Studies On Reverse Genetic Systems For Avian Influenza Virus And The Borna Disease Virus

Studies on reverse genetic systems for avian influenza virus and the Borna disease virusIn this study, a reverse genetic system was tried to establish for the avian influenza virus (AIV) A/Goose/Guangdong/1/96 (H5N1) and for the Borna disease virus (BDV) respectively.In the first part, eight Pol I plasmids for establishing a reverse genetic system for avian influenza virus A/Goose/Guangdong/ 1/96 (H5N1) have been developed. At least five plasmids were proved to be functional either by a direct (CAT-assay) or indirect (generation of a reassortant virus) method. The genes of the strain A/FPV/Rostock/34 (H7N1) (FPV) were used as a genetic background with only one gene of A/Goose/Guangdong/ 1/96 (H5N1) to rescue reassortant viruses. The rescued reassortant GD1NSFPV virus, that carries the A/Goose/Guangdong/1/96 (H5N1) NS gene and the other 7 genes of FPV, differs in its growth characteristics significantly from the wild type virus FPV. Furthermore the reassortant GDNS1FPV virus more effectively prevented the cellular interferon expression. The amino acids sequence of the NS1 protein of GD1NSFPV differs from that of FPV, specially in the RNA binding domain and in the effector domain. Moreover the differences in the RNA binding domain and the effector domain of both NS1 proteins result in a difference of hydrophilicity of both proteins.In the second part, according to new data for the BDV genomic 5'- and 3'-ends (the existence of an additional "A" residue at the extreme 3'-end of the single-strand genomic RNA of BDV and a "U" residue probably to be found at the 5 '-end), Pol I expression constructs starting with a hammerhead ribozyme sequence expressing the CAT gene were generated. The mini-transcript generated by the Pol I expression construct was used by the BDV polymerase for replication and transcription not only in a BDV-dependent system but also in a plasmid-based reverse genetic system. These results proved that the used 3'- and 5'-ends of the mini-genome are functional. Moreover it is the first time to prove that the putative polyadenylation signal in the 3 '-non coding region (NCR) is indeed used by the BDV polymerase. The expression of the reporter gene depends on the delicate ratio of the viral N and P proteins in a plasmid-based reverse genetic system .