Hailun Ma

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines

ABSTRACT 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 Schneiders 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.

Investigations of porcine circovirus type 1 (PCV1) in vaccine-related and other cell lines

Porcine circovirus type 1 (PCV1) is highly prevalent in swine and was recently reported in some rotavirus vaccines. Since animal-derived raw materials, such as cells, trypsin, and serum, can be a major source of introducing virus contamination in biological products, we have investigated PCV1 in several cell lines obtained from ATCC that have broad use in research, diagnostics, or vaccine development. It is expected that these cell lines have been exposed to bovine and porcine viruses during their establishment and passage history due to the use of serum and trypsin that was not qualified according to current testing guidances or processed using new virus-inactivation methods. This study showed that Vero, MRC-5, and CEFs, which represent cell substrates used in some U.S. licensed vaccines, and other cell lines used in investigational vaccines, such as MDCK, HEK-293, HeLa, and A549, were negative for PCV1 using a nested PCR assay; some were also confirmed negative by infectivity analysis. However, MDBK cells, which are used for some animal vaccines, contained PCV1 sequences, although no virus was isolated. Although the results showed that PCV infection may not have occurred under previous culture conditions, the recent cases of vaccine contamination emphasizes the need for continued efforts to reduce the likelihood of introducing viruses from animal-derived materials used in product manufacture.

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

ABSTRACT 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.

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.

Evaluation of the broad-range PCR-electrospray ionization mass spectrometry (PCR/ESI-MS) system and virus microarrays for virus detection.

Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences.

New Technologies and Challenges of Novel Virus Detection

The use of new cell substrates for the development of biologicals, particularly tumorigenic and tumor-derived cell lines, can pose a major regulatory challenge due to safety concerns related to the presence of novel viruses, latent and occult viruses including oncogenic viruses, and endogenous retroviruses, since these may not be detected by the currently recommended conventional assays. This report is a summary of our laboratorys experiences using advanced nucleic acid-based technologies to evaluate a Madin-Darby canine kidney (MDCK) cell line and the insect Sf9 cell line derived from Spodoptera frugiperda, and presents some ongoing efforts to address the challenges of novel virus detection.

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.

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

ABSTRACT 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%.

Detection of Latent Retroviruses in Vaccine-related Cell Substrates: Investigation of RT Activity Produced by Chemical Induction of Vero Cells

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1–3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA) The detection of known and novel viruses is important for cell substrate and vaccine safety. A major challenge is detection of latent viruses such as endogenous retroviruses and oncogenic DNA viruses. We have evaluated activation of endogenous retroviruses in a Vero cell line using chemical induction and various conventional and emerging methods for virus detection and characterization. In addition, infectivity studies were done to determine whether any induced virus particles were replication competent. This approach may be used for enhancing vaccine safety by assessing the presence of potential chemically-inducible, latent viruses in cell substrates to be used for vaccine manufacture.

Optimization of chemical induction for evaluation of endogenous retroviruses in different species

Background Chemical inducers such as 5-azacytidine (AzaC) and 5’iodo-2’deoxyuridine (lUdR) have been used to discover and characterize endogenous retroviruses from rodent and avian speciecs, for example KBALB mouse cells. We found that induction conditions that have been optimized for mouse cells were not successful in inducing retrovirus from cell lines of other species, including nonhuman primates. Therefore, we developed a stepwise strategy based upon identification of critical parameters for endogenous retrovirus induction in mouse cells for optimizing induction conditions for non-murine cells. Using this approach, we have determined optimum conditions for investigating inducible endogenous retroviruses from Vero cells, which are of African green monkey (AGM) origin, a species that has never been reported to produce endogenous retroviruses [1].