Characterization of Sf9 cell clones with differential susceptibilities to Sf-rhabdovirus X+3.7 and Sf-rhabdovirus X- replication.

Our laboratory previously discovered a novel rhabdovirus in the Spodoptera frugiperda Sf9 insect cell line that was designated as Sf-rhabdovirus. Using limiting dilution, this cell line was found to be a mixed population of cells infected by Sf-rhabdovirus variants containing either the full length X accessory gene with a 3.7 kb internal duplication (designated as Sf-rhabdovirus X+3.7) or lacking the duplication and part of the X gene (designated as Sf-rhabdovirus X-), and cells that were negative for Sf-rhabdovirus. In this paper, we found that the Sf-rhabdovirus negative cell clones had sub-populations with different susceptibilities to the replication of Sf-rhabdovirus X+3.7 and X- variants: cell clone Sf9-13F12 was more sensitive to replication by both virus variants compared to Sf9-3003; moreover, Sf9-3003 showed more resistance to X+3.7 replication than to X- replication. RNA-Seq analysis indicated significant differentially expressed genes in the Sf9-13F12 and Sf9-3003 cell clones further supporting that distinct sub-populations of virus-negative cells co-exist in the parent Sf9 cell line.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

Report of the third conference on next-generation sequencing for adventitious virus detection in biologics for humans and animals.

Next-generation sequencing (NGS) has been proven to address some of the limitations of the current testing methods for adventitious virus detection in biologics. The International Alliance for Biological Standardization (IABS), the U.S. Food and Drug Administration (FDA), and the European Directorate for the Quality of Medicines and Healthcare (EDQM) co-organized the "3rd Conference on Next-generation Sequencing for Adventitious Virus Detection in Biologics for Humans and Animals", which was held on September 27-28, 2022, in Rockville, Maryland, U.S.A. The meeting gathered international representatives from regulatory and public health authorities and other government agencies, industry, contract research organizations, and academia to present the current status of NGS applications and the progress on NGS standardization and validation for detection of viral adventitious agents in biologics, including human and animal vaccines, gene therapies, and biotherapeutics. Current regulatory expectations were discussed for developing a scientific consensus regarding using NGS for detection of adventitious viruses. Although there are ongoing improvements in the NGS workflow, the development of reference materials for facilitating method qualification and validation support the current use of NGS for adventitious virus detection.

IABS/DCVMN webinar on next generation sequencing.

Next Generation Sequencing (NGS) is a new technology that could overcome some of the limitations of the current viral testing methods for demonstrating the absence of adventitious agents in biologics. This report is for the webinar that was organized by the International Alliance for Biological Standardization (IABS) and the Developing Countries Vaccine Manufacturers Network (DCVMN), held on July 20, 2022, as an introduction to the technical and bioinformatics concepts of NGS and to some of the strengths and limitations of using the technology for those working in vaccine production or development. The current state of scientific knowledge and readiness of NGS to replace or supplement the current viral tests was further discussed in the 3rd Conference on NGS for Adventitious Virus Detection in Biologics for Humans and Animals that was held in Rockville, Maryland, USA, on September 27-28, 2022. The application of NGS to supplement or replace current in vivo and in vitro assays in adventitious virus testing during vaccine production is promising; however, assay performance (sensitivity, specificity, and reproducibility) needs to be demonstrated, which may include laboratory and bioinformatics work. Efforts from regulatory authorities, industry, and researchers are ongoing to facilitate validation and establishment of NGS as a new method for virus detection.

Report of the second international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals.

The IABS-EU, in association with PROVAXS and Ghent University, hosted the "2nd Conference on Next Generation Sequencing (NGS) for Adventitious Virus Detection in Human and Veterinary Biologics" held on November 13th and 14th 2019, in Ghent, Belgium. The meeting brought together international experts from regulatory agencies, the biotherapeutics and biologics industries, contract research organizations, and academia, with the goal to develop a scientific consensus on the readiness of NGS for detecting adventitious viruses, and on the use of this technology to supplement or replace/substitute the currently used assays. Participants discussed the progress on the standardization and validation of the technical and bioinformatics steps in NGS for characterization and safety evaluation of biologics, including human and animal vaccines. It was concluded that NGS can be used for the detection of a broad range of viruses, including novel viruses, and therefore can complement, supplement or even replace some of the conventional adventitious virus detection assays. Furthermore, the development of reference viral standards, complete and correctly annotated viral databases, and protocols for the validation and follow-up investigations of NGS signals is necessary to enable broader use of NGS. An international collaborative effort, involving regulatory authorities, industry, academia, and other stakeholders is ongoing toward this goal.

Complete Genome Sequence of Feline Leukemia Virus Kawakami-Theilen Strain KT-FeLV-UCD-1.

This full-length genome sequence of a feline leukemia virus Kawakami-Theilen strain (designated KT-FeLV-UCD-1), produced from the chronically infected FL74-UCD-1 cell line, was obtained using high-throughput sequencing. It consisted of 8,464 bp and had a genomic organization similar to that of other gammaretroviruses, containing long terminal repeats and open reading frames for Gag, Pol, and Env.

Genome Analysis and Replication Studies of the African Green Monkey Simian Foamy Virus Serotype 3 Strain FV2014.

African green monkey (AGM) spumaretroviruses have been less well-studied than other simian foamy viruses (SFVs). We report the biological and genomic characterization of SFVcae_FV2014, which was the first foamy virus isolated from an African green monkey (AGM) and was found to be serotype 3. Infectivity studies in various cell lines from different species (mouse, dog, rhesus monkey, AGM, and human) indicated that like other SFVs, SFVcae_FV2014 had broad species and cell tropism, and in vitro cell culture infection resulted in cytopathic effect (CPE). In Mus dunni (a wild mouse fibroblast cell line), MDCK (Madin-Darby canine kidney cell line), FRhK-4 (a fetal rhesus kidney cell line), and MRC-5 (a human fetal lung cell line), SFVcae_FV2014 infection was productive resulting in CPE, and had delayed or similar replication kinetics compared with SFVmcy_FV21 and SFVmcy_FV34[RF], which are two Taiwanese macaque isolates, designated as serotypes 1 and 2, respectively. However, in Vero (AGM kidney cell line) and A549 (a human lung carcinoma cell line), the replication kinetics of SFVcae_FV2014 and the SFVmcy viruses were discordant: In Vero, SFVcae_FV2014 showed rapid replication kinetics and extensive CPE, and a persistent infection was seen in A549, with delayed, low CPE, which did not progress even upon extended culture (day 55). Nucleotide sequence analysis of the assembled SFVcae_FV2014 genome, obtained by high-throughput sequencing, indicated an overall 80-90% nucleotide sequence identity with SFVcae_LK3, the only available full-length genome sequence of an AGM SFV, and was distinct phylogenetically from other AGM spumaretroviruses, corroborating previous results based on analysis of partial env sequences. Our study confirmed that SFVcae_FV2014 and SFVcae_LK3 are genetically distinct AGM foamy virus (FV) isolates. Furthermore, comparative infectivity studies of SFVcae_FV2014 and SFVmcy isolates showed that although SFVs have a wide host range and cell tropism, regulation of virus replication is complex and depends on the virus strain and cell-specific factors.

Report of the 2019 NIST-FDA workshop on standards for next generation sequencing detection of viral adventitious agents in biologics and biomanufacturing.

Adventitious virus testing assures product safety by demonstrating the absence of viruses that could be unintentionally introduced during the manufacturing process. The capabilities of next-generation sequencing (NGS) for broad virus detection in biologics have been demonstrated by the detection of known and novel viruses that were previously missed using the recommended routine assays for adventitious agent testing. A meeting was co-organized by the National Institute of Standards and Technology and the U.S. Food and Drug Administration on September 18-19, 2019 in Gaithersburg, Maryland, USA, to facilitate standardization of NGS technologies for applications of adventitious virus testing in biologics. The goal was to assess the currently used standards for virus detection by NGS and their public availability, and to identify additional needs for different types of reference materials and standards (natural and synthetic). The meeting focused on the NGS processes from sample preparation through sequencing but did not thoroughly cover bioinformatics, since this was considered to be the topic of a separate meeting.

The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones.

The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X+ in this paper). These results were extended by other reports describing an Sf-rhabdovirus variant in Sf9 cells, which lacked 320 nucleotides encompassing the X-gene and adjacent intergenic region (designated as X- in this paper), and the development of an Sf-rhabdovirus negative cell line. Here, we report that the Sf9 cell line is a mixed-cell population, based upon isolation of cell clones with distinct phenotypes: Sf-rhabdovirus-negative, X+, and X-. We also show that Sf-rhabdovirus X+ and X- variants replicate independently in Sf-rhabdovirus-negative cells. These results shed light on the detection of different rhabdovirus variants by different laboratories using Sf9-derived cell clones and confirm that both X+ and X- viruses are infectious in rhabdovirus-negative Sf9 cells.

Twelfth International Foamy Virus Conference-Meeting Report.

The 12th International Foamy Virus Conference took place on August 30⁻31, 2018 at the Technische Universität Dresden, Dresden, Germany. The meeting included presentations on current research on non-human primate and non-primate foamy viruses (FVs; also called spumaretroviruses) as well as keynote talks on related research areas in retroviruses. The taxonomy of foamy viruses was updated earlier this year to create five new genera in the subfamily, Spumaretrovirinae, based on their animal hosts. Research on viruses from different genera was presented on topics of potential relevance to human health, such as natural infections and cross-species transmission, replication, and viral-host interactions in particular with the immune system, dual retrovirus infections, virus structure and biology, and viral vectors for gene therapy. This article provides an overview of the current state-of-the-field, summarizes the meeting highlights, and presents some important questions that need to be addressed in the future.

Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection: Upstream Sample Processing and Library Preparation.

A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG) The topics include: use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.

Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection.

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS.

Report of the international conference on next generation sequencing for adventitious virus detection in biologicals.

A fundamental aspect of biological product safety is to assure absence of adventitious agents in the final product. Next-generation or high-throughput sequencing (NGS/HTS) has recently demonstrated detection of viruses that were previously missed using the recommended routine assays for adventitious agent testing of biological products. This meeting was co-organized by the International Alliance for Biological Standardization (IABS) and the U.S. Food and Drug Administration (FDA) to assess the current status and discuss the readiness of NGS for adventitious virus detection in biologics. The presentations included efforts for standardization, case studies on applications in biologics, comparison with routine virus detection assays, and current regulatory thinking. Participants identified the need for standard reference reagents, well-annotated databases, large data storage and transfer capacity, personnel with relevant expertise, particularly in bioinformatics; and harmonization of international regulations for testing biologic products and reagents used for their manufacturing. We hope this meeting summary will be of value to regulators and industry for considerations of NGS applications for adventitious virus detection in biologics.

A Reference Viral Database (RVDB) To Enhance Bioinformatics Analysis of High-Throughput Sequencing for Novel Virus Detection.

Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. IMPORTANCE To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection.

Spumaretroviruses: Updated taxonomy and nomenclature.

Spumaretroviruses, commonly referred to as foamy viruses, are complex retroviruses belonging to the subfamily Spumaretrovirinae, family Retroviridae, which naturally infect a variety of animals including nonhuman primates (NHPs). Additionally, cross-species transmissions of simian foamy viruses (SFVs) to humans have occurred following exposure to tissues of infected NHPs. Recent research has led to the identification of previously unknown exogenous foamy viruses, and to the discovery of endogenous spumaretrovirus sequences in a variety of host genomes. Here, we describe an updated spumaretrovirus taxonomy that has been recently accepted by the International Committee on Taxonomy of Viruses (ICTV) Executive Committee, and describe a virus nomenclature that is generally consistent with that used for other retroviruses, such as lentiviruses and deltaretroviruses. This taxonomy can be applied to distinguish different, but closely related, primate (e.g., human, ape, simian) foamy viruses as well as those from other hosts. This proposal accounts for host-virus co-speciation and cross-species transmission.

Complete Genome Sequence of the African Green Monkey Simian Foamy Virus Serotype 3 Strain FV2014 (SFVcae_FV2014).

The full-length sequence of simian foamy virus serotype 3 (SFV-3) strain FV2014, an African green monkey (Chlorocebus aethiops) isolate, was obtained using high-throughput sequencing. SFVcae_FV2014 consisted of 13,127 bp and had a genomic organization similar to those of other SFVs but was distinct from SFV strain LK3, isolated from the same monkey species.

A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection.

The capability of high-throughput sequencing (HTS) for detection of known and unknown viruses makes it a powerful tool for broad microbial investigations, such as evaluation of novel cell substrates that may be used for the development of new biological products. However, like any new assay, regulatory applications of HTS need method standardization. Therefore, our three laboratories initiated a study to evaluate performance of HTS for potential detection of viral adventitious agents by spiking model viruses in different cellular matrices to mimic putative materials for manufacturing of biologics. Four model viruses were selected based upon different physical and biochemical properties and commercial availability: human respiratory syncytial virus (RSV), Epstein-Barr virus (EBV), feline leukemia virus (FeLV), and human reovirus (REO). Additionally, porcine circovirus (PCV) was tested by one laboratory. Independent samples were prepared for HTS by spiking intact viruses or extracted viral nucleic acids, singly or mixed, into different HeLa cell matrices (resuspended whole cells, cell lysate, or total cellular RNA). Data were obtained using different sequencing platforms (Roche 454, Illumina HiSeq1500 or HiSeq2500). Bioinformatic analyses were performed independently by each laboratory using available tools, pipelines, and databases. The results showed that comparable virus detection was obtained in the three laboratories regardless of sample processing, library preparation, sequencing platform, and bioinformatic analysis: between 0.1 and 3 viral genome copies per cell were detected for all of the model viruses used. This study highlights the potential for using HTS for sensitive detection of adventitious viruses in complex biological samples containing cellular background. IMPORTANCE Recent high-throughput sequencing (HTS) investigations have resulted in unexpected discoveries of known and novel viruses in a variety of sample types, including research materials, clinical materials, and biological products. Therefore, HTS can be a powerful tool for supplementing current methods for demonstrating the absence of adventitious or unwanted viruses in biological products, particularly when using a new cell line. However, HTS is a complex technology with different platforms, which needs standardization for evaluation of biologics. This collaborative study was undertaken to investigate detection of different virus types using two different HTS platforms. The results of the independently performed studies demonstrated a similar sensitivity of virus detection, regardless of the different sample preparation and processing procedures and bioinformatic analyses done in the three laboratories. Comparable HTS detection of different virus types supports future development of reference virus materials for standardization and validation of different HTS platforms.

Complete Genome Sequence of a Naturally Occurring Simian Foamy Virus Isolate from Rhesus Macaque (SFVmmu_K3T).

The full-length genome sequence of a simian foamy virus (SFVmmu_K3T), isolated from a rhesus macaque (Macaca mulatta), was obtained using high-throughput sequencing. SFVmmu_K3T consisted of 12,983 bp and had a genomic organization similar to that of other SFVs, with long terminal repeats (LTRs) and open reading frames for Gag, Pol, Env, Tas, and Bet.

Whole-Genome Sequence of the Spodoptera frugiperda Sf9 Insect Cell Line.

The draft whole-genome sequence of the Spodoptera frugiperda Sf9 insect cell line was obtained using long-read PacBio sequence technology and Canu assembly. The final assembled genome consisted of 451 Mbp in 4,577 contigs, with 12,716× mean coverage and a G+C content of 36.53%.