A chemiluminescence detection method of hantaviral antigens in neutralisation assays and inhibitor studies.

A protocol for chemiluminescence detection of hantaviral antigens in infected cell foci is described. This focus detection is based on the conversion of a substrate into a luminescent product by peroxidase-antibody conjugates; the emitted light of infected cell foci can easily be recorded by autoradiography or video imaging providing a hard copy for documentation. The main advantage of this method as compared to conventional immunochemical staining is a higher detection sensitivity due to the inherent magnification effect of luminescence causing an obvious boost in focal image and intensity. This enables reduction of (i) incubation time of virus-infected cells and (ii) amount of needed antibody for antigen detection in foci. This method is applied to a chemiluminescence focus reduction assay for the serotyping of hantavirus-specific neutralising antibodies in infected persons and for the determination of activity of antiviral agents against hantavirus.

A novel link between stress and human cytomegalovirus (HCMV) infection: sympathetic hyperactivity stimulates HCMV activation.

Recently, inflammatory mediators such as TNFalpha were identified as triggering active human cytomegalovirus (HCMV) infection. Here, we demonstrate that a highly stressful event in the absence of systemic inflammation, as observed in patients with acute myocardial infarction, leads to the development of an active HCMV infection in latently infected patients. Elucidating the molecular mechanism of virus activation, we could show that catecholamines directly stimulate the HCMV immediate-early (IE) enhancer/promoter in monocytic cells via beta-2 adrenergic receptors. Subsequent activation of the cAMP/PK-A-signaling pathway results in enhanced synthesis and binding of the transcription factor CREB-1/ATF-1 to the cAMP-responsive elements within the IE enhancer. Epinephrine also enhanced HCMV gene expression in infected THP-1 cells by about 50% in three of four experiments. These data suggest that HCMV, like HSV-1 and VZV, can be (re)activated under stress conditions.

Pentoxifylline promotes replication of human cytomegalovirus in vivo and in vitro.

OKT3 monoclonal antibody (MoAb) therapy is well established in the prevention and therapy of acute rejection in transplant patients. Unfortunately, this therapy is associated with several short-term (cytokine release syndrome) and long-term (infections, EBV-related lymphoma) side effects. Recently, we were able to demonstrate an association between the TNF alpha release following the first OKT3 MoAb infusions and the appearance of human cytomegalovirus (HCMV) reactivation several days later. In order to prevent this TNF alpha associated HCMV reactivation patients were additionally treated with pentoxifylline (PTX), a methylxanthine derivative that has been shown to suppress TNF alpha induction. Although the TNF alpha peak plasma level following OKT3 MoAb treatment was markedly reduced, the incidence of HCMV reactivation and HCMV disease was not influenced. In transient transfection experiments using HCMV immediate early enhancer/promoter CAT reporter gene constructs PTX enhanced the promoter activity independently from TNF alpha in premonocytic cells. Furthermore, PTX acted synergistically with TNF alpha. In virus-infected human embryonal lung fibroblasts HCMV replication was triggered in the presence of both PTX and TNF alpha, while either substance alone had only marginal effects. The stimulatory effect of PTX on the immediate early (IE) enhancer/promoter was mediated via CREB/ ATF, a eukaryotic transcription factor that binds to the 19 bp sequence motif in the enhancer region, while TNF alpha stimulation was mediated by activation of the transcription factor NF-kappaB and its binding to the 18 bp sequence motif in the enhancer. These data suggest a potential side effect of cAMP-elevating drugs such as PTX.