Michael Sheppard

Fowl adenovirus recombinant expressing VP2 of infectious bursal disease virus induces protective immunity against bursal disease

SummaryThe right hand end Nde I fragment 3 (90.8–100 map units) of the fowl adenovirus serotype 10 (FAV-10) was characterised so as to allow the location of an insertion site for recombinant vector construction. Infectious bursal disease virus (IBDV) VP2 gene from the Australian classical strain 002/73, under the control of the FAV-10 major late promoter/leader sequence (MLP/LS) was inserted into a unique Not I site that was generated at 99.5 map units. This recombinant virus was produced without deletion of any portion of the FAV-10 genome. When administered to specific pathogen free (SPF) chickens intravenously, intraperitoneally, subcutaneously or intramuscularly, it was shown that the FAV-10/VP2 recombinant induced a serum VP2 antibody response and protected chickens against challenge with IBDV V877, an intermediate virulent classical strain. Birds were not protected when the recombinant was delivered via the conjunctival sac.

Identification of an infectious laryngotracheitis virus gene encoding an immunogenic protein with a predicted Mr of 32 kilodaltons

The nucleotide sequence of an infectious laryngotracheitis virus (ILTV) gene which maps immediately upstream from the glycoprotein 60 (gp60) gene was determined. The gene, designated p32, encodes a predicted polypeptide of 298 amino acids with an estimated M(r) of 32,000 daltons. The predicted protein sequence has four potential N-glycosylation sites and a signal sequence at the N-terminal region. Amino acid residues in the NH2-terminal region of the p32 protein exhibit similarity to glycoprotein X (gX) of pseudorabies virus (PRV) and its homolog in equine herpesvirus type 1 (EHV-1). Within the conserved (N-terminus) region, one putative N-linked glycosylation site and four cysteine residues are aligned in these proteins. These common structural features of the gX-like proteins were also found in glycoprotein G (gG) of human herpes simplex virus type 2 (HSV-2) and equine herpesvirus type 4 (EHV-4). High level bacterial production of the p32 protein was achieved by cloning the p32 open reading frame into a pGEX-2T expression vector. Western blot analysis of the fusion protein produced in E. coli using immune chicken sera confirms that p32 protein is of viral origin and is an immunogen in birds with infectious laryngotracheitis (ILT). An antiserum from chicken immunized with the fusion protein detected a substantial amount of p32 protein in the medium of ILTV-infected cells in Western blotting. Moreover tunicamycin treatment of cells infected with the virus indicated that p32 was glycosylated. This allows us to conclude that p32 is a glycoprotein and like gX of PRV accumulates in the medium of infected cells.

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.

Nucleotide sequence of infectious laryngotracheitis virus (gallid herpesvirus 1) ICP4 gene.

The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the ICP4 protein of herpes simplex virus (HSV) has been mapped to the inverted repeat region. The complete nucleotide sequence of ILTV ICP4 has been determined. The ILTV ORF encoding ICP4 is 4386 nucleotides long, calculated from the first of four ATG codons, and has an overall G+C content of 59%. The ILTV ICP4 contains two domains of high homology which have been reported in other studies to be conserved in the ICP4 homologues of alphaherpesviruses, and to be functionally important. Several regulatory features were identified including a serine-rich domain in region one. A more extensive serine-rich domain was located in region five which is also found in varicella-zoster virus (VZV) and bovine herpesvirus 1. A 5.4 kb immediate early transcript was identified in infected primary kidney cells.

Immunological and molecular comparison of fowl adenovirus serotypes 4 and 10

SummarySome discussion has centered on whether fowl adenovirus (FAV) serotypes 4 and 10 are distinct serotypes or in fact should be reclassified as a single serotype. We have undertaken a detailed characterisation of representatives of both serotypes in order to determine if types 4 and 10 should be grouped together or retained as distinct serotypes. Examination at the genomic level has revealed considerable similarities and few differences between these 2 serotypes. DNA cross-hybridization failed to distinguish between them and restriction enzyme analysis demonstrated limited sequence differences. In vivo studies demonstrated the cross-protection afforded by a natural route vaccination with serotype 4 FAV when chickens were challenged with serotype 10 FAV. On the basis of these studies it is suggested that these FAV serotypes be combined in future FAV classification. Physical maps for both serotype 4 and 10 have been constructed using the restriction enzymesHpa I,Dra I,Nde I,Xba I andNot I for both serotypes and in additionEco RI,Sfi I,Sma I andBgl II for serotype 10.

ICP27 immediate early gene, glycoprotein K (gK) and DNA helicase homologues of infectious laryngotracheitis virus (gallid herpesvirus 1) SA-2 strain

SummaryA 4.8 kilobase segment located at the left-terminal in the unique long (UL) region of infectious laryngotracheitis virus (ILTV) SA-2 strain contained three open reading frames (ORFs). The first of 421 amino acids (aa) was located at map units 0.065 to 0.07, and its predicted 48 kiloDaltons (kDa) protein product has significant homology to the immediate early regulatory protein ICP27 (UL54) of herpes simplex virus type-1 (HSV-1), to varicella-zoster virus (VZV) ORF4 and to equine herpesvirus 1 (EHV-1) ORF5. The zinc finger conserved in the C-terminal of the proteins from HSV-1, VZV and EHV-1, is poorly conserved in ILTV homologue. The second ORF of 336 aa, located at map units 0.075 to 0.08, has a predicted molecular weight (MW) of 38 kDa with significant homology to glycoprotein K (gK) of HSV-1 (UL53), ORF5 of VZV and ORF6 of EHV-1. ILTV gK has features characteristic of a membrane-bound glycoprotein. The 3′ region of a third ORF was located at map units 0.08 to 0.095. Translation of the sequence revealed significant homology to the 3′-region of the DNA helicase-primase complex protein (UL52) of HSV-1, ORF6 of VZV and ORF 7 of EHV-1. Northern blot analyses were used to characterize the ILTV ICP27, gK and DNA helicase mRNAs. The data revealed that ILTV ICP27 is an immediate early gene that encodes a 1.6 kb mRNA, ILTV gK encodes a late transcript of 1.8 kb, while ILTV DNA helicase encodes a late transcript of 3.7 kb.

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.

Characterization of the avian adenovirus penton base

A portion of the fowl adenovirus 10 (FAV-10) penton base gene was located within a Sau3A genomic DNA fragment by the combination of a plasmid expression library and colony immunoassays with rabbit anti-FAV-10-sera. The coding portion of the sequence contained in the expression vector was mapped to a 11.6-kb Bg/II fragment and more precisely mapped to the right-hand end of the 11.6-kb fragment at map units 37.7 to 41.3. Nucleotide sequence analysis of the region revealed an open reading frame of 1575 bp with translation of the sequence producing a polypeptide of 525 amino acids in length. The polypeptide predicted from the open reading frame would have a molecular weight of 57.4 kDa. Analysis of the amino acid sequence revealed an overall homology of 41.8% with the human adenovirus 2 (HAV-2) penton base. However, a region near the center of the polypeptide (amino acids 219 to 311) showed a significantly greater level of homology to the HAV-2 penton base (71%). Time course experiments using mRNA confirmed that this gene is expressed at late times postinfection. Upon probing genomic DNA from other FAV serotypes the penton base coding region hybridized to all six different serotypes tested, indicating a relatively conserved DNA sequence among a variety of FAVs.

Nucleotide sequence of the gene encoding infectious laryngotracheitis virus glycoprotein B

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb EcoRI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the UL region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated Mr of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.

Use of λgt11 and monoclonal antibodies to map the gene for the 60,000 dalton glycoprotein of infectious laryngotracheitis virus

To localize the gene encoding the 60 kD glycoprotein (gp60) of infectious laryngotracheitis virus (ILTV), a library of the ILTV genome was constructed in the λgt11 expression vector. Twelve recombinant bacteriophages expressing gp60 epitopes as fusion products with β-galactosidase were detected by immunoscreening with monoclonal antibodies specific for gp60. The ILTV DNA sequence contained in one of these recombinants λ24-4 was used as a hybridization probe for mapping the insert sequence on the viral genome. The gene for the gp60 was located at map unit 0.72–0.77 in the unique long region (UL) of the ILTV genome. The DNA sequence of the 1.2 kb insert of λ24-4 containing the gp60 epitope was determined. The majority of deduced gp60 amino acid sequence has no homology with any of the known alphaherpesvirus glycoproteins.