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Title:   Author  item
                                            Jenkins, Markitem
                                            Obrien, Celiaitem
                                            Santin-Duran, Monicaitem
                                            Fayer, Ronald
  Submitted to: Parasitology Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 2/24/2015
                                    Publication  Date: 6/1/2015
                                    Citation: Jenkins, M.C., Obrien, C.N., Santin, M., Fayer, R. 2015. Changes in the Levels of Cryspovirus During In Vitro Development of Cryptosporidium parvum. Parasitology Research. 114:2063-2068.Interpretive Summary: Cryptosporidiosis is an intestinal parasitic disease caused by various species of Cryptosporidium.  The disease is transmitted by a fecal-oral route of infection, usually involving the consumption of water contaminated with C. parvum or C. hominis oocysts.  An interesting feature of both C. parvum and C. hominis is the presence of a viral symbiont, named cryspovirus, that resides inside the parasite.  The function of this virus is unknown, partly because culture conditions to attain parasite development and molecular assays for detecting the virus are not available.  In the present study, a cell culture system is described that allows for C. parvum development, as well as molecular assays for measuring levels of cryspovirus during parasite growth.  Applying these techniques, it was shown that cryspovirus is released into culture medium during C. parvum development, but that cryspovirus levels remain nearly the same inside host cells.  This improved cell culture system and molecular assays for tracking the virus will be invaluable to future studies to discern a role of cryspovirus in Cryptosporidium.  It is possible that by inhibiting replication of the virus, one can alter the pathogenicity of C. parvum and C. hominis, and thus diminish the clinical effects in humans and animals.
                                Technical Abstract: The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan.  Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C. parvum sporozoites, followed by harvest of cells and culture medium  at 2, 24, 48, and 72 hr post-infection.  Changes in viral RNA levels were detected by RT-PCR using primers specific for RNA encoding the 40 kDa capsid protein (CP) or RNA-dependent RNA polymerase (RdRp). Parasite or host DNA was quantified by PCR specific for C. parvum or human glyceraldehyde-3-phosphate dehydrogenase (HuGAPDH).An internal standard (competitor) was incorporated into all assays as a control for PCR inhibition.  Intracellular levels of C. parvum DNA increased between 2 and 48 hr post-infection, and then decreased at 72 hr. Culture medium overlying these C. parvum-infected cells displayed a similar increase in CP and RdRp signal, reaching peak levels at 48 hr. However, the CP and RdRp levels in cellular RNA displayed only a modest increase between 2 and 48 hr, and exhibited no change (CP) or decreased (RdRp) at 72 hr. These data suggest that during the first 48 hr of C. parvum in vitro development, cryspovirus is released into the media overlying cells, but remains at fairly constant levels within infected cells.


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