The importance of the quasispecies nature of hepatitis C virus (HCV) for the evolution of HCV populations in patients: study on a single source outbreak of HCV infection.

The quasispecies populations of hepatitis C virus (HCV) in the infectious source (IS) and in patients of a single source outbreak were characterized. A predominant variant AD78-1 and some minor variants of HCV 5' NCR were identified in IS. Different complex HCV populations emerged rapidly in patients though HCV populations in chronic infection were rather simple and stable. AD78-1 remained predominant in 17/24 chronically infected patients while new predominant variants with distinct nucleotide substitutions persisted in 7 other patients. Thus, only distinct variants are involved in viral persistence and the genetic heterogeneity of HCV does not successively increase with time.

Does the concurrent administration of an inactivated hepatitis A vaccine influence the immune response to other travelers vaccines?

BACKGROUND: Travelers seeking protection from hepatitis A also often need protection against other infections, prevalent at their destinations. METHODS: A total of 396 volunteers received not only a hepatitis A vaccine but also either a vaccine against polio, hepatitis B, diphtheria, tetanus, yellow fever, Japanese encephalitis, typhoid fever or rabies according to their individual needs. We investigated the potential influence of the hepatitis A vaccination on the immune response to the other travelers vaccines that were administered concurrently. RESULTS: With seroprotection rates of 100% for yellow fever, Japanese encephalitis and rabies immunization and tetanus boosters our data demonstrate that the concurrent administration of hepatitis A vaccine does not compromise the immune response of these vaccines. Also for oral typhoid, hepatitis B and diphtheria vaccination we did not detect a negative influence of concurrent hepatitis A vaccine administration as compared with respective vaccinations when given alone. Prior to vaccination, more than one third of our subjects lacked protective antibody levels against diphtheria and only 44% of initially seronegative travelers seroconverted to an anti-diphtheria titer > or = 0.01 mIU/mL, supporting a need for an additional dose. Furthermore, only two thirds of the vaccinees tested prior to vaccination were protected against polio type 3, and the seroconversion rate following the administration of oral polio vaccine, was lower for viral type 3 (80%), as has been previously demonstrated in settings without concurrent other vaccinations. CONCLUSION: No negative effect of concurrent travelers vaccinations on the immune response of a hepatitis A vaccine has been detected in a previous report, and, likewise our data suggest no impairment of the antibody response of these travelers vaccines by the concurrent administration of the hepatitis A vaccine.

Immunization of woodchucks with plasmids expressing woodchuck hepatitis virus (WHV) core antigen and surface antigen suppresses WHV infection.

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 microgram of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 microgram of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 10(4) or 10(5) of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.

Distinct prevalence of antibodies to the E2 protein of GB virus C/hepatitis G virus in different parts of the world.

Since the identification of the new human virus, GB virus C (GBV-C)/hepatitis G-virus (HGV), in 1995/1996, reverse transcription polymerase chain reaction remained the sole available diagnostic tool for GBV-C/HGV infection. Recently, a serologic test based on the detection of antibodies to the putative envelope protein 2 (anti-E2) has been introduced. We used this assay for a seroepidemiological survey including 3,314 healthy individuals from different parts of the world, 123 patients from Germany who were suspected to have an increased risk of acquiring GBV-C/HGV infection, 128 multiple organ donors, and 90 GBV-C/HGV RNA positive persons. In European countries, anti-E2 seropositivity ranged from 10.9% (Germany) to 15.3% (Austria). In South Africa (20.3%) and Brazil (19.5%), even higher anti-E2 prevalence rates were recorded. In Asian countries like Bhutan (3.9%), Malaysia (6.3%), and the Philippines (2.7%), anti-E2 positivity was significantly lower. GBV-C/HGV anti-E2 prevalence in potential "risk groups," i.e., patients on hemodialysis and renal transplant recipients, did not vary significantly from anti-E2 seroprevalence in German blood donors. Anti-E2 and GBV-C/HGV RNA were found to be mutually exclusive, confirming the notion that anti-E2 has to be considered as a marker of past infection.

GB virus C infection in patients who underwent liver transplantation.

To elucidate the impact of an infection with the recently discovered GB virus C (GBV-C) on the clinical course after orthotopic liver transplantation (OLT), we studied eight patients who were GBV-C RNA positive after transplantation. Five individuals had been viraemic before transplantation, three became GBV-C RNA positive thereafter. A control group comprised eight patients without pre- or post-transplant GBV-C infection. GBV-C RNA was detected by reverse-transcription followed by nested polymerase-chain-reaction (PCR) with primers corresponding to the NS5 genome region. Nested PCR products were sequenced directly. The five patients infected with GBV-C before transplantation remained GBV-C RNA positive throughout the time of observation. Pre- and post-transplant GBV-C RNA titres were almost identical. Phylogenetic analysis revealed a very close relationship between the pre- and post-transplant viral nucleotide sequences indicating persistent GBV-C infection. No signs of hepatitis could be detected after transplantation in all GBV-C infected patients. However, four out of eight GBV-C RNA positive patients had a clinical course complicated by severe cholestasis, which was not observed in the control group. Although GBV-C infection does not lead to an increase in the rate of post-transplant hepatitis, it might be associated with severe unexplained cholestatic courses after OLT.

Booster immunization of low- and non-responders after a standard three dose hepatitis B vaccine schedule--results of a post-marketing surveillance.

Seventy-nine low-responders and 83 non-responders after a previous three-dose hepatitis B (HB) vaccine course at 0.1, and 6 months were enrolled to receive additional 20 micrograms recombinant HB vaccine doses every 2 months until all had anti-HBs levels > or = 100 mIU ml-1. After the first booster, 65.4% had anti-HBs levels > or = 100 mIU ml-1, 17.9% were low-responders (10-99 mIU ml-1), and 16.7% remained non-responders (< 10 mIU ml-1). All complying non-responders developed anti-HBs levels > or = 100 mIU ml-1 after the third booster at the latest, whereas all low-responders reached this level after the second booster. Although body mass index affected the response to the first hepatitis B booster, when full compliance to regular revaccination was ensured, all non- and low-responders eventually reached sufficient anti-HBs levels.

Immunogenicity of a recombinant hepatitis B vaccine in adults.

OBJECTIVES: To evaluate the immunogenicity and reactogenicity of a recombinant hepatitis B vaccine in health care staff under routine use and unselected conditions and to investigate factors that influence the response to vaccination. METHODS: This prospective postmarketing surveillance study was performed in unselected health care staff and their relatives (age range, 12-60 years) at 58 hospitals. Overall, 880 subjects were administered a 20-microgram dose of a vaccine at 0, 1, and 6 months according to the prescribing information and under routine hospital practice, and they were tested for antibody to hepatitis B surface antigen after the third dose at the hospitals routine laboratory. The principal outcome measures were antibody to hepatitis B surface antigen titers that were expressed as the seroprotection rate (SPR) (SPR [given as a percentage], > or = 10 mlU/ mL), spontaneously reported adverse events, and geometric mean titers (in milli-international units per milliliter). RESULTS: The compliance to the 3-dose schedule under routine hospital practice was 98.1%. The immune response was good in all age groups, and the overall SPR was 97.8% at 1 month after the third dose in field conditions with unselected health care workers. The SPR in vaccinees (age range, 40-59 years) was close to 95%. Age (P < .001), smoking (> or = 10 cigarettes per day) (P < .001), Broca index (> 110%) (P < .001), antibody to hepatitis B surface antigen testing (> 8 weeks after the last dose) (P = .03), chronic underlying disease (P = .04), and male gender (P = .04) were factors associated with lower geometric mean titers in routine vaccine use. No serious adverse events were reported. CONCLUSION: The large immune response that was elicited by this hepatitis B vaccine in adults under daily routine field conditions reflected reality, with a high SPR also found in elderly and other persons with risk factors associated with a lower immune response.

Immunogenicity of an inactivated hepatitis A vaccine administered according to two different schedules and the interference of other "travellers" vaccines with the immune response.

A total of 2036 persons consulting vaccination centers in Germany were vaccinated with an inactivated hepatitis A vaccine (containing 720 ELISA units of antigen) either according to the standard schedule (two vaccinations given 4 weeks apart) or to an abbreviated schedule (two vaccinations given 2 weeks apart) in a controlled clinical study. The abbreviated schedule induced a similar rate of seroconversion and geometric mean antibody titre as compared to the standard schedule. The incidence of reactions reported after vaccination was similar in both groups. When other "travellers" vaccines were given simultaneously neither the immunogenicity nor the reactogenicity of the hepatitis A vaccine were influenced. These findings have considerable practical importance in the prevention of hepatitis A in travellers.

Analysis of sequence and pathogenic properties of two variants of encephalomyocarditis virus differing in a single amino acid in VP1.

The encephalomyocarditis (EMC) virus-induced diabetes-like syndrome in mouse inbred strains was used as a model to study the insulin-dependent diabetes mellitus (IDDM). Our investigations were performed with two EMC virus variants, PV2 and PV7. After infection of SJL mice with 10(5) PFU of PV2 about 70% of the animals developed a diabetes-like syndrome, whereas the PV7 infected mice appeared healthy. Histological examination and in situ experiments revealed that the islets of Langerhans are a main target of PV2, whereas PV7 infection leads to only modest changes of the islets. Sequence analysis of both variants revealed one amino acid exchange within the capsid protein VP1. Hence, we describe the first diabetogenic and non-diabetogenic EMCV variants differing in only one single amino acid.

Interleukin-1 alpha (IL-1 alpha) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) as potential inducers of supravital chemotaxis.

The phenomenon of artificially induced local leucocyte reactions during the supravital period could be of practical importance, but has not yet been comprehensively investigated. For a more detailed evaluation, experiments with the chemotactic agents interleukin-1 alpha (IL-1 alpha) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) were performed by subcutaneous injection into various anatomical regions (back, abdomen, limbs) of NMRI-mice (National Medical Research Institute) and pigs 0-5 min after circulatory arrest. Phosphate buffered saline (PBS) without effective components was administered to equivalent areas of the animals as a control. Tissue specimens were collected at 6 h postmortem (mice) and 12-14 h postmortem (pigs), cut into serial sections, stained with H & E and examined under the microscope. A leucocyte reaction did not develop in pigs (n = 10, 30 tissue samples) following injection of FMLP, however, dermal, subcutaneous and perivascular infiltration of leucocytes (in particular mononuclear cells and a few granulocytes) was found in 3 out of 30 tissue specimens in murine experiments. In addition intravascular cell accumulations were detected in 2 out of 30 samples. The injection of IL-1 alpha to mice gave similar results, i.e., aggregations of leucocytes and intravascular cell accumulations in 4 out of 30 and 3 out of 30 tissue samples, respectively. In negative controls no leucocyte reaction was detectable. This shows that potent chemotactic factors such as IL-1 alpha and FMLP administered in the early supravital period can induce moderate local leucocyte reactions in animal models in at least some cases. A clear morphological differentiation between vital and supravital chemotaxis does not seem to be possible. The supravitally stimulated accumulations of leucocytes are interpreted as an aggregation of resident macrophages in combination with a slight migration of blood leucocytes. Presumably, these alterations are restricted to the very early supravital period as long as sufficient energy reserves are available. It must be stated that the observed changes are reactions, not spontaneous actions, so that the general validity of the phenomenon of leucocyte infiltration as a vital parameter is not affected.

The complete nucleotide sequence and construction of an infectious cDNA clone of a highly virulent encephalomyocarditis virus.

The nucleotide sequence of a highly variant of the encephalomyocarditis virus, PV21, was determined. Discounting the poly(A) tail the viral genome is 7.861 kb in length, including a poly(C) region of 157 or 158 nucleotides. The sequence differs in 38 positions from another virulent EMCV variant, EMCV-R (1). However, the PV21 sequence shows only 85% identity with two nonlethal variants isolated in this laboratory (2). A cDNA clone covering the complete virus sequence including a poly(C) region of approximately 150 nucleotides was constructed. The recombinant virus was shown to be infectious in NMRI mice.

A rapid and reliable assay for testing acyclovir sensitivity of clinical herpes simplex virus isolates independent of virus dose and reading time.

The determination of acyclovir (ACV) sensitivity of clinical herpes simplex virus (HSV) isolates was studied by means of a cytopathic effect (CPE) inhibitory assay (CIA). Medium of HSV-infected Vero cells was supplemented with different ACV concentrations. The CPE was read quantitatively by light microscopy. The inhibitory dose for 50% CPE reduction (ID50) was calculated by applying 1st or 3rd degree regression lines, and aspects of different methods for calculation are discussed. The CIA proved highly reproducible. Surprisingly, the obtained ACV-ID50 values were independent of reading time and virus dose used in the test. In comparison to dye-uptake assay, CIA could be evaluated earlier. Therefore, CIA provides simple, rapid, and precise determination of ACV sensitivity of clinical HSV-isolates which can be achieved within 1 or 2 days after the virus has been isolated out of a clinical specimen.

HPLC is an effective and fast method for analysis of viral proteins: a study of encephalomyocarditis virus mutants differing in pathogenicity.

We investigated the use of HPLC in analysis of picornavirus variants by comparing structural polypeptides of three stable mutants of encephalomyocarditis virus (EMCV). The variants are known to differ in their pathogenicity for mice: plaque variant 2 (PV2) is diabetogenic, PV7 is non-diabetogenic and PV21 induces a generalized lethal infection. We first used HPLC to separate the structural proteins at high purity levels. Detailed analysis of these structural proteins by HPLC-peptide mapping revealed differences in all four viral proteins of PV21 as compared with mutants PV2 and PV7. A single amino acid exchange was found in viral protein 1 between PV2 and PV7. Altered peaks were identified by calculating retention times of tryptic peptides using sequence data and a computer program. Since peak alterations could be attributed to the observed amino acid exchanges, the results correlate well with cDNA sequencing data. Thus HPLC proved to be a useful and fast tool for primary or additional characterization of picornavirus variants at the level of whole virus proteins.

Outbred mice infected by an encephalomyocarditis virus variant: a model for studying chronic viral heart disease.

Male 8 to 20-week-old NMRI mice (an outbred strain) infected with the encephalomyocarditis virus (EMCV) plaque variant (PV) 7 consistently develop a distinct myocarditis with a relatively low mortality (21%). Myocarditis occurs in essence independent of the virus dose applied, and other internal organs are not affected. Nevertheless, 3.5-week-old NMRI mice perished within 5 days of virus inoculation and exhibited disseminated myofibrillar degeneration (MFD); this obviously virus-induced myocardial damage was accompanied by scanty inflammatory infiltrates. EMCV PV7 infection of adult male C57Bl/6 and DBA/2 mice causes myocarditis comparable to that seen in NMRI mice. In DBA/2 mice, however, the virus-induced myocardial necrosis is complicated by subtotal calcification. This strain has a genetically determined "spontaneous" calcification of the myocardium, as shown by the study of uninfected controls. EMCV PV7-infected NMRI mice appear a promising model for study of long-term effects of viral myocarditis, possibly including cardiomyopathy. Furthermore, this outbred mouse strain offers the possibility of examining the pathogenesis of direct viral cytolysis and its relation to MFD as well as immunologically mediated cell damage.

[Perimyocarditis in influenza A virus infection]. / Perimyokarditis bei Influenza A Virus-Infektion.

A 48-year old man was admitted with suspected acute myocardial infarction because of severe precordial pain and monophasic ST-elevations in the ECG. The patient's history of an ongoing infection, the localization, extent, and course of the ECG changes as well as the development of a pericardial effusion suggested viral perimyocarditis. The diagnosis was supported by a significant rise of antibodies (seroconversion) against influenza A virus.

[Viral myocarditis and classical virology. Clinico-virologic and experimental data]. / Virusmyokarditis und klassische Virologie. Klinisch-virologische und experimentelle Daten.

A viral etiology of myocarditis has been proven stringently only in a few cases and mostly enteroviruses have been shown to be involved. In fact, a virological diagnosis of viral myocarditis is still rarely possible today. Virus isolation from heart-tissue is hard to achieve and biopsies, if at all, are often performed too late. Successful virus isolation from other materials (e.g. faeces, throat swab) or a serological diagnosis of acute virus infection cannot easily be correlated etiologically with cardial symptoms, depending of course also on the kind of virus infection diagnosed. To prove an acute infection serologically a fourfold rise in antibody titer or for some viruses specific IgM has to be detected. A group specific diagnosis of "acute enterovirus infection" is not reliably possible at the moment. The neutralization test is the only relatively type specific test in enterovirus serology. Intraserotypic virus variants cannot be detected by normal serology. Even when determining neutralizing IgM-antibodies, interpretation of results raises problems due to long persistence and possible cross reactivity. In case of suspected viral myocarditis clinicians should institute early and broad virological diagnostic measures. Virologists have to establish methods for virus or antigen determinations by molecular biological means. Furthermore, a fast and easy serological method for the diagnosis of an "acute enterovirus infection' would be very helpful. For research on the pathogenesis established animal models have to be used.

Virological examinations of patients with AIDS-related complex/Walter-Reed 5 enrolled in a double-blind placebo-controlled study with intravenous gammaglobulin administration. Prognostic value of anti-p24 determination. The ARC-IVIG Study Group.

Thirty patients with AIDS-related complex/Walter-Reed 5 enrolled in a placebo-controlled double-blind study with high-dose intravenous gammaglobulin administration were tested by quantitating HIV Western blot and other serological tests for viral antibodies. Furthermore, conventional virus isolation attempts were performed. Absence or loss of p24 antibodies during the study period was associated with progression to AIDS (p = 0.01) and thereby was an earlier prognostic parameter of a poor prognosis than T4 cell count. Neither changes in antibody patterns against other HIV polypeptides, HIV titers in the immunofluorescence test nor demonstration of HIV antigen were significantly associated with progression to AIDS. Cytomegalovirus (CMV) could be isolated from two duodenal biopsies of a patient who developed AIDS at the same time, but a concomitant serological diagnosis of CMV infection was not successful. Though signs in the serology of human herpesviruses (herpes simplex virus, CMV, Epstein-Barr virus), possibly indicating a reactivation of latent infections, could be observed in some instances, a correlation with clinical symptoms or the clinical outcome was not feasible, perhaps also because of a poor standardization of some of the test kits used. All patients were positive for IgG antibodies against the three herpesviruses when entering the study. High prevalence of hepatitis B virus (HBV) markers was found (83% anti-HBc positive), only 1 patient being chronically infected and highly infectious, as shown by HBV-DNA hybridization. No significant difference between treatment and placebo group was observed with the parameters tested in this study.

[Comparative test of the soluble toxicity of hydroxyapatite ceramics using human and animal osteoblasts]. / Toxizität von Hydroxylapatitkeramiken auf menschliche und tierische Osteoblasten.

The soluble toxicity of 5 different hydroxylapatite granulates was tested in osteoblast cell cultures. We established a human osteoblast-like cell culture and a culture of rat (Lewis) osteoblast-like cells. The granulates were also tested in cultures of human gingival fibroblasts as a conventional system. It is demonstrated that the osteoblast-like cell cultures are of higher sensitivity than fibroblast cultures which do not show any reaction to the extracts of hydroxylapatite granulates. Only 2 hydroxylapatite granulates show some toxic. The human osteoblast-like cells reacted slightly more sensitive to toxic substances of the materials tested than rat osteoblast-like cells. It is demonstrated that the osteoblast-like cell cultures are a highly sensitive test system for materials with low toxicity.