[Show abstract][Hide abstract] ABSTRACT: Many viral pathogens cycle between humans and insects. These viruses must have evolved strategies for rapid adaptation to different host environments. However, the mechanistic basis for the adaptation process remains poorly understood. To study the mosquito-human adaptation cycle, we examined changes in RNA structures of the dengue virus genome during host adaptation. Deep sequencing and RNA structure analysis, together with fitness evaluation, revealed a process of host specialization of RNA elements of the viral 3'UTR. Adaptation to mosquito or mammalian cells involved selection of different viral populations harvesting mutations in a single stem-loop structure. The host specialization of the identified RNA structure resulted in a significant viral fitness cost in the non-specialized host, posing a constraint during host switching. Sequence conservation analysis indicated that the identified host adaptable stem loop structure is duplicated in dengue and other mosquito-borne viruses. Interestingly, functional studies using recombinant viruses with single or double stem loops revealed that duplication of the RNA structure allows the virus to accommodate mutations beneficial in one host and deleterious in the other. Our findings reveal new concepts in adaptation of RNA viruses, in which host specialization of RNA structures results in high fitness in the adapted host, while RNA duplication confers robustness during host switching.
[Show abstract][Hide abstract] ABSTRACT: The principal mosquito innate immune response to virus infections, RNA interference (RNAi), differs substantially from the immune response to bacterial and fungal infections. The exo-siRNA pathway constitutes the major anti-arboviral RNAi response and its essential genetic components have been identified. Recent research has also implicated the Piwi-interacting RNA pathway in mosquito anti-arboviral immunity, but Piwi gene-family components involved are not well-defined. Arboviruses must evade or suppress RNAi without causing pathogenesis in the vector to maintain their transmission cycle, but little is known about mechanisms of arbovirus modulation of RNAi. Genetic manipulation of mosquitoes to enhance their RNAi response can limit arbovirus infection and replication and could be used in novel strategies for interruption of arbovirus transmission and greatly reduce disease.
[Show abstract][Hide abstract] ABSTRACT: Alphaviruses can be serious, sometimes lethal human pathogens that belong to the family Togaviridae. Structures of human Venezuelan equine encephalitis virus (VEEV), an alphavirus, in complex with two strongly neutralizing antibody Fab fragments (F5 and 3B4C-4) have been determined using a combination of cryo-electron microscopy (cryo-EM) and homology modeling. Here we characterize these monoclonal antibody Fab fragments known to abrogate VEEV infectivity by binding to the E2 (envelope) surface glycoprotein. Both these antibody Fab fragments cross-link the surface E2 glycoproteins and, therefore, probably inhibit infectivity by blocking the conformational changes that are required for making the virus fusogenic. The F5 Fab fragment cross-links E2 proteins within one trimeric spike, whereas the 3B4C-4 Fab fragment cross-links E2 proteins from neighboring spikes. Furthermore, F5 probably blocks the receptor-binding site, whereas 3B4C-4 sterically hinders the exposure of the fusion loop at the end of the E2 B-domain.
[Show abstract][Hide abstract] ABSTRACT: Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE.
Virology 05/2014; s 456–457:238–246. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Yellow fever virus (YFV) causes significant human disease and mortality in tropical regions of South and Central America and Africa, despite the availability of an effective vaccine. No specific therapy for YF is available. We previously showed that the humanized monoclonal antibody (MAb) 2C9-cIgG provided prophylactic and therapeutic protection from mortality in interferon receptor-deficient strain AG129 mice challenged with YF 17D-204 vaccine. In this study we tested the prophylactic and therapeutic efficacy of this MAb against virulent YFV infection in an immunocompetent hamster model. Intraperitoneal (ip) administration of a single dose of MAb 2C9-cIgG 24 h prior to YFV challenge resulted in significantly improved survival rates in animals treated with 380 or 38 μg of MAb compared to untreated animals. Treatment with the higher dose also resulted in significantly improved weight gain and reductions in serum alanine aminotransferase (ALT) and virus titers in serum and liver. Prophylactic treatment with 2C9-cIgG 24 h prior to virus challenge prevented the development of a virus-neutralizing antibody (vnAb) response in hamsters. Administration of a single ip dose of 380 μg of 2C9-cIgG as late as 72 h post-YFV challenge also resulted in significant improvement in survival rates. Hamsters treated at 4 to 72 h post-virus challenge developed a robust vnAb response. Enhanced survival and improvement of various disease parameters in the hamster model when MAb 2C9-cIgG is administered up to 3 days after virus challenge demonstrate the clinical potential of specific antibody therapy for YF.
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV) is the most common cause of viral encephalitis in Asia, and it is increasingly a global public health concern due to its recent geographic expansion. While commercial vaccines are available and used in some endemic countries, JEV continues to be a public health problem, with 50,000 cases reported annually. Research with virulent JEV in mouse models to develop new methods of prevention and treatment is restricted to BSL-3 containment facilities, confining these studies to investigators with access to these facilities. We have developed an adult small animal peripheral challenge model using interferon-deficient AG129 mice and the JEV live-attenuated vaccine SA14-14-2, thus requiring only BSL-2 containment. A low dose of virus (10PFU/0.1ml) induced 100% morbidity in infected mice. Increased body temperatures measured by implantable temperature transponders correlated with an increase in infectious virus and viral RNA in serum, spleen and brain as well as an increase in pro-inflammatory markers measured by a 58-biomarker multi-analyte profile (MAP) constructed during the course of infection. In the future, the MAP measurements can be used as a baseline for comparison in order to better assess the inhibition of disease progression by other prophylactic and therapeutic agents. The use of the AG129/JEV SA14-14-2 animal model makes vaccine and therapeutic studies feasible for laboratories with limited biocontainment facilities.
[Show abstract][Hide abstract] ABSTRACT: Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.
[Show abstract][Hide abstract] ABSTRACT: Abstract Mosquito salivary proteins inoculated during blood feeding modulate the host immune response, which can contribute to the pathogenesis of viruses transmitted by mosquito bites. Previous studies with mosquito bite-naïve mice indicated that exposure to arthropod salivary proteins resulted in a shift toward a Th2-type immune response in flavivirus-susceptible mice but not flavivirus-resistant animals. In the study presented here, we tested the hypothesis that immunization with high doses of Culex tarsalis salivary gland extracts (SGE) with an adjuvant would prevent Th2 polarization after mosquito bite and enhance resistance to mosquito-transmitted West Nile virus (WNV). Our results indicate that mice immunized with Cx. tarsalis SGE produced increased levels of Th1-type cytokines (IFNγ and TNFα) after challenge with mosquito-transmitted WNV and exhibited both a delay in infection of the central nervous system (CNS) and significantly lower WNV brain titers compared to mock-immunized mice. Moreover, mortality was significantly reduced in the SGE-immunized mice, as none of these mice died, compared to mortality of 37.5% of mock-vaccinated mice by 8 days after infected mosquito bite. These results suggest that development of a mosquito salivary protein vaccine might be a strategy to control arthropod-borne viral pathogens such as WNV.
[Show abstract][Hide abstract] ABSTRACT: Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.
We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.
Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.
[Show abstract][Hide abstract] ABSTRACT: Epidemic dengue has emerged throughout the tropical world. In the continued absence of a vaccine against dengue virus (DENV), mosquito vector surveillance and control programs are essential to reduce human infections. An effective test to detect DENV in infected mosquitoes would be a valuable addition to the surveillance effort. We investigated DENV detection in infected Aedes aegypti using a commercially available DENV non-structural protein 1 (NS1) ELISA kit (Platelia Dengue NS1 Ag), and by reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation assays. The DENV-infected mosquitoes were subjected to field-relevant conditions and assayed individually and pooled with uninfected mosquitoes. Overall, DENV NS1 antigen was detected in 98% of infected mosquitoes/pools versus 79% for RT-PCR and 29% for virus isolation. Our results indicate that NS1 is an excellent analyte for detection of DENV in Ae. aegypti and that the tested NS1 antigen kit provides a sensitive, rapid, and convenient test for DENV surveillance in mosquitoes.
The American journal of tropical medicine and hygiene 11/2012; · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RNA silencing, or RNA interference (RNAi) in metazoans mediates development, reduces viral infection and limits transposon mobility. RNA silencing involves 21-30 nucleotide RNAs classified into microRNA (miRNA), exogenous and endogenous small interfering RNAs (siRNA), and Piwi-interacting RNA (piRNA). Knock-out, silencing and mutagenesis of genes in the exogenous siRNA (exo-siRNA) regulatory network demonstrate the importance of this RNAi pathway in antiviral immunity in Drosophila and mosquitoes. In Drosophila, genes encoding components for processing exo-siRNAs are among the fastest evolving 3% of all genes, suggesting that infection with pathogenic RNA viruses may drive diversifying selection in their host. In contrast, paralogous miRNA pathway genes do not evolve more rapidly than the genome average. Silencing of exo-siRNA pathway genes in mosquitoes orally infected with arboviruses leads to increased viral replication, but little is known about the comparative patterns of molecular evolution among the exo-siRNA and miRNA pathways genes in mosquitoes. We generated nearly complete sequences of all exons of major miRNA and siRNA pathway genes dicer-1 and dicer-2, argonaute-1 and argonaute-2, and r3d1 and r2d2 in 104 Aedes aegypti mosquitoes collected from six distinct geographic populations and analyzed their genetic diversity. The ratio of replacement to silent amino acid substitutions was 1.4 fold higher in dicer-2 than in dicer-1, 27.4 fold higher in argonaute-2 than in argonaute-1 and similar in r2d2 and r3d1. Positive selection was supported in 32% of non-synonymous sites in dicer-1, in 47% of sites in dicer-2, in 30% of sites in argonaute-1, in all sites in argonaute-2, in 22% of sites in r3d1 and in 55% of sites in r2d2. Unlike Drosophila, in Ae. aegypti, both exo-siRNA and miRNA pathway genes appear to be undergoing rapid, positive, diversifying selection. Furthermore, refractoriness of mosquitoes to infection with dengue virus was significantly positively correlated for nucleotide diversity indices in dicer-2.
PLoS ONE 09/2012; 7(9):e44198. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutation of the West Nile virus-like particle (WN VLP) prM protein (T20D, K31A, K31V, or K31T) results in undetectable VLP secretion from transformed COS-1 cells. K31 mutants formed intracellular prM-E heterodimers; however these proteins remained in the ER and ER-Golgi intermediary compartments of transfected cells. The T20D mutation affected glycosylation, heterodimer formation, and WN VLP secretion. When infectious viruses bearing the same mutations were used to infect COS-1 cells, K31 mutant viruses exhibited delayed growth and reduced infectivity compared to WT virus. Epitope maps of WN VLP and WNV prM were also different. These results suggest that while mutations in the prM protein can reduce or eliminate secretion of WN VLPs, they have less effect on virus. This difference may be due to the quantity of prM in WN VLPs compared to WNV or to differences in maturation, structure, and symmetry of these particles.
[Show abstract][Hide abstract] ABSTRACT: We established a laboratory colony of Culex pipiens mosquitoes from eggs collected in Colorado and discovered that mosquitoes in the colony are naturally infected with Culex flavivirus (CxFV), an insect-specific flavivirus. In this study we examined transmission dynamics of CxFV and effects of persistent CxFV infection on vector competence for West Nile virus (WNV). We found that vertical transmission is the primary mechanism for persistence of CxFV in Cx. pipiens, with venereal transmission potentially playing a minor role. Vector competence experiments indicated possible early suppression of WNV replication by persistent CxFV infection in Cx. pipiens. This is the first description of insect-specific flavivirus transmission dynamics in a naturally infected mosquito colony and the observation of delayed dissemination of superinfecting WNV suggests that the presence of CxFV may impact the intensity of enzootic transmission of WNV and the risk of human exposure to this important pathogen.
[Show abstract][Hide abstract] ABSTRACT: Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne pathogen that requires wild-type (wt), virulent strains to be handled at biosafety level (BSL) 3, with HEPA-filtration of room air exhaust (BSL3+). YFV is found in tropical regions of Africa and South America and causes severe hepatic disease and death in humans. Despite the availability of effective vaccines (17D-204 or 17DD), YFV is still responsible for an estimated 200,000 cases of illness and 30,000 deaths annually. Besides vaccination, there are no other prophylactic or therapeutic strategies approved for use in human YF. Current small animal models of YF require either intra-cranial inoculation of YF vaccine to establish infection, or use of wt strains (e.g., Asibi) in order to achieve pathology. We have developed and characterized a BSL2, adult mouse peripheral challenge model for YFV infection in mice lacking receptors for interferons α, β, and γ (strain AG129). Intraperitoneal challenge of AG129 mice with 17D-204 is a uniformly lethal in a dose-dependent manner, and 17D-204-infected AG129 mice exhibit high viral titers in both brain and liver suggesting this infection is both neurotropic and viscerotropic. Furthermore the use of a mouse model permitted the construction of a 59-biomarker multi-analyte profile (MAP) using samples of brain, liver, and serum taken at multiple time points over the course of infection. This MAP serves as a baseline for evaluating novel therapeutics and their effect on disease progression. Changes (4-fold or greater) in serum and tissue levels of pro- and anti-inflammatory mediators as well as other factors associated with tissue damage were noted in AG129 mice infected with 17D-204 as compared to mock-infected control animals.
[Show abstract][Hide abstract] ABSTRACT: Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations ≥1.27 μg/mouse; however, the 2C9-cIgM did not protect even at a dose of 127 μg/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice.
Antiviral research 02/2012; 94(1):1-8. · 3.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.
[Show abstract][Hide abstract] ABSTRACT: In a previous study, a new flavivirus genome sequence was identified in Culex tarsalis mosquitoes obtained in Alberta, Canada and was shown to be genetically related to but distinct from members of the insect-specific flaviviruses. Nonstructural protein 5-encoding sequences amplified from Cx. tarsalis pools from western Canada have shown a high similarity to genome sequences of novel flaviviruses isolated from mosquitoes in California and Colorado. Despite wide distribution of this virus, designated Calbertado virus, strains demonstrate a high degree of nonstructural protein 5 nucleotide (> 90%) and amino acid (> 97%) identity. The ecology and geographic range of Calbertado virus warrants further study because it may potentially influence transmission of mosquito-borne flaviviruses, including important human pathogens such as West Nile and Saint Louis encephalitis viruses.
The American journal of tropical medicine and hygiene 07/2011; 85(1):162-8. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mosquitoes were collected in Colorado during 2006 and 2007 to examine spatial and seasonal patterns of risk for exposure to Culex vectors and West Nile virus. We used universal flavivirus primers to test pools of Culex mosquitoes for viral RNA. This led to the detection and subsequent isolation of two insect-specific flaviviruses: Culex flavivirus (CxFV), which was first described from Japan, and a novel insect flavivirus, designated Calbertado virus (CLBOV), which has also been detected in California and Canada. We recorded both viruses in Cx. tarsalis and Cx. pipiens from Colorado. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of CxFV RNA in Cx. pipiens eggs and larvae from a laboratory colony established in 2005 and naturally infected with CxFV, suggesting vertical transmission as a means of viral maintenance in natural Culex populations. Finally, we present phylogenetic analyses of the relationships between insect-specific flaviviruses and other selected flaviviruses.
The American journal of tropical medicine and hygiene 07/2011; 85(1):169-77. · 2.53 Impact Factor