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.

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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans and animals and is therefore a pathogen of grave concern within a One Health framework. Identifying animal-adaptive mutations is critical to preserving One Health, as these mutations could also lead to the persistence of SARS-CoV-2 in animal reservoirs with continual spillover to humans. Therefore, we sought to pair experimental evolution and epidemiological data to identify putative human- and animal-adaptive viral residues and determine their impact on replication-competent SARS-CoV-2 in both human and animal cells. We passaged SARS-CoV-2 in cells expressing human, dog, cat, mink, and white-tailed deer ACE2 and sequenced the passaged populations. In addition, we searched SARS-CoV-2 sequences for mutations following patterns of convergent evolution that were common to both human- and animal-derived SARS-CoV-2 sequences. We identified the epidemiologically relevant Spike A222V mutation from our passaging experiment in cells expressing cat ACE2, a mutation that has also arisen independently across eight lineages of SARS-CoV-2 from human- and animal-derived sequences. To assess its impact on replication in human and animal cells, we constructed SARS-CoV-2 Spike A222V in the Wuhan-Hu-1 backbone with Spike D614G; this virus replicated similarly to the WT SARS-CoV-2 in human lung epithelial cells. In contrast, SARS-CoV-2 Spike A222V demonstrated an advantage in replication in primary deer lung cells, which was not mediated by the deer ACE2 receptor. Infection via the human, dog, cat, and mink ACE2 receptor resulted in reduced replication of SARS-CoV-2 Spike A222V. Our experiments identified Spike A222V as a putatively deer-adaptive mutation. Future studies should assess Spike A222V’s relevance to transmission within deer and to other animal species in contact with deer.

Chikungunya virus (CHIKV) is an emerging virus responsible for millions of infections globally within the last 15 years and has the potential to become endemic in the US. CHIK disease is characterized by severe febrile illness, with 30–60% of cases leading to debilitating chronic joint pain. No licensed treatments are available to protect against CHIK disease; thus, there is a tremendous need to generate a safe and effective vaccine. Live-attenuated vaccines (LAVs) are an appealing immunization strategy because they typically generate long-term protection from a single dose. However, LAVs often cause post-vaccination side effects and produce systemic viral replication, which can potentially lead to reversion to a pathogenic phenotype or transmission to mosquitoes; thus, safer LAV platforms are needed. To that end, we sought to improve the traditional LAV platform by combining attenuating strategies; as a vaccine backbone, we used a previously developed chimera of CHIKV and the closely related Semliki Forest virus (SFV) where we replaced the E2 domain C region of CHIKV with the corresponding domain from SFV (CHIKV-SFV/DomC) that was highly attenuated in mice and mosquitoes. To further attenuate the backbone, we inserted IFN-γ or IL-21, important antiviral cytokine genes, into the viral genome. The IFN-γ- and IL-21-expressing candidates were significantly attenuated post-vaccination, generating 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. This novel dual attenuation strategy combining an attenuated chimeric backbone and an antiviral cytokine has the possibility to be applied for the attenuation of any RNA virus.