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.