A plasmid-based reverse genetics system for animal double-stranded RNA viruses.

Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.

Cell line cross-contamination: how aware are Mammalian cell culturists of the problem and how to monitor it?

HeLa was the first human cell line established (1952) and became one of the most frequently used lines because of its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast-growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these "HeLa contaminants" prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without being aware of their true identity and how many were not using available means to ensure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines; yet, only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969-2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using HeLa contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations.