Replication of cowpox virus in macrophages is dependent on the host range factor p28/N1R.

Zoonotic orthopoxvirus infections continue to represent a threat to human health. The disease caused by distinct orthopoxviruses differs in terms of symptoms and severity, which may be explained by the unique repertoire of virus factors that modulate the host's immune response and cellular machinery. We report here on the construction of recombinant cowpox viruses (CPXV) which either lack the host range factor p28 completely or express truncated variants of p28. We show that p28 is essential for CPXV replication in macrophages of human or mouse origin and that the C-terminal RING finger domain of p28 is necessary to allow CPXV replication in macrophages.

Cryopreservation of cellular products in a closed-bag system with an incorporated dimethyl sulfoxide-resistant sterile filter outside of cleanroom facilities.

BACKGROUND: Manipulations, for example, cryopreservation, of cellular therapeutics carried out in an open system must be performed in a class A environment with surrounding class B environment. To avoid cleanroom facilities, a new closed-bag system with an incorporated dimethyl sulfoxide-resistant sterile filter for cryopreservation of cellular products was evaluated at two different centers. STUDY DESIGN AND METHODS: A total of 44 different products (22 buffy coats [BCs] and 22 leukapheresis [LK] products) were split and cryopreserved in parallel in cleanroom facilities (Method I) and with the closed system on the bench of a "normal" laboratory (Method II). Viability analyzed by 7-aminoactinomycin D staining and flow cytometric analysis and sterility of the products were analyzed. RESULTS: Independent of the cellular source (BC or LK), the median viability of CD45+ cells decreased significantly (p < 0.01) during cryopreservation: namely, in BCs, -15.8 percent with both methods, and in LK products, -5.4 percent with Method I and -4.8 percent with Method II, respectively. CD3+ as well as CD14+ cells exhibited a similar pattern and were also found significantly (p < 0.01) diminished after thawing independent of the handling system. For CD19+ cells, the small decrease of viability was only for the BC group significant (p = 0.027) when the cells had been processed with Method I. No bacterial contamination was detected neither in fresh products nor in products after cryopreservation. CONCLUSION: The closed system for cryopreservation of cellular products appears to be equivalent to cleanroom-based methods regarding cellular integrity and sterility when appropriate quality of sterile filters is assured.