Internal poly(A) replacement strategy within 3′ UTR for live-attenuated tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV), a prominent orthoflavivirus transmitted by ticks, poses a risk of potentially fatal encephalitis in humans. Currently, the only available inactivated vaccines have limited usage, underscoring the need for developing novel vaccine strategies. The 3' untranslated region (UTR) of TBEV can be divided into variable and conserved regions, where structured RNA elements play crucial roles in viral replication and virulence. In this study, we found that deleting sequences in both the variable region and most of the conserved region within the 3'UTR eliminated viral replication. However, inserting a poly(A) tract effectively restored viral propagation. Notably, the recovered virus variants, Δ1-642/poly(A), exhibited high attenuation in mice. A single-dose immunization with Δ1-642/poly(A) induced robust TBEV-specific IgG, neutralizing antibody, and T-cell immune responses, providing complete protection against lethal challenges with wild-type virus. Collectively, our findings demonstrate that poly(A) replacement represents a viable strategy for developing live-attenuated TBEV vaccines.IMPORTANCEOrthoflaviviruses, encompassing mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Zika virus (ZIKV), West Nile virus (WNV), and tick-borne encephalitis virus (TBEV), represent a substantial global public health and economic burden. The 3' UTR is critical for viral replication and pathogenesis, as exemplified by the ongoing clinical development of the tetravalent Butantan dengue vaccine (with deletion in 3' UTR). Our previous study on WNV-poly(A) variants proposed a novel live-attenuated vaccine design via 3' UTR engineering, yet its broad applicability remained unclear due to extensive 3' UTR heterogeneity. In this study, we successfully rescued a TBEV poly(A) virus using an analogous strategy. This virus was highly attenuated in mice, and a single immunization provided long-term complete protection against lethal wild-type virus challenge. Collectively, our data validate the poly(A) replacement approach as a potentially universal platform for developing live-attenuated vaccines against both mosquito-borne and tick-borne orthoflaviviruses.