Complete Genome Sequence Of A DsRNA Virus In E. Stiedai And Construction Of Viral Transfer Vector

Rabbit coccidiosis is parasitic protozoan diseases caused by Eimeria spp., which has an enormous impact on rabbit breeding industry. In the domestic, 17 kinds of rabbit coccidiosis have been reported. Eimeria stiedai(E. stiedai) is the most virulent and harm, which invades the hepatobiliary epithelial cells of domestic rabbits and causes intensive hepatic coccidiosis. Currently, coccidiosis control in rabbit has relied mainly on chemoprophylaxis. However, the increasing drugs resistance and the problem of chemical medicines residue, it is difficult to solve the threat of coccidiosis from foundation. The main causes of due to little knowledge of cellular and molecular biological characteristics of rabbit coccidian. The discovered of E. stiedai virus provide a new direction for further research on the molecular biological characteristics of rabbit coccidian.Double-stranded RNA(ds RNA) viruses, belonging to the family Totiviridae, have been discovered in several protozoan parasites, for example, Giardia lamblia, Trichomonas vaginalis, Leishmania braziliensis and the Eimeria species. It has been demonstrated that Leishmania RNA virus-1(LRV-1) increases the inflammatory response through triggering Toll-like receptor 3(TLR3) signaling. In addition, a number of reports suggest that the ds RNA viruses might enhance the pathogenicity of protozoa. Furthermore, the viral RNA-mediated transfection research has been successfully implemented the study of gene expression regulation and protein function in Giardia lamblia. Although, virus-like particles have been discovered in E. stiedai, however, the complete genome sequence and taxonomic status of E. stiedai virus are still no reports. In the present study, the complete genome of E. stiedai virus was first cloned, sequenced, and analyzed. And then E. stiedai viral vector was constructed. This study provides a new idea for understand the molecular biological characteristics of rabbit coccidian.A full length c DNA clone of E. stiedai virus and sequence analysis Degenerate primers were designed according to the conserved sequence of known virus sequence. We successfully obtained the complete sequence of E. stiedai virus by RT-PCR and SMART RACE. The sequencing results showed that the complete sequence of E. stiedai ds RNA was 6219 bp in length and contained two open reading frames(ORFs) with a tetra-nucleotide overlap(AUGA). ORF1 had a length of 2400 bp and encoded a putative coat protein(799 amino acids); ORF2 was 3303 bp in length and encode a putative RNA-dependent RNA polymerase(1100 amino acids). BLASTp analysis showed that E. stiedai virus exhibited a high level of amino acid identity with E. tenella RNA virus 1. The complete genome sequence of E. stiedai virus resembled the features of the Totiviridae family, indicating that this virus was a new member of Totiviridae.Bioinformatics analysis of E. stiedai viral sequence Phylogenetic analysis of Es RV genome sequence and other Totiviridae virus genome sequences was performed, used multiple sequence alignments and phylogenetic tree. The phylogram results showed that the E. stiedai virus for a distinct branch with Et RV1 and Eb RV1 and separated from the reported protozoa viruses. This indicated that the Eimeriaviruses should be considered a novel genus of Totiviridae. Codon bias analysis showed that Mycoviruses have higher host adaptability than protozoa viruses, while, the host adaptability of GLV and Et RV1 are far more than any other protozoa viruses. The Cp G island and promoter scan test indicated the putative promoter region located within the upstream region of the coat and Rd Rp gene of Es RV. Rd Rp protein structure prediction showed that the palm, fingers and thumb domains.Study the viral proteins expression of different development stages in E. stiedai The schizonts, gametophyte, unsporulated oocysts and sporulated oocysts of E. stiedai were colleceted from rabbit liver of infected E. stiedai in different period, then extracted m RNA and protein samples, respectively. RT-PCR and Western blot analysis were used to detecting virus from the extracted samples. The results showed that virus' Rd Rp protein was expressed in four development stages in E. stiedai, while coat protein only expression in oocyst formation and process of oocyst sporulation.The construction of E. stiedai viral transfer vector According to the construction strategy of the related viral vectors which Eimeria stiedai viral vector was constructed. E. stiedai viral transfer vector by using green fluorescent protein(GFP) to replace the partial gene encoding region of the ds RNA. The constructed E. stiedai viral transfer vector was electroporated into E. stiedai unsporulated oocysts. The expression of GFP in second generation and third generation of E. stiedai sporulated oocysts were confirmed by laser scanning confocal microscope, flow cytometry and immunoblot.In summary, this study has the important significant to understand the basic characteristic of E. stiedai virus and develop gene manipulation tools of E. stiedai. Meanwhile, construction of E. stiedai viral vector will lay the foundation for further study the cellular and molecular biological features of rabbit coccidian.