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- Title
Changes in the levels of Cryspovirus during in vitro development of Cryptosporidium parvum.
- Authors
Jenkins, M.; O'Brien, C.; Santin, M.; Fayer, R.
- Abstract
The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of C ryspovirus, 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.
- Subjects
CRYPTOSPORIDIUM parvum; POLYMERASE chain reaction; PROTOZOA; CANCER cells; SPOROZOITES; CELL culture; RNA polymerases
- Publication
Parasitology Research, 2015, Vol 114, Issue 6, p2063
- ISSN
0932-0113
- Publication type
Article
- DOI
10.1007/s00436-015-4390-6