Evolution of high pathogenicity of H5 avian influenza virus: haemagglutinin cleavage site selection of reverse-genetics mutants during passage in chickens.

Low pathogenicity avian influenza viruses (LPAIVs) are generally asymptomatic in their natural avian hosts. LPAIVs can evolve into highly pathogenic forms, which can affect avian and human populations with devastating consequences. The switch to highly pathogenic avian influenza virus (HPAIV) from LPAIV precursors requires the acquisition of multiple basic amino acids in the haemagglutinin cleavage site (HACS) motif. Through reverse genetics of an H5N1 HPAIV, and experimental infection of chickens, we determined that viruses containing five or more basic amino acids in the HACS motif were preferentially selected over those with three to four basic amino acids, leading to rapid replacement with virus types containing extended HACS motifs. Conversely, viruses harbouring low pathogenicity motifs containing two basic amino acids did not readily evolve to extended forms, suggesting that a single insertion of a basic amino acid into the cleavage site motif of low-pathogenic viruses may lead to escalating selection for extended motifs. Our results may explain why mid-length forms are rarely detected in nature. The stability of the short motif suggests that pathogenicity switching may require specific conditions of intense selection pressure (such as with high host density) to boost selection of the initial mid-length HACS forms.

Antibody fragments, expressed by a fowl adenovirus vector, are able to neutralize infectious bursal disease virus.

Single-chain variable fragments (scFv) contain the heavy and light chain variable domains of immunoglobulin, joined by a short peptide linker. Previously, our laboratory has produced neutralizing scFv to epitopes of infectious bursal disease virus (IBDV). The in vitro delivery and expression of one of these scFv with and without the C(H)2-C(H)4 Fc domain of chicken IgY attached (scFv-Fc) by a serotype 8 fowl adenovirus (FAdV-8) vector was investigated in the present study. A panel of FAdV-8 vectors was constructed, each containing a different transgene (scFv or scFv-Fc), a different promoter to drive scFv and scFv-Fc transcription (CMVie or the fowl adenovirus major late promoter), and a different sized, right-hand end genomic deletion (52 bp or 2.3 kb). This panel was used to establish what effect these variables had on protein production, viral replication and scFv transcription, as measured by enzyme-linked imunosorbent assay and real-time polymerase chain reaction. Our results showed that, using a FAdV-8 vector containing the optimal CMVie promoter/2.3 kb deletion combination, we successfully expressed a secreted form of both scFv and scFv-Fc that were able to neutralize IBDV both in vitro and in ovo. These studies indicate that the FAdV-8 vector may be a promising candidate to deliver and express therapeutic molecules such as scFv and scFv-Fc in vivo in poultry.

Restriction fragment length polymorphism analysis of the VP2 gene of Australian strains of infectious bursal disease virus.

Twenty-four Australian strains of infectious bursal disease virus (IBDV) were characterized by reverse transcription/polymerase chain reaction-restriction fragment length polymorphism and compared with previously published overseas strains. A primer pair designed to amplify a 743 base pair fragment of the VP2 gene was used and restriction fragment length polymorphism profiles were determined for each strain using three restriction enzymes, Bst NI, MboI and SspI. Australian strains comprised 12 molecular groups that were unique and distinct from overseas IBDV strains. A specific SspI site that is used to predict a very virulent IBDV phenotype was absent from all Australian strains, contrary to a previous finding by Jackwood and Sommer (1999). One Australian strain (N1/99) contained an SspI site; however, this was located at a different position than that found in very virulent IBDV strains. The results demonstrate that restriction fragment length polymorphism can be used to rapidly differentiate Australian IBDV strains from overseas strains. However, the existence of a large number of molecular groups might preclude its effectiveness for inter-strain differentiation.