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Zika virus (ZIKV) is a flavivirus that is primarily transmitted by Aedes aegypti, the mosquito vector also important in transmission of the flaviviruses responsible for dengue fever, yellow fever, and chikungunya. Because of occurrence in the same geographic regions, serologic cross-reactivity, and similar but often less severe clinical manifestations, such as dengue and chikungunya infections, ZIKV infection likely has gone undetected, misdiagnosed, or both for many years. ZIKV is somewhat unique among flaviviruses in its ability to also be transmitted through sexual contact, nonsexual body fluids, and perinatally. The relatively recent detection of the link between ZIKV infection and Guillain-Barré syndrome and fetal neurological defects, including microcephaly, has prompted intense efforts aimed at the development of new and specific diagnostic tests. Infection with ZIKV has been postulated to lead to a more severe clinical course from other structurally related viruses, especially dengue, and vice versa because of a phenomenon termed antibody-dependent enhancement. Inactivated whole virus, DNA, RNA, and vectored vaccine approaches to prevent ZIKV infection are in development, as are treatments for active disease that are safe in pregnant women. Here we summarize the important epidemiologic and clinical features of ZIKV infection, as well as the progress and challenges in developing rapid point-of-care diagnostic tests and vaccines to prevent disease. We used electronic databases to identify relevant published data regarding ZIKV MeSH searches.

The Republic of Angola is a priority country for onchocerciasis and lymphatic filariasis (LF) elimination, however, the co-distribution of the filarial parasite Loa loa (loiasis) is a significant impediment, due to the risk of severe adverse events (SAEs) associated with ivermectin used in mass drug administration (MDA) campaigns. Angola has a high risk loiasis zone identified in Bengo Province where alternative interventions may need to be implemented; however, the presence and geographical overlap of the three filarial infections/diseases are not well defined. Therefore, this study conducted a rapid integrated filarial mapping survey based on readily identifiable clinical conditions of each disease in this risk zone to help determine prevalence and co-distribution patterns in a timely manner with limited resources. In total, 2007 individuals from 29 communities in five provincial municipalities were surveyed. Community prevalence estimates were determined by the rapid assessment procedure for loiasis (RAPLOA) and rapid epidemiological mapping of onchocerciasis (REMO) together with two questions on LF clinical manifestations (presence of lymphoedema, hydrocoele). Overall low levels of endemicity, with different overlapping distributions were found. Loiasis was found in 18 communities with a prevalence of 2.0% (31/1571), which contrasted to previous results defining the area as a high risk zone. Onchocerciasis prevalence was 5.3% (49/922) in eight communities, and LF prevalence was 0.4% for lymphoedema (8/2007) and 2.6% for hydrocoeles (20/761 males) in seven and 12 communities respectively. The clinical mapping survey method helped to highlight that all three filarial infections are present in this zone of Bengo Province. However, the significant difference in loiasis prevalence found between the past and this current survey suggests that further studies including serological and parasitological confirmation are required. This will help determine levels of infection and risk, understand the associations between clinical, serological and parasitological prevalence patterns, and better determine the most appropriate treatment strategies to reach onchocerciasis and LF elimination targets in the loiasis co-endemic areas. Our results also suggest that the utility of the earlier RAPLOA derived maps, based on surveys undertaken over a decade ago, are likely to be invalid given the extent of population movement and environmental change, particularly deforestation, and that fine scale micro-mapping is required to more precisely delineate the interventions required defined by these complex co-endemicities. info:eu-repo/semantics/publishedVersion

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20-hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20-hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development.

The prevalence of Loa loa, Onchocerca volvulus and Wuchereria bancrofti infections in an under-surveyed area of Bengo Province, Angola, was determined by surveying 22 communities with a combination of clinical, serological and DNA diagnostics. Additional information was collected on participants' duration of residency, access to mass drug administration, knowledge of insect vectors and use of bednets. A total of 1616 individuals (38.1% male: 61.9% female), with an average age of 43 years, were examined. For L. loa, 6.2% (n = 100/16616) individuals were found to have eyeworm, based on the rapid assessment procedure for loiasis (RAPLOA) surveys, and 11.5% (n =178/1543) based on nested PCR analyses of venous blood. L. loa prevalences in long-term residents (>10 years) and older individuals (>60 years) were significantly higher, and older men with eyeworm were better informed about Chrysops vectors. For O. volvulus, 4.7% (n = 74/1567) individuals were found to be positive by enzyme-linked immunosorbent assay (Ov 16 ELISA), with only three individuals reporting to have ever taken ivermectin. For W. bancrofti, no infections were found using the antigen-based immunochromatographic test (ICT) and real-time PCR analysis; however, 27 individuals presented with lymphatic filariasis (LF) related clinical conditions (lymphoedema = 11, hydrocoele = 14, both = 2). Just under half (45.5%) of the participants owned a bednet, with the majority (71.1%) sleeping under it the night before. Our approach of using combination diagnostics reveals the age-prevalence of loiasis alongside low endemicity of onchocerciasis and LF. Future research foci should be on identifying opportunities for more cost-effective ways to eliminate onchocerciasis and to develop innovative surveillance modalities for clinical LF for individual disease management and disability prevention.

Eastern equine encephalitis (EEE) is a mosquito-borne viral disease that is often fatal to humans and horses. Some species including white-tailed deer and passerine birds can survive infection with the EEE virus (EEEV) and develop antibodies that can be detected using laboratory techniques. In this way, collected serum samples from free ranging white-tailed deer can be used to monitor the presence of the virus in ecosystems. This study developed and tested a risk index model designed to predict EEEV activity in white-tailed deer in a three-county area of Michigan. The model evaluates EEEV risk on a continuous scale from 0.0 (no measurable risk) to 1.0 (highest possible risk). High risk habitats are identified as those preferred by white-tailed deer that are also located in close proximity to an abundance of wetlands and lowland forests, which support disease vectors and hosts. The model was developed based on relevant literature and was tested with known locations of infected deer that showed neurological symptoms. The risk index model accurately predicted the known locations, with the mean value for those sites equal to the 94th percentile of values in the study area. The risk map produced by the model could be used refine future EEEV monitoring efforts that use serum samples from free-ranging white-tailed deer to monitor viral activity. Alternatively, it could be used focus educational efforts targeted toward deer hunters that may have elevated risks of infection.

The complement of the Brugia malayi microRNA-71 was inserted into the 3' untranslated region of a reporter plasmid, resulting in a decrease in reporter activity. Mutation of the seed sequence restored activity. Insertion of the 3' untranslated regions from two algorithm-predicted putative target genes into the reporter resulted in a similar decrease in activity; mutation of the predicted target sequences restored activity. These experiments demonstrate that B. malayi microRNA targets may be predicted using current algorithms and describe a functional assay to confirm predicted targets.

A GIS-based risk index model was developed to quantify EEEV transmission risk to horses in the State of Florida. EEEV is a highly pathogenic arbovirus that is endemic along the east coast of the United States, and it is generally fatal to both horses and humans. The model evaluates EEEV transmission risk at individual raster cells in map on a continuous scale of 0 to 1. The risk index is derived based on local habitat features and the composition and configuration of surrounding land cover types associated with EEEV transmission. The model was verified and validated using the locations of documented horse cases of EEEV. These results of the verification and validation indicate that the model is able to predict locations of EEEV transmission to horses broadly across the state. The model is relatively robust to regional variation in EEEV transmission and habitat conditions in Florida, and it accurately predicted nearly all verification and validation cases in the Panhandle, North, and Central regions of the state. The model performed less accurately in the South, where relatively few cases are documented. Despite these differences, the model provides a useful way to assess EEEV risk both from a regional perspective and at more localized scales. The resulting predictive maps are designed to guide EEEV surveillance and prevention efforts by county mosquito control districts.

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesize that they might be involved in the Wolbachia-Brugia symbiotic relationship. Notably, immunolocalization studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localization by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.

Previous studies of Brugia malayi promoters have suggested that they are unusual in that they lack the CAAT or TATAA boxes that are often emblematic of eucaryotic core promoter domains. Instead, the region surrounding the spliced leader (SL) addition site appears to function as the core promoter domain in B. malayi. To test the hypothesis that polymorphisms in this SL addition domain are important determinants of promoter activity, a series of domain swap mutants were prepared replacing the SL addition domain of the B. malayi 13 kDa large subunit ribosomal protein (BmRPL13) with those of other ribosomal protein (RP) promoters exhibiting a wide range of activities. These constructs were then tested for promoter activity in a homologous transient transfection system. On average, polymorphisms in the SL addition domain were found to be responsible for 80% of the variation in promoter activity exhibited by the RP promoters tested. Essentially all of this effect could be attributable to polymorphisms in the 10nt located directly upstream of the SL addition site. A comparison of the sequence of this domain to the promoter activity exhibited by the domain swap mutants suggested that promoter activity was related to the number of T residues present in the coding strand of the upstream domain. Confirming this, mutation of the upstream domain of the promoter of the BmRPS4 gene to a homogeneous stretch of 10 T residues resulted in a significant increase in promoter activity.

Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70 kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.

A 7-nt motif (the trans-splicing motif or TSM) was previously shown to be necessary and sufficient to direct trans-splicing of transgenic mRNAs in transgenic Brugia malayi embryos. Insertion of the TSM into two genes lacking a TSM homologue resulted in trans-splicing of transgenic mRNAs from one transgene but not the other, suggesting that local sequence context might affect TSM function. To test this hypothesis, constructs inserting the TSM into different positions of two B. malayi genes were tested for their ability to support trans-splicing of transgenic mRNAs. Transgenic mRNAs derived from constructs in which the insertion of the TSM did not result in a perturbation of the local predicted secondary structure were trans-spliced, while those in which the TSM perturbed the local secondary structure were not. These data suggest that local secondary structure plays a role in the ability of the TSM to direct trans-splicing.

Operons are a common mode of gene organization in Caenorhabditis elegans. Similar gene arrangements suggest that functional operons may exist in Brugia malayi. To definitively test this hypothesis, a bicistronic reporter vector consisting of an upstream firefly luciferase gene and a downstream renilla luciferase gene was constructed. The genome was then surveyed to identify 15 gene pairs that were likely to represent operons. Two of four domains upstream of the 5' gene from these clusters exhibited promoter activity. When constructs replicating the promoter and intergenic arrangement found in the native putative operon were transfected into embryos, both firefly and renilla activities were detected, while constructs with the promoter alone or intergenic region alone produced no activity from the downstream reporter. These data confirm that functional operons exist in B. malayi. Mutation of three U-rich element homologues present in one of the operons resulted in a decrease in downstream renilla reporter activity, suggesting that these were important in mRNA maturation. Hemi-nested reverse transcriptase-PCR assays demonstrated that while the mRNA encoding the native downstream open reading frame of one operon contained an SL1 spliced leader at its 5' end, the renilla gene mRNA produced from the corresponding transgenic construct did not.

Many genes in parasitic nematodes are both cis- and trans-spliced. Previous studies have demonstrated that a 7 nt element encoded in the first intron of the Brugia malayi 70 kDa heat shock protein (BmHSP70) gene was necessary to permit trans-splicing of transgenic mRNAs in embryos transfected with constructs encoding portions of the BmHSP70 gene. Here we demonstrate that this element (the B. malayi HSP70 trans-splicing motif, or BmHSP70 TSM) is necessary and sufficient to direct trans-splicing of transgenic mRNAs derived from two genes naturally containing this motif. Mutations introduced into any position of the BmHSP70 TSM abrogated its ability to direct trans-splicing. Transgenic mRNAs derived from embryos transfected with constructs containing promoters and associated downstream domains from two normally trans-spliced genes that lack a BmHSP70 TSM homologue (the B. malayi 12 kDa small subunit ribosomal protein (BmRPS12) gene and the B. malayi RNA-binding protein (BmRBP1) gene), were not trans-spliced. Transfer of the BmHSP70 TSM into the first intron of the BmRPS12 gene rendered it competent for trans-splicing. Insertion of the BmHSP70 TSM into the single intron of the BmRBP1 gene did not render it trans-splicing competent. However, tagged constructs of the full-length BmRBP1 gene were trans-splicing competent. An analysis of the first exons and introns of over 200 trans-spliced B. malayi genes found homologues for the BmHSP70 TSM in roughly 25%. Thus, while the BmHSP70 TSM is necessary and sufficient to direct trans-splicing in some genomic contexts, independent trans-splicing signals are employed by other genes.

Pneumocystis pneumonia (PCP), caused by infection with Pneumocystis jirovecii, remains an important opportunistic infection in humans. A reverse transcriptase polymerase chain reaction assay has been shown to specifically detect viable P. jirovecii organisms. In the current study, we evaluated this assay on different types of respiratory samples. The assay had a diagnostic sensitivity of 100% and a specificity of 86% when applied to bronchoalveolar lavage samples. The assay's performance declined when applied to less invasive induced sputum and oropharyngeal wash (OPW) samples. The sensitivity, when applied to OPWs, was improved by examining multiple sequential OPW samples and was affected by clinical sampling parameters that could increase or decrease the number of potential organisms in the oropharynx. When used in conjunction with an optimized clinical sampling protocol, this assay may become a useful tool for detecting and monitoring P. jirovecii in minimally invasive clinical samples.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Eastern equine encephalitis virus (EEEV) is one of several arthropod-borne viruses (arboviruses) endemic to the United States. Interactions between arthropod (mosquito) vectors and avian amplification host populations play a significant role in the dynamics of arboviral transmission. Recent data have suggested the hypothesis that an increased rate of successful feeding on young-of-the-year (YOY) birds might play a role in the dynamics of EEEV transmission. To test this hypothesis, we developed a model to explore the effect of the interactions of the vectors and avian host populations on EEEV transmission. Sensitivity analyses conducted using this model revealed eleven parameters that were capable of disproportionately affecting the predicted level of EEEV infection in the vertebrate reservoir and vector populations. Of these, four parameters were related to the interaction of the vector with young-of-the-year birds. Furthermore, adult birds could not substitute for young-of-the-year in initiating and maintaining a predicted enzootic outbreak of EEEV. Taken together, the model predicted that young-of-the-year birds play a key role in establishing and maintaining enzootic outbreaks of EEEV.

Epidemiological, clinical and genetic data have all suggested that the filarial parasite Onchocerca volvulus, the causative agent of onchocerciasis (or river blindness) exists as two strains in West Africa. The severe strain induces severe ocular disease in a large proportion of the infected population, while the mild strain induces little ocular disease. Although DNA probes based upon a non-coding repeat sequence family can distinguish the two strains, the underlying basis for this difference in pathogenicity is not understood. Recently, several studies have implicated products produced by the Wolbachia endosymbiotic bacterium of O. volvulus in the pathogenesis of onchocerciasis. This suggested the hypothesis that differences in the Wolbachia endosymbiont population might be responsible for the pathogenic differences noted in the two strains. To test this hypothesis, quantitative PCR assays were used to measure the amount of Wolbachia DNA per nuclear genome in a collection of well characterized samples of mild and severe strain O. volvulus. The median ratio of Wolbachia DNA to nuclear DNA was significantly greater in severe strain parasites than in mild strain parasites. These data support the hypothesis that the pathogenic differences seen in severe and mild strain O. volvulus may be a function of their relative Wolbachia burden and provide additional support to the hypothesis that Wolbachia products may play a central role in the pathogenesis of ocular onchocerciasis.

Trans splicing is a process found in a variety of organisms, including Leishmania [1], trypanosomes, a number of parasitic nematodes [2–6] and the free-living nematode Caenorhabditis elegans [7]. In nematodes, this process involves the addition of a 22 nt sequence, known as the spliced leader (SL), to the 5′ end of the nascent mRNAs [8]. In vitro biochemical systems employing nuclear extracts have been used to extensively dissect the trans splicing pathway in the intestinal parasite Ascaris suum [8–12]. These studies have resulted in the identification of a number of trans acting factors that are involved in trans splicing [11–13] and have also succeeded in identifying the structural factors in the SL pre-RNA that are necessary for correct processing of the nascent transcript [8]. Recently, a transient transfection system for the parasitic nematode Brugia malayi was employed to map the promoter domains in the sequences present upstream of the gene for the heat shock protein 70 (HSP70) homologue of B. malayi. The native HSP70 message is trans spliced in vivo, with the SL added at a site that is located 47 nt upstream of the translational start [14]. Surprisingly, mRNAs transcribed from B. malayi embryos transfected with a synthetic transgene consisting of 659 nt upstream of the HSP70 ORF (including the native SL addition site) fused to a luciferase reporter gene were not trans spliced [15]. However, transgenes consisting of in frame fusions of the HSP70 659 nt upstream domain, exon1, intron 1 and part of exon 2 were correctly cis and trans spliced [15]. Furthermore, subsequent experiments employing a similar construct in which most of exon 1

We wish to give special thanks to TIGR for the cluster analysis of the EST data. The authors would also like to recognize the financial support of the Edna McConnell Clark Foundation, the Blakeslee Fund for Genetics Research at Smith College and the generous support to Smith College of the A.K. Watson Trust.

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.

Although the mechanisms underlying the host inflammatory response in ocular onchocerciasis have been examined, the role of particular parasite proteins in this process remains largely unexplored. Recently, it was found that one of the most abundant expressed sequence tags in Onchocerca volvulus infective larvae encoded a protein with similarities to a component of vespid venom. This clone was designated O. volvulus Activation associated Secreted Protein -1 (Ov-asp-1). We report the characterization of three members of a family of proteins, designated the Ov-ASP family, of which Ov-ASP-1 is a member. Sequence based and phylogenetic analyses suggest that these proteins form a filarial specific protein family related to both the vespid venom antigen 5 and the vertebrate CRISP/Tpx family of proteins. The three members of the Ov-ASP family exhibit distinct patterns of expression in the life cycle of O. volvulus. Genomic Southern blot analyses indicate that several genes encoding sequences related to the Ov-asp family are present in the genome of O. volvulus. Recombinant proteins expressed from full length cDNAs encoding two members of the Ov-asp family were found to induce an angiogenic response after injection into corneas of naive mice, and vessel formation was associated with only minor inflammatory cell infiltration. These data suggest that Ov-ASP proteins may directly induce an angiogenic response and may therefore contribute to corneal neovascularization in onchocercal keratitis.

Steroids and retinoids are important regulators of development in invertebrates and vertebrates. The central mediators of action of these compounds are their cognate receptors, which together form a family of proteins known as the nuclear receptor family. Previous studies have demonstrated that the genome of Onchocerca volvulus encodes at least three members of the nuclear receptor family. Here, the characterization of one member of this family from O. volvulus, designated OvNR-2, is described. OvNR-2 was found to be most similar to a number of vertebrate retinoic acid receptors and to the Drosophila melanogaster EiP78c protein. Modeling studies suggest that OvNR-2 forms a boot shaped ligand-binding cavity of a shape and size that can bind steroids. Expression of the mRNA corresponding to OvNR-2 is tightly regulated in adult parasites, appearing only in the extended intrauterine microfilariae. The protein derived from expression of the OvNR-2 cDNA in a bacterial system is recognized by serum antibodies in a majority of individuals infected with O. volvulus.

Ocular onchocerciasis results from immune recognition of parasite proteins released into the eye by degenerating microfilariae. Previous studies have shown that pathology similar to human ocular onchocerciasis can be induced in sensitized mice by intracorneal injection with Onchocerca volvulus antigens. In the current study, we used this murine model to map the segments of O. volvulus protein disulfide isomerase (OvPDI) associated with the development of corneal pathology. Subclones of OvPDI were constructed encompassing one or more predicted T cell epitopes. Keratitis was induced in BALB/c mice after subcutaneous immunizations with OvPDI, followed by intracorneal challenge of OvPDI constructs. Truncated OvPDI proteins containing amino acids 450–481 of OvPDI were found to induce keratitis, whereas constructs that did not include this region did not induce corneal pathology. Consistent with this observation, two peptides derived from the 450–481 region stimulated T cell proliferation to a greater degree than control carrier protein. DNA sequence analysis of cDNAs encoding OvPDI from blinding and non-blinding strains of O. volvulus indicated no differences in the primary amino acid sequence of the 450–481 domain. Immunization of animals with OvPDI induced antibodies recognizing a 55 kDa host protein, identical to the predicted molecular weight of the mouse PDI homologue. Together, these data implicate specific antigenic epitopes of OvPDI in the development of O. volvulus mediated corneal pathology.

Onchocerca volvulus infected Simulium damnosum s.l. were analysed by directed heteroduplex analysis. Of 73 infected flies, 68 produced heteroduplex products identical to those previously identified. All 6 major sibling species, except S. leonense, were present in this group. In the 5 remaining flies, 2 new heteroduplex patterns were noted. Molecular phylogenetic analysis of these samples suggested that they belonged to the S. squamosum/S. yahense subcomplex. The ability to reliably genotype adult flies will permit studies of the vectorial capacity of the sibling species of S. damnosum s.l. for the blinding and non-blinding strains of O. volvulus.

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.