Molecular Epidemiology and Genetic Diversity of Norovirus in Young Children in Phnom Penh, Cambodia

This study investigated the genetic diversity of noroviruses identified from a previous surveillance study conducted at the National Pediatric Hospital in Phnom Penh, Cambodia, from 2004 to 2006. In the previous study, 926 stool samples were collected from children aged 3–60 months with acute diarrhea (cases) and without diarrhea (controls) with reported 6.7% of cases and 3.2% of controls being positive for norovirus. The initial norovirus diagnostic assay was performed with real-time reverse transcription-polymerase chain reaction (real-time RT PCR) which also distinguished between genogroups I and II (GI and GII). Norovirus infection was most commonly detected in children aged 12–23 months in both cases and controls. Norovirus Genotyping Tool and phylogenetic analysis of partial sequences of the 3′ end of the RNA-dependent RNA Polymerase (RdRp) and the capsid domain region were employed to assign genotypes of the norovirus strains. GII.4 was the most predominant capsid genotype detected at 39.5% followed by GII.6 at 14.9%. The GII.4 Hunter 2004 variant was the predominant strain detected. Six RdRP/capsid recombinants including GII.P7/GII.6, GII.P7/GII.14, GII.P7/GII.20, GII.P12/GII.13, GII.P17/GII.16, and GII.P21/GII.3 were also identified. This study of norovirus infection in young children in Cambodia suggests genetic diversity of norovirus as reported worldwide

A Schistosome cAMP-Dependent Protein Kinase Catalytic Subunit Is Essential for Parasite Viability

Eukaryotes, protozoan, and helminth parasites make extensive use of protein kinases to control cellular functions, suggesting that protein kinases may represent novel targets for the development of anti-parasitic drugs. Because of their central role in intracellular signaling pathways, cyclic nucleotide–dependent kinases such as cAMP-dependent protein kinase (PKA) represent promising new targets for the treatment of parasitic infections and neoplastic disorders. However, the role of these kinases in schistosome biology has not been characterized and the genes encoding schistosome PKAs have not been identified. Here we provide biochemical evidence for the presence of a PKA signaling pathway in adult Schistosoma mansoni and show that PKA activity is required for parasite viability in vitro. We also provide the first full description of a gene that encodes a PKA catalytic subunit in S. mansoni, named SmPKA-C. Finally we demonstrate, through RNA interference, that SmPKA-C contributes to the PKA activity we detected biochemically and that inhibition of SmPKA-C expression in adult schistosomes results in parasite death. Together our data show that SmPKA-C is a critically important gene product and may represent an attractive therapeutic target for the treatment and control of schistosomiasis

An Atlas for Schistosoma mansoni Organs and Life-Cycle Stages Using Cell Type-Specific Markers and Confocal Microscopy

Schistosomiasis (bilharzia) is a tropical disease caused by trematode parasites (Schistosoma) that affects hundreds of millions of people in the developing world. Currently only a single drug (praziquantel) is available to treat this disease, highlighting the importance of developing new techniques to study Schistosoma. While molecular advances, including RNA interference and the availability of complete genome sequences for two Schistosoma species, will help to revolutionize studies of these animals, an array of tools for visualizing the consequences of experimental perturbations on tissue integrity and development needs to be made widely available. To this end, we screened a battery of commercially available stains, antibodies and fluorescently labeled lectins, many of which have not been described previously for analyzing schistosomes, for their ability to label various cell and tissue types in the cercarial stage of S. mansoni. This analysis uncovered more than 20 new markers that label most cercarial tissues, including the tegument, the musculature, the protonephridia, the secretory system and the nervous system. Using these markers we present a high-resolution visual depiction of cercarial anatomy. Examining the effectiveness of a subset of these markers in S. mansoni adults and miracidia, we demonstrate the value of these tools for labeling tissues in a variety of life-cycle stages. The methodologies described here will facilitate functional analyses aimed at understanding fundamental biological processes in these parasites

Eukaryotic Protein Kinases (ePKs) of the Helminth Parasite Schistosoma mansoni

<p>Abstract</p> <p>Background</p> <p>Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The <it>Schistosoma mansoni </it>genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the <it>S. mansoni </it>predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets.</p> <p>Results</p> <p>We have identified 252 ePKs, which corresponds to 1.9% of the <it>S. mansoni </it>predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that <it>S. mansoni </it>has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in <it>S. mansoni </it>or belong to an expanded family in this parasite. Only 16 <it>S. mansoni </it>ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite.</p> <p>Conclusions</p> <p>Our approach has improved the functional annotation of 40% of <it>S. mansoni </it>ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of <it>S. mansoni </it>in response to diverse environments during the parasite development, vector interaction, and host infection.</p

PKA activity is essential for adult <i>S. mansoni</i> viability <i>in vitro</i>.

<p>Adult <i>S. mansoni</i> worm pairs were maintained in medium containing varying concentrations of H-89 (A and B) or PKI 14–22 amide (C and D). Survival in the presence of each inhibitor was plotted against time (A and C). For H-89, concentrations of inhibitor are as follows: 0 and 1 µM (•); 10 µM (♦); 25 µM (▪); 50, 100, 250, and 500 µM (▾). For PKI 14–22 amide concentrations of inhibitor are as follows: 0, 1, 10, 25, and 50 µM (•); 100 µM (♦); 250 and 500 µM (▪). Treatment groups containing 6 worm pairs each (12 worms total) were used for each concentration. B and D, micrographs of representative worm pairs incubated in the presence of 100 µM H-89 (B) or100 µM PKI 14–22 amide (D). Scale bars = 1 mm. e, parasite eggs. Data are representative of three independent experiments.</p

Schistosome PKA activity is sensitive to adenylyl cyclase modulation.

<p>Kinase activity was measured in kinase reactions containing Sm lysate from adult worm pairs (A and C) that were previously treated with SQ22536 (A) or forskolin (C). Treatment groups contained 10 worm pairs (20 worms total) each and were performed in triplicate. Kinase reactions containing recombinant human PKA-Cα (B and D) were treated directly with SQ22536 (B) or forskolin (D). ▴, inhibitor-treated; •, no inhibitor. Data are representative of two independent experiments.</p

SmPKA-C encodes for an essential kinase activity.

<p>A, Electroporation of adult worms with 30 µg of SmPKA-C dsRNA (▪) resulted in 75% mortality while worms treated with control dsRNA (•) exhibited 100% survival. B, Three days after electroporation, SmPKA-C transcript levels were decreased in worms electroporated with 10 µg of SmPKA-C dsRNA, while transcript levels were unaffected in control dsRNA-treated worms, as determined by real-time PCR. C, 7 days after electroporation, SmPKA-C dsRNA-treated (•) and control dsRNA-treated worms (▴) were incubated for 2 h in 100 µM forskolin. PKA activity was significantly decreased after electroporation with 10 µg SmPKA-C dsRNA compared to control dsRNA-treated worms.</p

Identification of SmPKA-C as a PKA-C subunit from <i>S. mansoni</i>.

<p>A, Amino acid alignment of both SmPKA-C and putative splice variant peptide sequences with PKA-C sequences from <i>Caenorhabditis elegans</i> (Cekin-1; NP_493605), <i>Drosophila melanogaster</i> (DmPKA; NP_476977), <i>Mus musculus</i> (MmPKAa; P05132) and <i>Homo sapiens</i> (HsPKAa; P17612). B, Phylogenetic analysis comparing both SmPKA-C amino acid sequences to the following PKA-C sequences from other organisms: <i>Homo sapiens</i> PKA-Cα (HsPKAa; P17612), <i>H. sapiens</i> PKA-Cβ (HsPKAb; P22694), <i>H. sapiens</i> PKA-Cγ (HsPKAg; P22612), <i>Mus musculus</i> PKA-Cα (MmPKAa; P05132), <i>M. musculus</i> PKA-Cβ (MmPKAb; P68181), <i>Rattus norvegicus</i> PKA-Cα (RnPKAa; P27791), <i>R. norvegicus</i> PKA-Cβ (RnPKAb; P68182), <i>D. melanogaster</i> (DmPKA; NP_476977), <i>Aplysia californica</i> (AcPKA; CAA45014), and <i>Acyrthosiphon pisum</i> (ApPKA; XP_001946114). C, SmPKA-C transcript is expressed in cDNA of all the life cycle stages of <i>S. mansoni</i>. 1, egg, 2, miracidium, 3, sporocyst, 4, cercaria, 5, schistosomulum, 6, adult female, 7, adult male.</p