Abstract
Banyangvirus is a new genus (Phenuiviridae family, Bunyavirales order) that comprises a group of emerging tick-borne viruses with severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) as virulent representatives. As segmented RNA viruses, bunyaviruses may have genome reassortment potential, increasing the concern about new life-threatening bunyavirus emergence. Using a series of combinatory minigenome reporter assays based on transfection and superinfection, we showed that replication machinery proteins of designated banyangviruses can recognize genomic untranslated regions (UTRs) of other banyangviruses and assemble heterogenous minigenomes into functional ribonucleoproteins (RNPs). Moreover, both heterogenous and heterozygous RNPs were efficiently packaged by viral glycoproteins into infectious virus-like particles, manifesting remarkable reassortment potential of banyangviruses. Meanwhile, UTR promoter strength of the three banyangvirus segments appeared to be M > L > S. Secondary structure analysis revealed a conservative non-basepairing protruding nucleotide in the terminal UTR panhandles of M and L (but not S) segments of all banyangviruses and some related phleboviruses (Phlebovirus genus). Furthermore, not only a conserved panhandle region but also the protruding nucleotide proved important for UTR function. Removal of the protruding nucleotide abated M and L UTR activities and compatibilities with heterogenous viral proteins, and introduction of a protruding nucleotide into S panhandle, conversely, enhanced UTR promoter strength and compatibility, revealing the significance of the protruding nucleotide as a new signature of the genomic panhandle structure in both UTR activity and reassortment potential. The study demonstrates not only banyangvirus reassortment potential but also the notable role of the protruding nucleotide in UTR function and reassortment, providing clues to viral evolution and replication mechanisms and perhaps benefiting disease control and prevention in the future.
Highlights
Banyangviruses are a group of emerging tick-borne viruses (ICTV, 2018; Abudurexiti et al, 2019)
We here exploited RNA polymerase I (pol I) system for banyangvirus reverse genetic studies and firstly, murine pol I-driven minigenome systems for all the three segments of Severe fever with thrombocytopenia syndrome virus (SFTSV) were developed
SFTSV L, M, and S segment-based minigenome reporter plasmids were constructed by cloning the antisense firefly luciferase or enhanced green fluorescent protein (EGFP) cassette flanked by the untranslated regions (UTRs) of SFTSV L, M, and S segments into pRF42 under the control of pol I promoter to allow the generation of viral genome-like RNA segments, i.e., minigenomes, after transfection into cells (Figure 1A)
Summary
Banyangviruses are a group of emerging tick-borne viruses (ICTV, 2018; Abudurexiti et al, 2019). Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV) are both emerging virulent representatives of Banyangvirus (ICTV, 2018; Abudurexiti et al, 2019). SFTSV and HRTV are geographically isolated, they both can spread by tick bites and cause similar clinical symptoms in patients including severe fever, thrombocytopenia, leukocytopenia, gastrointestinal symptoms, hemorrhagic signs, and multiorgan dysfunction, associated with high case fatality rates (Yu et al, 2011; McMullan et al, 2012; Muehlenbachs et al, 2014; Center for Disease Control and Prevention [CDC], 2018). Following SFTSV and HRTV, Guertu virus (GTV) which was isolated from Dermacentor nuttalli ticks in China in 2014 by our research group is the third representative species of Banyangvirus listed by ICTV (Shen et al, 2018). Banyangviruses have posed severe threats to worldwide human health, there are no licensed vaccines or specific antivirals available currently
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