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Highly pathogenic avian influenza (AI) H5N1 viruses have been spreading from Asia since late 2003. Early detection and classification are paramount for control of the disease because these viruses are lethal to birds and have caused fatalities in humans. Here, we described TaqMan reverse transcriptase-polymerase chain reaction assays for rapid detection of all AI viruses (influenza type A) and for identification of H5N1 of the Eurasian lineage. The assays were sensitive and quantitative over a 10(5)-10(6) linear range, detected all of the tested AI viruses, and enabled differentiation between H5 and H7 subtypes. These tests allow definitive confirmation of an AI virus as H5 within hours, which is crucial for rapid implementation of control measures in the event of an outbreak.

Two fowlpox virus recombinants were constructed which expressed the host-protective antigen, VP2, of infectious bursal disease virus (IBDV). Recombinant FPV-VP 2.4.3 contained the gene for the VP 2-VP4-VP3 polyprotein under the control of the vaccinia virus late promoter P.L 11 inserted within the thymidine kinase (TK) gene of FPV. In infected chicken embryo skin (CES) cells VP2 and VP3 proteins were correctly processed from the polyprotein precursor molecule. Recombinant FPV-VP2 contained only the VP2 encoding region under the control of the fowlpox early/late promoter P.E/L inserted immediately downstream of the TK gene. The expression level of VP2 from FPV-VP2 was approximately 5 times higher than from FPV-VP2.4.3. Wing web inoculation of birds resulted in the development of typical fowlpox lesions and the development of antibodies to FPV with either of the recombinants, but only birds vaccinated with FPV-VP2 developed antibodies to IBDV. When challenged with IBDV (strain 002-73), a significant level of protection was provided by FPV-VP2 vaccination, although the level was lower than the protection provided by an oil adjuvanted inactivated whole IBDV vaccine. Birds vaccinated with FPV-VP2.4.3 were not protected from infection as assessed by ELISA for the presence of IBD virus in bursae.

Phage-displayed recombinant antibody libraries derived from splenic mRNA of chickens immunized with an Australian strain of infectious bursal disease virus (IBDV) were constructed as single chain variable fragments (scFv) by either overlap extension polymerase chain reaction (PCR) or sequential ligation of the individual heavy (V(H)) and light (V(L)) chain variable gene segments. Sequential cloning of the individual V(H) and V(L) genes into a newly constructed pCANTAB-link vector containing the synthetic linker sequence (Gly(4)Ser)(3) was more efficient than cloning by overlap extension PCR, increasing the library size 500 fold. Eighteen IBDV specific antibodies with unique scFv sequences were identified after panning the library against the immunizing antigen. Eight of the clones contained an identical V(H) gene but unique V(L) genes. In ELISA analysis using a panel of Australian and overseas IBDV strains, one scFv antibody was able to detect all strains, whilst 3 others could discriminate between Australian and overseas strains, classical and variant strains and Australian field strains and vaccine strains. In addition, some scFvs showed significant neutralization titres in vitro. This report shows that generation of chicken antibodies in vitro by recombinant means has considerable potential for producing antibodies of diverse specificity and neutralizing capacity.

An Indonesian very virulent (vv) strain of infectious bursal disease virus (IBDV), designated Tasik94, was characterised both in vivo and at the molecular level. Inoculation of Tasik94 into 5-week-old specific-pathogen-free (SPF) chickens resulted in 100% morbidity and 45% mortality. The complete nucleotide and predicted amino acid sequences of genomic segments A and B were determined. Across each of the three deduced open reading frames (ORFs), Tasik94 shared the greatest nucleotide homology to Dutch vv strain D6948. Phylogenetic analyses were performed using 15 full-length polyprotein sequences and a total of 105 VP2 hypervariable region sequences from geographically and pathogenically diverse strains. In each case, Tasik94 grouped closely with vv strains, particularly those from Europe. The deduced VP1, VP2, VP3, VP4 and VP5 protein sequences of Tasik94 were aligned with those from published strains and putative virulence determinants were identified in VP2, VP3 and VP4. Alignment of additional protein sequences across the VP2 hypervariable region confirmed that residues Ile[242], Ile[256] and Ile[294] were highly-conserved amongst vv strains, and may account for their enhanced virulence.

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