title: Changes in the levels of Cryspovirus during in vitro development of Cryptosporidium parvum.
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-h 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-h post-infection, and then decreased at 72 h. Culture medium overlying these C. parvum-infected cells displayed a similar increase in CP and RdRp signal, reaching peak levels at 48 h. However, the CP and RdRp levels in cellular RNA displayed only a modest increase between 2 and 48 h, and exhibited no change (CP) or decreased (RdRp) at 72 h. These data suggest that during the first 48 h of C. parvum in vitro development, Cryspovirus is released into the media overlying cells but remains at fairly constant levels within infected cells.
authors.affiliation: , Building 1040, BARC-EAST, Beltsville, MD, 200705, USA, mark.jenkins@ars.usda.gov.
authors.forename: M C
authors.initials: MC
authors.lastname: Jenkins
authors.forename: C N
authors.initials: CN
authors.lastname: O'Brien
authors.forename: M
authors.initials: M
authors.lastname: Santin
authors.forename: R
authors.initials: R
authors.lastname: Fayer
mesh_terms: D045744:Cell Line, Tumor; D016785:Cryptosporidium parvum / Q000254:growth & development / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction; D012328:RNA Viruses / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics
chemical_list: D012367:RNA, Viral