Enhanced attenuation of chikungunya vaccines expressing antiviral cytokines.

Alphaviruses are vector-borne, medically relevant, positive-stranded RNA viruses that cause disease in animals and humans worldwide. Of this group, chikungunya virus (CHIKV) is the most significant human pathogen, responsible for generating millions of infections leading to severe febrile illness and debilitating chronic joint pain. Currently, there are limited treatments to protect against alphavirus disease; thus, there is a tremendous need to generate safe and effective vaccines. Live-attenuated vaccines (LAVs) are cost-effective and potent immunization strategies capable of generating long-term protection in a single dose. However, LAVs often produce systemic viral replication, which can lead to unwanted post-vaccination side effects and pose a risk of reversion to a pathogenic phenotype and transmission to mosquitoes. Here, we utilized a chimeric infectious clone of CHIKV engineered with the domain C of the E2 gene of Semliki Forest virus (SFV) to express IFNγ and IL-21-two potent antiviral and immunomodulatory cytokines-in order to improve the LAV's attenuation while maintaining immunogenicity. The IFNγ- and IL-21-expressing vaccine candidates were stable during passage and significantly attenuated post-vaccination, as mice experienced reduced footpad swelling with minimal systemic replication and dissemination capacity compared to the parental vaccine. Additionally, these candidates provided complete protection to mice challenged with WT CHIKV. Our dual attenuation strategy represents an innovative way to generate safe and effective alphavirus vaccines that could be applied to other viruses.

An in vitro workflow to create and modify infectious clones using replication cycle reaction.

Reverse genetics systems are critical tools in combating emerging viruses which enable a better understanding of the genetic mechanisms by which viruses cause disease. Traditional cloning approaches using bacteria are fraught with difficulties due to the bacterial toxicity of many viral sequences, resulting in unwanted mutations within the viral genome. Here, we describe a novel in vitro workflow that leverages gene synthesis and replication cycle reaction to produce a supercoiled infectious clone plasmid that is easy to distribute and manipulate. We developed two infectious clones as proof of concept: a low passage dengue virus serotype 2 isolate (PUO-218) and the USA-WA1/2020 strain of SARS-CoV-2, which replicated similarly to their respective parental viruses. Furthermore, we generated a medically relevant mutant of SARS-CoV-2, Spike D614G. Results indicate that our workflow is a viable method to generate and manipulate infectious clones for viruses that are notoriously difficult for traditional bacterial-based cloning methods.

The E2 glycoprotein holds key residues for Mayaro virus adaptation to the urban Aedes aegypti mosquito.

Adaptation to mosquito vectors suited for transmission in urban settings is a major driver in the emergence of arboviruses. To better anticipate future emergence events, it is crucial to assess their potential to adapt to new vector hosts. In this work, we used two different experimental evolution approaches to study the adaptation process of an emerging alphavirus, Mayaro virus (MAYV), to Ae. aegypti, an urban mosquito vector of many other arboviruses. We identified E2-T179N as a key mutation increasing MAYV replication in insect cells and enhancing transmission after escaping the midgut of live Ae. aegypti. In contrast, this mutation decreased viral replication and binding in human fibroblasts, a primary cellular target of MAYV in humans. We also showed that MAYV E2-T179N generates reduced viremia and displays less severe tissue pathology in vivo in a mouse model. We found evidence in mouse fibroblasts that MAYV E2-T179N is less dependent on the Mxra8 receptor for replication than WT MAYV. Similarly, exogenous expression of human apolipoprotein receptor 2 and Mxra8 enhanced WT MAYV replication compared to MAYV E2-T179N. When this mutation was introduced in the closely related chikungunya virus, which has caused major outbreaks globally in the past two decades, we observed increased replication in both human and insect cells, suggesting E2 position 179 is an important determinant of alphavirus host-adaptation, although in a virus-specific manner. Collectively, these results indicate that adaptation at the T179 residue in MAYV E2 may result in increased vector competence-but coming at the cost of optimal replication in humans-and may represent a first step towards a future emergence event.

De Novo Assembly and Annotation of 11 Diverse Shrub Willow (Salix) Genomes Reveals Novel Gene Organization in Sex-Linked Regions.

Poplar and willow species in the Salicaceae are dioecious, yet have been shown to use different sex determination systems located on different chromosomes. Willows in the subgenus Vetrix are interesting for comparative studies of sex determination systems, yet genomic resources for these species are still quite limited. Only a few annotated reference genome assemblies are available, despite many species in use in breeding programs. Here we present de novo assemblies and annotations of 11 shrub willow genomes from six species. Copy number variation of candidate sex determination genes within each genome was characterized and revealed remarkable differences in putative master regulator gene duplication and deletion. We also analyzed copy number and expression of candidate genes involved in floral secondary metabolism, and identified substantial variation across genotypes, which can be used for parental selection in breeding programs. Lastly, we report on a genotype that produces only female descendants and identified gene presence/absence variation in the mitochondrial genome that may be responsible for this unusual inheritance.