Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X⁺ Genome Contains a 3.7 kb Internal Duplication

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X⁺ since it contained a unique accessory gene X. The Sf-rhabdovirus X⁺ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X⁺ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X⁺ virus. Thus, the X⁺ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X^+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X⁺ variant virus. Furthermore, the Sf-rhabdovirus X^+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.