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Petermann P.,University of Cologne | Petermann P.,Charles River Biopharmaceutical Services GmbH | Rahn E.,University of Cologne | Thier K.,University of Cologne | And 6 more authors.
Journal of Virology | Year: 2015

The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) can both mediate the entry of herpes simplex virus 1 (HSV-1). We have recently shown how these receptors contribute to infection of skin by investigating HSV-1 entry into murine epidermis. Ex vivo infection studies reveal nectin-1 as the primary receptor in epidermis, whereas HVEM has a more limited role. Although the epidermis represents the outermost layer of skin, the contribution of nectin-1 and HVEM in the underlying dermis is still open. Here, we analyzed the role of each receptor during HSV-1 entry in murine dermal fibroblasts that were deficient in expression of either nectin-1 or HVEM or both receptors. Because infection was not prevented by the absence of either nectin-1 or HVEM, we conclude that they can act as alternative receptors. Although HVEM was found to be highly expressed on fibroblasts, entry was delayed in nectin-1-deficient cells, suggesting that nectin-1 acts as the more efficient receptor. In the absence of both receptors, entry was strongly delayed leading to a much reduced viral spread and virus production. These results suggest an unidentified cellular component that acts as alternate but inefficient receptor for HSV-1 on dermal fibroblasts. Characterization of the cellular entry mechanism suggests that HSV-1 can enter dermal fibroblasts both by direct fusion with the plasma membrane and via endocytic vesicles and that this is not dependent on the presence or absence of nectin-1. Entry was also shown to require dynamin and cholesterol, suggesting comparable entry pathways in keratinocytes and dermal fibroblasts. © 2015, American Society for Microbiology.

Petermann P.,University of Cologne | Petermann P.,Charles River Biopharmaceutical Services GmbH | Thier K.,University of Cologne | Rahn E.,University of Cologne | And 9 more authors.
Journal of Virology | Year: 2015

Skin keratinocytes represent a primary entry site for herpes simplex virus 1 (HSV-1) in vivo. The cellular proteins nectin-1 and herpesvirus entry mediator (HVEM) act as efficient receptors for both serotypes of HSV and are sufficient for disease development mediated by HSV-2 in mice.HowHSV-1 enters skin and whether both nectin-1 andHVEMare involved are not known.Weaddressed the impact of nectin-1 during entry of HSV-1 into murine epidermis and investigated the putative contribution of HVEM. Using ex vivo infection of murine epidermis, we showed that HSV-1 entered the basal keratinocytes of the epidermis very efficiently. In nectin-1-deficient epidermis, entry was strongly reduced. Almost no entry was observed, however, in nectin-1-deficient keratinocytes grown in culture. This observation correlated with the presence ofHVEMon the keratinocyte surface in epidermis and with the lack ofHVEM expression in nectin-1-deficient primary keratinocytes. Our results suggest that nectin-1 is the primary receptor in epidermis, while HVEMhas a more limited role. For primary murine keratinocytes, on which nectin-1 acts as a single receptor, electron microscopy suggested that HSV-1 can enter both by direct fusion with the plasma membrane and via endocytic vesicles. Thus, we concluded that nectin-1 directs internalization into keratinocytes via alternative pathways. In summary, HSV-1 entry into epidermis was shown to strongly depend on the presence of nectin-1, but the restricted presence ofHVEMcan potentially replace nectin-1 as a receptor, illustrating the flexibility employed by HSV-1 to efficiently invade tissue in vivo. © 2015, American Society for Microbiology.

Asper M.,Charles River Biopharmaceutical Services GmbH | Hanrieder T.,Charles River Biopharmaceutical Services GmbH | Quellmalz A.,Uppsala University | Mihranyan A.,Uppsala University
Biologicals | Year: 2015

The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of 100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100nm latex beads and 50nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10 TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins. © 2015 The International Alliance for Biological Standardization.

Gavasane M.,RandD Group | Ali A.U.,RandD Group | Samagod A.,RandD Group | Kaundinya J.,RandD Group | And 9 more authors.
BioPharm International | Year: 2013

Demonstration of viral clearance is crucial for the recombinant proteins produced from mammalian cell culture. Size exclusion-based filtration is one of the methods for viral clearance valid for different types of mammalian viruses. In this study, the initial hydraulic performance of the virus filtration has enabled the development of a validation protocol and high-log reduction values (LRVs) for an appropriate panel of mammalian viruses (during spiking studies) with the desired throughput. This is essential to minimize the filter area, the cost of this unit operation, and to achieve a robust process. All of the four panel viruses (i.e., MuLV, PRV, Reo-3, and MVM) were evaluated during spiking studies. A high throughput of 1250 L/m 2 was obtained with a high flux because of low protein concentration. This was validated for a predefined throughput of 387 L/m 2 with high values of LRVs (>5) achieved resulting in the relatively small area of virus filter (0.04m 2) for a 50-L batch volume to be processed in 2 h. This study on a recombinant human follicle stimulating hormone (rhFSH) demonstrates the use of virus filters to reduce the risk of contamination and provide a robust process of virus filtration. © 2013 Advanstar Communications, Inc.

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