Virome Characterization in Commercial Bovine Serum Batches—A Potentially Needed Testing Strategy for Biological Products

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies’ established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.

Human and Animal RNA Virus Diversity Detected by Metagenomics in Cameroonian Clams

Many recent pandemics have been recognized as zoonotic viral diseases. While their origins remain frequently unknown, environmental contamination may play an important role in emergence. Thus, being able to describe the viral diversity in environmental samples contributes to understand the key issues in zoonotic transmission. This work describes the use of a metagenomic approach to assess the diversity of eukaryotic RNA viruses in river clams and identify sequences from human or potentially zoonotic viruses. Clam samples collected over 2years were first screened for the presence of norovirus to verify human contamination. Selected samples were analyzed using metagenomics, including a capture of sequences from viral families infecting vertebrates (VirCapSeq-VERT) before Illumina NovaSeq sequencing. The bioinformatics analysis included pooling of data from triplicates, quality filtering, elimination of bacterial and host sequences, and a deduplication step before de novo assembly. After taxonomic assignment, the viral fraction represented 0.8–15% of reads with most sequences (68–87%) remaining un-assigned. Yet, several mammalian RNA viruses were identified. Contigs identified as belonging to the Astroviridae were the most abundant, with some nearly complete genomes of bastrovirus identified. Picobirnaviridae sequences were related to strains infecting bats, and few others to strains infecting humans or other hosts. Hepeviridae sequences were mostly related to strains detected in sponge samples but also strains from swine samples. For Caliciviridae and Picornaviridae, most of identified sequences were related to strains infecting bats, with few sequences close to human norovirus, picornavirus, and genogroup V hepatitis A virus. Despite a need to improve the sensitivity of our method, this study describes a large diversity of RNA virus sequences from clam samples. To describe all viral contaminants in this type of food, and being able to identify the host infected by viral sequences detected, may help to understand some zoonotic transmission events and alert health authorities of possible emergence.

VIRAL CONTAMINANTS OF FOOD PRODUCTS AND METHODS OF THEIR DETECTION

There were summarized data on epidemiology and the properties of several groups of viral diseases, actually or potentially capable of implementation of the food route of transmission of infection (noroviruses, hepatitis viruses A and E, adenoviruses, astroviruses, rotaviruses, “avian” and “swine” flu viruses). There were mentioned most well-known massive outbreaks of enterovirus infections in countries of South-East Asia, India, China, Europe and other regions. The importance of products of animal and vegetable origin, and also water biological resources as factors of transmission of viral infections are shown. The analysis of available methods of detection of foodborne viruses shows the execution of analysis to demand for matching of methods for extraction and concentration of samples. An important criterion of the suitability of the used variant of the extraction must be its compatibility with demands for molecular methods - the minimum number of stages of sample processing with chemical reagents, neutral pH, preservation of antigenic properties and the intact viral RNA of pathogen. With consideration of the genetic diversity of food viruses, their detection requires the assortment of effective combinations of several types of primers, probes and conditions for the amplification. The methods of the rapid control should be based on the use of most modern types of analysis, including multi-primer PCR, hybridization on nucleotide microchips, immunochromatography and ELISA. Prior to the introduction into the practice, internal and external comparative tests of express methods should be executed to confirm their resolution and interlaboratory reproducibility. The introduction of comprehensive methods for the analysis of food viruses, the creation of a monitoring system on their basis, including the order and organization of research, the collection and exchange of information by competent organizations in real time regimen, can significantly increase the effectiveness of investigating outbreaks of viral infections with food transmission, reduce the risk of cross contamination in food enterprises, reduce the likelihood of using raw materials contaminated with viral pathogens in the production process, and improve the safety of food products

Combating viral contaminants in CHO cells by engineering STAT1 mediated innate immunity

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells, the most commonly used cell line in biomanufacturing, to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited increased resistance to the prototype RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

Enhanced Removal of Norovirus Surrogates, Murine Norovirus and Tulane Virus, from Aqueous Systems by Zero-Valent Iron

ABSTRACTViral contamination can compromise the safety of water utilized for direct consumption, produce irrigation, and postharvest washing of produce. Zero-valent iron (ZVI) is used commercially for chemical remediation of water and has been demonstrated to remove some biological contaminants from water in laboratory and field studies. This study investigated the efficacy of ZVI to remove human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV), from water and to characterize the reversibility and nature of viral association with ZVI. Genomic material of TV and MNV recovered from the effluent of inoculated water treatment columns containing a 1:1 mixture of ZVI and sand was 2 and 3 log, respectively, less than that recovered from the effluent of treatment columns containing only sand. Elution buffers (citrate buffers, pH 4 and 7, and virus elution buffer, pH 9.5, with and without added 1 M NaCl) did not increase recovery of infectious TV and MNV from ZVI as compared with elution with water alone. TV-inoculated lettuce washed with water in the presence of ZVI yielded 1.5 to 2 log fewer infectious TV from washwater as compared with lettuce washed with water alone or in the presence of sand. These data demonstrate the enhanced removal of human norovirus surrogates, TV and MNV, from water by ZVI and provide indications that unrecovered viruses are not readily disassociated from ZVI by buffers of various pH and ionic strength. These findings warrant further investigation into larger-scale simulations of water remediation of viral contaminants for potential application in the treatment of water used for drinking, irrigation, and food processing.