The alphaviruses Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV) are arthropod-borne positive-strand RNA viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. VEEV was weaponized during the Cold War and is recognized as a select agent. Currently, there are no FDAapproved vaccines or therapeutics for these viruses. The spread of VEEV and other members of this family due to climate change-mediated vector range expansion underscores the need for research aimed at developing medical countermeasures. These viruses utilize programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) to synthesize the viral trans-frame (TF) protein, which has previously been shown to be important for neuropathogenesis in the related Sindbis virus. Here, the alphavirus Ϫ1 PRF signals were characterized, revealing novel Ϫ1 PRF stimulatory structures. Ϫ1 PRF attenuation mildly affected the kinetics of VEEV accumulation in cultured cells but strongly inhibited its pathogenesis in an aerosol infection mouse model. Importantly, the decreased viral titers in the brains of mice infected with the mutant virus suggest that the alphavirus TF protein is important for passage through the blood-brain barrier and/or for neuroinvasiveness. These findings suggest a novel approach to the development of safe and effective live attenuated vaccines directed against VEEV and perhaps other closely related Ϫ1 PRF-utilizing viruses.IMPORTANCE Venezuelan equine encephalitis virus (VEEV) is a select agent that has been weaponized. This arthropod-borne positive-strand RNA virus causes acute and fatal encephalitis in many mammals, including humans. There is no vaccine or other approved therapeutic. VEEV and related alphaviruses utilize programmed Ϫ1 ribosomal frameshifting (Ϫ1 PRF) to synthesize the viral trans-frame (TF) protein, which is important for neuropathogenesis. Ϫ1 PRF attenuation strongly inhibited VEEV pathogenesis in mice, and viral replication analyses suggest that the TF protein is critical for neurological disease. These findings suggest a new approach to the development of safe and effective live attenuated vaccines directed against VEEV and other related viruses.

Noroviruses are the predominant cause of acute gastroenteritis, with a single genotype (GII.4) responsible for the majority of infections. This prevalence is characterized by the periodic emergence of new variants that present substitutions at antigenic sites of the major structural protein (VP1), facilitating escape from herd immunity. Notably, the contribution of intravariant mutations to changes in antigenic properties is unknown. We performed a comprehensive antigenic analysis on a virus-like particle panel representing major chronological GII.4 variants to investigate diversification at the inter- and intravariant level. Immunoassays, neutralization data, and cartography analyses showed antigenic similarities between phylogenetically related variants, with major switches to antigenic properties observed over the evolution of GII.4 variants. Genetic analysis indicated that multiple coevolving amino acid changes—primarily at antigenic sites—are associated with the antigenic diversification of GII.4 variants. These data highlight complexities of the genetic determinants and provide a framework for the antigenic characterization of emerging GII.4 noroviruses.

No abstract

Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus. CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed ؊1 ribosomal frameshift (؊1 PRF) signal located toward the 3 end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and ؊1 PRF. Analyses of RNA chemical modification data with selective 2-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV ؊1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.