Showing papers by "Reimar Johne published in 2004"

Nucleotide sequence analysis of a C1 gene fragment of psittacine beak and feather disease virus amplified by real-time polymerase chain reaction indicates a possible existence of genotypes.

Abstract: To investigate sequence diversity of psittacine beak and feather disease virus, samples collected from 31 psittacine species with or without clinical signs were tested for the presence of the viral genome A real-time polymerase chain reaction was developed amplifying a 202 base pair fragment of the region encoding the capsid protein C1 and detecting 100 to 1000 genome equivalents The nucleotide sequences of the polymerase chain reaction products showed 841 to 100% identity with no consistent pattern with regard to the infected bird species Amino acid exchanges were concentrated mainly in five of the 42 deduced positions Sequences obtained from an outbreak of acute beak and feather disease in lories clustered in a separate branch of a phylogenetic tree Sequences in samples from African grey parrots with feather disorders grouped together, whereas those from the same species with immunosuppression clustered in other branches These results indicate the possible existence of beak and feather disease virus genotypes

Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests.

Abstract: Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species. BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult. To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita). Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences. No protein was detected after induction of full-length C1 expression in Escherichia coli. However, deletion of an amino-terminal arginine-rich sequence facilitated expression. C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens. The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds. Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting. The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.

Nuclear Localization of Avian Polyomavirus Structural Protein VP1 Is a Prerequisite for the Formation of Virus-Like Particles

Abstract: Virions of polyomaviruses consist of the major structural protein VP1, the minor structural proteins VP2 and VP3, and the viral genome associated with histones. An additional structural protein, VP4, is present in avian polyomavirus (APV) particles. As it had been reported that expression of APV VP1 in insect cells did not result in the formation of virus-like particles (VLP), the prerequisites for particle formation were analyzed. To this end, recombinant influenza viruses were created to (co)express the structural proteins of APV in chicken embryo cells, permissive for APV replication. VP1 expressed individually or coexpressed with VP4 did not result in VLP formation; both proteins (co)localized in the cytoplasm. Transport of VP1, or the VP1-VP4 complex, into the nucleus was facilitated by the coexpression of VP3 and resulted in the formation of VLP. Accordingly, a mutant APV VP1 carrying the N-terminal nuclear localization signal of simian virus 40 VP1 was transported to the nucleus and assembled into VLP. These results support a model of APV capsid assembly in which complexes of the structural proteins VP1, VP3 (or VP2), and VP4, formed within the cytoplasm, are transported to the nucleus using the nuclear localization signal of VP3 (or VP2); there, capsid formation is induced by the nuclear environment.