Metagenomic sequencing has led to a recent and rapid expansion in the animal virome. It has uncovered a multitude of new virus lineages from under-sampled host lineages, including many that break up long branches among previously known clades, and many with genomes that display unexpected sizes and structures. Although there are challenges to inferring the existence of a virus from a virus-like sequence, the analysis of nucleic acid (including small RNAs) and sequence data can give us considerable confidence in the absence of an isolate. As a consequence, this period of ‘molecular natural history’ is helping to reshape our views of deep virus evolution. Nevertheless, there is a limit to what metagenomic discovery alone can tell us, and some open questions will require experimental isolates.

Advances in sequencing technologies and bioinformatics have greatly enhanced our knowledge of virus biodiversity. Currently, viromes of haematophagous invertebrates, such as mosquitoes and ixodid ticks, are actively studied. Tabanidae (Diptera) is a widespread family, with members mostly known for their persistent haematophagous behavior. They transmit viral, bacterial and other pathogens, both biologically and mechanically. However, tabanid viromes remain severely understudied. In this study, we used high-throughput sequencing to describe the viromes of several species of Hybomitra, Tabanus, Chrysops, and Haematopota genera, which were collected in Russia. We assembled 14 full coding genomes of the novel viruses, 4 partial coding genomes, as well as several fragmented viral sequences, which presumably belong to another 12 new viruses. All the discovered viruses were tested on their ability to replicate in the mammalian porcine embryo kidney (PEK), tick HAE/CTVM8, and mosquito C6/36 cell lines. In total, 16 viruses were detected in at least one cell culture after three passages (for PEK and C6/36) or 3 weeks of persistence in HAE/CTVM8. However, in the majority of cases, qPCR showed a decline in virus load over time. Nevertheless, four viruses were detected after six passages.