Generation and Characterization of Flavivirus Receptor-Binding Domain Mutants and Chimeras

Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of infections annually. Primarily transmitted by ticks and mosquitoes, their associated diseases range from mild febrile infections to encephalitis and hemorrhagic fever. The molecular basis of the differences in disease presentation between genetically related viruses is uncertain, but potentially involves the putative receptor-binding domain of the envelope glycoprotein, domain III (EIII). In order to determine the tolerance for heterologous sequences, and to characterize the role of EIII in virus growth and tissue tropism, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from a range of flaviviruses of increasing genetic diversity from WNV were substituted. Four versions of each chimera were generated using combinations of up- and downstream restriction sites. Of the constructs tested, chimeras containing EIII from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses. The JEV EIII chimeras behaved similarly to WNV both in vitro and in vivo. In contrast, the KOUV chimeras, despite having greater EIII sequence identity, were more attenuated, failing to successfully infect organs other than the kidney, despite generating a strong WNV-like viremia, suggesting a significant change in tissue tropism. The SLEV chimeras were particularly attenuated, with low peak viremia, suggesting a poor infection of early targets, despite strong WNV-like growth in vitro. The BAGV chimeras were similarly attenuated, with temperature sensitivity assays suggesting lower particle stability than the other viruses. In addition to the chimeras, in vivo characterization of a WNV EIII single-site G331A mutant was performed. This virus was significantly attenuated, even in immunodeficient mice, suggesting that receptor-binding had been significantly impaired. Mutations present in isolates from mouse brains suggested that the G331A mutant could be compensated for, presumably through the alteration of receptor usage. Taken together, the data demonstrate that mutations and substitutions of EIII can significantly affect virulence and tissue tropism in vivo, in keeping with the hypothesis that this region is the flavivirus receptor-binding domain.