A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.

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.

Gain without pain: adaptation and increased virulence of Zika virus in vertebrate host without fitness cost in mosquito vector.

IMPORTANCE: Previously, we modeled direct transmission chains of Zika virus (ZIKV) by serially passaging ZIKV in mice and mosquitoes and found that direct mouse transmission chains selected for viruses with increased virulence in mice and the acquisition of non-synonymous amino acid substitutions. Here, we show that these same mouse-passaged viruses also maintain fitness and transmission capacity in mosquitoes. We used infectious clone-derived viruses to demonstrate that the substitution in nonstructural protein 4A contributes to increased virulence in mice.

Obesity fosters severe disease outcomes in a mouse model of coronavirus infection associated with transcriptomic abnormalities.

Obesity has been identified as an independent risk factor for severe outcomes in humans with coronavirus disease 2019 (COVID-19) and other infectious diseases. Here, we established a mouse model of COVID-19 using the murine betacoronavirus, mouse hepatitis virus 1 (MHV-1). C57BL/6 and C3H/HeJ mice exposed to MHV-1 developed mild and severe disease, respectively. Obese C57BL/6 mice developed clinical manifestations similar to those of lean controls. In contrast, all obese C3H/HeJ mice succumbed by 8 days postinfection, compared to a 50% mortality rate in lean controls. Notably, both lean and obese C3H/HeJ mice exposed to MHV-1 developed lung lesions consistent with severe human COVID-19, with marked evidence of diffuse alveolar damage (DAD). To identify early predictive biomarkers of worsened disease outcomes in obese C3H/HeJ mice, we sequenced RNA from whole blood 2 days postinfection and assessed changes in gene and pathway expression. Many pathways uniquely altered in obese C3H/HeJ mice postinfection aligned with those found in humans with severe COVID-19. Furthermore, we observed altered gene expression related to the unfolded protein response and lipid metabolism in infected obese mice compared to their lean counterparts, suggesting a role in the severity of disease outcomes. This study presents a novel model for studying COVID-19 and elucidating the mechanisms underlying severe disease outcomes in obese and other hosts.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

Defective viral genomes from chikungunya virus are broad-spectrum antivirals and prevent virus dissemination in mosquitoes.

Defective viral genomes (DVGs) are truncated and/or rearranged viral genomes produced during virus replication. Described in many RNA virus families, some of them have interfering activity on their parental virus and/or strong immunostimulatory potential, and are being considered in antiviral approaches. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes spp. that infected millions of humans in the last 15 years. Here, we describe the DVGs arising during CHIKV infection in vitro in mammalian and mosquito cells, and in vivo in experimentally infected Aedes aegypti mosquitoes. We combined experimental and computational approaches to select DVG candidates most likely to have inhibitory activity and showed that, indeed, they strongly interfere with CHIKV replication both in mammalian and mosquito cells. We further demonstrated that some DVGs present broad-spectrum activity, inhibiting several CHIKV strains and other alphaviruses. Finally, we showed that pre-treating Aedes aegypti with DVGs prevented viral dissemination in vivo.

Insulin reduces the transmission potential of chikungunya virus and activates the toll pathway in Aedes aegypti mosquitoes.

Chikungunya virus (CHIKV) is an alphavirus that has re-emerged globally over the last two decades and has the potential to become endemic in the United States due to the presence of competent mosquito vectors, Aedes aegypti and Aedes albopictus. CHIK disease is characterised by fever, rash, and joint pain, and causes chronic debilitating joint pain and swelling in >50% of infected individuals. Given the disease severity caused by CHIKV and the global presence of vectors to facilitate its spread, strategies to reduce viral transmission are desperately needed; however, the human biological factors driving CHIKV transmission are poorly understood. Towards that end, we have previously shown that mosquitoes fed on alphavirus-infected obese mice have reduced infection and transmission rates compared to those fed on infected lean mice despite similar viremia in lean and obese mice. One of the many host factors that increase in obese hosts is insulin, which was previously shown to impact the infection of mosquitoes by several flaviviruses. However, insulin's impact on alphavirus infection of live mosquitoes is unknown and whether insulin influences mosquito-borne virus transmission has not been tested. To test this, we exposed A. aegypti mosquitoes to bloodmeals with CHIKV in the presence or absence of physiologically relevant levels of insulin and found that insulin significantly lowered both infection and transmission rates. RNA sequencing analysis on mosquito midguts isolated at 1-day-post-infectious-bloodmeal (dpbm) showed enrichment in genes in the Toll immune pathway in the presence of insulin, which was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We then sought to determine if the Toll pathway plays a role in CHIKV infection of Ae. aegypti mosquitoes; therefore, we knocked down Myd88, a critical immune adaptor molecule for the Toll pathway, in live mosquitoes, and found increased CHIKV infection compared to the mock knockdown control group. Overall, these data demonstrate that insulin reduces CHIKV transmission by Ae. aegypti and activates the Toll pathway in mosquitoes, suggesting that conditions resulting in higher serum insulin concentrations may reduce alphavirus transmission. Finally, these studies suggest that strategies to activate insulin or Toll signalling in mosquitoes may be an effective control strategy against medically relevant alphaviruses.

Genome Number and Size Polymorphism in Zika Virus Infectious Units.

Zika virus (ZIKV; Flaviviridae, Flavivirus) is an arthropod-borne infection that can result in severe outcomes, particularly in fetuses infected in utero It has been assumed that infection by ZIKV, as well as other viruses, is largely initiated by individual virus particles binding to and entering a cell. However, recent studies have demonstrated that multiple virus particles are frequently delivered to a cell simultaneously and that this collective particle delivery enhances infection. ZIKV is maintained in nature between Aedes aegypti mosquitos and vertebrate hosts, including humans. Human infection is initiated through the injection of a relatively small initial inoculum comprised of a genetically complex virus population. Since most mutations decrease virus fitness, collective particle transmission could benefit ZIKV and other arthropod-borne diseases by facilitating the maintenance of genetic complexity and adaptability during infection or through other mechanisms. Therefore, we utilized a barcoded ZIKV to quantify the number of virus genomes that initiate a plaque. We found that individual plaques contain a mean of 10 infecting viral genomes (range, 1 to 212). Few plaques contained more than two dominant genomes. To determine whether multigenome infectious units consist of collectively transmitting virions, infectious units of ZIKV were then separated mechanically by centrifugation, and heavier fractions were found to contain more genomes per plaque-forming unit, with larger diameters. Finally, larger/heavier infectious units reformed after removal. These data suggest that ZIKV populations consist of a variety of infectious unit sizes, likely mostly made up of aggregates, and only rarely begin with a single virus genome.IMPORTANCE The arthropod-borne Zika virus (ZIKV) infects humans and can cause severe neurological sequelae, particularly in fetuses infected in utero How this virus has been able to spread across vast geological ranges and evolve in new host populations is not yet understood. This research demonstrates a novel mechanism of ZIKV transmission through multigenome aggregates, providing insight into ZIKV evolution, immunologic evasion, and better future therapeutic design. This study shows that ZIKV plaques result from collections of genomes rather than individual genomes, increasing the potential for interactions between ZIKV genotypes.

Rapid evolution of enhanced Zika virus virulence during direct vertebrate transmission chains.

Zika virus (ZIKV) has the unusual capacity to circumvent natural alternating mosquito-human transmission and be directly transmitted human-to-human via sexual and vertical routes. The impact of direct transmission on ZIKV evolution and adaptation to vertebrate hosts is unknown. Here we show that molecularly barcoded ZIKV rapidly adapted to a mammalian host during direct transmission chains in mice, coincident with the emergence of an amino acid substitution previously shown to enhance virulence. In contrast, little to no adaptation of ZIKV to mice was observed following chains of direct transmission in mosquitoes or alternating host transmission. Detailed genetic analyses revealed that ZIKV evolution in mice was generally more convergent and subjected to more relaxed purifying selection than in mosquitoes or alternate passages. These findings suggest that prevention of direct human transmission chains may be paramount to resist gains in ZIKV virulence.Importance We used experimental evolution to model chains of direct and indirect Zika virus (ZIKV) transmission by serially passaging a synthetic swarm of molecularly barcoded ZIKV within and between mosquitoes and mice. We observed that direct mouse transmission chains facilitated a rapid increase in ZIKV replication and enhanced virulence in mice. These findings demonstrate that ZIKV is capable of rapid adaptation to a vertebrate host and indicate that direct human-to-human transmission could pose a greater threat to public health than currently realized.

Host nutritional status affects alphavirus virulence, transmission, and evolution.

Malnourishment, specifically overweight/obesity and undernourishment, affects more than 2.5 billion people worldwide, with the number affected ever-increasing. Concurrently, emerging viral diseases, particularly those that are mosquito-borne, have spread dramatically in the past several decades, culminating in outbreaks of several viruses worldwide. Both forms of malnourishment are known to lead to an aberrant immune response, which can worsen disease outcomes and reduce vaccination efficacy for viral pathogens such as influenza and measles. Given the increasing rates of malnutrition and spread of arthropod-borne viruses (arboviruses), there is an urgent need to understand the role of host nutrition on the infection, virulence, and transmission of these viruses. To address this gap in knowledge, we infected lean, obese, and undernourished mice with arthritogenic arboviruses from the genus Alphavirus and assessed morbidity, virus replication, transmission, and evolution. Obesity and undernourishment did not consistently influence virus replication in the blood of infected animals except for reductions in virus in obese mice late in infection. However, morbidity was increased in obese mice under all conditions. Using Mayaro virus (MAYV) as a model arthritogenic alphavirus, we determined that both obese and undernourished mice transmit virus less efficiently to mosquitoes than control (lean) mice. In addition, viral genetic diversity and replicative fitness were reduced in virus isolated from obese compared to lean controls. Taken together, nutrition appears to alter the course of alphavirus infection and should be considered as a critical environmental factor during outbreaks.

Nutritional status impacts dengue virus infection in mice.

BACKGROUND: Dengue virus (DENV) is estimated to infect 390 million people annually. However, few host factors that alter disease severity are known. Malnutrition, defined as both over- and undernutrition, is a growing problem worldwide and has long been linked to dengue disease severity by epidemiological and anecdotal observations. Accordingly, we sought to establish a mouse model to assess the impact of nutritional status on DENV disease severity. RESULTS: Using transiently immunocompromised mice, we established a model of mild dengue disease with measurable viremia. We then applied it to study the effects of healthy weight, obese, and low-protein diets representing normal, over-, and undernutrition, respectively. Upon infection with DENV serotype 2, obese mice experienced more severe morbidity in the form of weight loss and thrombocytopenia compared to healthy weight groups. Additionally, obesity altered cytokine expression following DENV infection. Although low protein-fed mice did not lose significant weight after DENV2 infection, they also experienced a reduction in platelets as well as increased spleen pathology and viral titers. CONCLUSIONS: Our results indicate that obese or undernourished mice incur greater disease severity after DENV infection. These studies establish a role for nutritional status in DENV disease severity.

Chikungunya Virus Vaccine Candidates with Decreased Mutational Robustness Are Attenuated In Vivo and Have Compromised Transmissibility.

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.

Molecularly barcoded Zika virus libraries to probe in vivo evolutionary dynamics.

Defining the complex dynamics of Zika virus (ZIKV) infection in pregnancy and during transmission between vertebrate hosts and mosquito vectors is critical for a thorough understanding of viral transmission, pathogenesis, immune evasion, and potential reservoir establishment. Within-host viral diversity in ZIKV infection is low, which makes it difficult to evaluate infection dynamics. To overcome this biological hurdle, we constructed a molecularly barcoded ZIKV. This virus stock consists of a "synthetic swarm" whose members are genetically identical except for a run of eight consecutive degenerate codons, which creates approximately 64,000 theoretical nucleotide combinations that all encode the same amino acids. Deep sequencing this region of the ZIKV genome enables counting of individual barcodes to quantify the number and relative proportions of viral lineages present within a host. Here we used these molecularly barcoded ZIKV variants to study the dynamics of ZIKV infection in pregnant and non-pregnant macaques as well as during mosquito infection/transmission. The barcoded virus had no discernible fitness defects in vivo, and the proportions of individual barcoded virus templates remained stable throughout the duration of acute plasma viremia. ZIKV RNA also was detected in maternal plasma from a pregnant animal infected with barcoded virus for 67 days. The complexity of the virus population declined precipitously 8 days following infection of the dam, consistent with the timing of typical resolution of ZIKV in non-pregnant macaques and remained low for the subsequent duration of viremia. Our approach showed that synthetic swarm viruses can be used to probe the composition of ZIKV populations over time in vivo to understand vertical transmission, persistent reservoirs, bottlenecks, and evolutionary dynamics.

Chikungunya Virus Overcomes Polyamine Depletion by Mutation of nsP1 and the Opal Stop Codon To Confer Enhanced Replication and Fitness.

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.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE: ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

Dissecting the Role of E2 Protein Domains in Alphavirus Pathogenicity.

UNLABELLED: Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE: Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.

Preexisting inter-serotype immunity drives antigenic evolution of dengue virus serotype 2.

Dengue virus (DENV) infects roughly 400 million people annually, causing febrile and hemorrhagic disease. While preexisting inter-serotype immunity (PISI) provides transient protection, it may drive severe disease over time. PISI's impact on virus evolution, however, is less understood. Retrospective epidemiological analyses suggest that PISI may drive DENV evolution. Using in vitro directed evolution, we explored how DENV2 evolves in the presence of DENV3/4 convalescent serum. Two post-passaging mutations (E-I6M and E-N203D) were then studied for fitness effects in mammalian and insect hosts and immune escape. E-I6M resisted neutralization, altered fitness in mammalian cell culture models, and had no effect in Aedes albopictus mosquitoes. E-N203D showed no change in neutralization sensitivity, reduced fitness in a DENV-naïve epithelial model, and no effects in the other models. These results align with surveillance data, where E-I6M emerged and disappeared, while E-203D and E-203 N cocirculate, thus suggesting that PISI can drive DENV evolution.

Forgotten Natural Products: Semisynthetic Development of Blasticidin S As an Antibiotic Lead.

Forgotten natural products offer value as antimicrobial scaffolds, providing diverse mechanisms of action that complement existing antibiotic classes. This study focuses on the derivatization of the cytotoxin blasticidin S, seeking to leverage its unique ribosome inhibition mechanism. Despite its complex zwitterionic properties, a selective protection and amidation strategy enabled the creation of a library of blasticidin S derivatives including the natural product P10. The amides exhibited significantly increased activity against Gram-positive bacteria and enhanced specificity for pathogenic bacteria over human cells. Molecular docking and computational property analysis suggested variable binding poses and indicated a potential correlation between cLogP values and activity. This work demonstrates how densely functionalized forgotten antimicrobials can be straightforwardly modified, enabling the further development of blasticidin S derivatives as lead compounds for a novel class of antibiotics.

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.