolymphocytotoxicitatstest in vitro und in vivo durch ihre immunsuppressive Wirkung bei Tumorverpflanzung (Mastocytoma P-815-x2 DBA/2 → NMRI) nachgewiesen.

Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients. Secretory aspartic proteases of Candida albicans , which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years. The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated. These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence. As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells. Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect. This inhibition was shown to be concentration dependent. The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases. On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors.

A specific and sensitive solid-phase radioimmunoassay has been developed for the detection of LCM virus antigens and anti-LCM virus antibodies. The test was performed in a microtiter system using polyvinylcholoride wells coated with anti-LCM virus rabbit hyperimmune serum. LCM virus antigens were allowed to bind to this antibody and afterwards detected by 125J-labeled anti-LCM virus-gamma-globulin. Anti-LCM virus antibodies were assayed by specific inhibition of these bound antigens. Since this technique is rapid and easy to perform the solid-phase radioimmunoassay is a valuable test for detecting LCM virus infections. Selected at random, 208 girls with, and 208 girls without, contact to Syrian hamsters were investigated for anti-LCM virus antibodies. Five (2.4%) of the hamster-exposed girls had anti-LCM virus antibodies (RIA-titer 1:8-1:32) in contrast to none of the control group (P=0.03).

Summary In a group of 238 homosexual men a hepatitis B vaccine developed at the National Reference Center for Viral Hepatitis (Gottingen/Germany) was evaluated. The vaccine was well tolerated. Three intramuscular injections of inactivated HBsAg (subtype ad; 11300 Natl. Units/dose) induced antibody production in 89.7 % of the subjects. Four participants exhibited signs of hepatitis B infection. Vaccine recipients younger than 30 years produced higher antibody levels than older vaccinees. More than one half of the subjects who had only anti-HBs (anti-HBc negative) prior to immunization did not show an anamnestic booster response but formed IgM-anti-HBs. Antibody levels were significantly lower in anti-HBs negative/anti-HBc positive recipients than in subjects who had only anti-HBs or no hepatitis B markers. Immunological studies of T and B cell function did not provide conclusive evidence for the underlying cause of the different immune responses.

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli . Core protein without pre-c, P22 c , assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25 e , instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25 e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HBeAg and the protective role of anti- HBe antibody.

The titer of antibody against core antigen of hepatitis B virus in the immunoglobulin M class (IgM anti-HBc) was determined by an IgM capture assay of reduced sensitivity (30 arbitrary units). The distribution of titers among 235 acute hepatitis patients who were hepatitis B surface antigen (HBsAg) positive suggested that 600 U forms a lower cutoff value for acute hepatitis B. Clinically apparent cases of acute hepatitis with high IgM anti-HBc and without HBsAg were rare (2.6%). Acute, non-B hepatitis in HBsAg carriers was more frequent (9.4%). In chronic hepatitis B, 39% of 174 biopsy-proven cases had moderate titers of 30 to 600 U, whereas healthy HBsAg carriers were rarely (4/84) positive. In mild or inapparent infections without HBsAg, titers were between 50 and 400 U. Thus, sufficiently accurate and sensitive quantitation of IgM anti-HBc allows for differentiation of acute and nonacute hepatitis B virus infection in acute hepatitis, partial differentiation between clinically symptomatic and asymptomatic chronic infections, and identification of recent subclinical infections.

The action of peritoneal exudate cells (PEC) from normal and vaccinia virus infected mice on infectious vaccinia virus particles was investigated in vitro. PEC from immune mice showed a significantly higher infectivity titre reduction (virus clearance, VC) than normal cells. This effect could be clearly attributed to the macrophage. Vaccinia virus multiplied in PEC from normal animals while there was no virus propagation in cells from immunized mice. The release of adsorbed or engulfed virus was reduced significantly in PEC from immunized animals. Anti-vaccinia-antibodies seem to activate normal macrophages to increased virus clearance. This stimulating effect was demonstrable only in the IgG fraction of the antiserum. The activity of macrophages from mice injected three times over a period of 14 days with vaccinia virus could be entirely blocked with anti-mouse-IgG, while PEC from mice injected one time six days previously were not inhibited.

Summary The intracellular state of Marek's disease virus (MDV) DNA was investigated in two permanent chicken cell lines: HPRS-1 and MSB-1. The HPRS-1 line was established from an ovarian lymphoma of a chicken with Marek's disease and is a virus-non-productive line, while the MSB-1 line originates from another animal with a Marek's disease splenic lymphoma and is a low producer line. By repeated isopycnic centrifugation in CsCl, MDV DNA in the HPRS-1 line showed properties of integrated DNA, whereas in cells of the productive MSB-1 line both integrated and free virus DNA appeared to be present. Under denaturing conditions (0.1 m-NaOH) the virus DNA remained associated with the cellular DNA as revealed by equilibrium centrifugation in CsCl and hybridization of the DNA in each single fraction with 32P-labelled complementary RNA transcribed from the DNA of the GA strain of MDV. Shearing of HPRS-1 DNA to a mol. wt. of about 8 × 106 released only part of the virus DNA to the density of free virus DNA, while a large proportion of MDV DNA could still be localized at the density of cellular DNA. Sedimentation velocity experiments with HPRS-1 and MSB-1 DNA originally fractionated on CsCl gradients revealed integrated virus DNA sequences in both cell lines and an additional peak of virus DNA at the position of free linear MDV DNA in the MSB-1 line. No fast-sedimenting virus DNA molecules with properties of covalently closed circular structures could be detected. Further evidence for the presence of integrated virus DNA sequences in MDV-transformed cells was provided by the Hirt (1967) precipitation procedure.

The relation of four different strains of MDV and two strains of HVT was analyzed by gel electrophoresis of viral DNA digested by various restriction endonucleases and by filter hybridization of viral DNA with complementary RNA. The four MDV strains showed fragment patterns completely different from those of HVT upon digestion of the viral DNA with Bam H I, Eco R I, Hind III, Hpa, I, and Xho and separation of fragments on agarose gels. The cleavage patterns of the four MDV strains showed great similarities among each other as well as some differences between the individual strains. In the cleavage patterns of HVT a similar close relationship was observed between the two HVT strains with slight divergence between both. Filter hybridizations of viral DNA with labelled complementary RNA prepared from the DNA of the GA strain of MDV or from the DNA of the PH-THV1 strain of HVT revealed no cross-hybridization between the MDV and the HVT strains. cRNA prepared from the DNA of an MDV strain hybridized only to restriction enzyme fragments of the MDV strains transferred to nitrocellulose filters, but not to fragments of HVT DNA, and vice versa.