Detection of Minute virus of mice strains in different cell lines: Implications for adventitious agent testing.

Minute virus of mice (MMV) has contaminated biotechnological processes in the past and specific MMV testing is therefore recommended, if the production cell line is known to be permissive for this virus. Testing is widely done using cell-culture-based adventitious virus assays, yet MMV strains may differ in their in vitro cell tropism. Here, we investigated the growth characteristics of different MMV strains on A9 and 324K cells and identified significant differences in susceptibility of these widely used indicator cell lines to infection by different strains of MMV, which has implications for MMV detectability during routine testing of biotechnology process harvests. An MMV-specific polymerase chain reaction was evaluated as a more encompassing method and was shown as suitable replacement for cell culture-based detection of the different MMV strains, with the additional benefit that detection is more rapid and can be extended to other rodent parvoviruses that might contaminate biotechnological processes. Although no MMV contamination event of human-derived cell lines has happened in the past, biotechnological processes that are based on these also need to consider MMV-specific testing, as, for example, HEK293, a human-derived cell line commonly used in biopharmaceutical manufacturing, was shown as susceptible to productive MMV infection in the current work.

Evolutionary dynamics of sex-biased gene expression in a young XY system: insights from the brown alga genus Fucus.

Sex-biased gene expression is considered to be an underlying cause of sexually dimorphic traits. Although the nature and degree of sex-biased expression have been well documented in several animal and plant systems, far less is known about the evolution of sex-biased genes in more distant eukaryotic groups. Here, we investigate sex-biased gene expression in two brown algal dioecious species, Fucus serratus and Fucus vesiculosus, where male heterogamety (XX/XY) has recently emerged. We find that in contrast to evolutionary distant plant and animal lineages, male-biased genes do not experience high turnover rates, but instead reveal remarkable conservation of bias and expression levels between the two species, suggesting their importance in sexual differentiation. Genes with consistent male bias were enriched in functions related to gamete production, along with sperm competition and include three flagellar proteins under positive selection. We present one of the first reports, outside of the animal kingdom, showing that male-biased genes display accelerated rates of coding sequence evolution compared with female-biased or unbiased genes. Our results imply that evolutionary forces affect male and female sex-biased genes differently on structural and regulatory levels, resulting in unique properties of differentially expressed transcripts during reproductive development in Fucus algae.

A closed, autologous bioprocess optimized for TCR-T cell therapies.

Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating engineered TCR-T cells. This bioprocess yielded 5-12 × 10e9 TCR-expressing T cells, transduced at low multiplicity of infections, within 7-10 days, and cells exhibited an enriched memory T-cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leukapheresed cells in a bioreactor without any T-cell or peripheral blood mononuclear cell enrichment steps had a high level of T-cell purity (~97%). Several critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale-up, lowering glycolysis through the addition of 2-deoxy- d-glucose, and modulating interleukin-2 levels, were investigated on their roles in regulating transduction efficiency, cell growth, and T-cell fitness such as T-cell memory phenotype and resistance to activation-induced cell death. The bioprocess described herein supports scale-out feasibility by enabling the processing of multiple patients' batches in parallel within a Grade C cleanroom.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Revisiting mouse minute virus inactivation by high temperature short time treatment.

In recent years, high temperature short time (HTST) treatment technology has been increasingly adopted for medium treatment to mitigate the potential risk of viral contamination in mammalian cell culture GMP manufacturing facilities. Mouse minute virus (MMV), also called minute virus of mice (MVM), implicated in multiple viral contamination events is commonly used as a relevant model virus to assess the effectiveness of HTST treatment of cell culture media. However, results from different studies vary broadly in inactivation kinetics as well as log reduction factors (LRFs) achieved under given treatment conditions. To determine whether the reported discrepancies stemmed from differences in MMV strains, laboratory-scale HTST devices, medium matrices, and/or experimental designs, we have taken a collaborative approach to systematically assess the effectiveness of HTST treatment for MMV inactivation. This effort was conceptualized based on a media treatment gap analysis conducted by the Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) under the MIT Center for Biomedical Innovation (CBI). Specifically, two different MMV strains were used to evaluate the effectiveness of HTST at various treatment conditions with regard to exposure temperature and hold time duration by two independent laboratories within two different companies. To minimize experimental variations, the two sites used the same batches of MMV stocks, the same commercially purchased medium, and the same model of thermocyclers as the laboratory-scale HTST device. The two independent laboratories yielded similar MMV inactivation kinetics and comparable LRF. No significant differences were observed between the two MMV strains evaluated, suggesting that the variations from prior studies were likely due to differences in equipment, medium matrices, or other factors. The data presented here indicate that MMV inactivation by HTST treatment obeys first-order kinetics and can be mathematically modeled using an Arrhenius equation. The model-based extrapolation provides a quantitative estimate of MMV inactivation by the current industry standard HTST condition (102°C for a hold time of 10 s) used for medium treatment. Finally, based on the data from the current study and the industry experience, it is recommended that any alternative virus barrier technologies adopted for medium treatment should provide a clearance of at least 3.0 LRF based on a worst-case model virus to effectively mitigate potential risks of viral contamination.

Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance.

BACKGROUND: Pteropods are planktonic gastropods that are considered as bio-indicators to monitor impacts of ocean acidification on marine ecosystems. In order to gain insight into their adaptive potential to future environmental changes, it is critical to use adequate molecular tools to delimit species and population boundaries and to assess their genetic connectivity. We developed a set of target capture probes to investigate genetic variation across their large-sized genome using a population genomics approach. Target capture is less limited by DNA amount and quality than other genome-reduced representation protocols, and has the potential for application on closely related species based on probes designed from one species. RESULTS: We generated the first draft genome of a pteropod, Limacina bulimoides, resulting in a fragmented assembly of 2.9 Gbp. Using this assembly and a transcriptome as a reference, we designed a set of 2899 genome-wide target capture probes for L. bulimoides. The set of probes includes 2812 single copy nuclear targets, the 28S rDNA sequence, ten mitochondrial genes, 35 candidate biomineralisation genes, and 41 non-coding regions. The capture reaction performed with these probes was highly efficient with 97% of the targets recovered on the focal species. A total of 137,938 single nucleotide polymorphism markers were obtained from the captured sequences across a test panel of nine individuals. The probes set was also tested on four related species: L. trochiformis, L. lesueurii, L. helicina, and Heliconoides inflatus, showing an exponential decrease in capture efficiency with increased genetic distance from the focal species. Sixty-two targets were sufficiently conserved to be recovered consistently across all five species. CONCLUSION: The target capture protocol used in this study was effective in capturing genome-wide variation in the focal species L. bulimoides, suitable for population genomic analyses, while providing insights into conserved genomic regions in related species. The present study provides new genomic resources for pteropods and supports the use of target capture-based protocols to efficiently characterise genomic variation in small non-model organisms with large genomes.

Proteomic and Transcriptomic Patterns during Lipid Remodeling in Nannochloropsis gaditana.

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.

Proteomics of HCV virions reveals an essential role for the nucleoporin Nup98 in virus morphogenesis.

Hepatitis C virus (HCV) is a unique enveloped virus that assembles as a hybrid lipoviral particle by tightly interacting with host lipoproteins. As a result, HCV virions display a characteristic low buoyant density and a deceiving coat, with host-derived apolipoproteins masking viral epitopes. We previously described methods to produce high-titer preparations of HCV particles with tagged envelope glycoproteins that enabled ultrastructural analysis of affinity-purified virions. Here, we performed proteomics studies of HCV isolated from culture media of infected hepatoma cells to define viral and host-encoded proteins associated with mature virions. Using two different affinity purification protocols, we detected four viral and 46 human cellular proteins specifically copurifying with extracellular HCV virions. We determined the C terminus of the mature capsid protein and reproducibly detected low levels of the viral nonstructural protein, NS3. Functional characterization of virion-associated host factors by RNAi identified cellular proteins with either proviral or antiviral roles. In particular, we discovered a novel interaction between HCV capsid protein and the nucleoporin Nup98 at cytosolic lipid droplets that is important for HCV propagation. These results provide the first comprehensive view to our knowledge of the protein composition of HCV and new insights into the complex virus-host interactions underlying HCV infection.

Ultrastructural analysis of hepatitis C virus particles.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins.

Differences by origin in methylome suggest eco-phenotypes in the kelp Saccharina latissima.

Most kelp species are of high ecological and economic importance worldwide, but are highly susceptible to rising ocean temperatures due to their sessile lifestyle. Due to interference with reproduction, development and growth, natural kelp forests have vanished in multiple regions after extreme summer heat waves. Furthermore, increasing temperatures are likely to decrease biomass production and, thus, reduce production security of farmed kelp. Epigenetic variation, and cytosine methylation as a heritable epigenetic trait, is a rapid means of acclimation and adaptation to environmental conditions, including temperature. While the first methylome of brown macroalgae has been recently described in the kelp Saccharina japonica, its functional relevance and contribution to environmental acclimation is currently unknown. The main objective of our study was to identify the importance of the methylome in the congener kelp species Saccharina latissima for temperature acclimation. Our study is the first to compare DNA methylation in kelp between wild populations of different latitudinal origin, and the first to investigate the effect of cultivation and rearing temperature on genome-wide cytosine methylation. Origin appears to determine many traits in kelp, but it is unknown to what extent the effects of thermal acclimation may be overruled by lab-related acclimation. Our results suggest that seaweed hatchery conditions have strong effects on the methylome and, thus, putatively on the epigenetically controlled characteristics of young kelp sporophytes. However, culture origin could best explain epigenetic differences in our samples suggesting that epigenetic mechanisms contribute to local adaptation of eco-phenotypes. Our study is a first step to understand whether DNA methylation marks (via their effect on gene regulation) may be used as biological regulators to enhance production security and kelp restoration success under rising temperatures, and highlights the importance to match hatchery conditions to origin.