Evaluation of a viral transcriptome Next Generation Sequencing assay as an alternative to animal assays for viral safety testing of cell substrates.

The viral safety of biological products is ensured by tests throughout the production chain, and, for certain products, by steps in the manufacturing process enabling the elimination or inactivation of viruses. Current testing programs include sample inoculation in animals and embryonic eggs. Following the 3Rs principles of replacement, reduction, and refinement of animal-use methods, such techniques are intended to be replaced not only for ethical reasons but also because of their inherent technical limitations, their long turnaround times, and their limits in virus detection. Therefore, we have compared the limit and range of sensitivity of in vivo tests used for viral testing of cells with a transcriptomic assay based on Next Generation Sequencing (NGS). Cell cultures were infected with a panel of nine (9) viruses, among them only five (5) were detected, with variable sensitivity, by in vivo tests. The transcriptomic assay was able to detect one (1) infected cell among 103 to 107 non-infected cells for all viruses assessed, including those not detected by the conventional in vivo tests. Here we show that NGS extends the breath of detection of viral contaminants compared to traditional testing. Collectively, these results support the replacement of the conventional in vivo tests by an NGS-based transcriptomic assay for virus safety testing of cell substrates.

A high-risk retinoblastoma subtype with stemness features, dedifferentiated cone states and neuronal/ganglion cell gene expression.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas.

High-Throughput Sequencing (HTS) of newly synthetized RNAs enables one shot detection and identification of live mycoplasmas and differentiation from inert nucleic acids.

Mycoplasma contamination threatens both the safety of biologics produced in cell substrates as well as the quality of scientific results based on cell-culture observations. Methods currently used to detect contamination of cells include culture, enzymatic activity, immunofluorescence and PCR but suffer from some limitations. High throughput sequencing (HTS) can be used to identify microbes like mycoplasmas in biologics since it enables an unbiased approach to detection without the need to design specific primers to pre-amplify target sequences but it does not enable the confirmation of microbial infection since this could reflect carryover of inert sequences. In order to unambiguously differentiate the presence of live or dead mycoplasmas in biological products, the present method was developed based on metabolic RNA labelling of newly synthetized mycoplasmal RNAs. HTS of labelled RNA detected A549 cell infection with Acholeplasma laidlawii in a manner similar to both PCR and culture and demonstrated that this technique can unambiguously identify bacterial species and differentiates infected cells from cells exposed to a high inoculum of heat-inactivated mycoplasmas. This method therefore combines the advantage of culture (that detects only live microorganisms) with those of molecular tests (rapidity) together with a very broad range of bacterial detection and identification.