Konstantin A. Tsetsarkin

A single mutation in chikungunya virus affects vector specificity and epidemic potential.

Chikungunya virus (CHIKV) is an emerging arbovirus associated with several recent large-scale epidemics. The 2005–2006 epidemic on Reunion island that resulted in approximately 266,000 human cases was associated with a strain of CHIKV with a mutation in the envelope protein gene (E1-A226V). To test the hypothesis that this mutation in the epidemic CHIKV (strain LR2006 OPY1) might influence fitness for different vector species, viral infectivity, dissemination, and transmission of CHIKV were compared in Aedes albopictus, the species implicated in the epidemic, and the recognized vector Ae. aegypti. Using viral infectious clones of the Reunion strain and a West African strain of CHIKV, into which either the E1–226 A or V mutation was engineered, we demonstrated that the E1-A226V mutation was directly responsible for a significant increase in CHIKV infectivity for Ae. albopictus, and led to more efficient viral dissemination into mosquito secondary organs and transmission to suckling mice. This mutation caused a marginal decrease in CHIKV Ae. aegypti midgut infectivity, had no effect on viral dissemination, and was associated with a slight increase in transmission by Ae. aegypti to suckling mice in competition experiments. The effect of the E1-A226V mutation on cholesterol dependence of CHIKV was also analyzed, revealing an association between cholesterol dependence and increased fitness of CHIKV in Ae. albopictus. Our observation that a single amino acid substitution can influence vector specificity provides a plausible explanation of how this mutant virus caused an epidemic in a region lacking the typical vector. This has important implications with respect to how viruses may establish a transmission cycle when introduced into a new area. Due to the widespread distribution of Ae. albopictus, this mutation increases the potential for CHIKV to permanently extend its range into Europe and the Americas.

Genome-scale phylogenetic analyses of chikungunya virus reveal independent emergences of recent epidemics and various evolutionary rates.

ABSTRACT Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.

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.

Sequential adaptive mutations enhance efficient vector switching by Chikungunya virus and its epidemic emergence.

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics.

Epistatic Roles of E2 Glycoprotein Mutations in Adaption of Chikungunya Virus to Aedes Albopictus and Ae. Aegypti Mosquitoes

Between 2005 and 2007 Chikungunya virus (CHIKV) caused its largest outbreak/epidemic in documented history. An unusual feature of this epidemic is the involvement of Ae. albopictus as a principal vector. Previously we have demonstrated that a single mutation E1-A226V significantly changed the ability of the virus to infect and be transmitted by this vector when expressed in the background of well characterized CHIKV strains LR2006 OPY1 and 37997. However, in the current study we demonstrate that introduction of the E1-A226V mutation into the background of an infectious clone derived from the Ag41855 strain (isolated in Uganda in 1982) does not significantly increase infectivity for Ae. albopictus. In order to elucidate the genetic determinants that affect CHIKV sensitivity to the E1-A226V mutation in Ae. albopictus, the genomes of the LR2006 OPY1 and Ag41855 strains were used for construction of chimeric viruses and viruses with a specific combination of point mutations at selected positions. Based upon the midgut infection rates of the derived viruses in Ae. albopictus and Ae. aegypti mosquitoes, a critical role of the mutations at positions E2-60 and E2-211 on vector infection was revealed. The E2-G60D mutation was an important determinant of CHIKV infectivity for both Ae. albopictus and Ae. aegypti, but only moderately modulated the effect of the E1-A226V mutation in Ae. albopictus. However, the effect of the E2-I211T mutation with respect to mosquito infections was much more specific, strongly modifying the effect of the E1-A226V mutation in Ae. albopictus. In contrast, CHIKV infectivity for Ae. aegypti was not influenced by the E2-1211T mutation. The occurrence of the E2-60G and E2-211I residues among CHIKV isolates was analyzed, revealing a high prevalence of E2-211I among strains belonging to the Eastern/Central/South African (ECSA) clade. This suggests that the E2-211I might be important for adaptation of CHIKV to some particular conditions prevalent in areas occupied by ECSA stains. These newly described determinants of CHIKV mosquito infectivity for Ae. albopictus and Ae. aegypti are of particular importance for studies aimed at the investigation of the detailed mechanisms of CHIKV adaptations to its vector species.

Novel Chikungunya Vaccine Candidate with an IRES-Based Attenuation and Host Range Alteration Mechanism

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that has recently caused devastating urban epidemics of severe and sometimes chronic arthralgia. As with most other mosquito-borne viral diseases, control relies on reducing mosquito populations and their contact with people, which has been ineffective in most locations. Therefore, vaccines remain the best strategy to prevent most vector-borne diseases. Ideally, vaccines for diseases of resource-limited countries should combine low cost and single dose efficacy, yet induce rapid and long-lived immunity with negligible risk of serious adverse reactions. To develop such a vaccine to protect against chikungunya fever, we employed a rational attenuation mechanism that also prevents the infection of mosquito vectors. The internal ribosome entry site (IRES) from encephalomyocarditis virus replaced the subgenomic promoter in a cDNA CHIKV clone, thus altering the levels and host-specific mechanism of structural protein gene expression. Testing in both normal outbred and interferon response-defective mice indicated that the new vaccine candidate is highly attenuated, immunogenic and efficacious after a single dose. Furthermore, it is incapable of replicating in mosquito cells or infecting mosquitoes in vivo. This IRES-based attenuation platform technology may be useful for the predictable attenuation of any alphavirus.

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.

Chikungunya virus: Evolution and genetic determinants of emergence

Chikungunya virus (CHIKV) causes a severe and often persistent arthralgic disease that is occasionally fatal. A mosquito-borne virus, CHIKV exists in enzootic, non-human primate cycles in Africa, but occasionally emerges into urban, human cycles to cause major epidemics. Between 1920 and 1950, and again in 2005, CHIKV emerged into India and Southeast Asia, where major urban epidemics ensued. Unlike the early introduction, the 2005 emergence was accompanied by an adaptive mutation that allowed CHIKV to exploit a new epidemic vector, Aedes albopictus, via an A226V substitution in the E1 envelope glycoprotein. However, recent reverse genetic studies indicate that lineage-specific epistatic restrictions can prevent this from exerting its phenotype on mosquito infectivity. Thus, the A. albopictus-adaptive A226V substitution that is facilitating the dramatic geographic spread CHIKV epidemics, was prevented for decades or longer from being selected in most African enzootic strains as well as in the older endemic Asian lineage.

Factors shaping the adaptive landscape for arboviruses: implications for the emergence of disease

Many examples of the emergence or re-emergence of infectious diseases involve the adaptation of zoonotic viruses to new amplification hosts or to humans themselves. These include several instances of simple mutational adaptations, often to hosts closely related to the natural reservoirs. However, based on theoretical grounds, arthropod-borne viruses, or arboviruses, may face several challenges for adaptation to new hosts. Here, we review recent findings regarding adaptive evolution of arboviruses and its impact on disease emergence. We focus on the zoonotic alphaviruses Venezuelan equine encephalitis and chikungunya viruses, which have undergone adaptive evolution that mediated recent outbreaks of disease, as well as the flaviviruses dengue and West Nile viruses, which have emerged via less dramatic adaptive mechanisms.

A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

ABSTRACT An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. IMPORTANCE The availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell-adapted version of it, which will be used for virus-host interaction studies and vaccine development. The availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell-adapted version of it, which will be used for virus-host interaction studies and vaccine development.