Grace Leal

Chikungunya virus emergence is constrained in Asia by lineage-specific adaptive landscapes

Adaptation of RNA viruses to a new host or vector species often results in emergence of new viral lineages. However, lineage-specific restrictions on the adaptive processes remain largely unexplored. Recently, a Chikungunya virus (CHIKV) lineage of African origin emerged to cause major epidemics of severe, persistent, debilitating arthralgia in Africa and Asia. Surprisingly, this new lineage is actively replacing endemic strains in Southeast Asia that have been circulating there for 60 y. This replacement process is associated with adaptation of the invasive CHIKV strains to an atypical vector, the Aedes albopictus mosquito that is ubiquitously distributed in the region. Here we demonstrate that lineage-specific epistatic interactions between substitutions at amino acid positions 226 and 98 of the E1 envelope glycoprotein, the latter of which likely resulted from a founder effect, have for 60 y restricted the ability of endemic Asian CHIKV strains to adapt to this new vector. This adaptive constraint appears to be allowing invasion of the unoccupied vector niche by Ae. albopictus-adapted African strains. These results underscore how different adaptive landscapes occupied by closely related viral genotypes can profoundly affect the outcome of viral evolution and disease emergence.

Multi-peaked adaptive landscape for chikungunya virus evolution predicts continued fitness optimization in Aedes albopictus mosquitoes

Host species-specific fitness landscapes largely determine the outcome of host switching during pathogen emergence. Using chikungunya virus (CHIKV) to study adaptation to a mosquito vector, we evaluated mutations associated with recently evolved sub-lineages. Multiple Aedes albopictus-adaptive fitness peaks became available after CHIKV acquired an initial adaptive (E1-A226V) substitution, permitting rapid lineage diversification observed in nature. All second-step mutations involved replacements by glutamine or glutamic acid of E2 glycoprotein amino acids in the acid-sensitive region, providing a framework to anticipate additional A. albopictus-adaptive mutations. The combination of second-step adaptive mutations into a single, ‘super-adaptive’ fitness peak also predicted the future emergence of CHIKV strains with even greater transmission efficiency in some current regions of endemic circulation, followed by their likely global spread. Supplementary information The online version of this article (doi:10.1038/ncomms5084) contains supplementary material, which is available to authorized users.

Attenuation of Chikungunya Virus Vaccine Strain 181/Clone 25 Is Determined by Two Amino Acid Substitutions in the E2 Envelope Glycoprotein

ABSTRACT Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

Variation in aedes aegypti mosquito competence for zika virus transmission

To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquitoes appear to be competent vectors of Zika virus in several regions of the Americas.

A chikungunya fever vaccine utilizing an insect-specific virus platform

Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

Venezuelan equine encephalitis virus activity in the Gulf Coast region of Mexico, 2003-2010.

Venezuelan equine encephalitis virus (VEEV) has been the causative agent for sporadic epidemics and equine epizootics throughout the Americas since the 1930s. In 1969, an outbreak of Venezuelan equine encephalitis (VEE) spread rapidly from Guatemala and through the Gulf Coast region of Mexico, reaching Texas in 1971. Since this outbreak, there have been very few studies to determine the northward extent of endemic VEEV in this region. This study reports the findings of serologic surveillance in the Gulf Coast region of Mexico from 2003–2010. Phylogenetic analysis was also performed on viral isolates from this region to determine whether there have been substantial genetic changes in VEEV since the 1960s. Based on the findings of this study, the Gulf Coast lineage of subtype IE VEEV continues to actively circulate in this region of Mexico and appears to be responsible for infection of humans and animals throughout this region, including the northern State of Tamaulipas, which borders Texas.

A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal aerosol challenge of eastern equine encephalitis virus.

Eastern equine encephalitis virus (EEEV) is a mosquito-borne alphavirus that causes sporadic, often fatal disease outbreaks in humans and equids, and is also a biological threat agent. Two chimeric vaccine candidates were constructed using a cDNA clone with a Sindbis virus (SINV) backbone and structural protein genes from either a North (SIN/NAEEEV) or South American (SIN/SAEEEV) strain of EEEV. The vaccine candidates were tested in a nonhuman primate (NHP) model of eastern equine encephalitis (EEE). Cynomolgus macaques were either sham-vaccinated, or vaccinated with a single dose of either SIN/NAEEEV or SIN/SAEEEV. After vaccination, animals were challenged by aerosol with a virulent North American strain of EEEV (NA EEEV). The SIN/NAEEEV vaccine provided significant protection, and most vaccinated animals survived EEEV challenge (82%) with little evidence of disease, whereas most SIN/SAEEEV-vaccinated (83%) and control (100%) animals died. Protected animals exhibited minimal changes in temperature and cardiovascular rhythm, whereas unprotected animals showed profound hyperthermia and changes in heart rate postexposure. Acute inflammation and neuronal necrosis were consistent with EEEV-induced encephalitis in unprotected animals, whereas no encephalitis-related histopathologic changes were observed in the SIN/NAEEEV-vaccinated animals. These results demonstrate that the chimeric SIN/NAEEEV vaccine candidate protects against an aerosol EEEV exposure.

Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States

Zika virus has recently spread throughout the Americas. Although Aedes aegypti mosquitoes are considered the primary vector, Culex quinquefasciatus and mosquitoes of other species may also be vectors. We tested Cx. quinquefasciatus and Ae. taeniorhynchus mosquitoes from the US Gulf Coast; both were refractory to infection and incapable of transmission.

A vaccine candidate for eastern equine encephalitis virus based on IRES-mediated attenuation

To develop an effective vaccine against eastern equine encephalitis (EEE), we engineered a recombinant EEE virus (EEEV) that was attenuated and capable of replicating only in vertebrate cells, an important safety feature for live vaccines against mosquito-borne viruses. The subgenomic promoter was inactivated with 13 synonymous mutations and expression of the EEEV structural proteins was placed under the control of an internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV). We tested this vaccine candidate for virulence, viremia and efficacy in the murine model. A single subcutaneous immunization with 10(4) infectious units protected 100% of mice against intraperitoneal challenge with a highly virulent North American EEEV strain. None of the mice developed any signs of disease or viremia after immunization or following challenge. Our findings suggest that the IRES-based attenuation approach can be used to develop a safe and effective vaccine against EEE and other alphaviral diseases.

Differential vector competency of aedes albopictus populations from the Americas for Zika Virus

To evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain.