Detlef Michel

Human cytomegalovirus-encoded chemokine receptor US28 promotes tumorigenesis.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a broad spectrum of chemokines and constitutively activates various pathways linked to proliferation. Our studies reveal that expression of US28 induces a proangiogenic and transformed phenotype by up-regulating the expression of vascular endothelial growth factor and enhancing cell growth and cell cycle progression. US28-expressing cells promote tumorigenesis when injected into nude mice. The G protein-uncoupled constitutively inactive mutant of US28, induces delayed and attenuated tumor formation, indicating the importance of constitutive receptor activity in the early onset of tumor development. Importantly, also in glioblastoma cells infected with the newly isolated clinical HCMV strain Titan, US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence, the constitutively activated chemokine receptor US28 might act as a viral oncogene and enhance and/or promote HCMV-associated tumor progression.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal.

Ganciclovir/Valganciclovir Prophylaxis Decreases Cytomegalovirus-Related Events and Bronchiolitis Obliterans Syndrome after Lung Transplantation

BACKGROUND Until recently, cytomegalovirus (CMV) infection represented a major threat to lung transplant recipients. Preliminary studies have shown that antiviral prophylaxis might improve the outcome for these patients. METHODS We extended our initial pilot trial of prolonged prophylaxis with either oral ganciclovir (1 g 3 times per day) or valganciclovir (450 mg twice per day). The trial included 96 patients who were at risk for CMV-related events. RESULTS CMV prophylaxis resulted in a significant decrease in CMV-related events (i.e., active infection and disease), from 75% in a control group and for 274 cases from the literature who did not receive prophylaxis to a cumulative incidence of 27% (P < .001). Only 11% of the prophylaxis recipients experienced CMV disease (P = .002). Moreover, at 5 years, there was a significant decrease in the rate of bronchiolitis obliterans syndrome, from 60% to 43% (P = .002), and an improved rate of survival, from 47% to 73% (P= .036), irrespective of the immunosuppressive regimen received. CMV strains with UL97 mutations were recovered from 7 of 12 analyzed cases, but the presence of this mutation had no impact on the severity of CMV disease. CONCLUSIONS A regimen of prolonged ganciclovir or valganciclovir prophylaxis decreased the rate of active CMV infection and disease, reduced the incidence of bronchiolitis obliterans syndrome, and improved the survival rate. Drug-resistant CMV strains may occur, but such strains appeared to have no impact on the outcome of CMV-related events.

Antiviral treatment of cytomegalovirus infection and resistant strains

This review discusses the management of resistant cytomegalovirus and prevention strategies for fatal therapy failures. Five drugs, ganciclovir/valganciclovir, cidofovir, foscarnet and fomivirsen, have been approved so far for the treatment of human cytomegalovirus (HCMV) diseases. Except for fomivirsen, all of the approved drugs share the same target molecule, the viral DNA polymerase. The emergence of drug-resistant HCMV has also been reported for all of them. For optimal care of patients, the clinical virologist has to provide the most meaningful assays for monitoring of therapy and early detection of emerging drug-resistant HCMV. Additionally, a quantitative drug monitoring would be helpful. New antiviral agents are urgently needed with less adverse effects, good oral bioavailability and possibly novel targets or mechanisms of action to avoid cross-resistance and to improve the ability to suppress the selection of resistant virus strains by combination therapy. Compounds like maribavir, leflunomide and artesunate, which exhibit anti-HCMV activity in vitro and in patients need to be evaluated in clinical studies. Besides these, new therapy approaches like immunotherapy or new diagnostic techniques like pyrosequencing have to be considered in the future.

HCV and HGV in B-cell non-Hodgkin's lymphoma

BACKGROUND/AIMS A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkins lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkins lymphoma. METHODS Serum samples from 69 patients with non-Hodgkins lymphoma were studied. Diagnosis of non-Hodgkins lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy. RESULTS Three of 69 (4.3%) patients with B-cell non-Hodgkins lymphoma were HCV-infected and nine non-Hodgkins lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkins lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins. CONCLUSIONS HCV seems to have no significance for the pathogenesis of non-Hodgkins lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkins lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane

ABSTRACT We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.

Constitutive Inositol Phosphate Formation in Cytomegalovirus-Infected Human Fibroblasts Is due to Expression of the Chemokine Receptor Homologue pUS28

ABSTRACT An open reading frame (ORF), US28, with homology to mammalian chemokine receptors has been identified in the genome of human cytomegalovirus (HCMV). Its protein product, pUS28, has been shown to bind several human CC chemokines, including RANTES, MCP-1, and MIP-1α, and the CX3C chemokine fractalkine with high affinity. Addition of CC chemokines to cells expressing pUS28 was reported to cause a pertussis toxin-sensitive increase in the concentration of cytosolic free Ca2+. Recently, pUS28 was shown to mediate constitutive, ligand-independent, and pertussis toxin-insensitive activation of phospholipase C via Gq/11-dependent signaling pathways in transiently transfected COS-7 cells. Since these findings are not easily reconciled with the former observations, we analyzed the role of pUS28 in mediating CC chemokine activation of pertussis toxin-sensitive G proteins in cell membranes and phospholipase C in intact cells. The transmembrane signaling functions of pUS28 were studied in HCMV-infected cells rather than in cDNA-transfected cells. Since DNA sequence analysis of ORF US28 of different laboratory and clinical strains had revealed amino acid sequence differences in the amino-terminal portion of pUS28, we compared two laboratory HCMV strains, AD169 and Toledo, and one clinical strain, TB40/E. The results showed that infection of human fibroblasts with all three HCMV strains led to a vigorous, constitutively enhanced formation of inositol phosphates which was insensitive to pertussis toxin. This effect was critically dependent on the presence of the US28 ORF in the HCMV genome but was independent of the amino acid sequence divergence of the three HCMV strains investigated. The constitutive activity of pUS28 is not explained by expression of pUS28 at high density in HCMV-infected cells. The pUS28 ligands RANTES and MCP-1 failed to stimulate binding of guanosine 5′-O-(3-[35S]thiotriphosphate to membranes of HCMV-infected cells and did not enhance constitutive activation of phospholipase C in intact HCMV-infected cells. These findings raise the possibility that the effects of CC chemokines and pertussis toxin on G protein-mediated transmembrane signaling previously observed in HCMV-infected cells are either independent of or not directly mediated by the protein product of ORF US28.

Protein kinase inhibitors of the quinazoline class exert anti-cytomegaloviral activity in vitro and in vivo

Cytomegalovirus infection is associated with severe disease in immunocompromised individuals. Current antiviral therapy faces several limitations. In a search of novel drug candidates, we describe here the anti-cytomegaloviral properties of two compounds of the chemical class of quinazolines, gefitinib (Iressa) and Ax7396 (RGB-315389). Both compounds showed strong inhibitory effects in vitro against human and animal cytomegaloviruses with IC(50)s in a low micromolar range. Cytotoxicity did not occur at these effective concentrations. The antiviral mode of action was based on the inhibition of protein kinase activity, mainly directed to a viral target kinase (UL97/M97) in addition to cellular target candidates. This was demonstrated by a high sensitivity of the respective protein kinases in vitro and by infection experiments with viral mutants carrying genomic alterations in the ORF UL97/M97 modulating viral drug sensitivity. In a guinea pig model, gefitinib showed inhibition of cytomegaloviral loads in blood and lung tissue. Importantly, the rate of mortality of infected animals was reduced by gefitinib treatment. In contrast to the in vitro data, Ax7396 showed no significant antiviral activity in a mouse model. Further in vivo analyses have to assess the potential use of gefitinib in the treatment of cytomegalovirus disease.

Phosphorylation of aciclovir, ganciclovir, penciclovir and S2242 by the cytomegalovirus UL97 protein : a quantitative analysis using recombinant vaccinia viruses

We used recombinant vaccinia viruses (rVV) containing the UL97 open reading frame (ORF) of the human cytomegalovirus (HCMV) to investigate the UL97-dependent phosphorylation of different nucleoside analogs. The rVV T1 expressed the wild-type UL97 protein whereas rVV A5 contained a 12 bp deletion in the UL97 which had been known to be responsible for resistance of HCMV to ganciclovir (GCV). The rVV T1opal was generated which contained a stop codon at position 1089 of the UL97 ORF and which expressed a truncated UL97 protein. We quantitatively analyzed the capability of these rVVs to phosphorylate GCV, penciclovir (PCV), aciclovir (ACV) and 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl] purine (S2242) as well as the natural nucleosides deoxycytidine and deoxythymidine. Moreover, we compared their phosphorylating capability with that of herpes simplex virus type 1 strains. In thymidine kinase (TK)-deficient 143B cells infected with rVV T1, the three compounds GCV, ACV and PCV were phosphorylated with different efficiency whereas in cells infected with the rVV A5 a markedly reduced but not completely abolished phosphorylation of these compounds was observed. In rVV T1opal-infected cells no specific phosphorylation of the compounds was detectable at all. Neither S2242 nor the natural substrates of TKs were phosphorylated by any of the vaccinia recombinants. The rVVs proved to be a suitable tool for analysis of UL97-dependent phosphorylation of nucleoside analogs and also allowed to quantitatively study the influence of UL97 mutations on drug phosphorylation.

Hantavirus outbreak, Germany, 2007.

To the Editor: Hantavirus disease (for review see [1]) has been reportable in Germany since 2001, according to the Federal Infection Protection Act. In this country, Puumala virus (PUUV) causes most clinical hantavirus cases, although Dobrava-Belgrade virus and Tula virus also circulate (1). From 2001 through 2006, an average of ≈220 cases were reported per year (incidence 0.267/100,000) with a maximum of 448 cases in 2005. In contrast, 1,687 cases were reported in 2007 (2). Whereas in 2005 the highest incidence of infection was in metropolitan areas (3), the current outbreak is focused in the rural areas in southern and western Germany. Clinical case-patients exhibit key characteristics of hantavirus disease (nephropathia epidemica): acute high fever; pain in the back, head, and/or abdomen; proteinuria; rise of serum creatinine; thrombocytopenia; and renal failure (1). The outbreak provided considerable numbers of clinical samples from the viremic phase and thus has enabled a molecular epidemiologic analysis of the circulating virus. At the National Consultation Laboratory for Hantavirus Infections (Berlin), we received early-phase serum specimens from the outbreak regions for confirmation assays. In enzyme immunoassays and Western blot tests (4), 80 samples from patients during the early clinical phase were positive for PUUV-specific immunoglobulin (Ig) M antibodies. All IgM data were accompanied by simultaneous or subsequent detection of PUUV-specific IgG. The samples were screened for hantavirus RNA by reverse transcription–PCR (RT-PCR) (5). Of the 80 early-phase serum samples, 42 (53%) were RT-PCR positive. For a subset of 14 of the 42 samples, a 557-nt segment of the nucleocapsid (S) gene underwent nucleotide sequence analysis as described previously (6). The Figure, panel A, shows a map of Germany with the residences of those patients from whom virus sequences were amplified (marked by letter H in front of the specimen number). In the phylogenetic analysis, despite a substantial evolutionary distance to PUUV strains from other parts of Europe, the virus sequences unambiguously grouped within the PUUV species (Figure, panel B). The few previously known human PUUV sequences from individual clinical case-patients in Germany, “Berkel” from Munsterland (7) and “Heidelberg” from a region located between Swabian Jura and Spessart Forest (8), as well as human-derived strains from a small 2004 outbreak in the Bavarian Forest (6), were included in this analysis. The results showed a clustering of the new viral sequences strictly according to residential areas of the patients, forming the following 4 clades: Swabian Jura (SJ), Spessart Forest (SF), Munsterland (ML), and Bavarian Forest (BF). Two different single sequences, Karlsruhe (from a region in northwestern Swabian Jura) and Essen (in southern Munsterland), represent 2 putative additional lineages. Figure A) Map of Germany showing origins of viral sequences from the 2007 outbreak. H, sequences of human origin; M, sequences of rodent origin (Myodes glareolus). Dotted circles mark the outbreak regions characterized by particular virus sequence clusters; ... Most sequences in this study were obtained from Swabian Jura, the region with the highest illness rate of the outbreak (incidence 32.9/100,000). The Swabian Jura was previously identified as a hantavirus-endemic area characterized by higher seroprevalence rates in the population compared with the rest of Germany (9). Sequence alignments within this clade showed a nucleotide sequence diversity of up to 5.5%. Within the BF clade, the diversity is up to 4%. However, between the 4 phylogenetic clades mentioned above (SJ, SF, ML, and BF), a sequence variability of 12%–18% was found. The natural reservoir of PUUV is the bank vole, Myodes glareolus; the virus is transmitted to humans by the aerosolized excreta of these rodents (1). Sequence comparisons showed a tight correlation between human- and rodent-derived PUUV sequences obtained from the same regional provenance (nucleotide identity >98%) and high variability of sequences originating from different geographic regions (nucleotide identity ≈85%). Neighbor-joining analyses confirmed the direct clustering of human- and rodent-derived sequences in the different phylogenetic clades (Figure, panel B). In this study we focused on the analysis of a 557-nt S-segment region. For more detailed studies, analysis of the complete S and M sequences of the virus strains will be necessary. Nevertheless, our results demonstrate a high variability among the German PUUV strains but a strong clustering of viral sequences of human and rodent origin in the same geographic region. The diversity of the PUUV clusters suggests their separate evolutionary history in the different regions of Germany. In contrast, within these particular geographic areas, only slight sequence differences were found in longitudinal analysis over several years. This conclusion is supported by the novel human Waldkirchen sequence (H72), which is almost identical to the BF strains from 2004 (6,10) and the similarity of newly derived human sequences from Munsterland (H208, H303) to the Berkel strain from 1994 (7). The molecular characterization of the viral sequences of patient and rodent origin from the outbreak areas demonstrates that PUUV is the causative agent of the current outbreak.