Chikungunya (CHIK) virus (CHIKV) is a reemerging arboviral pathogen that has recently caused explosive urban outbreaks involving millions of persons in Africa and Asia. The virus was first isolated from a human in Tanzania in 1953 during a major epidemic 
, and derives its name from a Makonde word meaning “that which bends up,” which describes the posture observed in afflicted persons. CHIKV typically causes a febrile illness and severe joint pain, which is clinically similar to dengue fever. These 2 viruses also share similar endemic distributions in the Eastern Hemisphere, resulting in many CHIKV cases being misdiagnosed when laboratory testing is not available 
. Large CHIK outbreaks were described during the 1950's and 60's in India and Southeast Asia 
. However, it was not until 2005 that CHIKV gained widespread public attention due to massive outbreaks on islands of the Indian Ocean 
and later in India 
and Southeast Asia 
. In total, several million persons have been affected 
. On the Island of Reunion alone, ca. 300,000 persons or one-third of the population was affected 
. Another factor driving the resurgence of interest in CHIK is the detection of occasional fatal cases, which were not documented before. Previously, individuals who became severely ill typically presented with hemorrhagic manifestations and occasionally shock 
. However, the recent outbreaks have been linked to thousands of deaths in Reunion and India due to neurologic disease 
CHIKV exists in two transmission cycles: an enzootic or sylvatic cycle and an endemic/epidemic urban cycle. The African sylvatic cycle likely involves several arboreal Aedes
mosquitoes as vectors and nonhuman primates as reservoir/amplifying hosts 
. African outbreaks occur from direct enzootic spillover or when CHIKV is introduced into an urban areas inhabited by the anthropophilic mosquito vector, Aedes aegypti
. More permanent endemic/epidemic transmission cycles were established when the virus was introduced into Asia ca. 1950, and into the Indian Ocean region, India and then Southeast Asia since 2005 
. A mutation in the E1 envelope glycoprotein gene that results in an A226V amino acid substitution dramatically increased the infectivity of some epidemic strains for an alternative urban vector, Ae. albopictus 
. The nearly ubiquitous distribution of Ae. aegypti
, and the expanding distribution of Ae. albopictus
into tropical and temperate regions of both hemispheres has raised concern that CHIKV may spread outside of its previous endemic region into the Western Hemisphere and Europe. The latter scenario was realized in 2007 during a small epidemic in Italy 
and during autochthonous transmission in southern France during 2010 (ProMED archive 20100926.3495).
CHIKV belongs to the family Togaviridae
, genus Alphavirus
, whose members are enveloped virions that contain a positive sense, single stranded, RNA genome of ~12 kb. The genome encodes 4 non-structural proteins (nsP1-4) and 3 major structural proteins (Capsid, E1, and E2 envelope glycoproteins)(). During replication, two distinct RNA's are produced: the genomic and subgenomic RNAs. A negative sense template RNA is also produced. The nonstructural polyprotein open reading frame (ORF) is translated via a cap-dependant mechanism from the genomic RNA, whereas the structural protein gene ORF is translated from the subgenomic RNA, also in a cap-dependent manner. The subgenomic RNA is transcribed late during infection from its promoter, which is found in the 3′ end of the nsP4 gene 
Genetic organization of wild-type CHIKV and the sequence of the subgenomic promoter.
There is no licensed vaccine or therapeutic CHIK, so outbreaks can only be controlled by preventing the exposure of people to infected mosquito vectors. Scientists at the Walter Reed Army Institute of Research produced an investigational vaccine called 181/clone 25 (hereafter called 181/25) during the 1980s. This live-attenuated strain was generated via serial plaque-to-plaque passages of a wild-type Thai CHIKV strain using MRC-5 cells 
. The virus is attenuated in both rodents and non-human primates and is highly immunogenic in humans. However, during phase II trials, strain 181/25 caused mild, transient arthralgia in 5 of 59 vaccinees 
. Also, strain 181/25 can be transmitted experimentally by the natural mosquito vector, Ae. aegypti 
To be effective in resource-limited nations that are endemic for CHIK as well as to combat an epidemic, an ideal CHIK vaccine would induce rapid and long-lived immunity after a single dose, have a low risk of reactogenicity and reversion to virulence, and be inexpensive. Vaccines against arboviral diseases should also have a low risk of transmission from vaccinated persons via mosquitoes in the event that viremia occurs, especially those used in non-endemic regions. Although replication-defective vaccine candidates have been described that emphasize safety 
, none has been shown to induce rapid or long-lived immunity after a single dose, and some may be expensive to produce. In contrast, live-attenuated vaccines like the yellow fever 17D vaccine 
have been spectacularly successful in preventing disease in developing tropical regions.
To generate a safer and more effective live-attenuated CHIK vaccine that meets the criteria outlined above, we previously produced and tested a series of chimeric alphaviruses containing either Venezuelan equine encephalitis virus (VEEV), eastern equine encephalitis or Sindbis virus non-structural protein genes along with the CHIKV structural protein genes 
. These vaccines produce robust neutralizing antibody (Ab) responses and provide complete protection against disease after CHIKV challenge. However, some residual ability to infect potential mosquito vectors remains, and attenuation is dependent on an intact murine interferon response (SCW, unpublished). To overcome these limitations, we developed a new attenuation strategy and conducted proof-of-principle studies with another alphavirus, VEEV vaccine strain TC-83. Both attenuation and elimination of mosquito infectability relied on the inactivation of the subgenomic promoter, and addition of a encephalomyocarditis virus (EMCV) internal ribosome entry sequence (IRES) to drive translation of the structural protein genes 
. Chimeric alphaviruses incorporating the IRES element have also been generated as vaccine candidates 
. The EMCV IRES also mediates inefficient translation in arthropod cells 
, rendering these mutants unable to infect mosquitoes. However, starting with the attenuated TC-83 strain, the IRES-based attenuation resulted in inadequate immunogenicity and the lack of a neutralizing Ab response.
Here, we implemented this IRES-based vaccine design for CHIKV using a cDNA clone generated from the wild-type La Reunion strain 
. Testing of this novel vaccine candidate in several murine models indicated that it is highly attenuated, even in the absence of an intact murine IFN response, is immunogenic and efficacious in preventing CHIK disease, and is unable to infect mosquitoes.