Antigenic investigation of genetically different strains of Beak and feather disease virus

[摘要] English: Psittacine beak and feather disease (PBFD) is a dermatological condition that affects both captive and wild psittacine birds worldwide. In Southern Africa, 10 �?20% of breeding stocks are lost due to the disease each year. PBFD threatens the survival of the indigenous endangered Cape parrot (Poicephalus robustus) as well as the black-cheeked lovebird (Agapornis nigrigenis).The disease is characterized by roughly symmetrical feather loss, feather abnormalities, anorexia and immunosuppression. In advanced cases of the disease beak and claw deformities are present. The causative agent of PBFD is Beak and feather disease virus (BFDV), a circovirus belonging to a diverse group of circoviruses within the family Circoviridae. BFDV has a circular single stranded DNA genome consisting of seven open reading frames (ORFs); three of these ORFs are conserved amongst all isolates of BFDV. ORF 1 encodes the Rep protein, ORF 2 the coat protein (CP) which is also the epitopic protein of the virus and ORF 5, whose function remains unclear.BFDV cannot be cultivated in tissue/cell culture or in embryonated eggs. The inability to cultivate the virus has hampered the development of diagnostic tests and a vaccine as preventative measure against the disease.BFDV is a genetically diverse virus. Researchers have demonstrated that there are at least eight different lineages of BFDV, where Southern African isolates group into three unique genotypes. Many studies have been performed which indicated the diversity of BFDV, but so far no studies have been done which link this genetic diversity to the possibility of the existence of more than one strain of BFDV. This led to the aims of the present study which were to investigate antigenicity of BFDV isolates belonging to different genotypes and then the subsequent bacterial expression of six isolates of BFDV that were genetically different.The entire CP genes of six isolates were amplified with polymerase chain reaction (PCR) and subsequently sequenced. Phylogenetic analysis of sequence data showed that the isolates from this study grouped into lineage one as was described by Heath and co-workers (2004). Amino acid sequences from the isolates from each lineage was applied in an in silico prediction algorithm in order to establish the possibility of more than one strain of BFDV. The predictions indicated that isolates from Australia and South Africa had the same antigenic profile. However, isolate BCL1-ZAM and LK-VIC each produced their own antigenic profile. This indicated the distinct possibility that there is more than one strain of BFDV and that at least one antigenic determinant was situated at the N-terminus of the CP. However, these results have to be confirmed by conducting in vitro studies.Attempts were made to express the full length CP genes of six isolates in BL21(DE3) Escherichia coli with the pET-28b(+) vector. Neither polyacrylamide gel electrophoresis (PAGE) nor Western blotting indicated the presence of recombinantly expressed protein in any of the studies conducted. The codon adaptation index (CAI) for BFDV was calculated to be 0.250, which indicated that the CP had a 25% possibility of being expressed in E. coli due to codon incompatibility. From this study it can be suggested that before attempting expression of any gene in E. coli the CAI should be calculated. It was also concluded from this work that no further attempts to express the CP gene in E. coli should be conducted.

[发布机构] University of the Free State