Faculty Bibliography

Chikungunya virus (CHIKV) is a reemerged arbovirus, a member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKVs by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population toward deleterious evolutionary trajectories. When the candidate viruses were tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found that the vaccine candidates elicited protective immunity related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naive mice, vaccine candidates could be transmitted by mosquito bite, leading to asymptomatic infection in mice with compromised dissemination. Using deep-sequencing technology, we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering the viruses promising vaccine candidates.IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently, there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection, and had low risk of reversion to pathogenic phenotypes.

Understanding the fundamental mechanisms of arbovirus transmission and pathogenesis is essential to develop strategies for treatment and prevention. We previously took an in vivo evolution-based approach and identified the chikungunya virus E1 glycoprotein residue 80 to play a critical role in viral transmission and pathogenesis. In this study, we address the genetic conservation and function of position 80 and demonstrate that this residue is a key determinant in alphavirus infectivity and dissemination through modulation of viral fusion and cholesterol dependence. In addition, in studying the evolution of position 80, we identified a network of glycoprotein residues, including epidemic determinants, that regulate virus dissemination and infectivity. These studies underscore the importance of taking evolution-based approaches to not only identify key viral determinants driving arbovirus transmission and pathogenesis but also to uncover fundamental aspects of arbovirus biology.

Arthropod-borne viruses represent a significant public health threat worldwide yet there are few antiviral therapies or prophylaxis targeting these pathogens. In particular, the development of novel antivirals for high-risk populations such as pregnant women is essential to prevent devastating disease such as that which was experienced with the recent outbreak of Zika virus (ZIKV) in the Americas. One potential avenue to identify new and pregnancy-acceptable antiviral compounds is to repurpose well-known and widely used FDA approved drugs. In this study, we addressed the antiviral role of atovaquone, a FDA Pregnancy Category C drug and pyrimidine biosynthesis inhibitor used for the prevention and treatment of parasitic infections. We found that atovaquone was able to inhibit ZIKV and chikungunya virus virion production in human cells and that this antiviral effect occurred early during infection at the initial steps of viral RNA replication. Moreover, we were able to complement viral replication and virion production with the addition of exogenous pyrimidine nucleosides indicating that atovaquone is functioning through the inhibition of the pyrimidine biosynthesis pathway to inhibit viral replication. Finally, using an ex vivo human placental tissue model, we found that atovaquone could limit ZIKV infection in a dose-dependent manner providing evidence that atovaquone may function as an antiviral in humans. Taken together, these studies suggest that atovaquone could be a broad-spectrum antiviral drug and a potential attractive candidate for the prophylaxis or treatment of arbovirus infection in vulnerable populations, such as pregnant women and children.IMPORTANCE The ability to protect vulnerable populations such as pregnant women and children from Zika virus and other arbovirus infections is essential to preventing the devastating complications induced by these viruses. One class of antiviral therapies may lie in known pregnancy-acceptable drugs that have the potential to mitigate arbovirus infections and disease yet this has not been explored in detail. In this study, we show that the common antiparasitic drug, atovaquone, inhibits arbovirus replication through intracellular nucleotide depletion and can impair ZIKV infection in an ex vivo human placental explant model. Our study provides a novel function for atovaquone and highlights that the rediscovery of pregnancy-acceptable drugs with potential antiviral effects can be the key to better addressing the immediate need for treating viral infections and preventing potential birth complications and future disease.

Background. Atovaquone, a hydroxynaphthoquinone, FDA Pregnancy category C, used for the treatment and prevention of pneumocysits jirovecii pneumonia (PCP), toxoplasmosis, babesiosis and malaria has in vitro activity against Zika virus (ZIKV). The mechanism of action against Plasmodium spp. and other parasites is based in the inhibition of mitochondrial cytochrome bc1 complex which further collapses parasite- mitochondrial membrane potential. But to date, antiviral activity of this drug has not been described. Methods. Vero cells (monkey kidney epithelial cells) were seeded. At 24 hours of incubation, the cells were pretreated with ribavirin and brequinar (known antiviral drugs) and atovaquone at different concentrations for 1 hour and then infected with ZIKV Brazilian strain and Ugandan strain, and subsequently treated with drugs again. After incubation for 72 hours virus antigen Env-protein production was quantified by immunodetection. The concentration of atovaquone that decreased the level of Env-protein production by 50% was calculated by non-linear regression analysis (CC50). Cell viability was measured using the CellTiter 96 aqueous one solution cell proliferation assay (Promega, Madison, WI), according to the manufacturers protocol. Viral infection was rescued adding uracil to Vero cells pre-treated with ribavirin, brequinar and atovaquone. Experiment was repeated with Chikungunya virus (CHIKV). Results. We found that atovaquone inhibits ZIKV infection in Vero cells at smaller concentration (CC50 = 0.52 muM) than those used for parasitic killing. The effect is more prominent in the Brazilian strain when compared with the Ugandan strain. No cytotoxic effect was found in Vero cells up to 15 muM; above this concentration atovaquone formed crystals. Uracil rescues ZIKV infection after treatment with atovaquone. Atovaquone also inhibited CHIKV infection in Vero cells. Conclusion. Atovaquone has antiviral activity against ZIKV likely via depletion of nucleotides blocking pyrimidine biosynthesis. Furthermore, the antiviral effect is applicable to other arboviruses which makes atovaquone a broad-spectrum antiviral drug and a potential attractive candidate for the treatment of ZIKV infection in vulnerable population such pregnant women and children. (Figure Presented)

The 3'untranslated region (UTR) in alphavirus genomes functions in virus replication and plays a role in determining virus host range. However, the molecular evolution of virus UTRs is understudied compared to the evolution of protein-coding regions. Chikungunya virus (CHIKV) has the longest 3'UTR among the alphaviruses (500-700 nt), and 3'UTR length and sequence structure vary substantially among different CHIKV lineages. Previous studies showed that genomic deletions and insertions are key drivers of CHIKV 3'UTR evolution. Inspired by hypothesized deletion events in the evolutionary history of CHIKV, we used experimental evolution to examine CHIKV adaptation in response to a large 3'UTR deletion. We engineered a CHIKV mutant with a 258 nt deletion in the 3'UTR (ΔDR1/2). This deletion reduced viral replication on mosquito cells, but did not reduce replication on mammalian cells. To examine how selective pressures from vertebrate and invertebrate hosts shape CHIKV evolution after a deletion in the 3'UTR, we passaged ΔDR1/2 virus populations strictly on primate cells, strictly on mosquito cells, or with alternating primate/mosquito cell passages. We found that virus populations passaged on a single host cell line increased in fitness relative to the ancestral deletion mutant on their selected host, and viruses that were alternately passaged improved on both hosts. Surprisingly, whole genome sequencing revealed few changes in the 3'UTR of passaged populations. Rather, virus populations evolved improved fitness through mutations in protein coding regions that were associated with specific hosts.

Polyamines, which are small positively charge molecules present in all cells, play important roles in the replication of DNA and RNA viruses. Chikungunya virus (CHIKV) relies on polyamines for translation of the viral genome upon viral entry, and pharmacological depletion of polyamines limits viral replication. However, the potential development of antiviral resistance necessitates a better understanding of how polyamines function and can be targeted via compounds that alter polyamine levels. We have isolated CHIKV that is resistant to polyamine depletion and contains two mutations in the nonstructural protein 1 (nsP1)-coding region in combination with a mutation to the opal stop codon preceding nsP4. These mutations, in addition to promoting viral replication in polyamine-depleted cells, confer enhanced viral replication in vitro and in vivo The nsP1 mutations enhance membrane binding and methyltransferase activities, while the stop codon mutation allows increased downstream translation. These mutations, when combined, enhance viral fitness, but individual mutants are attenuated in mosquitoes. Together, our results suggest that CHIKV can evolve resistance to polyamine depletion and that pharmaceuticals targeting the polyamine biosynthetic pathway may be best used in combination with other established antivirals to mitigate the development of resistance.IMPORTANCE Chikungunya virus is a mosquito-borne virus that has infected millions worldwide. Its expansion into the Americas and rapid adaptation to new mosquito hosts present a serious threat to human health, which we can combat with the development of antiviral therapies as well as understanding how these viruses will mutate when exposed to antiviral therapies. Targeting polyamines, small positively charged molecules in the cell, may be a potential strategy against RNA viruses, including chikungunya virus. Here, we have described a virus that is resistant to polyamine depletion and has increased fitness in cells and in full organisms. Mutations in viral genome capping machinery, membrane binding activity, and a stop codon arise, and their altered activities enhance replication in the absence of polyamines. These results highlight strategies by which chikungunya virus can overcome polyamine depletion and emphasize continued research on developing improved antiviral therapies.

Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes.

Polyamines are small, positively charged molecules derived from ornithine and synthesized through an intricately regulated enzymatic pathway. Within cells, they are abundant and play several roles in diverse processes. We find that polyamines are required for the life cycle of the RNA viruses chikungunya virus (CHIKV) and Zika virus (ZIKV). Depletion of spermidine and spermine via type I interferon signaling-mediated induction of spermidine/spermine N1-acetyltransferase (SAT1), a key catabolic enzyme in the polyamine pathway, restricts CHIKV and ZIKV replication. Polyamine depletion restricts these viruses in vitro and in vivo, due to impairment of viral translation and RNA replication. The restriction is released by exogenous replenishment of polyamines, further supporting a role for these molecules in virus replication. Thus, SAT1 and, more broadly, polyamine depletion restrict viral replication and suggest promising avenues for antiviral therapies.