Enhanced arbovirus surveillance with deep sequencing: Identification of novel rhabdoviruses and bunyaviruses in Australian mosquitoes

Abstract

Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥ 50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans.

Keywords: ABLV; AHSV; AINOV; AKAV; ALFV; ALMV; African horse sickness virus; Aino virus; Akabane virus; Alfuy virus; Almpiwar virus; Arbovirus surveillance; Australia; Australian bat lyssavirus; BANV; BASV; BEAUV; BEFV; BFV; BYSM; Banna virus; Barmah Forest virus; Bas-Congo virus; Beaumont virus; Bunyavirus; CCHFV; CHIKV; CHVV; CPV; Charleville virus; Coastal Plains virus; Crimean-Congo hemorrhagic fever virus; DAff sigma virus; DENV; DMel sigma virus; DOUV; DObs sigma virus; Deep sequencing; Douglas virus; Drosophila affinis sigma virus; Drosophila melanogaster sigma virus; Drosophila obscura sigma virus; EBLV; EHDV; EHV; EUBV; Edge Hill virus; Eubenangee virus; European bat lyssavirus; FPV; Facey's Paddock virus; GGV; Gan Gan virus; HDOOV; Humpty Doo virus; IHNV; JEV; Japanese encephalitis virus; KDV; KOKV; KUNV; Kadiporo virus; Kokobera virus; Kunjin virus; LEAV; LNV; LNYV; LYMoV; Leanyer virus; Liao Ning virus; MAPV; MFSV; MURBV; MVEV; Mapputta virus; Mosquito; Murray Valley encephalitis virus; Murrumbidgee virus; NCMV; NORCV; North Creek virus; Northern cereal mosaic virus; Novel virus; PEAV; PHSV; Peaton virus; Peruvian horse sickness virus; RRV; RVFV; Reovirus; Rhabdovirus; Rift Valley fever virus; Ross River virus; SASHV; SFTSV; SINV; SLOV; STRV; SVCV; Salt Ash virus; Sindbis virus; Stratford virus; Stretch Lagoon orbivirus; THIV; TIBV; TILV; TINV; TRUV; Thimiri virus; Tibrogargan virus; Tilligerry virus; Tinaroo virus; Trubanaman virus; VHSV; VSV; Virus discovery; WALV; WARV; WNV; Wallal virus; Warrego virus; West Nile virus; barley yellow striate mosaic; bovine ephemeral fever virus; chikungunya virus; dengue virus; epizootic hemorrhagic disease virus; infectious hematopoietic necrosis virus; lettuce necrotic yellows virus; lettuce yellow mottle virus; maize fine streak virus; severe fever with thrombocytopenia syndrome virus; spring viremia of carp virus; vesicular stomatitis virus; viral hemorrhagic septicemia virus.

Figure 1

Rhabdovirus maximum likelihood L gene open reading frame phylogeny with novel viruses highlighted in red. Numbers left of branches show statistical significance of tree topologies based on 1000 bootstrap re-sampling iterations. Symbols indicate geographic distributions and organism pictures show host ranges. Colored boxes show clade groupings; underlined groups denote established genera. L gene sequences used were 1568 aa (BEAUV), 2120 aa (NORCV, complete gene), ≈2100 aa (complete gene) for GenBank sequences.

Figure 2

Figure 2A, B: Orthobunyavirus maximum likelihood phylogenies of the L (A) and M (B) segment open reading frames with novel viruses highlighted in red. Sequences for M and L from GenBank were derived from the same isolate, when possible. Numbers to the left of branches show statistical significance of tree topologies based on 1000 bootstrap re-sampling iterations. Neighbor joining trees (not shown) exhibited similar topologies. Colored boxes show established orthobunyavirus groups defined based on genetic or serologic relatedness. Insect pictures show primary vectors. Gene sequences used were L: 2212 aa for SASHV, complete 2242 aa for MURBV, complete ≈2200 aa for GenBank sequences, M: complete 1377 aa for SASHV, complete 1371 aa for MURBV, complete ≈1400 aa for GenBank sequences.

Figure 2

Figure 2A, B: Orthobunyavirus maximum likelihood phylogenies of the L (A) and M (B) segment open reading frames with novel viruses highlighted in red. Sequences for M and L from GenBank were derived from the same isolate, when possible. Numbers to the left of branches show statistical significance of tree topologies based on 1000 bootstrap re-sampling iterations. Neighbor joining trees (not shown) exhibited similar topologies. Colored boxes show established orthobunyavirus groups defined based on genetic or serologic relatedness. Insect pictures show primary vectors. Gene sequences used were L: 2212 aa for SASHV, complete 2242 aa for MURBV, complete ≈2200 aa for GenBank sequences, M: complete 1377 aa for SASHV, complete 1371 aa for MURBV, complete ≈1400 aa for GenBank sequences.

Figure 3

Reoviridae RdRp gene maximum likelihood phylogeny with viruses detected here highlighted in red. Numbers to the left of branches show statistical significance of tree topologies based on 1000 bootstrap re-sampling iterations. Colored boxes show established reovirus genera. Pictures show primary vector types. Full ≈1250 aa RdRp gene sequences we used for viruses sequenced in this study, some shorter ≈130 aa sequences from GenBank were used when full sequences were not available.

Figure 4

Map of Australia showing mosquito collection locations for arboviruses detected in this study (red) as well as sites where other Australian arboviruses have been isolated previously (black and shaded regions) (–62); Akabane virus (AKAV, purple) (63), SLOV (yellow) (19, 62), and GGV (green) (62). EHV, KOKV, and RRV are not shown since they exhibit diffuse continental distributions. LNV has not been previously isolated in Australia. NSW, New South Wales; NT, Northern Territory; QLD, Queensland; SA, South Australia; VIC, Victoria, WA, Western Australia.