Hepatitis B virus surface antigen as a reporter of promoter activity

The coding sequence for the hepatitis B virus surface antigen (HBsAg) was used as a new reporter gene for studies on eukaryotic promoter activity and upstream regulatory sequences. The data observed in transfection assays were comparable to results obtained with conventional chloramphenicol acetyltransferase (CAT) assays, as was demonstrated using various transcriptional regulation sequences. The expression of HBsAg as a reporter protein offered some advantages: (i) In transient expression assays, a time course of promoter activity depending on variable culture conditions could be monitored over a period of time, since the HBsAg was secreted into the culture supernatant. (ii) Evaluation of HBsAg from supernatant aliquots and quantification of the corresponding promoter activities could be performed easily, using the very sensitive and readily available diagnostic HBsAg kits. (iii) In contrast to the conventional CAT assay, the cells remained available for further tests, e.g., Western blot, immunofluorescence or transcript analysis. Characteristics of several Epstein-Barr virus (EBV) promoters, depending on the virus state of EBV-positive B-cells (latency, chemical induction, lytic superinfection, trans-activation), were assayed using the HBsAg reporter system

Subcellular distribution and life cycle of Epstein-Barr virus in keratinocytes of oral hairy leukoplakia

The authors investigated the life cycle of Epstein-Barr virus (EBV) in keratinocytes of oral hairy leukoplakia by combining immunohistochemistry. DNA in situ hybridization, and lectin histochemistry with electron microscopy. Diffuse-staining components of the EBV early antigen complex (EA-D), EBV 150-kd capsid antigen (VCA), EBV membrane antigen (gp350/220), and double-stranded DNA were labeled with monoclonal antibodies. An EBV-DNA probe was used to locate EBV DNA. Wheat-germ agglutinin (WGA) was employed to distinguish Golgi-associated compartments. The authors found EBV proteins and EBV DNA only in keratinocytes with apparent viral assembly. In situ hybridization showed EBV DNA in free corelike material and in electron-dense cores of mature nucleocapsids. Monoclonal antibodies to nonspecific double-stranded DNA attached to the same structures and to marginated chromatin. Components of EA-D were dispersed throughout the nuclei but accumulated near condensed chromatin and in 'punched-out' regions of the chromatin. Epstein-Barr virus 150-kd capsid antigen was found only in the nuclei, where it appeared preferentially on mature nucleocapsids. As yet unexplained arrays of intranuclear particles that remained unlabeled with all EBV-specific probes reacted intensely with an antiserum against common papillomavirus antigen. Gp350/220 was detectable in various cellular membrane compartments and was highly concentrated on EBV envelopes in peripheral Golgi-associated secretory vesicles. It was less abundant on the extracellular EBV, indicating that viral membrane antigen partly dissociates from the mature virus. Combined lectin-binding histochemistry and electron microscopy demonstrated for the first time that EBV is processed in the Golgi apparatus, which eventually releases the virus by fusion with the plasma membrane. These results provide insight into the biologic events that occur during complete EBV replication in vivo

The BI'LF4 trans-activator of Epstein-Barr virus is modulated by type and differentiation of the host cell

We have analyzed the activity and regulated expression of a new Epstein-Barr virus (EBV) trans-activator (I'ta) encoded by left reading frame 4 (BI'LF4) of the BamHI I'fragment. The gene was detected in all genomes of established EBV strains and individual isolates, with the exception of B95-8, where the type-specific deletion of this open reading frame is tolerated in vitro. Specific trans-activation of two EBV promoters (early MS and I'ta promoter) could be shown in cotransfection assays. The I'ta product affected autoactivation but had no influence on heterologous target promoters. The I'ta promoter segment was shown to be costimulated in the process of host cell differentiation in the absence of other EBV gene products. Expression of the reading frame in bacteria identified a 48-kDa protein as a stable gene product. I'ta-specific antibodies were detected in sera from EBV-positive persons (nasopharyngeal carcinoma). When expressed with suitable eucaryotic vectors, a nuclear protein could be immunostained in transfected cells. Our experiments suggest a cell type-specific requirement for I'ta in the lytic cycle of EBV at a determined differentiation stage of the host cell

Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock