K. J. Fahey

Sequence Analysis and Expression of the Host-protective Immunogen VP2 of a Variant Strain of Infectious Bursal Disease Virus Which Can Circumvent Vaccination with Standard Type I Strains

The host-protective antigen VP2 of a variant strain of infectious bursal disease virus (IBDV) which emerged from a vaccinated flock and is able to circumvent vaccination with classic type I strains of IBDV, was cloned and its nucleotide sequence determined. Virus-neutralizing monoclonal antibodies (MAbs) raised against the Australian 002-73 strain of IBDV did not react or reacted only very weakly with the expression product of the variant virus. The deduced amino acid sequence of VP2 from the variant strain differed in 17 residues from that of the Australian strain and in eight positions from a consensus sequence compiled from six type I strains of IBDV. All the amino acid changes mapped within the central, variable region of VP2, which forms the conformational epitope recognized by virus-neutralizing MAbs. Changes in the two hydrophilic regions at either end of this fragment were unique to the variant virus and were crucial for its ability to escape the virus-neutralizing antibodies induced by vaccination with a standard type I vaccine.

The characterization and molecular cloning of the double-stranded RNA genome of an Australian strain of infectious bursal disease virus

The genome of infectious bursal disease virus (IBDV) strain 002-73 was found to consist of two segments of double-stranded (ds) RNA which were 3400 bp (MW 2.06 X 10(6)) and 2900 bp (MW 1.76 X 10(6)) long, respectively. The ds IBDV RNA could be translated, in vitro, only after extensive denaturation. The small RNA segment was found to code for a single polypeptide of MW 90K, while the large RNA segment coded for three major polypeptides of MW 52K, 32K, and 28K, and two minor polypeptides of MW 41K and 16K. The large RNA segment could encode proteins of MW 125K while the MW of the translated products was 169K suggesting that a precursor-product relationship exists between some of the translation products. A method is described for the synthesis of ds cDNA from large ds RNA molecules. Analyses of recombinant colonies showed that inserts covering the entire IBDV genome had been cloned.

Characterization by Western blotting of the immunogens of infectious bursal disease virus.

The Australian isolate of infectious bursal disease (IBD) virus (002/73) was purified from infected bursae by rate-zonal and density-equilibrium centrifugation and characterized by polyacrylamide gel electrophoresis. Two major polypeptides having approximate mol. wt. of 32 000 (32K) and 37K and three other polypeptides of approximate mol. wt. 29K, 41.5K and 91.5K were present in all preparations of virus having a buoyant density of 1.33 g/ml. Western blotting of the polypeptides of IBD virus showed that the initial antibody response of chickens infected with live virus or injected with an inactivated oil-emulsion vaccine was directed primarily towards the 32K polypeptide. Only sera obtained late in the response to live virus or following hyperimmunization contained antibodies recognizing the 29K, 37K and 41.5K polypeptides. An antibody response to the 91.5K polypeptide was not detected routinely by this technique. It was concluded that the 32K polypeptide is a major immunogen of IBD virus.

Molecular characterization of highly virulent fowl adenoviruses associated with outbreaks of inclusion body hepatitis

Restriction enzyme analysis of DNA was used to characterize fowl adenoviruses (FAVs) consistently associated with outbreaks of acute inclusion body hepatitis. When low doses of these FAVs were administered via a natural route to chickens they caused IBH. A strong genomic relationship was demonstrated between these virulent FAVs. In contrast, the genomes of serologically related, but non-virulent or mildly virulent FAVs were found to differ substantially from those of the virulent FAVs.

Passive protection against infectious bursal disease virus by viral VP2 expressed in yeast

Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the worlds poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.

Gallid herpesvirus 1 (infectious laryngotracheitis virus): cloning and physical maps of the SA-2 strain.

SummaryClones representing 90% of the genome of Gallid herpesvirus 1 (infectious laryngotracheitis virus; ILTV) were obtained and used in hybridization experiments to constructEcoRI,KpnI amdSmaI physical maps. The genome was 155 kilobase pairs (kbp) and comprised of a long unique sequence (120 kbp) and a short unique sequence (17 kbp) bounded by repeat sequences each of 9 kbp. An unrelated second pair of repeat sequences was located at 0.67 and 0.88 map untis. A terminal repeat of the unique long region (UL) was also detected, but no isomerization of UL was detected.

The role of mucosal antibody in immunity to infectious laryngotracheitis virus in chickens

The role of mucosal antibody in recovery from a primary infection and resistance to reinfection with infectious laryngotracheitis (ILT) herpesvirus was studied in bursectomized chickens, which were unable to synthesize specific antibodies. Viral antigen in the infected trachea was assessed by indirect immunofluorescence on tissue sections and by ELISA. The ability of bursectomized chickens to resolve primary infections as effectively as intact chickens and of vaccinated-bursectomized chickens to prevent the replication of challenge virus without the participation of mucosal antibody, is evidence for the importance of local cell-mediated rather than humoral immune mechanisms in the outcome of infection with ILT virus.

Expression in Escherichia coli of cDNA fragments encoding the gene for the host-protective antigen of infectious bursal disease virus

The larger segment of the IBDV genome codes for a 32-kDa host-protective antigen. Inserts from a cDNA library in pBR 322, containing overlapping cDNA fragments of varying sizes and covering the entire large segment of the IBDV genome, were subcloned into a mixture of expression vectors pUR 290, 291, and 292. Clones expressing the host-protective antigen, or parts of it, were identified by an immunoblot assay and the fusion proteins were further characterized by Western blot analysis using a monoclonal antibody specific for the 32-kDa polypeptide. Hybridization of inserts from expressing clones to the original cDNA library led to the identification of the region of the IBDV genome that codes for the 32-kDa host-protective antigen. Clone D1 which encodes approximately 50% and clone D6 which encodes the entire 32-kDa protein were selected for further studies. The fusion proteins from clones D1 and D6 were affinity purified and tested for their immunogenicity in chickens. Both fusion proteins induced the synthesis of antibodies in both primed and unprimed chickens that reacted specifically with denatured 32-kDa viral protein, but less well with intact virus. It was concluded that the response to the fusion proteins was to linear rather than conformational epitopes on the 32-kDa viral protein.

Cloning and sequencing of the chicken anaemia virus (CAV) ORF-3 gene, and the development of an ELISA for the detection of serum antibody to CAV

Chicken anaemia virus (CAV) is a small, unclassified virus involved in anaemia and suspected of causing immunosuppression in young chickens. We have developed an ELISA for the detection of serum antibody to CAV based on cloned antigen. The gene for ORF-3 (the putative capsid protein) was cloned, sequenced and expressed in a bacterial expression system, pGEX. An ORF-3 fusion protein was used to produce an indirect ELISA.

Akabane virus infection in the pregnant ewe. 1. Growth of virus in the foetus and the development of the foetal immune response

Abstract Three different pools of the CSIRO 16 strain of Akabane virus differing in their laboratory passage histories were used to inoculate 39 ewes between 32 and 36 days pregnant; 22 pregnant ewes received inocula containing no virus. There was no difference in the development, duration and titre of the viraemia and neutralising antibody response between the three infected groups of ewes. Both infected and control ewes had 141% foetuses when autopsied at 69 to 105 days gestation. Of the 55 foetuses from infected ewes 44 (80%) had gross developmental abnormalities. At autopsy of the dams Akabane virus was isolated only from the uterine caruncle. From foetal samples virus was isolated from a wide range of tissues, from one foetus at 69 days and from the blood of four foetuses at 95 to 106 days gestation. Virus was also isolated from 24 of the choriolllantoic fluid samples and from 37 placentomes of the 44 foetuses with developmental defects, in concentrations ranging from 10 2 to 10 5.5 TCID 50 /ml or/g. No virus was isolated from the tissues of the control ewes or their foetuses. Neutralising antibody to Akabane virus was detected in 78% of the foetal sera from the infected group, titres ranging from 2 to 64. IgM and IgG 1 and neutralising antibody were detected in sera of 40 foetuses with developmental abnormalities including three that were of 76 to 78 days gestation. Neutralising antibody was detected only in serum that contained IgG 1 but may also have been associated with IgM in infected foetuses. IgM was detected in the serum of most foetuses including the non-infected controls, but sera from the control foetuses did not contain IgG 1 or neutralising antibody to Akabane virus. No IgG 2 or IgA were detected in any foetal serum.