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Humoral antibodies can be induced in cotton topped (CT) marmosets with killed Herpesvirus saimiri (HVS) vaccines. The serum antibodies delayed, but did not prevent, malignant lymphoma in actively and passively immunized monkeys after inoculation with 10(3.8)TCID50 of cell-free HVS. Experiments are in progress to determine whether the vaccines are able to protect against smaller challenge dosages of HVS. The immunized monkeys were not resistant against transplantation of tumor cells. Infection with Epstein-Barr virus (EBV) and Marek's disease virus (MDV) did not protect against the tumors caused by superinfection with HVS. Attenuation of HVS was not achieved by passaging the HVS genome through different monkey species.

Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.

Objective: Various laboratory methods for detection of cytomegalovirus (CMV) are available, but all suffer from limited sensitivity and specificity or are prone to sampling errors.

The risk of vertical transmission of the hepatitis C virus (HCV) from infected mothers to their children during pregnancy and delivery was determined in 120 children born to HCV-positive mothers. Methods included enzyme immunoassay and immunoblot for detection of HCV antibodies and reverse transcription polymerase chain reaction (RT-PCR) for detection of viral RNA. Six (5%) children were perinatally infected with HCV as shown by RT-PCR. None of the infected children had clinical signs of hepatitis. None of the pregnancies was complicated by abortion, stillbirth, premature birth, or malformation of the child. Special concern was given to the possibility of HCV transmission via breast milk. In no breast milk sample obtained from 34 HCV-infected mothers was HCV RNA detected. These observations indicate that HCV infection is not necessarily a contraindication for breast-feeding.

The reverse transcriptase V207I mutation within the hepatitis B virus (HBV) polymerase is associated with resistance to lamivudine in vitro. The prevalence of this mutation in treatment-naïve patients was 1% (1/96). A follow-up of the patient carrying this mutation prior to treatment revealed no loss of sensitivity of HBV to lamivudine in vivo.

Human cytomegalovirus (HCMV) is a major pathogen in newborn children and immunosuppressed individuals. There are various diagnostic methods for HCMV, but all test systems either suffer from limited sensitivity and specificity or are prone to sampling errors. Due to its unequaled sensitivity, strong hopes were pinned on the polymerase chain reaction (PCR) for the early diagnosis of active HCMV infections. However, when monitoring immunocompromised patients, unwanted positive results are often obtained without further evidence of active HCMV infection. To better discriminate between latent and active infections, different methods for the quantitation of viral genomes in clinical samples by PCR have been devised in the past years. Here we discuss the current standing of quantitative PCR in HCMV diagnostics.

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.

To determine whether the persistent presence of antibodies to recombinant antigens of the hepatitis C virus (HCV) corresponds to the presence of hepatitis C virus RNA in the same serum, 85 anti-HCV positive patients were studied by the polymerase chain reaction (PCR). The focus of the research was on patients with chronic hepatitis. Eighty- three patients were found to be positive by PCR; only two were negative. In addition, liver biopsies taken from seven patients positive for anti-HCV were shown to contain HCV-specific RNA. Sera collected from three patients suspected to have NANB hepatitis on the basis of clinical symptoms were negative both for HCV antibodies and HCV RNA. The correlation between HCV antibody positivity and detection of HCV RNA was 97.6%.

summary. The prevalence of a newly described DNA virus (SENV-H) was examined in a population of 599 individuals by polymerase chain reaction (PCR). All individuals were assigned to a nonrisk or a risk group depending on the presence of historical or serological factors indicating an increased risk for parenterally transmitted diseases. In a group of 226 healthy blood donors, 38 (16.8%) were found to be SENV-H viraemic. The highest prevalence of SENV-H viraemia was observed among patients infected by HIV (28 of 63; 44.4%). Contrarily, of 78 individuals on maintenance haemodialysis, only 10 (12.8%) were found positive in the SENV-H PCR. Our results demonstrate that SENV-H viraemia is widespread in the general population. Therefore, it seems to be questionable if parenteral transmission is the main route for spreading SENV-H. The hepatitis-inducing capacity of SENV-H is unclear. However, taking our clinical and epidemiological data into account it seems unlikely that this virus is responsible for hepatitis.

Known and potential virulence factors of enterococcal blood culture isolates were studied using 89 Enterococcus faecalis and 24 Enterococcus faecium isolates. The prevalence of the respective factors was (Enterococcus faecalis vs. Enterococcus faecium): hemolysin 16% vs. 0%, gelatinase 55% vs. 0%, aggregation substance 63% vs. 13%, lipase 35% vs. 4%, hemagglutinin 97% vs. 0%. Deoxyribonuclease was not detected in any isolate. The study showed that hemagglutinin and lipase may represent additional virulence factors of Enterococcus faecalis but not Enterococcus faecium. The significance of these factors in the pathogenesis of enterococcal infection needs to be elucidated in further studies.

The detection of HCV-PCR amplification products by DNA enzyme immunoassay (DEIA) was compared with conventional hybridization carried out with a 32P-labelled oligonucleotide probe. The detection limit of both methods was shown to be between 100 pg and 1 ng of amplicon. All serum samples of 40 HCV-seropositive patients were positive after PCR in autoradiography, but only 38 with the DEIA technique (sensitivity 95%). There were no false-positive reactions by either method. The advantage of the DEIA method was the fast and non-radioactive detection of HCV amplicons. DEIA combines the specificity of the hybridization event with the speed of an ELISA procedure and is suitable for HCV-PCR.

The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany. The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli. The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA). Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined. In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests. In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR. In follow-up studies conducted over 1 year, these results did not change. In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative. All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later. In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0. In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR. All three patients were free of complaints. The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%). This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.

Virus infections are common among drug abusers and the medical complications are severe [1, 2, 3, 4]. Many infections are due to an insterile technique of injection (needle sharing); disease transmission via blood or body fluids may also be associated with sexual intercourse.

4000 sera were tested for antibodies against hepatitis C virus (HCV) by means of an ELISA using the C100-3 antigen. 38.9% of patients with non-A, non-B hepatitis following blood transfusion (n = 108) had HCV antibodies. Among patients with chronic liver damage of unknown origin (n = 316) 30.4% were anti-HCV positive, and in 2,506 patients with transitional or chronic elevation of transaminases 14.8% showed HCV antibodies. Haemophiliacs (n = 26) with 65.4% anti-HCV positives and drug addicts (n = 46) with 56.5% anti-HCV positives had the highest prevalence among high risk groups. Addicts dying from drug abuse (n = 216) and HIV 1 positives (n = 127) were anti-HCV positive in 37.5% and 26.0%, respectively. Patients on haemodialysis (n = 331) had antibodies against HCV in 12.4%. Health care workers (n = 217) appear to be at a comparably low risk with only 2.8% anti-HCV positives. Up to now we could not find a single case of intrafamilial spread of HCV in 46 examined cases. We suggest that HCV infectivity of contaminated body fluids and blood is lower than that of hepatitis B virus or human immunodeficiency virus type 1 carriers. In suspected non-A, non-B hepatitis negative test results should be confirmed in a second sample because it may take three to six months after infection before HCV antibodies occur. However, about 10% of chronic HCV infections are not detectable with the presently available test. This may change when new tests become available using HCV specific antigens other than C100-3.

Needle sharing among drug addicts leads to the transmission of infectious diseases such as hepatitis B and AIDS. After development of a test system based on gene technology against the hepatitis C virus (HCV), drug addicts have been regarded as an important reservoir for hepatitis C. In our study 113 (40.1%) out of 282 addicts who died from drug abuse in Hamburg between 1988 and 1990 had antibodies against HCV (anti-HCV). The prevalence of anti-HCV differed in various age groups; the highest prevalence was found in addicts aged 30-34 years. Co-infections with hepatitis B and hepatitis C virus were found in 57 drug addicts (59.4%) out of 96 deceased with antibodies to hepatitis B (anti-HBc), whereas only 8 out of 23 HIV-infected were anti-HCV positive (34.8%).

Needle sharing among drug addicts leads to the transmission of infectious diseases such as hepatitis B and AIDS. Since the identification of the hepatitis C virus (HCV) drug addicts have been regarded as an important reservoir for hepatitis C. The hepatitis C virus is a major cause of the chronic non-A, non-B hepatitis. HCV is a single stranded encapsulated RNA virus probably belonging to the flaviviruses with a diameter of 50–60 nm (Grob and Joller-Gemelka 1990). However, until now only one HCV associated antigen is available for detection of specific antibodies in infected patients. Seroconversion may take 3 to 6 months or more and may occur after onset of clinical symptoms. We have little knowledge about the persistence of HCV antibodies in cases of chronic or resolved non-A, non-B hepatitis. A negative test result therefore does not exclude HCV infection as the patient may have antibodies against other antigen determinants of the virus. Confirmatory tests are necessary to give further information on infectiosity and clinical outcome of patients with antibodies against HCV (anti-HCV), (Marwick and Skolnick 1990).

The prevalence of hepatitis G virus (HGV) and hepatitis C virus (HCV) infection was determined by reverse transcription-PCR in 777 individuals with and without risk factors for viral transmission via blood. From our results we conclude that transmission of HGV and that of HCV are favored by similar risk factors.

For up to 20 to 40% of patients chronically infected with hepatitis C virus (HCV), the mode of transmission is still unknown. We demonstrate that tear fluid contains HCV RNA-carrying material with the properties of infectious virus and conclude that smear infection with tear fluid may play a role in HCV transmission.

In 1989 blood samples from 2581 fatalities investigated at the Institute of Forensic Medicine in Hamburg were screened for HIV-1-antibodies. Sera from 13 corpses were confirmed positive for HIV-1 (prevalence rate approx 0.5 %).-Viable HIV was found in blood cultures of 4 cadavers stored under non-refrigerated conditions up to 36 hours after death.

By means of an ELISA the level of hepatitis B e-antigen (HBeAg) titre was determined in 51 patients with acute and 123 patients with persistent hepatitis B virus (HBV) infection. We also investigated 120 sera of these patients for infectivity by detecting HBV-specific DNA polymerase (DNA-P). Additional determination of the HBeAg titre revealed that the detection of HBeAg at a dilution of at least 1:50 correlates with the detection of DNA-P in 95% of cases, while in sera with an HBeAg titre of 1:25 or less DNA-P is only detectable in up to 5% of cases. Since the detection of DNA-P indicates high infectivity, certain titres of HBeAg can be employed to differentiate three different degrees of infectivity: (1) Sera without HBeAg show no or only little infectivity; (2) sera containing HBeAg≧1:50 are highly infectious; (3) HBeAg titres up to 1:25 indicate a medium degree of infectivity. These three markers for different degrees of infectivity were applied to study the course of infectivity during acute and persistent forms of HBV infection. In acute hepatitis B the period for the elimination of HBeAg is independent of the HBeAg titre in the first serum sample. In all cases the elimination of HBeAg in acute hepatitis B did not exceed 4 months. Patients with persistent HBV infection and an initial HBeAg titre ≧1:50 remained at this high degree of infectivity for at least 18–30 months in 70% of cases. Only 30% of this collective showed a decrease in infectivity. However, 40% of the patients with persistent hepatitis B with an initial low titre of HBeAg showed an increase in infectivity, while 60% remained low or converted to anti-HBe. This confirms that an initially high degree of infectivity in the persistent form of hepatitis B is an unfavourable sign. Also, the present study demonstrates that the correlation of the amount of Dane particles and HBeAg titre is not due to conversion of spontaneously released hepatitis B core antigen (HBcAg) from circulating Dane particles to HBeAg. By means of the ELISA the half-life of HBeAg in vitro was determined to be 12–24 h.

An enzyme-linked immunosorbent assay (ELISA) using a highly purified soluble antigen prepared from pathogenic Treponema pallidum (Nichols) was carried out to detect specific IgM antibodies in syphilis. Serum specimens from 365 patients with untreated or treated syphilis were tested by the ELISA and by the 19S(IgM) fluorescent treponemal antibody-absorbed test. In the ELISA sera were preabsorbed with aggregated human IgG to eliminate any rheumatoid factor present. Sera from 331 healthy blood donors served as controls. The results of both tests correlated well. The specificity and sensitivity of the treponemal ELISA were both greater than 97%. The assay was easy to perform and may be automated.

The viruses of equine arteritis, rubella, bovine viral diarrhea and hog cholera have been concentrated and purified. In preparations negatively stained with uranyl acetate they appeared as roughly spherical particles about 60 nm across. Within their envelopes isometrical cores were detected after spontaneous disruption or upon pretreatment; they are considered to be the viral nucleocapsids. The lower coefficients of variation calculated for the nucleocapsids as compared with those of the respective virus particles indicate that the pronounced heterogeneity in virion size must be attributed to the envelope. Ringlike morphological subunits observed on the surface of the spherical nucleocapsids and the presence of centra] core components suggest that the four viruses should be classified as togaviruses (lipid-containing RNA viruses with cubic symmetry of the nucleocapsid), together with arbo-A-viruses. The bovine viral diarrhea and hog cholera viruses differ from the other viruses studied in their lower sedimentation and density values, their lesser nucleocapsid diameters and an unusual “rosary” envelope structure. A further subdivision of the togavirus group is discussed.

The simultaneous detection test gave no evidence for the presence of RNA tumour viruses in herpesvirus induced malignant lymphomas of non-human primates. The 12 tumours tested were obtained from three different monkey species inoculated with Herpesvirus saimiri or herpesvirus ateles. Particles encapsulating RNA-instructed DNA polymerase and high mol. wt. virus-related RNA were easily demonstrated in tumours of the mouse induced by type-C or type-B oncornaviruses and in human lymphoid cells infected with simian sarcoma virus type I which were examined in parallel. Attempts to demonstrate partial expression of an oncornavirus genome in the herpesvirus induced tumours and attempts to detect an interspecies antigen related to monkey oncornaviruses were negative and strengthened the observations made with the simultaneous detection test.

HERPESVIRUSES are suspected to be causal agents in human malignant neoplasia. At the present level of knowledge, clinical trials in man with viral vaccines against oncogenic herpesviruses cannot be initiated, but the oncogenic herpesviruses derived from monkeys1,2 can be used in vivo to study the problems of herpesvirus cancer vaccines. The oncogenic herpesvirus saimiri (HVS) regularly induces malignant lymphoma in cotton topped (CT) marmoset monkeys (S. oedipus). A killed vaccine from HVS was developed in our laboratory and tested for safety and efficacy in this primate model system.

In January 1985 we started to screen all blood donors of the blood bank of the University clinics in Hamburg Eppendorf (UKE) for LAV/HTLV-III antibodies [1, 2]. Of the 7624 donors examined up until July 1985, 10 were positive in the enzyme-linked immunosorbent assay (ELISA), the IFT, and the Western blot analysis. All of the LAV/HTLV-III-positive donors were male; the mean age was 34 years; nine out of the ten LAV/HTLV-III positives were homosexual and one out of the ten had had multiple contacts with prostitutes.

The periods of persisting immunoglobulin M class antibodies to hepatitis B core antigen in 28 patients with acute hepatitis B infections and in 134 patients with chronic hepatitis B infections were studied by using an enzyme-linked immunosorbent assay and an indirect immunofluorescence technique. We showed that the clearance of antibodies to hepatitis B core antigen is independent of the degree of viremia in both forms of hepatitis.

HERPESVIRUSES are the most important candidates for human cancer agents. The Epstein–Barr virus (EBV) in particular is suspected of being the causal agent in Burkitt's lymphoma and nasopharyngeal carcinoma1. Immunological principles, well understood from vaccine prophylaxis of viral diseases, provide the potential for the eventual control of herpesvirus-induced neoplasia. The development of safe and efficient cancer vaccines with the human candidate herpesviruses is, however, difficult, as there are no reliable in vitro or experimental animal methods for detecting oncogenic potential in preparations which might be applicable to the human subject. We are studying the problems of herpesvirus cancer vaccines in non-human primates. Two members of the herpesvirus family, H. saimiri (HVS) and H. ateles (HVA), induce malignant lymphoma and leukemia in several New World monkey species2,3. HVS-induced lymphoma in cotton topped (CT) marmoset monkeys (Saguinus oedipus) has been used in this study as all monkeys inoculated with HVS regularly develop malignant lymphoma within two months4,5. This investigation describes the effect of a killed herpesvirus cancer vaccine in protecting non-human primates against malignant lymphoma.