Fowl Adenovirus Type Research Articles

Structure of the C-terminal head domain of the fowl adenovirus type 1 long fiber

Avian adenovirus CELO (chicken embryo lethal orphan virus, fowl adenovirus type 1) incorporates two different homotrimeric fiber proteins extending from the same penton base: a long fiber (designated fiber 1) and a short fiber (designated fiber 2). The short fibers extend straight outwards from the viral vertices, whilst the long fibers emerge at an angle. In contrast to the short fiber, which binds an unknown avian receptor and has been shown to be essential to the invasiveness of this virus, the long fiber appears to be unnecessary for infection in birds. Both fibers contain a short N-terminal virus-binding peptide, a slender shaft domain and a globular C-terminal head domain; the head domain, by analogy with human adenoviruses, is likely to be involved mainly in receptor binding. This study reports the high-resolution crystal structure of the head domain of the long fiber, solved using single isomorphous replacement (using anomalous signal) and refined against data at 1.6 A (0.16 nm) resolution. The C-terminal globular head domain had an anti-parallel beta-sandwich fold formed by two four-stranded beta-sheets with the same overall topology as human adenovirus fiber heads. The presence in the sequence of characteristic repeats N-terminal to the head domain suggests that the shaft domain contains a triple beta-spiral structure. Implications of the structure for the function and stability of the avian adenovirus long fiber protein are discussed; notably, the structure suggests a different mode of binding to the coxsackievirus and adenovirus receptor from that proposed for the human adenovirus fiber heads.

The complete nucleotide sequence of fowl adenovirus type 8.

The fowl adenovirus type 8 (FAdV-8) genome was sequenced and found to be 45063 nucleotides in length, the longest adenovirus (AdV) genome for which the complete nucleotide sequence has been determined so far. No regions homologous to early regions 1, 3 and 4 (E1, E3 and E4) of mastadenoviruses were recognized. Gene homologues for early region 2 (E2) proteins, intermediate protein IVa2 and late proteins were found by their similarities to protein sequences from other AdVs. However, sequences homologous to intermediate protein IX and late protein V could not be identified. Sequences for virus-associated RNA could also not be recognized. Two regions of repeated sequences were found on the FAdV-8 genome. The shorter repeat region contained five identical and contiguous direct repeats that were each 33 bp long, while the longer repeat region was made of 13 identical and contiguous, 135 bp long repeated subunits.

Sequence and transcriptional analysis of terminal regions of the fowl adenovirus type 8 genome.

Fowl adenovirus (FAdV) type 1, CELO strain has no homologies to mastadenovirus E1A, E1B, E3 and E4, which regulate virus gene expression, DNA replication and virus-host interaction. Similarly, the right 5 kb and left 15 kb ends of CELO virus DNA are non-homologous to mastadenoviruses. To compare CELO virus with another FAdV, 7.5 kb of the left and 17 kb of the right ends of FAdV type 8 (strain A-2A) were sequenced and nine and 17 open reading frames (ORFs), respectively, were found. This FAdV-8 genome was similar to CELO virus in that (1) the central region contained the major structural protein genes including the fibre, pVIII, 100K, late 33K and pIVa2 genes, which were in the same order as in mastadenoviruses, (2) no homologues of mastadenovirus E1A, E1B, E3 and E4 were found in the ends, and (3) the left 6 kb and the right 13 kb ends showed no homology to mastadenoviruses. Several genomic features were unique to FAdV-8 compared to CELO virus. FAdV-8 contained one fibre gene in contrast to two in CELO virus. Three of eight unassigned ORFs in the left and five of 13 unassigned ORFs in the right ends were unique compared to CELO virus. Two sets of tandem repeats, one with five identical 33 bp repeats and the other with more than ten identical 135 bp repeats, mapped between 4.5 and 7.5 kb from the right terminus. No virus-associated RNA gene was found. Fifteen of 16 unique FAdV-8 ORFs tested were, as determined by RT-PCR, transcribed early.

Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line

Fowl adenoviruses, many of which appear to be non-pathogenic, are ubiquitous in birds. In addition, the genome of these viruses is large, making them ideal candidates for construction as vectors for foreign genes. Current methods to cultivate fowl adenoviruses use primary cell cultures derived from embryonated chicken eggs. In order to provide a more suitable culture method, the growth of fowl adenovirus type 8 (FAdV-8) was investigated in CH-SAH, a continuous hepatoma cell line. A one step growth curve demonstrated release of extracellular virus beginning by 18 h p.i. and with a final yield about 100 fold higher than that in chicken embryo liver cells. Viral DNA synthesis was first detected 8 h prior to this. The CH-SAH cell line supported the production of progeny viruses similar to the wild-type virus after being transfected with purified FAdV-8 DNA. This study demonstrated that the continuous hepatoma cell line is an appropriate in vitro host for FAdV-8.

Genes for fowl adenovirus CELO penton base and core polypeptides.

A 3.5-kilobase DNA fragment of the fowl adenovirus type 1 (CELO), located between map units 31.1 and 39.4 has been determined. The sequence contains the probable CELO equivalents of the IIIa protein, penton base, pVII and pV core protein genes of human adenovirus (HAV). The CELO penton base and major core protein (analog HAV pVII) were found to consist of 514 (56.8 kDa) and 72 amino acids (8.4 kDa), respectively.

The fowl adenovirus type 1 (CELO) virus-associated RNA-encoding gene: a new ribozyme-expression vector

A new system for hammerhead ribozyme (Rz) expression was examined in which fowl adenovirus type 1 (CELO) virus-associated RNA (CELO VA RNA) was used as a vector for the incorporation of Rz to target the mRNA of secreted alkaline phosphatase (SEAP) both in vitro and in vivo. The Rz gene was integrated into the CELO VA RNA between the internal promoter boxes A and B; apparently this did not interfere with its transcription. Rz integrated into CELO VA RNA and, lacking the viral sequences, exhibited the same activity in vitro. Consequently, CELO VA RNA sequences did not inhibit the integrated Rz activity in vitro. In vivo experiments were carried out with human 293 cells by co-transfection with plasmids containing Rz and SEAP. Inhibition of enzyme activity was 50% in 48 h. We conclude that CELO VA RNA may be used for effective expression of hammerhead Rz.

The oncogenicity of avian adenoviruses IV. Confirmatory evidence for recombination between viral and cellular DNA sequences and repetition of the recombinant in cells of a tumor line

On the basis of hybridization analyses with viral DNA fragments as probes, we previously concluded that, in DNAs of many fowl adenovirus type 1-induced rodent tumors, units consisting of viral DNA segments flanked on both sides by cellular DNA segments are repeated more than 100 times per diploid genome of cell. Molecular cloning of virus—cellular junction fragments and analyses of their sequence arrangement in the present study confirmed that the previous conclusion was basically correct.

Preferential clustering of viral DNA sequences at or near the site of chromosomal rearrangement in fowl adenovirus type 1 DNA-transformed cell lines.

All six transformants obtained by inoculating fowl adenovirus type 1 (CELO virus) DNA or its fragments into a rat cell line of normal karyotype had more than 50 copy-equivalents of viral DNA sequences. Each of the transformants had almost all if not all of these viral DNA sequences clustered on a marker chromosome(s). Although the marker chromosome(s) differed from one cell line to another, viral DNA sequences preferentially clustered in or near the achromatic (or light-stained) region of the G-banded marker chromosomes where chromosomal rearrangement or translocation occurred. These results indicate that no particular chromosome is required to act as the integration site of viral DNA for the transformation of cells, but chromosomal rearrangement at or near the cluster of viral DNA sequences might contribute to the transformation.

The structural proteins of chick embryo lethal orphan virus (fowl adenovirus type 1).

Chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contains at least 14 structural proteins with polypeptide molecular weights ranging from 100K to about 6K. A nomenclature of the CELO virion polypeptides is presented and the molar proportion of each polypeptide has been estimated. The CELO virus pentons were specifically released from the virion by dialysis against borate-based calcium-magnesium saline. The penton base (polypeptide III, mol. wt. 92K) and two fibres were separated, characterized and their polypeptides were correlated with their morphological positions in the virion. Peptide mapping suggested that the long fibre (polypeptide IV, mol. wt. 65K), and the short fibre (polypeptide VII, mol. wt. 44.5K) were not related in their primary sequences and are therefore probably encoded by separate genes. The time course of synthesis of the CELO virion polypeptides indicated that, like their mammalian adenovirus counterparts, they are synthesized late (after viral DNA replication).

DNA-binding proteins of chick embryo lethal orphan virus: lack of complementation between early proteins of avian and human adenoviruses.

Chick cells infected by chick embryo lethal orphan (CELO) virus (fowl adenovirus type 1) contained four prominent virus-specific, structurally related DNA-binding proteins with mol. wt. of 74K, 64K, 56K, 52K, and two minor forms. The CELO virus DNA-binding proteins were phosphorylated, delayed-early nuclear proteins. CELO virus early proteins were expressed in BHK cells, but did not complement human adenovirus type 5 mutants with lesions E1A, E2A or E2B. Moreover, CELO virus DNA-binding proteins were not produced in 293 cells, which express human adenovirus E1 genes. These results suggest that activation of transcription by adenovirus E1A genes involves specific interactions between the E1A gene products and viral early promoters.

Sequence homology between avian and human adenoviruses.

Studies of hybridization between fowl adenovirus type 1 (chicken embryo lethal orphan virus) DNA and human adenovirus type 2 DNA revealed two short but distinct regions which cross-hybridized under stringent conditions. One of the homologous regions was located between map positions 18.1 and 19.3 and did not correspond to any gene recognized so far. The second region mapped in the hexon gene between position 57 and 58.

Characterization of Two Strains of Fowl Adenovirus Type 1

An adenovirus isolated from the trachea of a chicken with respiratory disease was compared with an isolate obtained from the trachea of a clinically normal chicken. The isolates caused similar respiratory disease experimentally but differed in plaque morphology and fluorescent-antibody reactions in infected chick embryos.

Studies on the antigenic relationship between an isolate (127) from the egg drop syndrome 1976 and a fowl adenovirus

Birds infected with fowl adenovirus type 1 (FAV-1) developed precipitating antibody to FAV-1 but not to haemagglutinating virus 127 isolated from flocks with a depressed egg production syndrome. Once the precipitating antibody to FAV-1 was no longer detectable, the birds were then infected with 127 virus. They developed precipitating and haemagglutination-inhibiting antibody to 127 virus. They also developed precipitating antibody to the adenovirus group antigen. Birds infected only with 127 virus did not develop adenovirus group antibody. Birds which had been infected with FAV-1, and subsequently infected with infectious bronchitis virus, when without detectable FAV-1 antibody, did not show a recall of adenovirus group antibody. It is concluded that 127 and FAV-1 must share an antigen. This is further evidence that 127 is an avian adenovirus.

Biochemical Studies on a Virus Associated with Egg Drop Syndrome 1976

SUMMARY A number of hitherto undescribed, serologically identical viruses have been isolated from a syndrome of depressed egg production in broiler breeder flocks (Egg Drop Syndrome 1976). One of these, 127 virus, was purified after growth in chick embryo liver cells. Three particle types B1, B2 and B3 with densities of 1.32, 1.30 and 1.28 g/ml, respectively, were separated in CsCl equilibrium density gradients. B1 and B2 particles possessed infectivity and were labelled with 3H-thymidine. However, they differed morphologically and B2 particles agglutinated chicken erythrocytes whereas B1 particles did not. B3 particles were not infectious, were not labelled with 3H-thymidine but agglutinated chicken erythrocytes. They were penetrated by stain when examined by electron microscopy and probably correspond to empty particles. B1, B2 and B3 particles differed in their polypeptide compositions; seven of the polypeptides in B1 and B2 particles had counterparts in purified fowl adenovirus type 1. The soluble haemagglutinin from 127 virus, which did not sediment under centrifugation conditions sufficient to pellet virus particles, was purified using DEAE cellulose chromatography and gel filtration. Purified soluble haemagglutinin contained two polypeptides (mol. wt. 67000 and 65000), which co-migrated in polyacrylamide gels with two of those present in purified 127 virus particles. The soluble haemagglutinin sedimented heterogeneously (20 to 50S) in sucrose density gradients and had a density of 1.24 g/ml in CsCl. It was inactivated by trypsin, urea and pyridine. Electron microscopy of purified soluble haemagglutinin showed rod-like structures with lengths of 25 to 30 nm, which radiated from a central area measuring approx. 10 nm in diam. It is suggested that 127 virus is an adenovirus.