No-known-vector flaviviruses exhibit diverse replication and virulence phenotypes in mice

The Orthoflavivirus genus (flaviviruses) includes globally significant arboviruses, which cycle between arthropod vectors (mosquitoes and ticks) and vertebrate hosts. In contrast, no-known-vector flaviviruses (NKVFVs) have been isolated from rodents and bats, but not arthropods, so they are thought to spread by vector-independent routes. However, little is known about the host range and pathogenic mechanisms of these viruses. To evaluate NKVFV pathogenesis, we infected wild-type, Ifnar1^-/-, and Ifnar1^-/- Ifngr1^-/- mice by footpad inoculation with 12 NKVFVs: Entebbe bat virus (ENTV), Sokuluk virus (SOKV), Yokose virus (YOKV), Modoc virus (MODV), Apoi virus (APOIV), Jutiapa virus (JUTV), Sal Vieja virus (SVV), Dakar bat virus (DBV), Rio Bravo virus (RBV), Montana myotis leukoencephalitis virus (MMLV), Phnom Penh bat virus (PPBV), and Tamana bat virus (TABV). We compared these NKVFVs to the mosquito-borne Zika virus and Kedougou virus and to the tick-borne Langat virus and Kadam virus. We monitored disease signs and measured viremia. Seven NKVFVs (ENTV, SOKV, YOKV, MODV, APOIV, DBV, RBV) were virulent in Ifnar1^-/- mice, causing 100% lethality within 9 days post-infection, accompanied by viremia. All viruses tested were virulent in Ifnar1^-/- Ifngr1^-/- mice and produced greater viremia compared to Ifnar1^-/- mice. No viremia or disease signs were detected in wild-type mice. We further evaluated RBV, MMLV, and PPBV replication in mouse primary fibroblasts and bone marrow-derived macrophages, as well as in cell lines from three bat species. Altogether, our results provide new information about the virulence and replication phenotypes of NKVFVs, supporting future studies investigating NKVFV-specific and pan-flavivirus pathogenic mechanisms.IMPORTANCEFlaviviruses that cause human disease are transmitted by mosquitoes and ticks (e.g., West Nile virus, yellow fever virus, tick-borne encephalitis virus). But there are related flaviviruses that are not known to infect arthropods, so are thought to spread by vector-independent routes (no-known-vector flaviviruses, NKVFVs). Not much is known about NKVFVs, but they provide an opportunity to understand flavivirus replication, tropism, and pathogenesis more broadly. We evaluated a panel of 12 NKVFVs for their ability to cause disease in mice with and without antiviral interferon responses as well as their replication in mouse cells. Our findings provide new information about these under-studied viruses and demonstrate which mouse models may be appropriate to use for further studies with NKVFVs.