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

Coccidiosis in chicken is a worldwide disease caused by the genus Eimeria, which resulted in about $800 million losses annually in global poultry production(Williams, 1998). At present, prevention and treatment of clinical coccidiosis depend on salinomycin, Maduramycin and other synthetic drugs. However, due to drug residues and the resistance of chicken coccidia, application of chemical drugs has been extremely limited. In addition, the vaccine is currently an important means of prevention of coccidiosis in chickens. However, due to the complex composition of chicken coccidias, vaccine preventive effect is not ideal. Because of using the attenuated live vaccines,the risk of vaccine strains recovery toxicity and spread of infection is unexpected. In recent years, genetically engineered chicken coccidiosis vaccine become a hot topic, but no one has been applied to the actual production. The root reason of these problems is the biological characteristics of chicken coccidia are still unclear. Therefore, the study of molecular biology chicken coccidia and effective application of genetic engineering techniques for genetic manipulation of chicken coccidiosis have become an important direction of chicken coccidiosis prevention.Parasitic protozoa double-stranded RNA virus(ds RNA viruses) has been found in Trichomonas vaginalis, Giardia lamblia, Leishmania and other parasitic protozoa. In this study, a ds RNA virus-like particle is discovered in E. tenella(strain Et611) isolated from Changchun, and successfully sequencing the genome of the virus. The research of chicken coccidia virus is beneficial to understanding the biological characteristics of chicken coccidia, finding antigenic protein, developing more reasonable and effective drugs and vaccines against chicken coccidiosis.The complete genome sequence of Et RV1 A three-step strategy was designed to sequence the complete genome of this novel ds RNA virus. The sequencing results showed that the complete genome sequence was 6006 bp containing two open reading frames(ORFs)(2367bp for ORF1 and 3216 bp for ORF2) with a five-nucleotide overlap(UGA/UG). BLASTp analysis showed that amino acid sequences of E. tenella virus shared similarity with that of E. brunetti RNA virus. The complete genome sequence of E. tenella virus resembled the features of the Totiviridae family, indicating that this virus was a novel member of Totiviridae. According to classification and nomenclature of viruses of the international committee on taxonomy of viruses, this virus was named E.tenella RNA virus 1.The detection of virus in Eimeria different developmental stages We used RT-PCR and western blotting to detecting virus from coccidiosis chicken cecal epithelium(containing tachyzoites), unsporulated oocysts and sporulated oocysts.The expression of virus’ Rd Rp in E. tenella was confirmed by RT-PCR and immunoblot. In addition, coat did not expressed in chinken cecum, and the process of expression of coat followed with the process of sporulation.The bioinformatic analysis of Et RV1 To further study the virus, phylogenetic analysis of Et RV1 genome sequence and other Totiviridae virus genome sequences was performed, used Multiple sequence alignments and phylogenetic tree. The result showed that E. tenella RNA virus 1, E. brunetti RNA virus 1, and Mycoviruses were clustered into the genus Victorivirus and separated from the reported protozoa viruses, strongly suggesting a novel Eimeriaviruses subgenus. Coccidia viruses and their hosts exhibits coevolution. The Promoter Scan test indicated the putative promoter region located within the upstream region of the Rd Rp gene of Et RV1, and the putative promoter was also found in a similar region within several of the Victoriviruses.The construction of viral transfer vector Basic research of Et RV1 provides a theoretical basis for the development of viral vector. Based on the putative promoter and viral protein expression period, Eimeria viral vectors were constructed. e GFP and RFP were used as reporter genes. The in vitro transcribed double stranded RNAs were electroporated into E. tenella unsporulated oocysts. Fluorescent parasites were isolated by cell sorting and reproduced in vivo. The expression of e GFP or e GFP-RFP in E. tenella oocysts from the second generation to the forth generation was confirmed by flow cytometry, immunoblot and laser scanning confocal microscope. The overall proportion of fluorescent protein-expressing coccidiosis stable at around 60%. This new vector system based on E. tenella ds RNA virus promoter will be useful in E. tenella studies.