[Show abstract][Hide abstract] ABSTRACT: After a 75-year absence from Florida, substantial local transmission of dengue virus (DENV) occurred in Key West, Monroe County, Florida in 2009 and continued in 2010. The outbreak culminated in 85 reported cases. In 2011 and 2012, only isolated cases of local DENV transmission were reported in Florida; none were reported in Key West. In 2013, a new outbreak occurred, but this time in Martin County about 275 miles north of Key West with 22 reported cases. As the Key West and Martin County outbreaks involved DENV serotype 1 (DENV-1), we wanted to investigate whether the same strain or a different strain of DENV was responsible for the outbreaks. In this study, we report the sequence and phylogenetic analysis of the E gene region from a patient diagnosed with dengue in Martin County. Our results indicate that the 2013 Martin County DENV-1 strain is distinct from the 2009–2010 Key West DENV-1 and that it is most closely related to viruses from a recent expansion of South American DENV-1 strains into the Caribbean. We conclude that the 2013 Martin County outbreak was the result of a new introduction of DENV-1 in Florida.
[Show abstract][Hide abstract] ABSTRACT: Background. Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironrnent to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. Methods. We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8(+) T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. Results. We showed that YF-17D-induced CD8(+) T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8(+) T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. Conclusion. Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity.
[Show abstract][Hide abstract] ABSTRACT: Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA.
PLoS ONE 11/2012; 7(11):e50995. DOI:10.1371/journal.pone.0050995 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infections, yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at sub-neutralizing concentrations. Neutralization activity of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.
Journal of Virology 10/2012; 87(1). DOI:10.1128/JVI.02273-12 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To the Editor: Except for sporadic cases along the Texas-Mexico border, local transmission of dengue virus (DENV) has not occurred in the contiguous United States since 1946. In 2009, DENV was diagnosed in a vacationer to Key West, Florida (1). Subsequently, 25 other cases were reported that year, transmission was confirmed by detection of DENV serotype 1 (DENV-1) in local mosquitoes, and a random serosurvey showed evidence of recent DENV infection in 5.4% of Key West residents (1). Transmission continued in 2010, and an additional 63 cases were confirmed (2). We used PCR amplification and sequence analysis of virus identified from mosquito collections during 2010 to identify the closest relatives, probable geographic origin, and divergence time of the Key West DENV.
[Show abstract][Hide abstract] ABSTRACT: Severe dengue virus (DENV) disease symptoms, including dengue hemorrhagic fever and dengue shock syndrome, have been correlated with the presence of pre-existing antibodies that enhance rather than neutralize infections in Fc receptor bearing cells. These antibodies can originate from previous infection with a different serotype of dengue, or from waning antibody titers that occur in infants and young children as they are weaned from breast milk that contains protective dengue-specific antibodies. Despite the apparent importance of this antibody dependent enhancement (ADE) effect, there has been no description of any specific inhibitors of this process. We explored DENV entry inhibitors as a potential strategy to block ADE. Two different peptide entry inhibitors were tested for the ability to block antibody-mediated DENV-2 infection of human, FcRII bearing K562 cells in vitro. Both peptides were able to inhibit ADE, showing that entry inhibitors are possible candidates for the development of specific treatment for severe DENV infection.
Antiviral research 01/2011; 89(1):71-4. DOI:10.1016/j.antiviral.2010.11.008 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Virus surface proteins mediate interactions with target cells during the initial events in the process of infection. Inhibiting these proteins is therefore a major target for the development of antiviral drugs. However, there are a very large number of different viruses, each with their own distinct surface proteins and, with just a few exceptions, it is not clear how to build novel molecules to inhibit them. Here we applied a computational binding optimization strategy to an atomic resolution structure of dengue virus serotype 2 envelope protein to generate peptide sequences that should interact strongly with this protein. We picked dengue virus as a target because it is the causative agent for the most important mosquito transmitted viral disease. Out of a small number of candidates designed and tested, we identified two different highly inhibitory peptides. To verify our results, we showed that these peptides block virus:cell binding, interfere with a step during viral entry, alter the surface structure of dengue viral particles, and that they interact directly with dengue virus envelope protein. We expect that our approach may be generally applicable to other viral surface proteins where a high resolution structure is available.
[Show abstract][Hide abstract] ABSTRACT: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection.
Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection.
HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.
[Show abstract][Hide abstract] ABSTRACT: Background: Dengue Virus (DENV) causes over 50 million cases of Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) each year. Antibody produced during infection provides homotypic immunity. However, prior infection or circulating maternal antibodies can also produce a non-protective antibody response that can intensify the course of disease in a subsequent heterotypic infection. This antibody-dependent enhancement (ADE) complicates vaccine development, as vaccine components could stimulate severe disease rather than provide protection. To date, most published anti-dengue monoclonal antibodies (MAbs) are of mouse origin. These reagents may not reflect repertoire and roles of antibody produced by humans during natural infection. Methods: Epstein-Barr Virus (EBV) transformation of B cells isolated from peripheral blood from a single patient was performed. B cell cultures were screened by ELISA for antibodies to Dengue E protein. ELISA positive cultures were cloned by limiting dilution. HMAbs were purified on Protein A columns from batch cultures. Binding specificity to E glycoprotein was verified by Western Blot. Neutralization, competition and affinity binding assays were conducted on each MAb. Results: Three IgG1 HMAbs were generated: 2.3D, 3.6D and 4.8A. All three bound to DENV-1, 2, and 3, but none reacted with DENV-4. MAb 4.8A bound best to DENV-1 and 3, while 2.3D and 3.6D bound best to DENV-1 and 2. Only 4.8A demonstrated neutralization against DENV-3. Competition assays suggest that these HMAbs bind to distinct epitopes on the E glycoprotein. Conclusions: HMAbs such as those described here will help to identify epitopes recognized by the human immune system during natural infection. Their role in neutralization of homotypic and heterotypic serotypes as well as their role in ADE will help develop safe and effective vaccines.
Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
[Show abstract][Hide abstract] ABSTRACT: The Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), are well known for their destruction of human dwellings and flora in the tropics and subtropics. A method to deliver foreign genes into termite cell cultures would provide a controlled environment to facilitate the study of key regulatory functions at the molecular and cellular level. Here a method for the establishment and cryopreservation of primary embryonic termite cell cultures is described. Evidence is presented of viral-mediated gene transfer in these cells and foreign gene expression using a recombinant vesicular stomatitis virus vector.
[Show abstract][Hide abstract] ABSTRACT: The anti-adhesive compound p-sulfoxy-cinnamic acid, zosteric acid (ZA), is derived from the temperate marine eelgrass, Zostera marina. ZA and five combinatorial chemistries based on ZA were evaluated for their anti-viral properties against dengue virus in a focus forming unit reduction assay. None of the compounds showed evidence of toxicity to the monkey kidney cell line LLCMK-2 over the concentration ranges tested. ZA showed a modest IC(50) of approximately 2.3 mM against DENV-2. Three other compounds showed IC(50) values of 2.5, 2.4, 0.3 mM, with a fourth not achieving a 50% inhibitory concentration against DENV-2. The most active compound, CF 238, showed IC(50) values of 24, 46, 14 and 47 microM against DENV-1, DENV-2, DENV-3 and DENV-4, respectively. CF 238 showed evidence of inhibition at an entry step in the viral life cycle and enhanced virus:cell binding as evidenced by a quantitative RT-PCR assay system. CF 238 may promote inappropriate virus:cell attachments common to all DENV strains that interfere with receptor interactions required for viral entry. These and other related chemistries may be useful as reagents for studying DENV entry, capturing and detecting DENV, and development of pharmaceuticals.
Antiviral research 07/2008; 80(2):135-42. DOI:10.1016/j.antiviral.2008.05.007 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Viral fusion proteins mediate cell entry by undergoing a series of conformational changes that result in virion-target cell membrane fusion. Class I viral fusion proteins, such as those encoded by influenza virus and human immunodeficiency virus (HIV), contain two prominent alpha helices. Peptides that mimic portions of these alpha helices inhibit structural rearrangements of the fusion proteins and prevent viral infection. The envelope glycoprotein (E) of flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), are class II viral fusion proteins comprised predominantly of beta sheets. We used a physio-chemical algorithm, the Wimley-White interfacial hydrophobicity scale (WWIHS) in combination with known structural data to identify potential peptide inhibitors of WNV and DENV infectivity that target the viral E protein. Viral inhibition assays confirm that several of these peptides specifically interfere with target virus entry with 50% inhibitory concentration (IC50) in the 10 microM range. Inhibitory peptides similar in sequence to domains with a significant WWIHS scores, including domain II (IIb), and the stem domain, were detected. DN59, a peptide corresponding to the stem domain of DENV, inhibited infection by DENV (>99% inhibition of plaque formation at a concentrations of <25 microM) and cross-inhibition of WNV fusion/infectivity (>99% inhibition at <25 microM) was also demonstrated with DN59. However, a potent WNV inhibitory peptide, WN83, which corresponds to WNV E domain IIb, did not inhibit infectivity by DENV. Additional results suggest that these inhibitory peptides are noncytotoxic and act in a sequence specific manner. The inhibitory peptides identified here can serve as lead compounds for the development of peptide drugs for flavivirus infection.
[Show abstract][Hide abstract] ABSTRACT: Summary 1. Differences in timing of development can greatly affect morphology and locomotor performance, which are of crucial importance in juvenile organisms. Early hatching in amphibians can occur in response to egg threats, and early hatched individuals may suffer the costs of physical immaturity posthatching. 2. We measured maximum jumping performance for early hatched (EH) and normal hatched (NH) froglets of the direct-developing frog Eleutherodactylus coqui on both a wet and a dry substrate. Snout-vent length, hindlimb length and mass were compared with jumping ability and behaviours on the wet substrate were scored. 3. EH froglets had lighter mass, shorter relative hindlimbs and performed more poorly on both substrates than NH individuals. Between groups, hindlimb length and snout-vent length scaled higher with mass than the expected geometric scale. EH froglets also engaged in more walking and swimming behaviour than did NH froglets on wet substrates. 4. We found that EH froglets have shorter relative hindlimbs and poorer jumping per- formance than NH froglets. EH froglets may compensate for their poor jumping ability by using other forms of locomotion. Limbs that are longer than theoretically predicted may help these terrestrial organisms escape forest-floor predators.
[Show abstract][Hide abstract] ABSTRACT: Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.
[Show abstract][Hide abstract] ABSTRACT: World-wide reports of amphibian population declines have led to increased interest in the reproductive biology of anurans. As a model system, here we present evidence for the effective cryoprotection of sperm from the Puerto Rican frog, Eleutherodactylus coqui, using mixtures of fetal bovine serum (FBS) and dimethylsulfoxide (Me(2)SO), glycerol or sucrose extenders. Using a fluorescent dye exclusion assay, we found that 53.9 and 50.4% of all sperm with intact membranes prior to freezing maintained membrane integrity after rapid freezing and thawing when protected with either a FBS/glycerol or FBS/sucrose solution, respectively. The methods reported here may be useful for similar work with many of the more than 700 other species in this genus.
[Show abstract][Hide abstract] ABSTRACT: The Sykes' monkey and related forms (Cercopithecus mitis) make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk). We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex.
The SIVsyk antigen-specific Western blot assay detected more serum samples as positive than either of the cross-reactive ELISA assays. Using this assay, we found that seroprevalence is higher than previously reported, but extremely variable in wild populations (from 0.0 to 90.9%). Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified.
We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission.
[Show abstract][Hide abstract] ABSTRACT: This study investigates ovulation and egg deposition behaviors in the anuran Eleutherodactylus coqui from Puerto Rico in response to stimulation with gonadotropin and gonadotropin releasing hormones. Five hormones were tested by injection over a range of doses, including mammalian LHRH, avian LHRH, fish LHRH, D-Ala6, des-Gly10 ethylamide LHRH and hCG. We report a low level of ovulation and egg deposition in response to all hormones, with the most complete and consistent results from the non-natural D-Ala6, des-Gly10 ethylamide LHRH derivative. To confirm the viability of eggs produced in this manner we performed in vitro fertilization experiments that resulted in the development of normal frogs. Reproductive behaviors in E. coqui are apparently not controlled by a mammalian form of LHRH as reported in other common laboratory anuran species. D-Ala6, des-Gly10 ethylamide LHRH induces ovulation and deposition of mature and fertilizable eggs in E. coqui.
[Show abstract][Hide abstract] ABSTRACT: The genome of Schistosoma mansoni contains a proviral form of a retrovirus-like long terminal repeat (LTR) retrotransposon, designated Boudicca. Sequence and structural characterization of the new mobile genetic element, which was found in bacterial artificial chromosomes
prepared from S. mansoni genomic DNA, revealed the presence of three putative open reading frames (ORFs) bounded by direct LTRs of 328 bp in length.
ORF1 encoded a retrovirus-like major homology region and a Cys/His box motif, also present in Gag polyproteins of related
retrotransposons and retroviruses. ORF2 encoded enzymatic domains and motifs characteristic of a retrovirus-like polyprotein,
including aspartic protease, reverse transcriptase, RNase H, and integrase, in that order, a domain order similar to that
of the gypsy/Ty3 retrotransposons. An additional ORF at the 3′ end of the retrotransposon may encode an envelope protein. Phylogenetic comparison
based on the reverse transcriptase domain of ORF2 confirmed that Boudicca was a gypsy-like retrotransposon and showed that it was most closely related to CsRn1 from the Oriental liver fluke Clonorchis sinensis and to kabuki from Bombyx mori. Bioinformatics approaches together with Southern hybridization analysis of genomic DNA of S. mansoni and the screening of a bacterial artificial chromosome library representing ≈8-fold coverage of the S. mansoni genome revealed that numerous copies of Boudicca were interspersed throughout the schistosome genome. By reverse transcription-PCR, mRNA transcripts were detected in the
sporocyst, cercaria, and adult developmental stages of S. mansoni, indicating that Boudicca is actively transcribed in this trematode.
Journal of Virology 07/2003; 77(11):6153-66. DOI:10.1128/JVI.77.11.6153-6166.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV-infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy-four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory-cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme previously had been identified only from prokaryotes. The 74 GSS included 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein-encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r = 0.79) and Shizosaccharomyces pombe (r = 0.70), but was markedly dissimilar to E. cuniculi (r = 0.19).