The use of minigenome systems as tools to “fill in the blanks” for uncultured emerging viruses and facilitate virus rescue: a Lloviu virus case study Adam J Hume1,2, Whitney A Manhart1,2, Jennifer R Pacheco1,2, Tessa N Cressey1,2, Elke Mühlberger1,2 1Department of Microbiology; 2National Emerging Infectious Disease Laboratories, Boston University School of Medicine, Boston, MA 02118; USA
Recently, traces of zoonotic viruses have been discovered in bats and other animals around the world, but despite repeated attempts full viral genomes have not been rescued. The difficulties of isolating infectious virus from specimens prevent research on their pathogenic potential for humans. Although reverse genetic systems have facilitated the rescue of a great many viruses, the absence of critical genetic sequences located at the genomic ends of these uncultured emerging viruses prevent their rescue. One example of these zoonotic pathogens is Lloviu virus (LLOV), a filovirus which is closely related to Ebola virus. LLOV has not yet been isolated and the reported LLOV genome is missing sequence at both the 3' and 5' termini based on alignments with other filovirus genomes. We have recently established chimeric LLOV minigenome systems based on sequence complementation from other filoviruses. Our results show that the LLOV replication and transcription mechanisms are, in general, more similar to ebolaviruses than to marburgviruses. We also show that a single nucleotide at the 3' genome end determines species specificity of the LLOV polymerase. The data obtained using these chimeric minigenomes are being used to create a reverse genetic system to facilitate the rescue of infectious LLOV clones for pathogenesis studies. These data highlight the usefulness of minigenome systems in complementing the missing sequences from uncultured emerging viruses in order to be able to rescue these viruses and assess their pathogenic potential.