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.

Fig 1. BLAST search of SfRV RNA sequence against the Sf21 draft whole genome shotgun sequence (GenBank, JQCY00000000.2).

SfRV genes and their position on the genome are presented under the scale bar. Sf21 genome DNA sequences showing homology to SfRV RNA are aligned on the map; open triangle, anti-sense (SfRV genome RNA strand); closed circle, chimera with Sf21 DNA sequence.

Fig 2. BLAST search of SfRV RNA sequence against GenBank EST database.

EST clone sequences showing homology to SfRV RNA are aligned on the map and represented by colored circles; yellow circle, Bombyx mori (Bm5 or BmN cells, or Nnor); green circle, BmNPV infected BmN cells; blue circle, Heliothis virescence; black circle, Aphid gossippi; closed circle, sense strand sequence; open circle, anti-sense strand sequence.

Fig 3

(A). RT-PCR of total RNA from Sf9 cells. Primers were designed to amplify 14 target sequences that cover 99.1% of the reported Sf-rhabdovirus RNA in an overlapping manner as indicated by the gene schematic. Sizes of 13 target sequences ranged from ~1.0 to 1.3 kb (lanes 1–13) and one target sequence was ~0.5 kb (lane 14). The sizes of amplicons matched the expected sizes of target sequences except for one amplicon (*, lane 7), which was amplified by primers targeting bp 6965–7134. (B). Sequencing of the amplicon revealed a deletion of 320 bp at position 6371–6690 in Sf-rhabdovirus RNA. The 320 bp deletion contains the 3’ region of ORF-X and a part of the intergenic region between ORF-X and ORF-L that includes rhabdovirus conserved transcription motifs for the X and L genes.

Fig 4. qRT-PCR of sucrose density gradient fractions using SfRV L gene primers.

Ct values were normalized to that of fraction 2 and are represented as a relative copy number of target SfRV L gene sequence. The photo shows the sucrose density gradient prior to fractionation. Left to right: top to bottom of the gradient.

Fig 5. Electron microscopy of the 1.14 g/ml fraction.

Bar indicates 100 μm in length. A, no incubation after loading the 1.14 fraction (diluted with distilled water) onto a grid. B, 1 min incubation after loading. Both grids were washed twice with distilled water and sample was stained with 1% uranyl acetate.

Fig 6. Identification of major protein components in the 1.14 g/ml sucrose density gradient fraction of Sf9 culture supernatant by LC MS/MS analysis.

Eleven groups containing up to 3 stained protein bands were analyzed. The major protein identified per band is listed on the right.