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 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.

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.

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.

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.

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

UNLABELLED: The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. IMPORTANCE: The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results.

Identification of recombination in the envelope gene of simian foamy virus serotype 2 isolated from Macaca cyclopis.

The full-length sequence of simian foamy virus serotype 2 (SFVmcy-2), isolated from a Taiwanese macaque, was determined. SFVmcy-2 was highly related to SFV serotype 1 (SFVmcy-1), an isolate from the same species, except in the putative receptor binding domain (RBD) in env, which contained novel sequences related to SFV serotype 3 (SFVagm-3), isolated from an African green monkey. The results identify a potential region of neutralization in SFVs and demonstrate recombination between genetically divergent foamy viruses.

No evidence of xenotropic murine leukemia virus-related virus transmission by blood transfusion from infected rhesus macaques.

The discovery of xenotropic murine leukemia virus-related virus (XMRV) in human tissue samples has been shown to be due to virus contamination with a recombinant murine retrovirus. However, due to the unknown pathogenicity of this novel retrovirus and its broad host range, including human cell lines, it is important to understand the modes of virus transmission and develop mitigation and management strategies to reduce the risk of human exposure and infection. XMRV transmission was evaluated by whole-blood transfusion in rhesus macaques. Monkeys were infected with XMRV to serve as donor monkeys for blood transfers at weeks 1, 2, and 3 into naïve animals. The donor and recipient monkeys were evaluated for XMRV infection by nested PCR assays with nucleotide sequence confirmation, Western blot assays for development of virus-specific antibodies, and coculture of monkey peripheral blood mononuclear cells (PBMCs) with a sensitive target cell line for virus isolation. XMRV infection was demonstrated in the virus-injected donor monkeys, but there was no evidence of virus transmission by whole-blood transfusion to naïve monkeys based upon PCR analysis of PBMCs using XMRV-specific gag and env primers, Western blot analysis of monkey plasma up to 31 to 32 weeks after transfusion, and coculture studies using monkey PBMCs from various times after transfusion. The study demonstrates the lack of XMRV transmission by whole-blood transfusion during the acute phase of infection. Furthermore, analysis of PBMC viral DNA showed extensive APOBEC-mediated G-to-A hypermutation in a donor animal at week 9, corroborating previous results using macaques and supporting the possible restriction of XMRV replication in humans by a similar mechanism.

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.

Chemical induction of endogenous retrovirus particles from the vero cell line of African green monkeys.

Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may be expressed as RNAs; however, the majority are defective for virus production. Although virus has been isolated from various Old World monkey and New World monkey species, there has been no report of endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al., Biologicals 37:196-201, 2009). Based upon this strategy, optimum conditions were determined for investigating the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse transcriptase activity was produced with 5-azacytidine (AzaC) and with 5'-iodo-2'-deoxyuridine (IUdR); none was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol, and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious, viruslike particles.

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines.

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10(5) cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells.

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.

Role of neutralizing antibodies in controlling simian foamy virus transmission and infection.

BACKGROUND: Human infections with simian foamy viruses (SFVs) have been reported after occupational and nonoccupational exposure to infected animals and their tissues, blood, and body fluids, although there is no evidence for human-to-human transmission. We previously demonstrated SFV transmission in monkeys by blood transfusion with whole blood from one donor animal that had a low neutralizing antibody (NAb) endpoint titer, whereas blood transfusion from a second donor monkey that had a high NAb titer failed to transmit virus. These results suggested a role for NAbs in SFV transmission and establishment of infection. STUDY DESIGN AND METHODS: Whole blood and antibody-reduced blood were transfused into SFV-negative rhesus macaques. SFV infection in recipient animals was monitored by detection of virus sequences using polymerase chain reaction assays with nucleotide sequence confirmation, by analysis for antibody development in Western blots, and by virus isolation in coculture assays. NAb titer was evaluated by endpoint dilution assays. RESULTS: SFV transmission by whole blood transfusion from a donor monkey with high NAb endpoint titer failed to establish infection in SFV-negative monkeys, whereas virus transmission was successful with transfer of antibody-reduced blood cells. CONCLUSIONS: Passive transfer of high-titer NAbs blocked SFV cell-associated transmission, indicating that NAbs may play a role in virus transmission to individuals exposed to SFV-infected blood and tissues.

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.

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.

Proposed algorithm to investigate latent and occult viruses in vaccine cell substrates by chemical induction.

The recent urgency to develop new vaccines for emerging and re-emerging diseases, such as pandemic influenza, has necessitated the use of cell substrates not previously used in the manufacture of licensed vaccines. A major safety concern in the use of novel cell substrates is the presence of potential adventitious agents, such as latent and occult viruses, that may not be detected by currently used conventional assays. In cases where the novel cell substrate is known to be tumorigenic, there are additional safety issues related to tumorigenicity of intact cells and oncogenicity of residual cellular DNA. We have developed a strategy for evaluating vaccine cell substrates for the presence of latent/occult viruses, including endogenous retroviruses, latent RNA viruses and oncogenic DNA viruses, by optimizing conditions for chemical induction of viruses and using a combination of broad and specific assays to enable detection of known and novel viruses.

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.