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1.
mSphere ; 4(3)2019 06 05.
Article in English | MEDLINE | ID: mdl-31167947

ABSTRACT

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.IMPORTANCE 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.


Subject(s)
DNA Contamination , DNA, Viral/analysis , RNA, Viral/analysis , Viruses/genetics , Animals , Cell Differentiation , Chlorocebus aethiops , Computational Biology , High-Throughput Nucleotide Sequencing , Vero Cells , Viruses/isolation & purification
2.
Biologicals ; 59: 29-36, 2019 May.
Article in English | MEDLINE | ID: mdl-30992161

ABSTRACT

The utilization of the current combination of in vitro, in vivo and PCR assays for the identification of adventitious viruses in production cells has a limited range of detection. While Next Generation Sequencing (NGS) has a broader breadth of detection, it is unable to differentiate sequences from replicating viruses versus background inert sequences. In order to improve NGS specificity, we have designed a new NGS approach which targets subsets of viral RNAs only synthesized during cell infection. In order to evaluate the performance of this approach for detecting low levels of adventitious viruses, we selected two difficult virus/cell systems. This included B95-8 cells persistently infected by Human herpesvirus 4 (HHV-4) and serially diluted into HHV-4 negative Ramos cells and Madin-Darby bovine kidney cells with an early infection produced via a low dose of Bovine viral diarrhea virus. We demonstrated that the sensitivity of our RNA NGS approach was equivalent to targeted PCR with an increased specificity for the detection of viral infection. We were also able to identify a previously undetected Murine Leukemia Virus contaminant in Ramos cells. Based on these results, we conclude that this new RNA NGS approach is suitable for conducting viral safety evaluations of cells.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , RNA, Viral/genetics , Sequence Analysis, RNA/methods , Viruses/genetics , Animals , Cattle , Cell Line , Cell Line, Tumor , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/physiology , Humans , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/physiology , Mice , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Virus Diseases/diagnosis , Virus Diseases/virology , Viruses/classification
3.
Blood Transfus ; 14(5): 400-7, 2016 09.
Article in English | MEDLINE | ID: mdl-27136432

ABSTRACT

BACKGROUND: 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 3.8×10(3) IU/mL of HBV-DNA and 1.7×10(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.


Subject(s)
Blood Donors , Metagenomics , Blood Safety , Blood Transfusion , HIV Infections/diagnosis , Hepacivirus/genetics , Hepatitis B/blood , Hepatitis C/diagnosis , Humans
4.
Biologicals ; 43(1): 31-6, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25466699

ABSTRACT

Heparin 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 Picornaviridae, 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.


Subject(s)
Heparin/biosynthesis , High-Throughput Screening Assays/methods , Intestines/virology , Mucus/virology , Viruses/classification , Animals , Base Sequence , DNA Primers , Real-Time Polymerase Chain Reaction , Swine
5.
Biologicals ; 42(4): 218-9, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24930452

ABSTRACT

Specific Pathogen Free (SPF) embryonated eggs are used for the production of many veterinary and human vaccines. We have used High Throughput Sequencing to screen allantoic fluids and embryos for the presence of encapsidated viral genomes and viral transcripts, respectively. SPF eggs from two different producers were tested. We evidenced sequences corresponding to known endogenous retroviruses and sequences of Avian Leukosis Virus, but no sequence that might suggest a productive infection of eggs with a virus even distant from known viruses. Our results strongly suggest that SPF eggs such as those used for this study represent a safe substrate for the production of vaccines.


Subject(s)
Eggs/analysis , Eggs/virology , High-Throughput Nucleotide Sequencing/methods , Specific Pathogen-Free Organisms , Animals , Avian Leukosis Virus/genetics , Chick Embryo , Chickens/virology , DNA, Viral/analysis , Endogenous Retroviruses/genetics , RNA, Viral/analysis , Vaccines/biosynthesis
6.
Biologicals ; 42(3): 145-52, 2014 May.
Article in English | MEDLINE | ID: mdl-24661556

ABSTRACT

Fetal 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 HTS 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.


Subject(s)
Diarrhea Viruses, Bovine Viral/classification , High-Throughput Nucleotide Sequencing/methods , Polyomavirus/classification , Animals , Cattle , Cells, Cultured , Diarrhea Viruses, Bovine Viral/genetics , Polymerase Chain Reaction , Polyomavirus/genetics
7.
J Virol ; 86(18): 10036-46, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22787214

ABSTRACT

During a study of the fecal microbiomes from two healthy piglets using high-throughput sequencing (HTS), we identified a viral genome containing an open reading frame encoding a predicted polyprotein of 2,133 amino acids. This novel viral genome displayed the typical organization of picornaviruses, containing three structural proteins (VP0, VP3, and VP1), followed by seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C(pro), and 3D(pol)). Given its particular relationship with Parechovirus, we propose to name it "Pasivirus" for Parecho sister clade virus, with "Swine pasivirus 1" (SPaV1) as the type species. Fecal samples collected at an industrial farm from healthy sows and piglets from the same herd (25 and 75, respectively) with ages ranging from 4 to 28 weeks 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 from healthy sows, indicating that SPaV1 circulates through enteric infection of healthy piglets. We propose that SPaV1 represents the first member of a novel Picornaviridae genus related to parechoviruses.


Subject(s)
Picornaviridae/genetics , Picornaviridae/isolation & purification , Sus scrofa/virology , Amino Acid Sequence , Animals , Base Sequence , Capsid Proteins/genetics , DNA, Viral/genetics , Feces/virology , Female , Genetic Variation , Genome, Viral , Male , Metagenome , Molecular Sequence Data , Parechovirus/classification , Parechovirus/genetics , Phylogeny , Picornaviridae/classification , Sequence Homology, Amino Acid , Virus Shedding
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