Scientific Publications by FDA Staff

J Virol 2014 Jun 15;88(12):6576-85

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

Ma H, Galvin TA, Glasner DR, Shaheduzzaman S, Khan AS

Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products and no viruses have been reported after extensive testing. We have 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 ORFs corresponding to the N, P, M, G, and L genes in other rhabdoviruses, and an unknown ORF of 111 amino acids located between G and L. BLAST searches indicated that Sf-rhabdovirus was related in a limited region of the L protein gene to Taastrup virus, a newly discovered member of 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 was 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 Sf9 parental cell line, Sf21, but not in other insect cell lines such as BT1-TN-5B1-4 (Tn5; High Five™) and Schneider's Drosophila Line 2 [D. Mel (2)., SL2], 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 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 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.