Michael A. Johnson

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.

Avian infectious laryngotracheitis: Virus‐host interactions in relation to prospects for eradication

This review examines the virology, immunology and molecular biology of infectious laryngotracheitis virus (ILTV) and its interactions with the chicken, in the context of assessing the feasibility of eradication. Establishment of the latent phase during infection of the host, its central role in biological survival of ILTV and the host-viral events that are associated with reactivation of infection, are considered. In counterpoint there are several features of the biology of ILTV in its natural mode of infection which can be exploited in eradicating this pathogen from intensive poultry production sites. These include the high degree of host-specificity of ILTV, dependence on contact for spread, the short-lived infectivity outside the chicken and the stability of the genome and lack of significant antigenic variation. Further, ILTV cannot replicate productively in its main target organ, the trachea, in the face of local specific cell-mediated immunity. Genetically-engineered vaccines that are capable of generating immunity, but without the ILTV latent infections induced by conventional modified-live ILT vaccine strains, are now well into development. This paper postulates that, used in conjunction with specific site quarantine and hygiene measures, such vaccines can provide the technological tools required to eradicate ILTV from production sites, and then regionally, in developed poultry industries from around the year 2000.

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.

Molecular evolution of infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1): An ancient example of the Alphaherpesviridae?

An analysis of two essential genes of infectious laryngotracheitis virus (ILTV), glycoprotein D (gD) and the immediate early gene, herpes simplex virus homologue ICP27, was performed with the equivalent gene homologues from several alphaherpesviruses. Amino acid (aa) sequence analysis revealed that these ILTV genes shared limited homology to other alphaherpesvirus equivalents and were distinct from the two other avian herpesviruses, Mareks disease virus (MDV) and herpesvirus of turkeys (HVT). Simplex and varicella group viruses are clearly separate from the avian group. The amino acid sequences of these ILTV genes will be presented with comparisons to the homologues from other alphaherpes viruses, contributing further evidence of the evolution of this group of viruses from a common progenitor and that ILTV could be an ancient example of the Alphaherpesvirinae.

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.

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.

The major late promoter and bipartite leader sequence of fowl adenovirus

SummaryThe region of the fowl adenovirus serotype 10 (FAV-10) genome con- taining the major late promoter (MLP) and leader sequences was determined and appropriate genomic fragments were cloned and sequenced. A TATA box was identified and the location of the putative transcription start site was determined. By using synthetic primers from the transcription start site in conjunction with oligonucleotides from the coding regions of the penton base and hexon genes, cDNA was produced from late mRNA isolated from cell cultures infected with FAV-10 at 24 h post-infection. The resulting cDNA was cloned and sequenced and the leader sequences thus identified. It was found that the FAV-10 MLP utilized only two leader sequences (a bipartite leader). By comparison with human adeno- viruses (HAVs) it appeared that the second leader in HAVs was absent from the FAV-10. The second leader sequences of FAV-10 was larger than either the second or third leaders of HAVs, but was 29 baseparirs shorter than the combined size of the leader sequences 2 and 3 from HAV-2. To confirm the transcription start site and leader sequences, single stranded cDNA was produced from mRNA using the primers from within the coding sequence for the penton base or hexon. A tail of dGTP’s was added and cDNA synthesis was completed using an oligonucleotide from within the hexon or penton base coding sequence and a second poly-dCTP oligonucleotide. Sequencing of the resultant G-tailed DNA confirmed the location of the transcription start site as an adenosine residue 24 basepairs upstream from the 3-prime (3’) end of the TATA box. Sequencing 5’ of the TATA box failed to reveal any sequence similarity with the human adenovirus upstream stimulatory factor (USF). Various plasmids were constructed which placed the determinedsequences of the MLP, leader, and the region upstream of the TATA box linked to the co-acetyl acid transferase (CAT) gene. These expression plasmids in transient expression assays of CAT activity in primary chicken kidney cell culture with or without FAV-10 co-infection were determined. These experiments showed that the cassette containing sequences 5′ of the TATA box expressed CAT to a much greater level than cassettes not containing this upstream region and that the presence of virus significantly increased the activity of the promoter following the onset of viral DNA replication. Without the 5′ region, cassettes failed to express above background levels. These results suggest that the basic structure of the fowl adenovirus MLP is similar to that of the human adenovirus although it utilizes a bipartite rather than a tripartite leader sequence.