Erin M. Borland

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.

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.

Probing the attenuation and protective efficacy of a candidate chikungunya virus vaccine in mice with compromised interferon (IFN) signaling.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes explosive outbreaks of febrile illness associated with rash, and painful arthralgia. The CHIK vaccine strain 181/clone25 (181/25) developed by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) was shown to be well-tolerated and highly immunogenic in phase I and II clinical trials although it induced transient arthralgia in some healthy adult volunteers. In an attempt to better understand the host factors that are involved in the attenuating phenotype of CHIK 181/25 vaccine virus we conducted studies in interferon (IFN)-compromised mice and also evaluated its immunogenic potential and protective capacity. Infection of AG129 mice (defective in IFN-α/β and IFN-γ receptor signaling) with CHIK 181/25 resulted in rapid mortality within 3-4 days. In contrast, all infected A129 mice (defective in IFN-α/β receptor signaling) survived with temporary morbidity characterized by ruffled appearance and body weight loss. A129 heterozygote mice that retain partial IFN-α/β receptor signaling activity remained healthy. Infection of A129 mice with CHIK 181/25 induced significant levels of IFN-γ and IL-12 while the inflammatory cytokines, TNFα and IL-6 remained low. A single administration of the CHIK 181/25 vaccine provided both short-term and long-term protection (38 days and 247 days post-prime, respectively) against challenge with wt CHIKV-La Reunion (CHIKV-LR). This protection was at least partially mediated by antibodies since passively transferred immune serum protected both A129 and AG129 mice from wt CHIKV-LR and 181/25 virus challenge. Overall, these data highlight the importance of IFNs in controlling CHIK 181/25 vaccine and demonstrate the ability of this vaccine to elicit neutralizing antibody responses that confer short-and long-term protection against wt CHIKV-LR challenge.

High Rates of O’Nyong Nyong and Chikungunya Virus Transmission in Coastal Kenya

Background 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. Methodology 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. Principal Findings 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. Conclusions/Significance 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.

PREVALENCE OF ANTIBODIES TO ALPHAVIRUSES AND FLAVIVIRUSES IN FREE-RANGING GAME ANIMALS AND NONHUMAN PRIMATES IN THE GREATER CONGO BASIN

Vector-borne and zoonotic pathogens have comprised a significant proportion of the emerging infectious diseases in humans in recent decades. The role of many wildlife species as reservoirs for arthropod-borne viral pathogens is poorly understood. We investigated the exposure history of various African wildlife species from the Congo basin to mosquito-borne flaviviruses and alphaviruses by testing archived serum samples. Sera from 24 African forest buffalo (Syncerus caffer nanus), 34 African elephants (Loxodonta africana), 40 duikers (Cephalophus and Philantomba spp.), 25 mandrills (Mandrillus sphinx), 32 mountain gorillas (Gorilla beringei beringei), five Grauer’s gorillas (Gorilla beringei graueri), two L’Hoest’s monkeys (Cercopithecus lhoesti), two golden monkeys (Cercopithecus kandti), and three chimpanzees (Pan troglodytes) sampled between 1991 and 2009 were tested for antibodies against chikungunya virus (CHIKV), o’nyong-nyong virus (ONNV), West Nile virus (WNV), dengue 2 virus (DENV-2), and yellow fever virus (YFV) by plaque reduction neutralization test. Specific neutralizing antibodies against ONNV were found in African forest buffalo in the Democratic Republic of the Congo (DRC) and Gabon, duikers in the DRC, and mandrills in Gabon, providing novel evidence of enzootic circulation of ONNV in these countries. African forest buffalo in the DRC and Gabon also demonstrated evidence of exposure to CHIKV, WNV, and DENV-2, while mandrills in Gabon were antibody positive for CHIKV, DENV-2, WNV, and YFV. All of the elephants tested had a strong neutralizing antibody response to WNV. We also document results from a survey of gorillas for arboviruses, of which 4/32 (13%) had antibody to an alphavirus or flavivirus. Overall, our results demonstrate a high prevalence of neutralizing antibodies against multiple arboviruses in wildlife in equatorial Africa.

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.

Molecular Determinants of Mouse Neurovirulence and Mosquito Infection for Western Equine Encephalitis Virus

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90–100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.

Sunguru virus: a novel virus in the family Rhabdoviridae isolated from a chicken in north-western Uganda

Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda, in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11056 nt in length and contains the five core rhabdovirus genes plus an additional C gene (within the ORF of a phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells with SUNV resulted in significant viral growth, with a peak titre of 7.8 log10 p.f.u. ml(-1) observed in baby hamster kidney (BHK) cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes having a 47.4% infection rate in the body but no dissemination of the virus to the salivary glands; this suggests that this novel virus is not arthropod borne as some other members of the family Rhabdoviridae.

Increased rates of Guillain-Barré syndrome associated with Zika virus outbreak in the Salvador metropolitan area, Brazil

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.

Arboviruses Isolated From Mosquitoes Collected in Uganda, 2008–2012

A large number of arthropod-borne viruses are endemic to East Africa. As a part of the process of undertaking a systematic characterization of the mosquito fauna of Uganda, we examined mosquitoes collected from 2008 through early 2012 for known and novel viruses. In all, 8,288 mosquito pools containing 157,554 mosquitoes were tested. Twenty-nine isolations of 11 different viruses were made from mosquitoes of nine distinct species and from pools identified only to genus Culex. Identified viruses were from family Togaviridae, alphaviruses Sindbis and Babanki viruses; family Rhabdoviridae, hapaviruses Mossuril and Kamese viruses; family Flaviviridae, flaviviruses West Nile and Usutu viruses; family Phenuiviridae, phlebovirus Arumowot virus; and family Peribunyaviridae, orthobunyaviruses Witwatersrand, Pongola, and Germiston viruses. In addition, a novel orthobunyavirus, provisionally named Mburo virus, was isolated from Coquillettidia metallica (Theobald). This is the first report of Babanki, Arumowot, and Mossuril virus isolation from Uganda.