Holger F. Rabenau

Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus

Summary The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

Treatment of SARS with human interferons

Summary Effective antiviral agents are needed to treat severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. We assessed the antiviral potential of recombinant interferons against two clinical isolates of SARS-CoV—FFM-1, from Frankfurt patients, and Hong Kong—replicated in Vero and Caco2 cells. Interferon β was five to ten times more effective in Caco2 cells. Interferon α effectively inhibited SARS-CoV replication, but with a selectivity index 50–90 times lower than that for interferon β. Interferon γ was slightly better than interferon α in Vero cell cultures, but was completely ineffective in Caco2 cell cultures. Interferon β could be useful alone or in combination with other antiviral drugs for the treatment of SARS.

Virological and immunological effects of treatment interruptions in HIV-1 infected patients with treatment failure.

ObjectiveTo analyse the immunological and virological effects of treatment interruptions in HIV-1-infected patients with treatment failure and multidrug-resistant virus. MethodsDrug susceptibility was assessed using Antivirogram and genotypic analysis was based on population and clonal sequencing for 48 patients who had interrupted treatment (⩾ 2 months). ResultsTreatment interruption resulted in viral load increases (mean 0.7 log10 copies/ml;P = 0.0001) and CD4 cell count decreases (mean 89 × 106 cells/l;P = 0.0001). A complete shift to wild-type virus at the phenotypic, genotypic and clonal level was observed in 28/45 patients. These patients differed from those that did not show a shift to wild type in baseline CD4 cell counts (192 versus 59 × 106 cells/l;P = 0.007) and in the relationship between baseline viral load and CD4 cell count (no correlation versus a significant negative correlation;P = 0.008). Response to re-initiation of treatment fell with increasing viral load [relative hazard (RH) 0.33;P = 0.001] and with increasing total number of drugs with reduced susceptibility (RH 0.51;P = 0.0003); it improved with the number of new drugs received (RH 2.12;P = 0.0002) and a shift to wild type (RH 5.22, P = 0.006). ConclusionsChanges in surrogate markers suggest that treatment provided benefit in spite of virological failure and resistant virus. Although patients with a shift to wild-type virus responded better in the short term to treatment re-initiation, the long-term effects are not known and the risk of immune deterioration needs to be carefully considered.

Seroprevalence of herpes simplex virus type 1 and type 2 in selected German populations—relevance for the incidence of genital herpes

This study was carried out to determine the prevalence of antibodies to herpes simplex virus types 1 (HSV‐1) and 2 (HSV‐2) in selected German populations, such as blood donors, hospital patients, and human immunodeficiency virus (HIV)‐seropositive individuals. Serum samples collected between 1996 and 1998 were tested by enzyme immunoassays using monoclonal antibody‐selected native gG1 and gG2 as antigens and an immunoblot using type‐specific recombinant glycoproteins. Equivocal results were resolved by an “in‐house” Western blot assay. The prevalence of HSV‐1 antibodies increased steadily with age and reached high levels of ≥88% among subjects 40 years of age or older. In the sample of patients and blood donors, the HSV‐2 seroprevalence was 12.8% (95% CI = 11.9–13.8%). About 81% of the HSV‐2 seropositive subjects were coinfected with HSV‐1. When adjusted for age, there was no difference in the HSV‐2 seroprevalence between hospital patients and blood donors. The HSV‐2 seroprevalence was significantly higher among women (15%) than among men (10.5%), yielding a female : male odds ratio of 1.5 for hospital patients and of 1.67 for blood donors. Among the HIV‐infected population, 91.1% were seropositive for HSV‐1 and 47.9% for HSV‐2. HIV‐infected women have a significantly higher risk of HSV‐2 infection than men (odds ratio [OR] = 3.22; 95% confidence ratio [CI] 1.99–5.20). In conclusion, although the rate of infections with HSV‐2 is relatively low in the German population, attention should be given to the further development in adolescents, especially in view of a possible decrease of HSV‐1 seroprevalence in childhood. J. Med. Virol. 61:201–207, 2000.

Influenza Vaccination Compliance Among Health Care Workers in a German University Hospital

Background:Since 1988, the Standing Committee on Vaccination (STIKO) at the Robert Koch-Institute, Berlin, has explicitly recommended that health-care workers (HCWs) should be vaccinated against seasonal influenza. However, acceptance of the influenza vaccination by medical personnel is low.Methods:This study analyzes factors associated with the compliance of HCWs with the seasonal influenza vaccination on the basis of three different anonymized questionnaires during two consecutive influenza seasons: 2006/2007 and 2007/2008. The questionnaires covered details of demographics, frequency of previous vaccinations, reasons for accepting or declining the vaccination, and the HCW’s knowledge of the influenza vaccine and influenza itself.Results:Our study showed that physicians were significantly more likely to have been vaccinated than nurses (38.8% vs 17.4%; p < 0.0001). The main reasons for noncompliance included: supposition of a low risk of infection, fear of side effects, the belief that the influenza vaccine might trigger the influenza virus infection, and scepticism about the effectiveness of the influenza vaccination.Conclusion:Our findings confirm the importance of a comprehensive approach to the vaccination, ensuring that HCWs are correctly informed about the vaccine and that it is convenient to receive it.

Multicenter Evaluation of a New Automated Fourth-Generation Human Immunodeficiency Virus Screening Assay with a Sensitive Antigen Detection Module and High Specificity

ABSTRACT Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody that were available on the international market until now have antigen detection modules with relatively poor sensitivity and produce a higher rate of false-positive results than third-generation enzyme immunoassays (EIAs). The new Cobas Core HIV Combi EIA with an improved sensitivity for HIV p24 antigen was compared to alternative fourth- and third-generation assays, the p24 antigen test, and HIV type 1 (HIV-1) RNA reverse transcriptase PCR (RT-PCR). A total of 94 seroconversion panels (n = 709 sera), samples from the acute phase of infection after seroconversion (n = 32), anti-HIV-1-positive specimens (n = 730) from patients in different stages of the disease, 462 subtyped samples from different geographical locations, anti-HIV-2-positive sera (n = 302), dilutions of cell culture supernatants (n = 62) from cells infected with different HIV-1 subtypes, selected performance panels from Boston Biomedica Inc., 7,579 unselected samples from blood donors, 303 unselected daily routine samples, 997 specimens from hospitalized patients, and potentially interfering samples (n = 1,222) were tested with Cobas Core HIV Combi EIA. The new assay showed a sensitivity comparable to that of the Abbott HIV-1 AG Monoclonal A for early detection of HIV infection in seroconversion panels. The mean time delay of Cobas Core HIV Combi EIA (last negative sample plus 1 day) in comparison to that for HIV-1 RT-PCR for 87 panels tested with both methods was 2.75 days. The diagnostic window was reduced with Cobas Core HIV Combi EIA by between 3.6 and 5.7 days from that for third-generation assays. The specificities of Cobas Core HIV Combi EIA in blood donors were 99.84 and 99.85% (after repeated testing). Overall, 30 repeatedly reactive false-positive results out of 10,031 HIV-negative samples were obtained with Cobas Core HIV Combi EIA. Our results show that a fourth-generation assay with improved specificity such as Cobas Core HIV Combi EIA is suitable for blood donor screening because of its low number of false positives and because it detects HIV p24 antigen with a sensitivity comparable to that of single-antigen assays.

Modulatory Effects of Human Cytomegalovirus Infection on Malignant Properties of Cancer Cells

Although there is no definitive evidence of the association of human cytomegalovirus (HCMV) infection with human cancers, the oncogenic potential of HCMV has been well established by in vitro studies demonstrating the ability of UV-irradiated or infectious virus to transform a variety of cells. After prolonged passaging the transformed cell type was maintained while HCMV DNA sequences were no more detectable. Three morphological transforming regions (mtr) of HCMV have been identified. The effects of HCMV on cellular functions which may be associated with the malignant phenotype include the expression of oncogenes and transcriptional activation of growth factors and interleukin synthesis. In infected cells, HCMV induces cytoskeletal alterations and changes in expression of cell surface receptors for extracellular matrix proteins which could result in increased motility and dissemination of cancer cells. Several human neuroblastoma cell lines undergo maturation in different neural crest derived cell types upon treatment with oncogenic potential agents, i.e. retinoic acid. The persistent HCMV infection of neuroblastoma cells (> 1 year) is accompanied by the increased expression of oncoproteins (i.e. N-myc) and decreased expression of tyrosine hydroxylase and dopamine-beta-hydroxylase. The activation of the cellular metabolism is due to HCMV binding to cellular receptors (prior to virus gene expression) and to the activity of HCMV immediate early (IE) gene products. IE proteins act directly as transcriptional activators or their activity is mediated by a variety of cellular transcription factors. HCMV infection may result in activation of promoters of cellular genes coding for cytokines, replication enzymes, proto-oncogenes and viral promoters. Recently it has been demonstrated that HCMV IE proteins block apoptosis probably by suppressing the ability of the antioncogene p53 to activate a reporter gene. The interactions of HCMV with tumor suppressor proteins such as p53 or retinoblastoma (pRb) susceptibility protein are reminiscent of those mediated by the oncoproteins of DNA tumor viruses. The acquisition of a fully malignant phenotype by normal cells is thought to require several mutations in a number of cellular genes. In this connection, HCMV may play the role of a nonobligate either direct or indirect cofactor for tumor genesis, e.g. by blocking apoptosis, which may be an essential requirement for tumor progression. Due to the stimulation of growth factors and/or inhibition of antioncogenes by its gene products, HCMV may modulate the malignant potential for tumor cells.

Laboratory Diagnosis of Norovirus: Which Method Is the Best?

Noroviruses (NV) are transmitted by fecally contaminated food, vomit, and person-to-person contact. They are one of the main causes of non-bacterial acute gastroenteritis in nursing, old people and children’s homes. NV outbreaks are characterized by a short incubation period (12–48 h), nausea, vomiting and diarrhea, and high secondary attack rates. The illness is generally mild and self-limiting. The aim of diagnostic procedures in viral gastroenteritis is to avoid nosocomial infections on the one hand and unnecessary antibiotic treatment on the other. Diagnostic procedures for NV are based on the detection of virus in stool samples by (immune) transmission electron microscopy (TEM), antigen ELISA, or polymerase chain reaction (PCR). In our study, a total of 244 stool samples obtained from 227 patients between March and May 2002 were tested by TEM, antigen ELISA and in-house PCR. Our data showed that PCR has the highest sensitivity (94.1%), followed by TEM (58.3%), and ELISA (31.3%), while specificity was highest for TEM (98.0%), followed by ELISA (94.9%), and PCR (92.4%). All three methods tested (TEM, ELISA and PCR) are useful for epidemiological investigations in gastroenteritis outbreaks; however, to maximize diagnostic validity for individual cases, at least two of the methods should be combined.

HIV protease inhibitor nelfinavir inhibits replication of SARS-associated coronavirus

Abstract A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.

CD4 Lymphocyte Count as a Predictor of the Duration of Highly Active Antiretroviral Therapy—Induced Suppression of Human Immunodeficiency Virus Load

The association between CD4 cell count and duration of virus load suppression was investigated in 558 patients in the Frankfurt HIV Clinic Cohort who had begun highly active antiretroviral therapy and who had virus load declines to </=500 copies/mL. The Kaplan-Meier method estimated viral rebound in 42.5% of patients by 24 weeks and in 64.3% by 84 weeks. Risk of viral rebound was independently associated with baseline and changes from baseline CD4 cell count. The achieved CD4 cell count was the most important factor for prediction of viral rebound (relative hazard, 5.4 for an updated CD4 cell count of <20 vs. >500 copies/mL; P=.0001). Baseline virus load was not associated with virus load rebound. Lower baseline CD4 cell counts were associated with increased risk of viral rebound; however, this risk was significantly reduced in persons with low baseline CD4 cell counts who experienced substantial increases in CD4 cell counts during follow-up.