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.

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. Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged since 2004 to cause millions of cases of severe and often persistent arthralgia. Because no licensed vaccine exists to prevent this disease, we utilized an attenuation approach to produce a live CHIKV vaccine candidate that elicits a robust, protective immune response yet causes no detectable disease in mice. It is also incapable of infecting mosquito vectors, an important safety feature for a live virus vaccine that may be used in nonendemic locations to immunize travelers or laboratory personnel. This vaccine approach, which exploits the attenuating effect of altering the expression of the alphavirus structural proteins with a picornavirus IRES, may be broadly applicable to other alphaviruses that cause important febrile diseases as well as encephalitis.

Chikungunya virus (CHIKV) and o’nyong nyong virus (ONNV) are mosquito-borne alphaviruses endemic in East Africa that cause acute febrile illness and arthritis. The objectives of this study were to measure the seroprevalence of CHIKV and ONNV in coastal Kenya and link it to demographics and other risk factors. Demographic and exposure questionnaires were administered to 1,848 participants recruited from two village clusters (Milalani-Nganja and Vuga) in 2009. Sera were tested for alphavirus exposure using standardized CHIKV IgG ELISA protocols and confirmed with plaque reduction neutralization tests (PRNT). Logistic regression models were used to determine the variables associated with seropositivity. Weighted K test for global clustering of houses with alphavirus positive participants was performed for distance ranges of 50–1,000 meters, and G* statistic and kernel density mapping were used to identify locations of higher seroprevalence. 486 (26%) participants were seropositive by IgG ELISA. Of 443 PRNT confirmed positives, 25 samples (6%) were CHIKV+, 250 samples (56%) were ONNV+, and 168 samples (38%) had high titers for both. Age was significantly associated with seropositivity (OR 1.01 per year, 95% C.I. 1.00–1.01); however, younger adults were more likely to be seropositive than older adults. Males were less likely to be seropositive (p<0.05; OR 0.79, 95% C.I. 0.64–0.97). Adults who owned a bicycle (p<0.05; OR 1.37, 95% C.I. 1.00–1.85) or motor vehicle (p<0.05; OR 4.64, 95% C.I. 1.19–18.05) were more likely to be seropositive. Spatial analysis demonstrated hotspots of transmission within each village and clustering among local households in Milalani-Nganja, peaking at the 200–500m range. Alphavirus exposure, particularly ONNV exposure, is common in coastal Kenya with ongoing interepidemic transmission of both ONNV and CHIKV. Women and adults were more likely to be seropositive. Household location may be a defining factor for the ecology of alphaviral transmission in this region. Alphaviruses, such as chikungunya and o’nyong nyong viruses, are likely important causes of human disease in endemic regions, but are often misdiagnosed as malaria in the acute care setting. Our objective was to uncover the burden of alphavirus exposure in our study region, rural coastal Kenya. Of 1848 participants tested, 26% were seropositive by screening ELISA, demonstrating intense transmission to humans in this area. Surprisingly, confirmatory PRNT testing revealed that the majority of alphavirus exposures were due to o’nyong nyong virus, rather than chikungunya virus. Both CHIKV and ONNV antibodies were confirmed in young children, demonstrating undocumented and ongoing transmission in this region. Of the examined risk factors, older age and female gender were associated with alphavirus seropositivity.

In mid-2015, Salvador, Brazil, reported an outbreak of Guillain-Barré syndrome (GBS), coinciding with the introduction and spread of Zika virus (ZIKV). We found that GBS incidence during April–July 2015 among those ≥12 years of age was 5.6 cases/100,000 population/year and increased markedly with increasing age to 14.7 among those ≥60 years of age. We conducted interviews with 41 case-patients and 85 neighborhood controls and found no differences in demographics or exposures prior to GBS-symptom onset. A higher proportion of case-patients (83%) compared to controls (21%) reported an antecedent illness (OR 18.1, CI 6.9–47.5), most commonly characterized by rash, headache, fever, and myalgias, within a median of 8 days prior to GBS onset. Our investigation confirmed an outbreak of GBS, particularly in older adults, that was strongly associated with Zika-like illness and geo-temporally associated with ZIKV transmission, suggesting that ZIKV may result in severe neurologic complications. Shortly following the introduction of Zika virus (ZIKV), a type of flavivirus transmitted by mosquitoes, into Brazil in early 2015, the Brazil Ministry of Health began receiving increased reports of a paralyzing condition known as Guillain-Barré syndrome (GBS). The areas with the greatest number of GBS cases appeared to correlate geographically and temporally with the areas reporting the highest rate of ZIKV infections. This association had been previously observed during a ZIKV outbreak in French Polynesia, however, this had not been systematically examined in a case-control investigation for the ZIKV outbreak in South America. In this investigation, the authors found that the occurrence of GBS in the affected population was nearly four times higher than would be expected, and the risk for GBS was particularly elevated among older adults. GBS was associated with ZIKV-like symptoms and with a combination of ZIKV-like symptoms plus laboratory evidence of a recent flavivirus infection. Taken together, these findings provide strong support for and greater understanding of the link between ZIKV and GBS.