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No description available.

1,10-phenanthroline and its non-chelating isomer, 1,7-phenanthroline were used to inhibit the replication of vaccinia virus (VV). Serial passage of VV in the presence of various concentrations of either 1,10-phenanthroline or 1,7-phenanthroline was carried out. No drug resistant mutants were isolated, suggesting that the observed inhibition was due to a cellular protease as opposed to the putative viral protease G1L. Cultures infected in the presence of the inhibitors, were radio labeled with �����S-methionine at various time points post infection, to determine which step of VV replication was inhibited. Infections in the presence of 1,10-phenanthroline proceeded only through early gene transcription, suggesting that the point of inhibition was uncoating. Finally, cells infected with VV with or without the inhibitors at time zero and eight hours post infection were used to generate transmission electron microscopic images. Taken together these results indicate that inhibition was occurring at the level of uncoating. / Graduation date: 1999

The Orthopoxviruses (OPVs) comprise a group of viruses that possess very similar genomes; they vary considerably, however, in virulence. Among them, rabbitpox virus (RPXV) and monkeypox virus (MPXV) are the focus of this thesis. RPXV is closely related to vaccinia virus (VACV) but is significantly more virulent in rabbits. The West African isolates of MPXV, which also caused the human monkeypox 2003 outbreak in the USA. have different disease profiles from the Central African MPXV. To determine the basis for these differences, the complete genomes of RPXV-UTR and three West African isolates of MPXV were sequenced and analyzed. The result of the RPXV study indicates that 3 RPXV genes. alone or in combination. likely play a key role in the enhanced RPXV-UTR virulence over VACV isolates. These genes encode: the RING finger protein (RPXV-UTR 008), an ankyrin repeat family protein (RPXV-UTR 180) and the chemokine binding protein (RPXV-UTR 001/184) in the inverted terminal repeats (ITR) of RPXV. Examination of the evolutionary relationship between RPXV-UTR and other OPVs was carried out using the central DNA sequence of the genome that is conserved among all completely sequenced OPVs and also the protein sequences derived from the 49 genes present in all completely sequenced Chordopoxviruses (ChPV). The results of these analyses both confirm the hypothesis that RPXV-UTR is most similar to VACV. An animal study found that the Central African MPXV isolate is more virulent than the West African MPXV isolate. The comparison of the three West African isolates MPXV-COP-58. MPXV-SL-V70. and MPXV-WRAIR, and the Congo basin (Central Africa) isolate MPXV-ZAI-96-I-16 shows that the MPXV-ZAI-96-I-16 ORF D14L, which encodes an inhibitor of human complement, is most likely the virulence gene responsible for the pathogenesis differences between the West and Central African isolates. These results explain the lack of fatalities in the 2003 MPXV outbreak in the USA, which was caused by importation of a West African MPXV isolate.

Nach der Eradikation der humanen Pockenerkrankung stellen zoonotische Orthopockenvirus-(OPV-)Infektionen heute eine mögliche Bedrohung der öffentlichen Gesundheit dar. Hierbei sind insbesondere Kuhpocken-(CPXV), Affenpocken-(MPXV) und Vaccinia Viren (VACV) von Bedeutung. In dieser Arbeit wurde das Genexpressionsprofil humaner (HeLa) Zellen nach Infektion mit CPXV, MPXV oder VACV untersucht. Es wurden sowohl zelluläre Gene identifiziert, welche generell von allen verwendeten Viren reguliert wurden, als auch Gene, die eine Virus-spezifische Regulation durch individuelle OPV aufwiesen. Gemeinsamkeiten zeigten sich insbesondere zwischen CPXV und MPXV, welche, im Gegensatz zu VACV, die Expression zahlreicher Cytokine und Chemokine induzierten. Insbesondere für Interleukin-6, -8 und CXCL1 konnte auch auf Proteinebene eine gesteigerte Sekretion durch CPXV-infizierte Zellen nachgewiesen werden. Vermutlich aufgrund dieser Induktion, trat in vitro eine verstärkte Rekrutierung von Monozyten und Makrophagen in Folge einer CPXV-, nicht aber einer VACV-Infektion auf. Makrophagen spielen eine kontroverse Rolle im Rahmen einer OPV-Infektion und sind sowohl für deren Bekämpfung, als auch, im infizierten Zustand, für die Ausbreitung der Viren im Organismus von Bedeutung. Daher wurde die Replikationsfähigkeit von CPXV und VACV in Makrophagen charakterisiert. Der Virulenzfaktor p28, welcher von den meisten VACV Stämmen nicht kodiert wird, konnte als essentiell für die Replikation von CPXV in einer murinen Makrophagen-Zelllinie, primären peritonealen Makrophagen der Ratte und in Makrophagen aus primären humanen PBMCs identifiziert werden. In Anbetracht der Bedeutung der Replikationsfähigkeit in Makrophagen für die Ausbreitung einer OPV-Infektion im Wirtsorganismus, deuten diese Ergebnisse darauf hin, dass CPXV, im Fall einer weiteren Adaption an den Menschen, ein höheres Bedrohungspotential im Vergleich zu VACV aufweisen könnten. / Today, following the eradication of human smallpox, zoonotic infections caused by orthopoxviruses (OPV) are emerging as a potential human health threat. Especially cowpox viruses (CPXV), vaccinia viruses (VACV), and monkeypox viruses (MPXV) are gaining importance as a cause of infectious disease of man and livestock. This study aimed to analyse and compare the gene expression profile of human (HeLa) cells following infection with CPXV, MPXV or VACV. Cellular genes were identified which were either commonly modulated by infection with any of the three viruses, or which were specifically modulated by one individual OPV. Particularly similar effects on cellular gene expression were observed in the case of CPXV and MPXV infection, which both induced the expression of several cytokine and chemokine genes. Especially interleukin-6, -8, and CXCL1 were strongly secreted by CPXV-infected cells but not by VACV-infected cells. Consequently, CPXV infection also induced a strong chemotactic recruitment of monocytes and macrophages in vitro in contrast to VACV infection. Especially macrophages are known to play a controversial role during OPV infection. On the one hand, macrophages are of importance for the control of the infection. On the other hand, infected macrophages also facilitate virus spread across the organism. Therefore, the capability of CPXV and VACV to replicate in macrophages was analysed. Thereby, the poxviral virulence factor p28, which is absent from most strains of VACV, was identified as an essential factor, allowing CPXV replication in a murine macrophage cell line, primary peritoneal rat macrophages and in human PBMC-derived macrophages. Concerning the importance of productively infected macrophages for OPV spread, these results suggest that CPXV, if further adapted to human beings as host species, may harbor a greater threat to human health when compared to VACV.

Indiana University-Purdue University Indianapolis (IUPUI) / Vaccinia virus (VV) is the prototypical member of the orthopoxvirus genus of the Poxviridae family, and is currently being evaluated as a vector for vaccine development and cancer cell-targeting therapy. Despite the importance of studying poxvirus effects on the human immune system, reports of the direct interactions between poxviruses and primary human leukocytes (PHLs) are limited. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias towards binding and infecting monocytes among PHLs. VV binding strongly co-localized with lipid rafts on the surface of these cell types, even when lipid rafts were relocated to the cell uropods upon cell polarization. In humans, monocytic and professional antigen-presenting cells (APCs) have so far only been reported to exhibit abortive infections with VV. We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, were permissive to VV replication. The majority of virions produced in MDMs were extracellular enveloped virions (EEV). Visualization of infected MDMs revealed the formation of VV factories, actin tails, virion-associated branching structures and cell linkages, indicating that infected MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. Classical activation of MDMs by LPS plus IFN-γ stimulation caused no effect on VV replication, whereas alternative activation of MDMs by IL-10 or LPS plus IL-1β treatment significantly decreased VV production. The IL-10-mediated suppression of VV replication was largely due to STAT3 activation, as a STAT3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data indicate that PHL subsets express and share VV protein receptors enriched in lipid rafts. We also demonstrate that primary human macrophages are permissive to VV replication. After infection, MDMs produced EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread.