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Abstract The flavivirus envelope protein is a class II fusion protein that drives flavivirus-cell membrane fusion. The membrane fusion process is triggered by the conformational change of the E protein from dimer in the virion to trimer, which involves the rearrangement of three domains, EDI, EDII, and EDIII. The movement between EDI and EDII initiates the formation of the E protein trimer. The EDI-EDII hinge region utilizes four motifs to exert the hinge effect at the interdomain region and is crucial for the membrane fusion activity of the E protein. Using West Nile virus (WNV) NY99 strain derived from an infectious clone, we investigated the role of eight flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region in the conformational change of E protein from dimer to trimer and viral entry. Single mutations of the E-A54, E-I130, E-I135, E-I196, and E-Y201 residues affected infectivity. Importantly, the E-A54I and E-Y201P mutations fully attenuated the mouse neuroinvasive phenotype of WNV. The results suggest that multiple flavivirus-conserved hydrophobic residues in the EDI-EDII hinge region play a critical role in the structure–function of the E protein and some contribute to the virulence phenotype of flaviviruses as demonstrated by the attenuation of the mouse neuroinvasive phenotype of WNV. Thus, as a proof of concept, residues in the EDI-EDII hinge region are proposed targets to engineer attenuating mutations for inclusion in the rational design of candidate live-attenuated flavivirus vaccines.

The genetic diversities of mammalian tick-borne flaviviruses are poorly understood. We used next-generation sequencing (NGS) to deep sequence different viruses and strains belonging to this group of flaviviruses, including Central European tick-borne encephalitis virus (TBEV-Eur), Far Eastern TBEV (TBEV-FE), Langat (LGTV), Powassan (POWV), Deer Tick (DTV), Kyasanur Forest Disease (KFDV), Alkhurma hemorrhagic fever (AHFV), and Omsk hemorrhagic fever (OHFV) viruses. DTV, AHFV, and KFDV had the lowest genetic diversity, while POWV strains LEIV-5530 and LB, OHFV, TBEV-Eur, and TBEV-FE had higher genetic diversities. These findings are compatible with the phylogenetic relationships between the viruses. For DTV and POWV, the amount of genetic diversity could be explained by the number of tick vector species and amplification hosts each virus can occupy, with low diversity DTV having a more limited vector and host pool, while POWV with higher genetic diversities has been isolated from different tick species and mammals. It is speculated that high genetic diversity may contribute to the survival of the virus as it encounters these different environments.

The Flavivirus genus contains many important human pathogens, including dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), West Nile (WN), yellow fever (YF) and Zika (ZIK) viruses. While there are effective vaccines for a few flavivirus diseases (JE, TBE and YF), the majority do not have vaccines, including WN and ZIK. The flavivirus nonstructural 1 (NS1) protein has an unusual structure–function because it is glycosylated and forms different structures to facilitate different roles intracellularly and extracellularly, including roles in the replication complex, assisting in virus assembly, and complement antagonism. It also plays a role in protective immunity through antibody-mediated cellular cytotoxicity, and anti-NS1 antibodies elicit passive protection in animal models against a virus challenge. Historically, NS1 has been used as a diagnostic marker for the flavivirus infection due to its complement fixing properties and specificity. Its role in disease pathogenesis, and the strong humoral immune response resulting from infection, makes NS1 an excellent target for inclusion in candidate flavivirus vaccines.