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Background: The full scope of the genes expressed by schistosomes during intramolluscan development has yet to be characterized. Understanding the gene products deployed by larval schistosomes in their snail hosts will provide insights into their establishment, maintenance, asexual reproduction, ability to castrate their hosts, and their prolific production of human-infective cercariae. Using the Illumina platform, the intramolluscan transcriptome of Schistosoma mansoni was investigated in field-derived specimens of the prominent vector species Biomphalaria pfeifferi at 1 and 3 days post infection (d) and from snails shedding cercariae. These S. mansoni samples were derived from the same snails used in our complementary B. pfeifferi transcriptomic study. We supplemented this view with microarray analyses of S. mansoni from B. glabrata at 2d, 4d, 8d, 16d, and 32d to highlight robust features of S. mansoni transcription, even when a different technique and vector species was used. Principal findings: Transcripts representing at least 7,740 (66%) of known S. mansoni genes were expressed during intramolluscan development, with the greatest number expressed in snails shedding cercariae. Many transcripts were constitutively expressed throughout development featuring membrane transporters, and metabolic enzymes involved in protein and nucleic acid synthesis and cell division. Several proteases and protease inhibitors were expressed at all stages, including some proteases usually associated with cercariae. Transcripts associated with G-protein coupled receptors, germ cell perpetuation, and stress responses and defense were well represented. We noted transcripts homologous to planarian anti-bacterial factors, several neural development or neuropeptide transcripts including neuropeptide Y, and receptors that may be associated with schistosome germinal cell maintenance that could also impact host reproduction. In at least one snail the presence of larvae of another digenean species (an amphistome) was associated with repressed S. mansoni transcriptional activity. Conclusions/Significance: This in vivo study, emphasizing field-derived snails and schistosomes, but supplemented with observations from a lab model, provides a distinct view from previous studies of development of cultured intramolluscan stages from lab-maintained organisms. We found many highly represented transcripts with suspected or unknown functions, with connection to intramolluscan development yet to be elucidated. [ABSTRACT FROM AUTHOR]

Lymphatic filariasis (LF), a morbid disease caused by the tissue-invasive nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, affects millions of people worldwide. Global eradication efforts have significantly reduced worldwide prevalence, but complete elimination has been hampered by limitations of current anti-filarial drugs and the lack of a vaccine. The goal of this study was to evaluate B. malayi intestinal UDP-glucuronosyltransferase (Bm-UGT) as a potential therapeutic target. To evaluate whether Bm-UGT is essential for adult filarial worms, we inhibited its expression using siRNA. This resulted in a 75% knockdown of Bm-ugt mRNA for 6 days and almost complete suppression of detectable Bm-UGT by immunoblot. Reduction in Bm-UGT expression resulted in decreased worm motility for 6 days, 70% reduction in microfilaria release from adult worms, and significant reduction in adult worm metabolism as detected by MTT assays. Because prior allergic-sensitization to a filarial antigen would be a contraindication for its use as a vaccine candidate, we tested plasma from infected and endemic normal populations for Bm-UGT-specific IgE using a luciferase immunoprecipitation assay. All samples (n = 35) tested negative. We then tested two commercially available medicines known to be broad inhibitors of UGTs, sulfinpyrazone and probenecid, for in vitro activity against B. malayi. There were marked macrofilaricidal effects at concentrations achievable in humans and very little effect on microfilariae. In addition, we observed that probenecid and sulfinpyrazone exhibit a synergistic macrofilaricidal effect when used in combination with albendazole. The results of this study demonstrate that Bm-UGT is an essential protein for adult worm survival. Lack of prior IgE sensitization in infected and endemic populations suggest it may be a feasible vaccine candidate. The finding that sulfinpyrazone and probenecid have in vitro effects against adult B. malayi worms suggests that these medications have promise as potential macrofilaricides in humans. [ABSTRACT FROM AUTHOR]

Background: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. Conclusions/Significance: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies. [ABSTRACT FROM AUTHOR]

Ticks successfully feed and transmit pathogens by injecting pharmacological compounds in saliva to thwart host defenses. We have previously used LC-MS/MS to identify proteins that are present in saliva of unfed Amblyomma americanum ticks that were exposed to different hosts. Here we show that A. americanum serine protease inhibitor (serpin) 27 (AAS27) is an immunogenic saliva protein that is injected into the host within the first day of tick feeding and is an anti-inflammatory protein that might act by blocking plasmin and trypsin functions. Although AAS27 is injected into the host throughout tick feeding, qRT-PCR and western blotting analyses indicate that the respective transcript and protein are present in high amounts within the first 24 h of tick feeding. Biochemical screening of Pichia pastoris-expressed recombinant (r) AAS27 against mammalian proteases related to host defense shows it is an inhibitor of trypsin and plasmin, with stoichiometry of inhibition indices of 3.5 and 3.8, respectively. Consistent with typical inhibitory serpins, rAAS27 formed heat- and SDS-stable irreversible complexes with both proteases. We further demonstrate that rAAS27 inhibits trypsin with ka of 6.46 ± 1.24 x 104 M-1 s-1, comparable to serpins of other tick species. We show that native AAS27 is part of the repertoire of proteins responsible for the inhibitory activity against trypsin in crude tick saliva. AAS27 is likely utilized by the tick to evade the hosts inflammation defense since rAAS27 blocks both formalin and compound 48/80-induced inflammation in rats. Tick immune sera of rabbits that had acquired resistance against tick feeding following repeated infestations with A. americanum or Ixodes scapularis ticks reacts with rAAS27. Of significant interest, antibody to rAAS27 blocks this serpin inhibitory functions. Taken together, we conclude that AAS27 is an anti-inflammatory protein secreted into the host during feeding and may represent a potential candidate for development of an anti-tick vaccine. [ABSTRACT FROM AUTHOR]

The 2013–2016 Ebola virus outbreak in West Africa was the largest and deadliest outbreak to date. Here we conducted a serological study to examine the antibody levels in survivors and the seroconversion in close contacts who took care of Ebola-infected individuals, but did not develop symptoms of Ebola virus disease. In March 2017, we collected blood samples from 481 individuals in Makeni, Sierra Leone: 214 survivors and 267 close contacts. Using commercial, quantitative ELISAs, we tested the plasma for IgG-specific antibodies against three major viral antigens: GP, the only viral glycoprotein expressed on the virus surface; NP, the most abundant viral protein; and VP40, a major structural protein of Zaire ebolavirus. We also determined neutralizing antibody titers. In the cohort of Ebola survivors, 97.7% of samples (209/214) had measurable antibody levels against GP, NP, and/or VP40. Of these positive samples, all but one had measurable neutralizing antibody titers against Ebola virus. For the close contacts, up to 12.7% (34/267) may have experienced a subclinical virus infection as indicated by detectable antibodies against GP. Further investigation is warranted to determine whether these close contacts truly experienced subclinical infections and whether these asymptomatic infections played a role in the dynamics of transmission. [ABSTRACT FROM AUTHOR]

The N-linked glycosylation motif at amino acid position 154–156 of the envelope (E) protein of West Nile virus (WNV) is linked to enhanced murine neuroinvasiveness, avian pathogenicity and vector competence. Naturally occurring isolates with altered E protein glycosylation patterns have been observed in WNV isolates; however, the specific effects of these polymorphisms on avian host pathogenesis and vector competence have not been investigated before. In the present study, amino acid polymorphisms, NYT, NYP, NYF, SYP, SYS, KYS and deletion (A’DEL), were reverse engineered into a parental WNV (NYS) cDNA infectious clone to generate WNV glycosylation mutant viruses. These WNV glycosylation mutant viruses were characterized for in vitro growth, pH-sensitivity, temperature-sensitivity and host competence in American crows (AMCR), house sparrows (HOSP) and Culex quinquefasciatus. The NYS and NYT glycosylated viruses showed higher viral replication, pH and temperature sensitivity than NYP, NYF, SYP, SYS, KYS and A’DEL viruses in vitro. Interestingly, in vivo results demonstrated asymmetric effects in avian and mosquito competence that were independent of the E-protein glycosylation status. In AMCRs and HOSPs, all viruses showed comparable viremias with the exception of NYP and KYS viruses that showed attenuated phenotypes. Only NYP showed reduced vector competence in both Cx. quinquefasciatus and Cx. tarsalis. Glycosylated NYT exhibited similar avian virulence properties as NYS, but resulted in higher mosquito oral infectivity than glycosylated NYS and nonglycosylated, NYP, NYF, SYP and KYS mutants. These data demonstrated that amino acid polymorphisms at E154/156 dictate differential avian host and vector competence phenotypes independent of E-protein glycosylation status. [ABSTRACT FROM AUTHOR]

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging human pathogen naturally transmitted by ticks, has spread widely since it was first detected in 2010. Although SFTSV-specific antibodies have been detected in wild birds, these natural reservoir and amplifying hosts for the virus have not been well studied. Methodology/Principle findings: Here we report an experimental infection of spotted doves (Streptopelia chinensis) with two strains of SFTSV, JS2010-14, a Chinese lineage strain, and JS2014-16, a Japanese lineage strain, which represent the main viral genotypes currently circulating in East Asia. In these studies, we have determined that spotted doves are susceptible to SFTSV and the severity of the viremia is dose-dependent. When challenged with 107 and 105 PFU, all doves developed viremia which peaked 3–5 days post infection (dpi). Only a subset (25–62.5%) of the birds developed viremia when challenged at 103 PFU. Virulence of SFTSV in spotted doves was strain dependent. Infection with 107 PFU of strain JS2014-16 resulted in 12.5% mortality over 6.8 days and mean peak viremia titers of 106.9 PFU/mL in experimentally inoculated birds. All doves inoculated with 107 PFU of the JS2010-14 strain survived infection with relatively lower mean viremia titers (105.6 PFU/mL at peak) over 6.1 days. Conclusions/Significance: Our results suggest that spotted doves, one of the most abundant bird species in China, could be a competent amplifying host for SFTSV and play an important role in its ecology. Between the two SFTSV strains, the strain of the Japanese lineage caused mortality, higher viremia titers in infected birds over a longer time period than did the Chinese strain. Our observations shed light on the ecology of SFTSV, which could benefit the implementation of surveillance and control programs. [ABSTRACT FROM AUTHOR]

Nipah virus (NiV) is a highly pathogenic re-emerging virus that causes outbreaks in South East Asia. Currently, no approved and licensed vaccine or antivirals exist. Here, we investigated the efficacy of ChAdOx1 NiVB, a simian adenovirus-based vaccine encoding NiV glycoprotein (G) Bangladesh, in Syrian hamsters. Prime-only as well as prime-boost vaccination resulted in uniform protection against a lethal challenge with NiV Bangladesh: all animals survived challenge and we were unable to find infectious virus either in oral swabs, lung or brain tissue. Furthermore, no pathological lung damage was observed. A single-dose of ChAdOx1 NiVB also prevented disease and lethality from heterologous challenge with NiV Malaysia. While we were unable to detect infectious virus in swabs or tissue of animals challenged with the heterologous strain, a very limited amount of viral RNA could be found in lung tissue by in situ hybridization. A single dose of ChAdOx1 NiVB also provided partial protection against Hendra virus and passive transfer of antibodies elicited by ChAdOx1 NiVB vaccination partially protected Syrian hamsters against NiV Bangladesh. From these data, we conclude that ChAdOx1 NiVB is a suitable candidate for further NiV vaccine pre-clinical development. [ABSTRACT FROM AUTHOR]

Background: Multiple mutations in the voltage-gated sodium channel have been associated with knockdown resistance (kdr) to DDT and pyrethroid insecticides in a major human disease vector Aedes aegypti. One mutation, V1016G, confers sodium channel resistance to pyrethroids, but a different substitution in the same position V1016I alone had no effect. In pyrethroid-resistant Ae. aegypti populations, V1016I is often linked to another mutation, F1534C, which confers sodium channel resistance only to Type I pyrethroids including permethrin (PMT), but not to Type II pyrethroids including deltamethrin (DMT). Mosquitoes carrying both V1016G and F1534C exhibited a greater level of pyrethroid resistance than those carrying F1534C alone. More recently, a new mutation T1520I co-existing with F1534C was detected in India. However, whether V1016I or T1520I enhances pyrethroid resistance of sodium channels carrying F1534C remains unknown. Methodology/Principal findings: V1016I, V1016G, T1520I and F1534C substitutions were introduced alone and in various combinations into AaNav1-1, a sodium channel from Aedes aegypti. The mutant channels were then expressed in Xenopus oocytes and examined for channel properties and sensitivity to pyrethroids using the two-electrode voltage clamping technique. The results showed that V1016I or T1520I alone did not alter the AaNav1-1 sensitivity to PMT or DMT. However, the double mutant T1520I+F1534C was more resistant to PMT than F1534C, but remained sensitive to DMT. In contrast, the double mutant V1016I+F1534C was resistant to DMT and more resistant to PMT than F1534C. Furthermore, V1016I/G and F1534C channels, but not T1520I, were resistant to dichlorodiphenyltrichloroethane (DDT). Cryo-EM structures of sodium channels suggest that T1520I allosterically deforms geometry of the pyrethroid receptor site PyR1 in AaNav1-1. The small deformation does not affect binding of DDT, PMT or DMT, but in combination with F1534C it increases the channel resistance to PMT and DDT. Conclusions/Significance: Our data corroborated the previously proposed sequential selection of kdr mutations in Ae. aegypti. We proposed that mutation F1534C first emerged in response to DDT/pyrethroids providing a platform for subsequent selection of mutations V1016I and T1520I that confer greater and broader spectrum of pyrethroid resistance. [ABSTRACT FROM AUTHOR]

Background: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. Methods and findings: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1–10 eggs per gram of feces that were undiagnosed by KK parasitological technique. Conclusions: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei causes melioidosis, a common source of pneumonia and sepsis in Southeast Asia and Northern Australia that results in high mortality rates. A caprine melioidosis model of aerosol infection that leads to a systemic infection has the potential to characterize the humoral immune response. This could help identify immunogenic proteins for new diagnostics and vaccine candidates. Outbred goats may more accurately mimic human infection, in contrast to the inbred mouse models used to date. B. pseudomallei infection was delivered as an intratracheal aerosol. Antigenic protein profiling was generated from the infecting strain MSHR511. Humoral immune responses were analyzed by ELISA and western blot, and the antigenic proteins were identified by mass spectrometry. Throughout the course of the infection the assay results demonstrated a much greater humoral response with IgG antibodies, in both breadth and quantity, compared to IgM antibodies. Pre-infection sera showed multiple immunogenic proteins already reactive for IgG (7–20) and IgM (0–12) in most of the goats despite no previous exposure to B. pseudomallei. After infection, the number of IgG reactive proteins showed a marked increase as the disease progressed. Early stage infection (day 7) showed immune reaction to chaperone proteins (GroEL, EF-Tu, and DnaK). These three proteins were detected in all serum samples after infection, with GroEL immunogenically dominant. Seven common reactive antigens were selected for further analysis using ELISA. The heat shock protein GroEL1 elicited the strongest goat antibody immune response compared to the other six antigens. Most of the six antigens showed the peak IgM reactivity at day 14, whereas the IgG reactivity increased further as the disease progressed. An overall MSHR511 proteomic comparison between the goat model and human sera showed that many immune reactive proteins are common between humans and goats with melioidosis. [ABSTRACT FROM AUTHOR]

Background: Mammarenaviruses are associated with human hemorrhagic fever diseases in Africa and America. Recently, a rodent mammarenavirus, Wēnzhōu virus (WENV) and related viruses, have been reported in China, Cambodia, and Thailand. Moreover, in Cambodia, these viruses were suspected to be associated with human disease. In China, Yunnan Province is famous for its abundant animal and plant diversity and is adjacent to several South-eastern Asia countries. Therefore, it is necessary to know whether WENV-related viruses, or other mammarenaviruses, are prevalent in this province. Methodology/Principal findings: Small mammals were trapped, euthanized, and sampled. Mammarenavirus RNA was detected using a nested reverse transcription polymerase chain reaction (RT-PCR) and quantified by real-time RT-PCR. A total of 1040 small mammals belonging to 13 genera and 26 species were trapped in Yunnan Province. WENV-related mammarenaviruses were detected in 41 rodent liver samples, mainly in brown rats (Rattus norvegicus) and oriental house rats (R. tanezumi).Viral nucleocapsid protein was detected in liver sections by indirect immunofluorescence assay. Full-length-genomes were amplified by RT-PCR and used for phylogenetic analysis with the MEGA package. Recombination analysis was performed using the SimPlot and Recombination Detection Program. Conclusions/Significance: WENV related viruses circulated in small mammals in Yunnan Province. Whole genome sequence analysis of five selected viral strains showed that these viruses are closely related to WENVs discovered in Asia and form an independent branch in the phylogenetic tree in the WENV clade. Paying attention to investigate the influence of these viruses to public health is essential in the epidemic regions. [ABSTRACT FROM AUTHOR]

Background: Hosts repeatedly bitten by sand flies develop antibodies against sand fly saliva and screening of these immunoglobulins can be employed to estimate the risk of Leishmania transmission, to indicate the feeding preferences of sand flies, or to evaluate the effectiveness of vector control campaigns. Previously, antibodies to sand fly saliva were detected using whole salivary gland homogenate (SGH) or recombinant proteins, both of which also have their disadvantages. This is the first study on sand flies where short peptides designed based on salivary antigens were successfully utilized for antibody screening. Methodology/Principal findings: Specific IgG was studied in hosts naturally exposed to Phlebotomus orientalis, the main vector of Leishmania donovani in East Africa. Four peptides were designed by the commercial program EpiQuest-B, based on the sequences of the two most promising salivary antigens, yellow-related protein and ParSP25-like protein. Short amino acid peptides were synthesised and modified for ELISA experiments. Specific anti-P. orientalis IgG was detected in sera of dogs, goats, and sheep from Ethiopia. The peptide OR24 P2 was shown to be suitable for antibody screening; it correlated positively with SGH and its specificity and sensitivity were comparable or even better than that of previously published recombinant proteins. Conclusions/Significance: OR24 P2, the peptide based on salivary antigen of P. orientalis, was shown to be a valuable tool for antibody screening of domestic animals naturally exposed to P. orientalis. We suggest the application of this promising methodology using species-specific short peptides to other sand fly-host combinations. [ABSTRACT FROM AUTHOR]

Background: Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed. Methodology/Principal findings: Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity. Conclusions/Significance: The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom. [ABSTRACT FROM AUTHOR]

Background: We examined relationships between schistosome infection, HIV transmission or acquisition, and all-cause death. Methods: We retrospectively tested baseline sera from a heterosexual HIV-discordant couple cohort in Lusaka, Zambia with follow-up from 1994–2012 in a nested case-control design. Schistosome-specific antibody levels were measured by ELISA. Associations between baseline antibody response to schistosome antigens and incident HIV transmission, acquisition, and all-cause death stratified by gender and HIV status were assessed. In a subset of HIV- women and HIV+ men, we performed immunoblots to evaluate associations between Schistosoma haematobium or Schistosoma mansoni infection history and HIV incidence. Results: Of 2,145 individuals, 59% had positive baseline schistosome-specific antibody responses. In HIV+ women and men, baseline schistosome-specific antibodies were associated with HIV transmission to partners (adjusted hazard ratio [aHR] = 1.8, p<0.005 and aHR = 1.4, p<0.05, respectively) and death in HIV+ women (aHR = 2.2, p<0.001). In 250 HIV- women, presence of S. haematobium-specific antibodies was associated with increased risk of HIV acquisition (aHR = 1.4, p<0.05). Conclusion: Schistosome infections were associated with increased transmission of HIV from both sexes, acquisition of HIV in women, and increased progression to death in HIV+ women. Establishing effective prevention and treatment strategies for schistosomiasis, including in urban adults, may reduce HIV incidence and death in HIV+ persons living in endemic areas. [ABSTRACT FROM AUTHOR]

Human rabies is an encephalitic disease transmitted by animals infected with lyssaviruses. The most common lyssavirus that causes human infection is rabies virus (RABV), the prototypic member of the genus. The incubation period of RABV in humans varies from few weeks to several months in some instances. During this prodromal period, neither antibodies nor virus is detected. Antibodies, antigen and nucleic acids are detectable only after the onset of encephalitic symptoms, at which point the outcome of the disease is nearly 100% fatal. Hence, the primary intervention for human RABV exposure and subsequent post-exposure prophylaxis relies on testing animals suspected of having rabies. The most widely used diagnostic tests in animals focus on antigen detection, RABV-encoded nucleoprotein (N protein) in brain tissues. N protein accumulates in the cytoplasm of infected cells as large and granular inclusions, which are visualized in infected brain tissues by immuno-microscopy using anti-N protein antibodies. In this study, we explored a mass spectrometry (MS) based method for N protein detection without the need for any specific antibody reagents or microscopy. The MS-based method described here is unbiased, label-free, requires no amplification and determines any previously sequenced N protein available in the database. The results demonstrate the ability of MS/MS based method for N protein detection and amino acid sequence determination in animal diagnostic samples to obtain RABV variant information. This study demonstrates a potential for future developments of rabies diagnostic tests based on MS platforms. [ABSTRACT FROM AUTHOR]

Background: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. Methodology/Principal findings: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. Conclusions/Significance: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus. [ABSTRACT FROM AUTHOR]

Background: Scavenger Receptors (SRs) from the host’s innate immune system are known to bind multiple ligands to promote the removal of non-self or altered-self targets. CD5 and CD6 are two highly homologous class I SRs mainly expressed on all T cells and the B1a cell subset, and involved in the fine tuning of activation and differentiation signals delivered by the antigen-specific receptors (TCR and BCR, respectively), to which they physically associate. Additionally, CD5 and CD6 have been shown to interact with and sense the presence of conserved pathogen-associated structures from bacteria, fungi and/or viruses. Methodology/Principal findings: We report herein the interaction of CD5 and CD6 lymphocyte surface receptors with Echinococcus granulosus sensu lato (s.l.). Binding studies show that both soluble and membrane-bound forms of CD5 and CD6 bind to intact viable protoscoleces from E. granulosus s.l. through recognition of metaperiodate-resistant tegumental components. Proteomic analyses allowed identification of thioredoxin peroxidase for CD5, and peptidyl-prolyl cis-trans isomerase (cyclophilin) and endophilin B1 (antigen P-29) for CD6, as their potential interactors. Further in vitro assays demonstrate that membrane-bound or soluble CD5 and CD6 forms differentially modulate the pro- and anti-inflammatory cytokine release induced following peritoneal cells exposure to E. granulosus s.l. tegumental components. Importantly, prophylactic infusion of soluble CD5 or CD6 significantly ameliorated the infection outcome in the mouse model of secondary cystic echinococcosis. Conclusions/Significance: Taken together, the results expand the pathogen binding properties of CD5 and CD6 and provide novel evidence for their therapeutic potential in human cystic echinococcosis. [ABSTRACT FROM AUTHOR]

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens. [ABSTRACT FROM AUTHOR]

The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis. [ABSTRACT FROM AUTHOR]

Background: Tanzania is among the Rift Valley fever (RVF) epizootic/endemic countries in sub Saharan Africa, where RVF disease outbreaks occur within a range of 3 to 17-year intervals. Detection of Rift Valley fever virus (RVFV) antibodies in animals in regions with no previous history of outbreaks raises the question of whether the disease is overlooked due to lack-of effective surveillance systems, or if there are strains of RVFV with low pathogenicity. Furthermore, which vertebrate hosts are involved in the inter-epidemic and inter-epizootic maintenance of RVFV? In our study region, the Kyela and Morogoro districts in Tanzania, no previous RVF outbreaks have been reported. Methodology: The study was conducted from June 2014 to October 2015 in the Kyela and Morogoro districts, Tanzania. Samples (n = 356) were retrieved from both the local breed of zebu cattle (Bos indicus) and Bos indicus/Bos Taurus cross breed. RVFV antibodies were analyzed by two enzyme-linked immunosorbent assay (ELISA) approaches. Initially, samples were analyzed by a RVFV multi-species competition ELISA (cELISA), which detected both RVFV IgG and IgM antibodies. All serum samples that were positive with the cELISA method were specifically analysed for the presence of RVFV IgM antibodies to trace recent infection. A plaque reduction neutralization assay (PRNT80) was performed to determine presence of RVFV neutralizing antibodies in all cELISA positive samples. Findings: Overall RVFV seroprevalence rate in cattle by cELISA in both districts was 29.2% (104 of 356) with seroprevalence rates of 33% (47/147) in the Kyela district and 27% (57/209) in the Morogoro district. In total, 8.4% (30/356) of all cattle sampled had RVFV IgM antibodies, indicating current disease transmission. When segregated by districts, the IgM antibody seroprevalence was 2.0% (3/147) and 12.9% (27/209) in Kyela and Morogoro districts respectively. When the 104 cELISA positive samples were analyzed by PRNT80 to confirm that RVFV-specific antibodies were present, the majority (89%, 93/104) had RVFV neutralising antibodies. Conclusion: The results provided evidence of widespread prevalence of RVFV antibody among cattle during an inter-epizootic/inter-epidemic period in Tanzania in regions with no previous history of outbreaks. There is a need for further investigations of RVFV maintenance and transmission in vertebrates and vectors during the long inter-epizootic/inter-epidemic periods. [ABSTRACT FROM AUTHOR]

The transmission dynamics of mosquito-vectored pathogens are, in part, mediated by mosquito host-feeding patterns. These patterns are elucidated using blood meal analysis, a collection of serological and molecular techniques that determine the taxonomic identities of the host animals from which blood meals are derived. Modern blood meal analyses rely on polymerase chain reaction (PCR), DNA sequencing, and bioinformatic comparisons of blood meal DNA sequences to reference databases. Ideally, primers used in blood meal analysis PCRs amplify templates from a taxonomically diverse range of vertebrates, produce a short amplicon, and avoid co-amplification of non-target templates. Few primer sets that fit these requirements are available for the cytochrome c oxidase subunit I (COI) gene, the species identification marker with the highest taxonomic coverage in reference databases. Here, we present new primer sets designed to amplify fragments of the DNA barcoding region of the vertebrate COI gene, while avoiding co-amplification of mosquito templates, without multiplexed or nested PCR. Primers were validated using host vertebrate DNA templates from mosquito blood meals of known origin, representing all terrestrial vertebrate classes, and field-collected mosquito blood meals of unknown origin. We found that the primers were generally effective in amplifying vertebrate host, but not mosquito DNA templates. Applied to the sample of unknown mosquito blood meals, > 98% (60/61) of blood meals samples were reliably identified, demonstrating the feasibility of identifying mosquito hosts with the new primers. These primers are beneficial in that they can be used to amplify COI templates from a diverse range of vertebrate hosts using standard PCR, thereby streamlining the process of identifying the hosts of mosquitoes, and could be applied to next generation DNA sequencing and metabarcoding approaches. [ABSTRACT FROM AUTHOR]

Japanese encephalitis is mainly considered a rural disease, but there is growing evidence of a peri-urban and urban transmission in several countries, including Cambodia. We, therefore, compared the epidemiologic dynamic of Japanese encephalitis between a rural and a peri-urban setting in Cambodia. We monitored two cohorts of 15 pigs and determined the force of infection–rate at which seronegative pigs become positive–in two study farms located in a peri-urban and rural area, respectively. We also studied the mosquito abundance and diversity in proximity of the pigs, as well as the host densities in both areas. All the pigs seroconverted before the age of 6 months. The force of infection was 0.061 per day (95% confidence interval = 0.034–0.098) in the peri-urban cohort and 0.069 per day (95% confidence interval = 0.047–0.099) in the rural cohort. Several differences in the epidemiologic dynamic of Japanese encephalitis between both study sites were highlighted. The later virus amplification in the rural cohort may be linked to the later waning of maternal antibodies, but also to the higher pig density in direct proximity of the studied pigs, which could have led to a dilution of mosquito bites at the farm level. The force of infection was almost identical in both the peri-urban and the rural farms studied, which shifts the classic epidemiologic cycle of the virus. This study is a first step in improving our understanding of Japanese encephalitis virus ecology in different environments with distinct landscapes, human and animal densities. [ABSTRACT FROM AUTHOR]

Background: Canine leishmaniasis (CanL) is a zoonotic disease, caused by Leishmania infantum and transmitted by Phlebotomus perniciosus in the Mediterranean basin. Previously, an ELISA based on the P. perniciosus salivary protein SP03B was proposed as a valid tool to screen for canine exposure to sand fly bites across regions endemic for CanL. Although this approach is useful in laboratory settings, a practical tool for immediate application in the field is needed. In this study we propose the rSP03B sero-strip, the first immunochromatographic test (ICT) in the field of vector exposure able to rapidly screen dogs living in endemic areas for the presence of P. perniciosus and to aid in the evaluation of vector control programs. Methodology/Principal findings: The ICT was prepared using the bacterially expressed recombinant protein rSP03B as antigen. For test optimization, pre-immune sera from non-bitten laboratory-bred Beagles were used as negative controls. In order to validate the test, sera from laboratory-bred Beagles experimentally exposed to P. perniciosus bites were used as positive controls. Additionally, all samples were tested by ELISA using whole salivary gland homogenate (SGH) and the rSP03B protein as antigen. An almost perfect degree of agreement was found between the ICT and the SGH-ELISA. Furthermore, the newly proposed rSP03B sero-strip showed a sensitivity of 100% and a specificity of 86.79%. Conclusions/Significance: We developed a simple and rapid ICT based on the P. perniciosus rSP03B salivary protein, able to replace the standard ELISA used in previous studies. Our rSP03B sero-strip showed to be highly sensitive and specific in the detection of antibodies (IgG) against P. perniciosus saliva. In the future, this test can be employed during large-scale epidemiological studies of CanL in the Mediterranean area to evaluate the efficacy of vector control programs. [ABSTRACT FROM AUTHOR]

Concerns regarding outbreaks of human monkeypox or the potential reintroduction of smallpox into an immunological naïve population have prompted the development of animal models and countermeasures. Here we present a marmoset model of monkeypox and smallpox disease utilizing a relevant poxvirus via a natural exposure route. We found that 1000 plaque forming units (PFU) of Monkeypox virus was sufficient to recapitulate smallpox disease, to include an incubation period of approximately 13 days, followed by the onset of rash, and death between 15 and 17 days. Temporally accurate manifestation of viremia and oral shedding were also features. The number of lesions ranged from no lesions to 299, the most reported in a marmoset exposed to a poxvirus. To both evaluate the efficacy of our antibodies and the applicability of the model system, marmosets were prophylactically treated with two monoclonal antibodies, c7D11 and c8A. Of three marmosets, two were completely free of disease and a single marmoset died 8 days after the mock (n = 1) or PBS control(s) (n = 2). Evaluation of the serum levels of the three animals provided a possible explanation to the animal succumbing to disease. Interestingly, more females had lesions (and a greater number of lesions) and lower viral burden (viremia and oral shedding) than males in our studies, suggesting a possible gender effect. [ABSTRACT FROM AUTHOR]

Background: Dengue is one of the most serious and rapidly spreading arboviral diseases in the world. Despite many acute febrile illnesses in Ethiopia, the burden of illness due to dengue in the country is largely unknown. Thus, the present study aimed to provide the first baseline data on seroprevalence and associated risk factors of dengue virus (DENV) infection in the country. Methods: A cross-sectional study of febrile patients who were visiting Metema and Humera hospitals in Northwest Ethiopia from March 2016 to May 2017 was conducted. Blood samples were collected from each participant and serum samples were separated and tested for IgM and IgG antibodies against DENV infection by enzyme-linked immunosorbent assay (ELISA). Risk factors associated with the prevalence of anti-DENV antibodies were tested using logistic regression analysis. Results: Of the 600 samples tested, the overall seroprevalence against DENV infection was 33.3%, while the seroprevalence by the study area was 40% in Metema and 27.5% in Humera. The overall prevalence of IgM and IgG antibodies against DENV infection was 19% and 21% respectively. Of these, 6.7% were positive for both IgM and IgG antibodies. Residence and occupational status were significantly associated with the prevalence of anti-DENV IgM seropositivity and anti-DENV IgM-/G+serostatus. The seasonal variation was significantly associated with the prevalence of anti-DENV IgM but not with anti-DENV IgM-/G+serostatus. The prevalence of anti-DENV IgM-/G+serostatus was significantly higher in Metema than Humera. High prevalence of anti-DENV IgM seropositivity was found in the summer and spring, with a peak in the month of August. The presence of uncovered water either indoor or outdoor and lack of mosquito net use was identified as risk factors for DENV infection. Conclusions: These findings provide the preliminary data on seroprevalence and associated risk factors of DENV infection in the country. The presence of antibodies against DENV infection indicates dengue as one of the causes of undifferentiated febrile illnesses in the study areas. This suggests that prevention and control measures should be designed considering the risk factors identified by this study. Furthermore, we recommend a large-scale study to include DENV infection in the differential diagnosis of all febrile illnesses in Ethiopia. [ABSTRACT FROM AUTHOR]

The dynamics of dengue virus (DENV) circulation depends on serotype, genotype and lineage replacement and turnover. In São José do Rio Preto, Brazil, we observed that the L6 lineage of DENV-1 (genotype V) remained the dominant circulating lineage even after the introduction of the L1 lineage. We investigated viral fitness and immunogenicity of the L1 and L6 lineages and which factors interfered with the dynamics of DENV epidemics. The results showed a more efficient replicative fitness of L1 over L6 in mosquitoes and in human and non-human primate cell lines. Infections by the L6 lineage were associated with reduced antigenicity, weak B and T cell stimulation and weak host immune system interactions, which were associated with higher viremia. Our data, therefore, demonstrate that reduced viral immunogenicity and consequent greater viremia determined the increased epidemiological fitness of DENV-1 L6 lineage in São José do Rio Preto. [ABSTRACT FROM AUTHOR]

Background: Trichinellosis is a serious zoonositc parasitosis worldwide. Because its clinical manifestations aren’t specific, the diagnosis of trichinellosis is not easy to be made. Trichinella spiralis muscle larva (ML) excretory–secretory (ES) antigens are the most widely applied diagnostic antigens for human trichinellosis, but the major drawback of the ES antigens for assaying anti-Trichinella antibodies is the false negative in the early Trichinella infection period. The aim of this study was to characterize the T. spiralis putative serine protease (TsSP) and to investigate its potential use for diagnosis of trichinellosis. Methodology/Principal findings: The full-length TsSP sequence was cloned and expressed, and recombinant TsSP (rTsSP) was purified by Ni-NTA-Sefinose Column. On Western blotting analysis the rTsSP was recognized by T. spiralis-infected mouse serum, and the natural TsSP was identified in T. spiralis ML crude and ES antigens by using anti-rTsSP serum. Expression of TsSP was detected at various T. spiralis developmental stages (newborn larvae, muscle larvae, intestinal infective larvae and adult worms). Immunolocalization identified the TsSP principally in cuticles and stichosomes of the nematode. The sensitivity of rTsSP-ELISA and ES-ELISA was 98.11% (52/53) and 88.68% (47/53) respectively (P > 0.05) when the sera from trichinellosis patients were examined. However, while twenty-one serum samples of trichinellosis patients’ sera at 19 days post-infection (dpi) were tested, the sensitivity (95.24%) of rTsSP-ELISA was distinctly higher than 71.43% of ES-ELISA (P < 0.05). The specificity (99.53%) of rTsSP-ELISA was remarkably higher than 91.98% of ES-ELISA (P < 0.01). Only one out of 20 serum samples of cysticercosis patients cross-reacted with the rTsSP. Specific anti-Trichinella IgG in infected mice was first detected by rTsSP-ELISA as soon as 7 dpi and antibody positive rate reached 100% on 10 dpi, whereas the ES-ELISA did not permit detection of 100% of infected mice before 16 dpi. Conclusions: The rTsSP is a potential early diagnostic antigen for human trichinellosis. [ABSTRACT FROM AUTHOR]

Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease estimated to affect over 200 million people worldwide. Praziquantel is the only antischistosomal currently available for treatment, and there is an urgent need for new therapeutics. Ion channels play key roles in physiology and are targets for many anthelmintics, yet only a few representatives have been characterized in any detail in schistosomes and other parasitic helminths. The transient receptor potential (TRP) channel superfamily comprises a diverse family of non-selective cation channels that play key roles in sensory transduction and a wide range of other functions. TRP channels fall into several subfamilies. Members of both the TRPA and TRPV subfamilies transduce nociceptive and inflammatory signals in mammals, and often also respond to chemical and thermal signals. We previously showed that although schistosomes contain no genes predicted to encode TRPV channels, TRPV1-selective activators such as capsaicin and resiniferatoxin elicit dramatic hyperactivity in adult worms and schistosomula. Surprisingly, this response requires expression of a S. mansoni TRPA1-like orthologue (SmTRPA). Here, we show that capsaicin induces a rise in intracellular Ca2+ in mammalian cells expressing either SmTRPA or a S. haematobium TRPA1 orthologue (ShTRPA). We also test SmTRPA and ShTRPA responses to various TRPV1 and TRPA1 modulators. Interestingly, in contrast to SmTRPA, ShTRPA is not activated by the TRPA1 activator AITC (allyl isothiocyanate), nor do S. haematobium adult worms respond to this compound, a potentially intriguing species difference. Notably, 4-hydroxynonenal (4-HNE), a host-derived, inflammatory product that directly activates mammalian TRPA1, also activates both SmTRPA and ShTRPA. Our results point to parasite TRPA1-like channels which exhibit atypical, mixed TRPA1/TRPV1-like pharmacology, and which may also function to transduce endogenous host signals. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that has caused large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Currently, no licensed vaccine or therapeutics exists to treat this potentially deadly disease. The explosive nature of RVFV outbreaks and the severe consequences of its accidental or intentional introduction into RVFV-free areas provide the impetus for the development of novel vaccine candidates for use in both livestock and humans. Rationally designed vaccine candidates using reverse genetics have been used to develop deletion mutants of two known RVFV virulence factors, the NSs and NSm genes. These recombinant viruses were demonstrated to be protective and immunogenic in rats, mice, and sheep, without producing clinical illness in these animals. Here, we expand upon those findings and evaluate the single deletion mutant (ΔNSs rRVFV) and double deletion mutant (ΔNSs-ΔNSm rRVFV) vaccine candidates in the common marmoset (Callithrix jacchus), a non-human primate (NHP) model resembling severe human RVF disease. We demonstrate that both the ΔNSs and ΔNSs-ΔNSm rRVFV vaccine candidates were found to be safe and immunogenic in the current study. The vaccinated animals received a single dose of vaccine that led to the development of a robust antibody response. No vaccine-induced adverse reactions, signs of clinical illness or infectious virus were detected in the vaccinated marmosets. All vaccinated animals that were subsequently challenged with RVFV were protected against viremia and liver disease. In summary, our results provide the basis for further development of the ΔNSs and ΔNSs-ΔNSm rRVFV as safe and effective human RVFV vaccines for this significant public health threat. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is a liver fluke that can dwell in the bile ducts of mammals. Bile acid transporters function to maintain the homeostasis of bile acids in C. sinensis, as they induce physiological changes or have harmful effects on C. sinensis survival. The organic solute transporter (OST) transports mainly bile acid and belongs to the SLC51 subfamily of solute carrier transporters. OST plays a critical role in the recirculation of bile acids in higher animals. In this study, we cloned full-length cDNA of the 480- amino acid OST from C. sinensis (CsOST). Genomic analysis revealed 11 exons and nine introns. The CsOST protein had a ‘Solute_trans_a’ domain with 67% homology to Schistosoma japonicum OST. For further analysis, the CsOST protein sequence was split into the ordered domain (CsOST-N) at the N-terminus and disordered domain (CsOST-C) at the C-terminus. The tertiary structure of each domain was built using a threading-based method and determined by manual comparison. In a phylogenetic tree, the CsOST-N domain belonged to the OSTα and CsOST-C to the OSTβ clade. These two domains were more highly conserved with the OST α- and β-subunits at the structure level than at sequence level. These findings suggested that CsOST comprised the OSTα- and β-subunits. CsOST was localized in the oral and ventral suckers and in the mesenchymal tissues abundant around the intestine, vitelline glands, uterus, and testes. This study provides fundamental data for the further understanding of homologues in other flukes. [ABSTRACT FROM AUTHOR]

Background: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied. Here, we characterize the structural and proteolytic attributes of serine protease 2 (SmSP2) from Schistosoma mansoni, one of the major species responsible for the tropical infectious disease, schistosomiasis. Methodology/Principal findings: SmSP2 comprises three domains: a histidine stretch, TSR-1 and a serine protease domain. The cleavage specificity of recombinant SmSP2 was determined using positional scanning and multiplex combinatorial libraries and the determinants of specificity were identified with 3D homology models, demonstrating a trypsin-like endopeptidase mode of action. SmSP2 displayed restricted proteolysis on protein substrates. It activated tissue plasminogen activator and plasminogen as key components of the fibrinolytic system, and released the vasoregulatory peptide, kinin, from kininogen. SmSP2 was detected in the surface tegument, esophageal glands and reproductive organs of the adult parasite by immunofluorescence microscopy, and in the excretory/secretory products by immunoblotting. Conclusions/Significance: The data suggest that SmSP2 is secreted, functions at the host-parasite interface and contributes to the survival of the parasite by manipulating host vasodilatation and fibrinolysis. SmSP2 may be, therefore, a potential target for anti-schistosomal therapy. [ABSTRACT FROM AUTHOR]

The anthelmintic praziquantel (±PZQ) serves as a highly effective antischistosomal therapy. ±PZQ causes a rapid paralysis of adult schistosome worms and deleterious effects on the worm tegument. In addition to these activities against the parasite, ±PZQ also modulates host vascular tone in blood vessels where the adult worms reside. In resting mesenteric arteries ±PZQ causes a constriction of basal tone, an effect mediated by (R)-PZQ activation of endogenous serotoninergic G protein coupled receptors (GPCRs). Here, we demonstrate a novel vasodilatory action of ±PZQ in mesenteric vessels that are precontracted by high potassium-evoked depolarization, an effect previously reported to be associated with agonists of the transient receptor potential melastatin 8 channel (TRPM8). Pharmacological profiling a panel of 17 human TRPs demonstrated ±PZQ activity against a subset of human TRP channels. Several host TRP channels (hTRPA1, hTRPC3, hTRPC7) were activated by both (R)-PZQ and (S)-PZQ over a micromolar range whereas hTRPM8 showed stereoselective activation by (S)-PZQ. The relaxant effect of ±PZQ in mesenteric arteries was caused by (S)-PZQ, not (R)-PZQ, and mimicked by TRPM8 agonists. However, persistence of both (S)-PZQ and TRPM8 agonist evoked vessel relaxation in TRPM8 knockout tissue suggested that canonical TRPM8 does not mediate this (S)-PZQ effect. We conclude that (S)-PZQ is vasoactive over the micromolar range in mesenteric arteries although the molecular mediators of this effect remain to be identified. These data expand our knowledge of the polypharmacology and host vascular efficacy of this clinically important anthelmintic. [ABSTRACT FROM AUTHOR]

Ticks salivate while feeding on their hosts. Saliva helps blood feeding through host anti-hemostatic and immunomodulatory components. Previous transcriptomic and proteomic studies revealed the complexity of tick saliva, comprising hundreds of polypeptides grouped in several multi-genic families such as lipocalins, Kunitz-domain containing peptides, metalloproteases, basic tail secreted proteins, and several other families uniquely found in ticks. These studies also revealed that the composition of saliva changes with time; expression of transcripts from the same family wax and wane as a function of feeding time. Here, we examined whether host immune factors could influence sialome switching by comparing sialomes of ticks fed naturally on a rabbit, to ticks artificially fed on defibrinated blood depleted of immune components. Previous studies were based on transcriptomes derived from pools of several individuals. To get an insight into the uniqueness of tick sialomes, we performed transcriptomic analyses of single salivary glands dissected from individual adult female I. ricinus ticks. Multivariate analysis identified 1,279 contigs differentially expressed as a function of time and/or feeding mode. Cluster analysis of these contigs revealed nine clusters of differentially expressed genes, four of which appeared consistently across several replicates, but five clusters were idiosyncratic, pointing to the uniqueness of sialomes in individual ticks. The disclosure of tick quantum sialomes reveals the unique salivary composition produced by individual ticks as they switch their sialomes throughout the blood meal, a possible mechanism of immune evasion. [ABSTRACT FROM AUTHOR]

Brucellosis is an important zoonotic disease causing huge economic losses worldwide. Currently no effective immunotherapy for Brucellosis or any biomarker to monitor the efficacy of therapy is available. Treatment is ineffective and animals remain carrier lifelong. S19 and RB51 are live attenuated vaccine strains of Brucella abortus. However, S19 induces only antibody, ineffective for intracellular pathogen. RB51 induces cell mediated immunity (CMI) but it is Rifampicin resistant. Both organisms are secreted in milk and can infect humans and cause abortions in animals. Phage lysed bacteria (lysates) retain maximum immunogenicity as opposed to killing by heat or chemicals. We report here the successful immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). The SL induced strong antibody response and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma production. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live virulent Brucella from Brucellosis affected cattle with plasma level of Brucella specific 223 bp amplicon undetectable by RT-PCR and blood negative for live Brucella by culture in 3 monthspost-immunization. This is the first report on minimally invasive monitoring of the efficacy of antibacterial therapy employing plasma RNA specific for live bacteria as a biomarker as well as on the use of RB51 phage lysate for successful immunotherapy of Brucellosis in cattle. [ABSTRACT FROM AUTHOR]

Background: Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. Methodology/Principal findings: We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. Conclusions/Significance: This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens. [ABSTRACT FROM AUTHOR]

Background: Current WHO recommendations for lymphatic filariasis (LF) surveillance advise programs to implement activities to monitor for new foci of transmission after stopping mass drug administration (MDA). A current need in the global effort to eliminate LF is to standardize diagnostic tools and surveillance activities beyond the recommended transmission assessment survey (TAS). Methodology: TAS was first conducted in American Samoa in 2011 (TAS 1) and a repeat TAS was carried out in 2015 (TAS 2). Circulating filarial antigen (CFA) and serologic results from both surveys were analyzed to determine whether interruption of LF transmission has been achieved in American Samoa. Principal findings: A total of 1,134 and 864 children (5–10 years old) were enrolled in TAS 1 and TAS 2, respectively. Two CFA-positive children were identified in TAS 1, and one CFA-positive child was identified in TAS 2. Results of both surveys were below the threshold for which MDA was warranted. Additionally, 1,112 and 836 dried blood spots from TAS 1 and TAS 2, respectively were tested for antibodies to Wb123, Bm14 and Bm33 by luciferase immunoprecipitation system (LIPS) assay and multiplex bead assay. In 2011, overall prevalence of responses to Wb123, Bm14, and Bm33 was 1.0%, 6.8% and 12.0%, respectively. In 2015, overall prevalence of positive Bm14 and Bm33 responses declined significantly to 3.0% (p<0.001) and 7.8% (p = 0.013), respectively. Conclusions/Significance: Although passing TAS 1 and TAS 2 and an overall decline in the prevalence of antibodies to Bm14 and Bm33 between these surveys suggests decreased exposure and infection among young children, there were persistent responses in some schools. Clustering and persistence of positive antibody responses in schools may be an indication of ongoing transmission. There is a need to better understand the limitations of current antibody tests, but our results suggest that serologic tools can have a role in guiding programmatic decision making. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is a highly evolutionarily-conserved cell-cell signaling pathway that regulates many events during development. It plays a pivotal role in the regulation of fundamental cellular processes, such as cell proliferation, stem cell maintenance, and differentiation during embryonic and adult development. However, functions of Notch signaling in Aedes aegypti, the major mosquito vector for dengue, are largely unknown. In this study, we identified a unique feature of A. aegypti Notch (AaNotch) in the control of the sterile-like phenotype in female mosquitoes. Silencing AaNotch with a reverse genetic approach significantly reduced the fecundity and fertility of the mosquito. Silencing AaNotch also resulted in the prevention of micropyle formation, which led to impaired fertilization. In addition, JNK phosphorylation (a signaling molecule in the non-canonical Notch signaling pathway) was inhibited in the absence of AaNotch. Furthermore, treatment with a JNK inhibitor in the mosquito resulted in impaired fecundity and fertility. Taken together, our results demonstrate that non-canonical Notch signaling is essential for controlling fertility in the A. aegypti mosquito. [ABSTRACT FROM AUTHOR]

The mosquito Aedes aegypti is the primary vector of human arboviral diseases caused by dengue, chikungunya and Zika viruses. Many studies have shown the potential roles of small RNA molecules such as microRNA, small interfering RNA and PIWI-interacting RNA in vector mosquitoes. The function of tRNA fragments (tRF), the newly discovered class of small RNAs, in mosquitoes is not known. In this study, we show that specific tRFs are expressed in significantly differential manner between males and females of Ae. aegypti strains. Specific tRFs also show differential response during developmental transition from larvae to adults, as well as after blood feeding of adult females. The expression pattern of tRFs upon blood feeding varied depending upon if the blood contained dengue virus, and also if the females were treated with antibiotic prior to feeding to cleanse of the gut bacteria. Our findings show that a single tRF derived from the precursor sequences of a tRNA-Gly was differentially expressed between males and females, developmental transitions and also upon blood feeding by females of two laboratory strains that vary in midgut susceptibility to dengue virus infection. The multifaceted functional implications of this specific tRF suggest that biogenesis of small regulatory molecules from a tRNA can have wide ranging effects on key aspects of Ae. aegypti vector biology. [ABSTRACT FROM AUTHOR]

Background: Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs). Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies. Methodology/principal findings: We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII), which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S) antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs). These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice. Conclusions/significance: Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent seroconversion. DSV4 has a significant potential to emerge as a safe, efficacious and inexpensive subunit dengue vaccine candidate. [ABSTRACT FROM AUTHOR]

Objectives: We determined the seroprevalence of chikungunya virus (CHIKV) infection in the adult resident population in Singapore following local outbreaks of chikungunya fever (CHIKF) in 2008–2009. Methods: Our cross-sectional study involved residual sera from 3,293 adults aged 18–79 years who had participated in the National Health Survey in 2010. Sera were tested for IgG antibodies against CHIKV and dengue virus (DENV) and neutralizing antibodies against CHIKV. Results: The prevalence of CHIKV-neutralizing antibodies among Singapore residents aged 18–79 years was 1.9% (95% confidence interval: 1.4%– 2.3%). The CHIKV seroprevalence was highest in the elderly aged 70–79 years at 11.5%, followed by those aged 30–39 years at 3.1%. Men had significantly higher CHIKV seroprevalence than women (2.5% versus 1.3%, p = 0.01). Among the three main ethnic groups, Indians had the highest seroprevalence (3.5%) compared to Chinese (1.6%) and Malays (0.7%) (p = 0.02 and p = 0.01, respectively). Multivariable logistic regression identified adults aged 30–39 years and 70–79 years, men, those of Indian ethnicity and ethnic minor groups, and residence on ground floor of public and private housing apartments as factors that were significantly associated with a higher likelihood of exposure to CHIKV. The overall prevalence of anti-DENV IgG antibodies was 56.8% (95% CI: 55.1%– 58.5%), while 1.5% (95% CI: 1.1%– 2.0%) of adults possessed both neutralizing antibodies against CHIKV and IgG antibodies against DENV. Conclusions: Singapore remains highly susceptible to CHIKV infection. There is a need to maintain a high degree of vigilance through disease surveillance and vector control. Findings from such serological study, when conducted on a regular periodic basis, could supplement surveillance to provide insights on CHIKV circulation in at-risk population. [ABSTRACT FROM AUTHOR]

Rabies is a neglected zoonotic disease that is preventable in humans by appropriate post-exposure prophylaxis (PEP). However, current PEP relies on polyclonal immune globulin products purified from pooled human (HRIG) or equine (ERIG) plasma that are either in chronic shortage or in association with safety concerns. Here, we present the development of an antibody cocktail, SYN023, made of two novel monoclonal antibodies (MAb) CTB011 and CTB012 that could serve as safer and more cost-effective alternatives to the current RIG products. Both CTB011 and CTB012 are humanized MAbs that bind to non-overlapping epitopes on the rabies virus (RABV) glycoprotein (G) with sub-nanomolar affinities. Sequence analysis revealed that many of the critical residues in binding are highly conserved across different species of lyssaviruses. When combined at a 1:1 ratio, CTB011/CTB012 exhibited neutralization capabilities equivalent or superior to HRIG against 10 North American street RABV isolates in vitro and 15 prevalent Chinese RABV strains in animal models. Finally, SYN023, at a dosage of 0.03 mg/kg, was able to offer the same degree of protection as standard HRIG administration (20 IU/kg) in Syrian hamsters challenged with a highly virulent bat (Tadarida brasiliensis) RABV variant. Taken together, the high-potency and broad-spectrum neutralization demonstrated by SYN023 make it an effective candidate for human rabies PEP consideration. [ABSTRACT FROM AUTHOR]

In Southeast Asia, envenoming resulting from cobra snakebites is an important public health issue in many regions, and antivenom therapy is the standard treatment for the snakebite. Because these cobras share a close evolutionary history, the amino acid sequences of major venom components in different snakes are very similar. Therefore, either monovalent or polyvalent antivenoms may offer paraspecific protection against envenomation of humans by several different snakes. In Taiwan, a bivalent antivenom—freeze-dried neurotoxic antivenom (FNAV)—against Bungarus multicinctus and Naja atra is available. However, whether this antivenom is also capable of neutralizing the venom of other species of snakes is not known. Here, to expand the clinical application of Taiwanese FNAV, we used an animal model to evaluate the neutralizing ability of FNAV against the venoms of three common snakes in Southeast Asia, including two ‘true’ cobras Naja kaouthia (Thailand) and Naja siamensis (Thailand), and the king cobra Ophiophagus hannah (Indonesia). We further applied mass spectrometry (MS)-based proteomic techniques to characterize venom proteomes and identify FNAV-recognizable antigens in the venoms of these Asian snakes. Neutralization assays in a mouse model showed that FNAV effectively neutralized the lethality of N. kaouthia and N. siamensis venoms, but not O. hannah venom. MS-based venom protein identification results further revealed that FNAV strongly recognized three-finger toxin and phospholipase A2, the major protein components of N. kaouthia and N. siamensis venoms. The characterization of venom proteomes and identification of FNAV-recognizable venom antigens may help researchers to further develop more effective antivenom designed to block the toxicity of dominant toxic proteins, with the ultimate goal of achieving broadly therapeutic effects against these cobra snakebites. [ABSTRACT FROM AUTHOR]

Scabies is an infestation of the skin by the mite Sarcoptes scabiei. It manifests with pruritic erythematous papules and excoriations, in addition to the pathognomonic burrows. Multiple drugs can be used for treatment, but resistance to conventional therapy is increasing throughout the years. This paper will review the mechanisms of resistance proposed in the literature and some of the potential solutions to this problem. [ABSTRACT FROM AUTHOR]

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller’s stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories. [ABSTRACT FROM AUTHOR]

Background: Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family. Both experimental and epidemiological data strongly support a role of these parasites in the etiology of the hepatobiliary pathologies and an increased risk of intrahepatic cholangiocarcinoma. Understanding a functional link between the infection and hepatobiliary pathologies requires a detailed description a host-parasite interaction on different levels of biological regulation including the metabolic response on the infection. The last one, however, remains practically undocumented. Here we are describing a host response on Opisthorchiidae infection using a metabolomics approach and present the first exploratory metabolomics study of an experimental model of O. felineus infection. Methodology and Principal findings: We conducted a Nuclear Magnetic Resonance (NMR) based longitudinal metabolomics study involving a cohort of 30 animals with two degrees of infection and a control group. An exploratory analysis shows that the most noticeable trend (30% of total variance) in the data was related to the gender differences. Therefore further analysis was done of each gender group separately applying a multivariate extension of the ANOVA—ASCA (ANOVA simultaneous component analysis). We show that in the males the infection specific time trends are present in the main component (43.5% variance), while in the females it is presented only in the second component and covers 24% of the variance. We have selected and annotated 24 metabolites associated with the observed effects and provided a physiological interpretation of the findings. Conclusions: The first exploratory metabolomics study an experimental model of O. felineus infection is presented. Our data show that at early stage of infection a response of an organism unfolds in a gender specific manner. Also main physiological mechanisms affected appear rather nonspecific (a status of the metabolic stress) the data provides a set of the hypothesis for a search of the more specific metabolic markers of the Opisthorchiidae infection. [ABSTRACT FROM AUTHOR]

Bacillus cereus biovar anthracis (Bcbva) is a member of the B. cereus group which carries both B. anthracis virulence plasmids, causes anthrax-like disease in various wildlife species and was described in several sub-Saharan African rainforests. Long-term monitoring of carcasses in Taï National Park, Côte d’Ivoire, revealed continuous wildlife mortality due to Bcbva in a broad range of mammalian species. While non-lethal anthrax infections in wildlife have been described for B. anthracis, nothing is known about the odds of survival following an anthrax infection caused by Bcbva. To address this gap, we present the results of a serological study of anthrax in five wildlife species known to succumb to Bcbva in this ecosystem. Specific antibodies were only detected in two out of 15 wild red colobus monkeys (Procolobus badius) and one out of 10 black-and-white colobus monkeys (Colobus polykomos), but in none of 16 sooty mangabeys (Cercocebus atys), 9 chimpanzees (Pan troglodytes verus) and 9 Maxwell’s duikers (Cephalophus maxwellii). The combination of high mortality and low antibody detection rates indicates high virulence of this disease across these different mammalian species. [ABSTRACT FROM AUTHOR]

West Nile Fever is a zoonotic disease caused by a mosquito-borne flavivirus, WNV. By its clinical sensitivity to the disease, the horse is a useful sentinel of infection. Because of the virus’ low-level, short-term viraemia in horses, the primary tools used to diagnose WNV are serological tests. Inter-laboratory proficiency tests (ILPTs) were held in 2010 and 2013 to evaluate WNV serological diagnostic tools suited for the European network of National Reference Laboratories (NRLs) for equine diseases. These ILPTs were designed to evaluate the laboratories’ and methods’ performances in detecting WNV infection in horses through serology. The detection of WNV immunoglobulin G (IgG) antibodies by ELISA is widely used in Europe, with 17 NRLs in 2010 and 20 NRLs in 2013 using IgG WNV assays. Thanks to the development of new commercial IgM capture kits, WNV IgM capture ELISAs were rapidly implemented in NRLs between 2010 (4 NRLs) and 2013 (13 NRLs). The use of kits allowed the quick standardisation of WNV IgG and IgM detection assays in NRLs with more than 95% (20/21) and 100% (13/13) of satisfactory results respectively in 2013. Conversely, virus neutralisation tests (VNTs) were implemented in 33% (7/21) of NRLs in 2013 and their low sensitivity was evidenced in 29% (2/7) of NRLs during this ILPT. A comparison of serological diagnostic methods highlighted the higher sensitivity of IgG ELISAs compared to WNV VNTs. They also revealed that the low specificity of IgG ELISA kits meant that it could detect animals infected with other flaviviruses. In contrast VNT and IgM ELISA assays were highly specific and did not detect antibodies against related flaviviruses. These results argue in favour of the need for and development of new, specific serological diagnostic assays that could be easily transferred to partner laboratories. [ABSTRACT FROM AUTHOR]

Background: Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro. Methodology/Principal findings: In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH). Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS) combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA), Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection. Conclusion/Significance: This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ), whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug’s anti-parasite efficacy and be involved in the development of resistance to re-infection. [ABSTRACT FROM AUTHOR]

Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by Monkeypox virus (MPXV), the most virulent orthopoxvirus affecting humans since the eradication of Variola virus (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (Funisciurus spp.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [,]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via in vivo bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions. [ABSTRACT FROM AUTHOR]