Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line

Abstract

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Infectivity of Sf-rhabdovirus variants in insect and mammalian cell lines

“…1B). This same 320 bp deletion was found in the Sf-rhabdovirus X and X/L sequences detected by Hashimoto and coworkers (2017) in Sf9 L5814 cells (Fig 1C). …”

“…The identification of particles with rhabdovirus-like morphology in concentrated Sf9 cell-free supernatants (Ma et al, 2014) further suggests these infections can be productive. However, in their recent study of Sf-rhabdovirus in Sf9 L5814 cells, Hashimoto and coworkers (2017) detected no particles with rhabdovirus-like morphology or size. They noted the Sf-rhabdovirus X/L sequences detected in these cells have the 320 bp deletion first described by Haynes (2015), and further noted this deletion eliminates the X and L gene transcriptional motifs originally predicted by Ma and coworkers (Hashimoto et al, 2017).…”

“…However, in their recent study of Sf-rhabdovirus in Sf9 L5814 cells, Hashimoto and coworkers (2017) detected no particles with rhabdovirus-like morphology or size. They noted the Sf-rhabdovirus X/L sequences detected in these cells have the 320 bp deletion first described by Haynes (2015), and further noted this deletion eliminates the X and L gene transcriptional motifs originally predicted by Ma and coworkers (Hashimoto et al, 2017). Thus, this 320 bp X/L deletion would be expected to impair or prevent L gene transcription, which would be expected to block Sf-rhabdoviral replication because the L gene encodes the viral RNA-dependent RNA polymerase.…”

“…1). Since the original discovery of this agent, other investigators have confirmed the presence of Sf-rhabdoviral RNA sequences in Sf9 (Haynes, 2015; Maghodia et al, 2016) and Sf9 L5814 (Hashimoto et al, 2017) cells. Thus, Sf-rhabdovirus is now widely recognized as a common contaminant of at least some Sf cell lines used as hosts for baculovirus-mediated recombinant protein production.…”

“…This cell line was used to isolate a subclone, which was designated Sf9 (Summers and Smith, 1987) and is widely used for research purposes today. Subsequently, Sf9 ATCC CRL-1711 lot 5814 (Sf9 L5814 ) was used to isolate Sf900+, an evolved derivative that was commercialized as expres SF + and is now used as the host in Protein Sciences Corporation’s baculovirus expression system (BEVS) platform technology (Adams, 2017; Hashimoto et al, 2017; Smith et al, 2011). …”