Identification And Its Intracellular Distribution Of Eimeria Tenella Virus

Identification and Its Intracellular Distribution of Eimeria tenella Virus Coccidiosis is a worldwide disease in poultry caused by members attributed to Eimeira genus which can lead to serious economical losses. E. tenella is one of the most important and harmful species causing coccidiosis in chickens. Up to now parasitic protozoa viruses or RNA segments (RNAs) associated with viruses have been described in Eimeria species including E. stiedae, E. nieschulzi, E. necatrix, E. maxima, E. acervulina, and E. brunette. However, virus-like RNAs and virus-like particles (VLPs) have not been reported in E. tenella with similar assays. In the present report, for the first time in our knowledge, E. tenella virus was discovered and identified in E. tenella sporulated oocysts isolated from Changchun, China. The intracellular localization of these viruses at different development stages was observed.Screening and purification of E. tenella strain carrying viruses: The unsporulated oocystes of E. tenella were collected from caecum from the clinical samples which was confirmed to be E. tenella infected. Sporulated oocysts were propagated through broilers. To identify putative virus in Changchun strain of E. tenella, total nucleic acids samples from sporulated oocysts were screened by native agarose gel electrophoresis. Oocysts which have extrachromosomal RNA segments (RNAs) were chosen for further analysis. E. tenella strain carrying RNAs was purified by single-oocyst isolation technique and verified by RT-PCR with species specific primers and total nucleic acids analysis. The results indicated that three bands with sizes of 1.4kb, 2.4 kb, and 3.6 kb appeared after agarose gel electrophoresis of total nucleic acids samples from E. tenella sporulated oocysts. Two E. tenella strains with virus-like RNAs were obtained after single-oocyst purification as evidenced by the results of RT-PCR and total nucleic acids extraction.Identification of E. tenella virus: To identify E. tenella viruses, total nucleic acids associated with RNAs were analyzed and virus morphology was observed under an Electron Microscopy. RNAs was analyzed by nuclease sensitivity assay, RNase-protection assay, and RNA-dependent RNA polymerase (RDRP) activity assay. The results of nuclease sensitivity analysis showed that three bands with sizes of 1.4kb, 2.4 kb, and 3.6 kb described above were DNase-resistant(100μg/mL), but were digested by RNase A (1.0μg/mL). These results suggested that the three bands were RNAs in nature. Moreover, the RNA bands were resistant to RNase A digestion at high salt concentration (0.3 M NaCl), but not at low salt concentration (0.015 M NaCl). These RNAs possessed RDRP activity as suggested by theα-32P-UTP incorporated assay. The results indicated that they were double-stranded RNAs (dsRNAs). E. tenella virus was separated by sucrose gradient centrifugation and examined under electron microscopy. The results showed that they have a homogeneous icosahedral shape with a diameter of approximately 38 nm, without an envelope.Intracellular distribution of E. tenella virus: Results from EM and analysis of total nucleic acids showed that E. tenella viral RNAs existed at different development stages including sporogony, merogony, and gametogony. However, viral paticles were only seen in those organisms at the sporogony stage under electron microscopy. To examine the intracellular distribution of E. tenella virus particles, sporulated oocysts of E. tenella were homogenized and different fractions were separated by centrifugation. By analysis of the total nucleic acids extracted from different fractions, the viral particles were identified to be localized in sporozoite cytoplasm of sporulated oocysts. However, viral particles were not found at merogony and gametogony stages under electronic microscope, which indicate that viral particles were not assembled at these two stages of E. tenella.The discovery of E. tenella virus will for sure enrich our knowledge of protozoa virus. This novel viral species could be a potential vector system for the study of E. tenella. This will certainly help our causes in the prevention and treatment of coccidiosis in poultry.