Molecular cloning and characterisation of the RESA gene, a marker of genetic diversity of Plasmodium falciparum

To identity immunodiagnostic antigen genes, a Plasmodium falciparum (Dd2 clone) expression library was screened using human immune sera. The ring-infected erythrocyte surface antigen (RESA) was isolated: this antigen of the resistant clone presents repeat tandem sequences like the 3D7 clone, albeit in different numbers. RESA has been studied as a marker of genetic diversity, with different sizes being observed in different isolates and clones of Plasmodium falciparum. The native protein was localised in cultures by western-blot and immuno-transmission electron microscopy. The antigenicity of RESA was evaluated by ELISA, using the carboxy-terminal repeat region as antigen. The assay’s sensitivity and specificity were 78.2 and 94% respectively

Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei.

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.MN is funded by grants from the Spanish Ministerio de Ciencia e Innovación, (SAF2012-40029), Junta de Andalucia (CTS-5841) and VI PN de I+D+I 2008–2011, Instituto de Salud Carlos III – Subdirección General de Redes y Centros de Investigación Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by ‘Fondo de Investigación Sanitaria’ PI10/01128. JR and MC were funded by a Wellcome Trust Project Grant 093008/Z/10/Z. Work in the Dundee laboratory was funded by the Wellcome Trust (program grant 093008/Z/10/Z) and the Medical Research Council.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/cmi.1245

Initial characterization of Pf62, a novel protein of Plasmodium falciparum identified by immunoscreening

In order to find new antigens from Plasmodium falciparum, a complementary DNA (cDNA) library was constructed and screened. The study of expression library of P. falciparum was performed in an attempt to identify new antigens that could have potential relevance for the falciparum-malaria diagnosis and/or protection. Between the positive clones detected (ring erythrocyte surface antigen, merozoite erythrocyte surface antigen, RHOP H3, CSP, LSA), a new gene that correspond to a new protein (Pf62) was isolated and characterized. This antigen was useful for the diagnosis of malaria in enzyme-linked immunosorbent assay tests. The cDNA corresponding to this antigen and structure of the gene were characterized. Pf62 is a single copy gene that contains one exon. The Pf62 cDNA has an open reading frame of 1,599 nucleotides that code for a putative protein of 532 amino acids with a predicted molecular mass of 62 kDa. The polypeptide contains in the central section two regions of repeats of 21 and 19 amino acids, respectively. The localization of the Pf62 protein was performed by immunoblot, indirect immunofluorescence assay and immunoelectron microscopy. Pf62 is localized in the cytoplasm of the parasite and also on the surface of the infected erythrocyte. Serologic assays by using synthetic peptides designed from different antigenic regions of the Pf62 protein resulted in acceptable data of sensitivity and specificity in symptomatic malaria patients.This study was financially supported by the Spanish Ministry of Public Health and the Instituto de Salud Carlos III within the Network of Tropical Diseases Research Centers (RICETRD06/0021) by a grant from Centro Nacional de Medicina Tropical, Instituto de Salud Carlos III. None of the authors has any conflict of interest to declare.S

Detection of a new insect flavivirus and isolation of Aedes flavivirus in Northern Italy

Abstract Background During recent years, numerous novel ‘insect flaviviruses’ have been discovered in natural mosquito populations. In a previous study we described the presence of flavivirus DNA sequences integrated in Aedes albopictus (Asian tiger mosquito) populations from Northern Italy in 2007. Methods During 2008 we collected and tested Aedes females for flavivirus presence and developed phylogenetic analysis, virus isolation, electron microscopy studies and RNAse treatments. Results We detected a high prevalence of flavivirus in Ae. albopictus (77.5%). The phylogenetic analysis identified the insect flavivirus sequences as Aedes flavivirus (AEFV) recently described in Japan, and that may have been introduced in Italy travelling with the tiger mosquito. Some of these pools grew in C6/36 cells, producing cytopathic effects, and the RNase treatment results showed the presence of the detected sequences in RNA forms. Furthermore, we detected a new insect flavivirus in one pool of Aedes cinereus/geminus mosquitoes. Phylogenetic analysis of this virus shows that it forms a distinct cluster within the clade of insect flavivirus. Conclusions This is the first study to report a high prevalence, to describe the seasonal activity and an isolation of the insect flavivirus Aedes flavivirus in Europe. Moreover we describe the detection of a new insect flavivirus detected from Ae. cinereus mosquitoes from Italy. These flavivirus may be common, ubiquitous and diverse in nature and we discuss the implications of the insect flavivirus group in virus evolution and transmission.This work was supported by The Autonomous Province of Trento, postdoctoral project Risktiger, “Risk assessment of new arbovirus diseases transmitted by Aedes albopictus (Diptera: Culicidae) in the Autonomous Province of Trento” and EuroWestNile EU grant HEALTH.2010.2.3.3-3 Project 261391.Peer Reviewe

Detection of a new insect flavivirus and isolation of <it>Aedes</it> flavivirus in Northern Italy

Abstract Background During recent years, numerous novel ‘insect flaviviruses’ have been discovered in natural mosquito populations. In a previous study we described the presence of flavivirus DNA sequences integrated in Aedes albopictus (Asian tiger mosquito) populations from Northern Italy in 2007. Methods During 2008 we collected and tested Aedes females for flavivirus presence and developed phylogenetic analysis, virus isolation, electron microscopy studies and RNAse treatments. Results We detected a high prevalence of flavivirus in Ae. albopictus (77.5%). The phylogenetic analysis identified the insect flavivirus sequences as Aedes flavivirus (AEFV) recently described in Japan, and that may have been introduced in Italy travelling with the tiger mosquito. Some of these pools grew in C6/36 cells, producing cytopathic effects, and the RNase treatment results showed the presence of the detected sequences in RNA forms. Furthermore, we detected a new insect flavivirus in one pool of Aedes cinereus/geminus mosquitoes. Phylogenetic analysis of this virus shows that it forms a distinct cluster within the clade of insect flavivirus. Conclusions This is the first study to report a high prevalence, to describe the seasonal activity and an isolation of the insect flavivirus Aedes flavivirus in Europe. Moreover we describe the detection of a new insect flavivirus detected from Ae. cinereus mosquitoes from Italy. These flavivirus may be common, ubiquitous and diverse in nature and we discuss the implications of the insect flavivirus group in virus evolution and transmission.</p