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Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence™, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC₅₀) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. The disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. They may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. In this study, we analysed the specific modulation of the host cell's gene expression profile by cowpox, monkeypox and vaccinia virus infection. We aimed to identify mechanisms that are either common to orthopoxvirus infection or specific to certain orthopoxvirus species, allowing a more detailed description of differences in virus-host cell interactions between individual orthopoxviruses. To this end, we analysed changes in host cell gene expression of HeLa cells in response to infection with cowpox, monkeypox and vaccinia virus, using whole-genome gene expression microarrays, and compared these to each other and to non-infected cells. Results: Despite a dominating non-responsiveness of cellular transcription towards orthopoxvirus infection, we could identify several clusters of infection-modulated genes. These clusters are either commonly regulated by orthopoxvirus infection or are uniquely regulated by infection with a specific orthopoxvirus, with major differences being observed in immune response genes. Most noticeable was an induction of genes involved in leukocyte migration and activation in cowpox and monkeypox virus-infected cells, which was not observed following vaccinia virus infection. Conclusion: Despite their close genetic relationship, the expression profiles induced by infection with different orthopoxviruses vary significantly. It may be speculated that these differences at the cellular level contribute to the individual characteristics of cowpox, monkeypox and vaccinia virus infections in certain host species. [ABSTRACT FROM AUTHOR]

Abstract: Orthopoxviruses are large DNA viruses which can cause disease in numerous host species. Today, after eradication of Variola virus and the end of vaccination against smallpox, zoonotic Orthopoxvirus infections are emerging as potential threat to human health. The most common causes of zoonotic Orthopoxvirus infections are Cowpox virus in Europe, Monkeypox virus in Africa and Vaccinia virus in South America. Although all three viruses are genetically and antigenically closely related, the human diseases caused by each virus differ considerably. This observation may reflect different capabilities of these viruses to modulate the hosts’ immune response. Therefore, we aimed at characterizing the specific cytokine response induced by Orthopoxvirus infection in vitro. We analysed the gene expression of nine human pro-inflammatory cytokines and chemokines in response to infection of HeLa cells and could identify an upregulation of cytokine gene expression following Cowpox virus and Monkeypox virus infection but not following Vaccinia virus infection. This was verified by a strong induction of especially IL-6, IL-8 and CXCL1 secretion into the cell culture supernatant following Cowpox virus infection. We could further show that supernatants derived from Cowpox virus-infected cells exhibit an increased chemotactic activity towards monocytic and macrophage-like cells. On the one hand, increased cytokine secretion by Cowpox virus-infected cells and subsequent monocyte/macrophage recruitment may contribute to host defence and facilitate clearance of the infection. On the other hand, given the assumed important role of circulating macrophages in viral spread, this may also point towards a mechanism facilitating delivery of the virus to further tissues in vivo. [Copyright &y& Elsevier]