Protein profile of FAdV-4 based on SDS-PAGE and Western blot isolated from chickens in India.

Fowl adenovirus-4 (FAdV-4) causes hydropericardium syndrome (HPS) in poultry worldwide. An understanding of viral structural protein composition is important for developing novel immunodiagnostics and immunoprophylactics. Here we report isolation, culture, molecular and protein profile of FAdV-4 isolates recovered from HPS outbreaks in chicken in the states of Himachal Pradesh and Tamil Nadu in India. We performed a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting-based protein profiling of FAdV-4 isolates against a reference FAdV-1 or Chicken Embryo Lethal Orphan (CELO) virus. SDS-PAGE analysis showed that seven protein bands in FAdV-4 isolates were similar to CELO expect an additional band of 110 kDa in CELO virus. On Western blotting, two protein fractions of 43 kDa and 78 kDa size were observed in FAdV-4 isolates. Overall, results show that FAdV-4 isolates recovered from different regions of the country had similar protein profile and possibly a common source of origin.

Coding potential and transcript analysis of fowl adenovirus 4: insight into upstream ORFs as common sequence features in adenoviral transcripts.

Recombinant fowl adenoviruses (FAdVs) have been successfully used as veterinary vaccine vectors. However, insufficient definitions of the protein-coding and non-coding regions and an incomplete understanding of virus-host interactions limit the progress of next-generation vectors. FAdVs are known to cause several diseases of poultry. Certain isolates of species FAdV-C are the aetiological agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS). In this study, we report the complete 45667 bp genome sequence of FAdV-4 of species FAdV-C. Assessment of the protein-coding potential of FAdV-4 was carried out with the Bio-Dictionary-based Gene Finder together with an evaluation of sequence conservation among species FAdV-A and FAdV-D. On this basis, 46 potentially protein-coding ORFs were identified. Of these, 33 and 13 ORFs were assigned high and low protein-coding potential, respectively. Homologues of the ancestral adenoviral genes were, with few exceptions, assigned high protein-coding potential. ORFs that were unique to the FAdVs were differentiated into high and low protein-coding potential groups. Notable putative genes with high protein-coding capacity included the previously unreported fiber 1, hypothetical 10.3K and hypothetical 10.5K genes. Transcript analysis revealed that several of the small ORFs less than 300 nt in length that were assigned low coding potential contributed to upstream ORFs (uORFs) in important mRNAs, including the ORF22 mRNA. Subsequent analysis of the previously reported transcripts of FAdV-1, FAdV-9, human adenovirus 2 and bovine adenovirus 3 identified widespread uORFs in AdV mRNAs that have the potential to act as important translational regulatory elements.

Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre.

There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.

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.

Sequence analysis of the left end of fowl adenovirus genomes.

Nucleotide sequence analysis of the left end of the genome of fowl adenoviruses (FAdV) representing species group C (FAdV-4 and -10), D (FAdV-2) and E (FAdV-8) were carried out, and the sequence data was compared to those of FAdV-1 (FAdV-A) and FAdV-9 (FAdV-D). The viruses were propagated in chicken hepatoma cell line for viral DNA isolation. Restriction endonuclease analysis was performed followed by hybridization with two DNA probes representing the left end of FAdV-9. The identified fragments were sequenced, and the generated data were compared with the GenBank database. Nucleotide sequence homology and amino acid sequence identities were high between members of the same species group, FAdV-2 and -9, and FAdV-4 and -10, whereas different degrees of variations were observed among all FAdVs. Gene arrangement and position of ORFs at the left end of FAdV genomes were largely conserved suggesting similar gene functions. All previously characterized left end ORFs in CELO virus and FAdV-9 were found in all analyzed FAdVs. However, ORF 1C was absent in FAdV-4 and -10, but additional ORFs, most likely corresponding to duplicates of ORF 14, were observed in these viruses.

Crystallization of the C-terminal head domain of the avian adenovirus CELO long fibre.

Avian adenovirus CELO contains two different fibres: fibre 1, the long fibre, and fibre 2, the short fibre. The short fibre is responsible for binding to an unknown avian receptor and is essential for infection of birds. The long fibre is not essential, but is known to bind the coxsackievirus and adenovirus receptor protein. Both trimeric fibres are attached to the same penton base, of which each icosahedral virus contains 12 copies. The short fibre extends straight outwards, while the long fibre emerges at an angle. The carboxy-terminal amino acids 579-793 of the avian adenovirus long fibre have been expressed with an amino-terminal hexahistidine tag and the expressed trimeric protein has been purified by nickel-affinity chromatography and crystallized. Crystals were grown at low pH using PEG 10,000 as precipitant and belonged to space group C2. The crystals diffracted rotating-anode Cu Kalpha radiation to at least 1.9 angstroms resolution and a complete data set was collected from a single crystal to 2.2 angstroms resolution. Unit-cell parameters were a = 216.5, b = 59.2, c = 57.5 angstroms, beta = 101.3 degrees, suggesting one trimer per asymmetric unit and a solvent content of 46%. The long fibre head does not have significant sequence homology to any other protein of known structure and molecular-replacement attempts with known fibre-head structures were unsuccessful. However, a map calculated using SIRAS phasing shows a clear trimer with a shape similar to known adenovirus fibre-head structures. Structure solution is in progress.