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

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.

此前，研究人员通过下一代测序（next generation sequencing）与全面的生物信息学分析，在草地贪夜蛾（Spodoptera frugiperda）细胞系Sf9细胞[ATCC CRL-1711，批次58078522]中鉴定出一株推定的新型弹状病毒（rhabdovirus，SfRV）。本研究针对本实验室保藏的Sf9细胞库（ATCC CRL-1711，批次5814，即Sf9L5814）开展了全面分析，以确认该病毒是否存在于1987年自ATCC获取的细胞中。对从Sf9 L5814细胞基因组DNA中提取的DNA进行反向PCR（inverse PCR）分析，结果显示SfRV序列整合至宿主细胞基因组中。对总RNA进行逆转录PCR（reverse transcription PCR，RT-PCR）分析发现，SfRV RNA存在一段320 nt的缺失区域，该区域包含X基因与L基因的转录基序。将浓缩后的细胞培养上清液进行蔗糖密度梯度离心（sucrose density gradient centrifugation）分析，在1.14 g/ml的密度处获得单一条带。进一步对该组分进行电子显微镜（electron microscopy）观察，可见无定形的颗粒状碎屑，其形态与大小均不符合弹状病毒的特征。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis，SDS-PAGE）分析证实，该组分的蛋白组成并不包含弹状病毒典型的五种结构蛋白；液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry，LC-MS/MS）分析则显示，其主要成分为外泌体标记蛋白、SfRV N蛋白以及截短形式的SfRV N、P与G蛋白。将1.14 g/ml的组分经乙醚处理后进行超速离心，从上清液中回收得到SfRV L基因片段的RNA序列，这表明SfRV L基因片段序列并不与耐乙醚的核衣壳（nucleocapsid）相关联。值得注意的是，该1.14 g/ml组分可将杆状病毒（baculovirus）DNA转入Sf9L5814细胞中，这与功能性外泌体的存在相符。本研究结果证实，ATCC CRL-1711批次5814的Sf9细胞中不存在病毒颗粒，这与此前一项研究的结论相悖——该研究称不同批次的Sf9细胞中存在具有感染性的弹状病毒颗粒。本研究揭示，不同谱系的细胞系可能呈现出不同的病毒相关组分，因此无法针对不同实验室来源的Sf9细胞得出普适性结论。