An Anechoic Recording of Demosthenes’ 1st Olynthic Oration in German

This data set contains two anechoic recordings of an excerpt of Demosthenes’ 1st Olynthic Oration in expressive and loud speech in German language.DFG, 194453117, EXC 1027: Bild Wissen Gestaltung. Ein interdisziplinäres Labo

An Anechoic Recording of Cicero’s 3rd Cataline Oration: Italian, Latin and German

This data set contains three anechoic recordings of an excerpt of Cicero's 3rd Cataline Oration in Italian, Latin and German language.DFG, 194453117, EXC 1027: Bild Wissen Gestaltung. Ein interdisziplinäres Labo

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.

Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories. The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories

Viral diversity in swine intestinal mucus used for the manufacture of heparin as analyzed by high-throughput sequencing

International audienceHeparin is one of the main pharmaceutical products manufactured from raw animal material. In order to describe the viral burden associated with this raw material, we performed high-throughput sequencing (HTS) on mucus samples destined for heparin manufacturing, which were collected from European pigs. We identified Circoviridae and Parvoviridae members as the most prevalent contaminating viruses, together with viruses from the Picomaviridae, Astroviridae, Reoviridae, Caliciviridae, Adenoviridae, Birnaviridae, and Anelloviridae families. Putative new viral species were also identified. The load of several known or novel small non-enveloped viruses, which are particularly difficult to inactivate or eliminate during heparin processing, was quantified by qPCR. Analysis of the combined HTS and specific qPCR results will influence the refining and validation of inactivation procedures, as well as aiding in risk analysis of viral heparin contamination

Adventitious Virus Detection in Cells by High-Throughput Sequencing of Newly Synthesized RNAs: Unambiguous Differentiation of Cell Infection from Carryover of Viral Nucleic Acids

The use of high-throughput sequencing (HTS) to identify viruses in biologicals differs from current molecular approaches, since its use enables an unbiased approach to detection without the need to design specific primers to preamplify target sequences. Its broad range of detection and analytical sensitivity make it an important tool to ensure that biologicals are free from adventitious viruses. Similar to other molecular methods, however, identification of viral sequences in cells by HTS does not prove viral infection, since this could reflect carryover of inert viral sequences from reagents or other sources or the presence of transcriptionally inactive cellular sequences. Due to the broad range of detection associated with HTS, the above can potentially be perceived as a drawback for the testing of pharmaceutical biological products using this method. In order to avoid the identification of inert viral sequences, we present a methodology based on metabolic RNA labeling and sequencing, which enables the specific identification of newly synthesized viral RNAs in infected cells, resulting in the ability to unambiguously distinguish active infection by DNA or RNA viruses from inert nucleic acids. In the present study, we report the ability to differentiate Vero cells acutely infected by a single-stranded positive-sense RNA virus (tick-borne encephalitis virus) from cells which have been in contact with nonreplicating virus particles. Additionally, we also found a laboratory contamination by the squirrel monkey retrovirus of our Vero cell line, which was derived from an Old World (African green) monkey, a type of contamination which until now has been identified only in cells derived from primates from the New World.The use of high-throughput sequencing (HTS) to identify viral contamination of biological products is extremely sensitive and provides a broad range of detection. Nevertheless, viral sequences identified can also be inert. Examples include contamination resulting from reagents or the presence of inactivated viruses in animal-derived components of the cell culture medium. We therefore developed a method that relies on the sequencing of newly synthesized RNAs, an unequivocal sign of the presence of a transcriptionally active virus. This improvement in the specificity of viral testing increases the acceptability of HTS as a standard test for cells used in manufacturing biologicals and in biotherapies

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

International audienceFetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by FITS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures

A Member of a New Picornaviridae Genus Is Shed in Pig Feces

During a study of the fecal microbiomes from two healthy piglets using high-throughput sequencing (HTS), we identified a viralgenome containing an open reading frame encoding a predicted polyprotein of 2,133 amino acids. This novel viral genome displayedthe typical organization of picornaviruses, containing three structural proteins (VP0, VP3, and VP1), followed by sevennonstructural proteins (2A, 2B, 2C, 3A, 3B, 3Cpro , and 3Dpol). Given its particular relationship with Parechovirus, we propose toname it “Pasivirus” for Parecho sister clade virus, with “Swine pasivirus 1” (SPaV1) as the type species. Fecal samples collected atan industrial farm from healthy sows and piglets from the same herd (25 and 75, respectively) with ages ranging from 4 to 28weeks were analyzed for the presence of SPaV1 by one-step reverse transcription (RT)-PCR targeting a 3D region of 151 bp.SPaV1 was detected in fecal samples from 51/75 healthy piglets (68% of the animals) and in none of the 25 fecal samples fromhealthy sows, indicating that SPaV1 circulates through enteric infection of healthy piglets. We propose that SPaV1 represents thefirst member of a novel Picornaviridae genus related to parechoviruses

Viral metagenomics applied to blood donors and recipients at high risk for blood-borne infections

International audienceBackground. Characterisation of human-associated viral communities is essential for epidemiological surveillance and to be able to anticipate new potential threats for blood transfusion safety. In high-resource countries, the risk of blood-borne agent transmission of well-known viruses (HBV, HCV, HIV and HTLV) is currently considered to be under control. However, other unknown or unsuspected viruses may be transmitted to recipients by blood-derived products. To investigate this, the virome of plasma from individuals at high risk for parenterally and sexually transmitted infections was analysed by high throughput sequencing (HTS). Materials and methods. Purified nucleic acids from two pools of 50 samples from recipients of multiple transfusions, and three pools containing seven plasma samples from either HBV-, HCV- or HIV-infected blood donors, were submitted to HTS. Results. Sequences from resident anelloviruses and HPgV were evidenced in all pools. HBV and HCV sequences were detected in pools containing 3.8x10(3) IU/mL of HBV-DNA and 1.7x10(5) IU/mL of HCV-RNA, respectively, whereas no HIV sequence was found in a pool of 150 copies/mL of HIV-RNA. This suggests a lack of sensitivity in HTS performance in detecting low levels of virus. In addition, this study identified other issues, including laboratory contaminants and the uncertainty of taxonomic assignment of short sequence. No sequence suggestive of a new viral species was identified. Discussion. This study did not identify any new blood-borne virus in high-risk individuals. However, rare and/or viruses present at very low titre could have escaped our protocol. Our results demonstrate the positive contribution of HTS in the detection of viral sequences in blood donations