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The nucleotide sequence at the right end of the genomes of fowl adenoviruses (FAdVs) representing species groups C (FAdV-4 CA and FAdV-10 C-2B), D (FAdV-2 CA), and E (FAdV-8 CA) was determined and analyzed and compared to FAdV-1 (species group A virus) and FAdV-9 A-2A (species group B virus). High nucleotide sequence identities and amino acid identities (74-94%) were found among viruses in the same species group. Homologues to known open reading frames (ORFs) of the right end of the reported FAdV genomes, such as lipase and Gam-1, were also found in all analyzed viral genomes. Homologues to a few FAdV ORFs were found in viruses other than FAdVs as well. Several novel FAdV ORFs were also found. Most identified ORFs have unknown function and thus further studies are necessary to establish their importance in virus replication. ORF content within the right end of the FAdV genomes varied among FAdVs, while the gene order and orientation of shared ORFs are conserved between different FAdVs.

Nanomaterial-based artificial enzymes or nanozymes exhibit superior properties such as stability, cost effectiveness and ease of preparation in comparison to conventional enzymes. However, the lower catalytic activity of nanozymes limits their sensitivity and thereby practical applications in the bioanalytical field. To overcome this drawback, herein we propose a very simple but highly sensitive, specific and low-cost dual enhanced colorimetric immunoassay for avian influenza A (H5N1) virus. 3,3´,5,5´- Tetramethylbenzidine (TMBZ) was used as a reducing agent to produce gold nanoparticles (Au NPs) with blue colored solution from a viral target-specific antibody-gold ion mixture at first step. The developed blue color from the sensing design was further amplified through catalytic activity of Au NPs in presence of TMBZ-hydrogen peroxide (H2O2) solution in second step. Hence, the developed dual enhanced colorimetric immunosensor enables the detection of avian influenza virus A (H5N1) with a limit of detection (LOD) of 1.11 pg/mL. Our results confirmed that the developed assay has superior sensitivity than the conventional ELISA method, plasmonic-based bioassay and commercial flu diagnostic kits. Proposed sensing method further showed its capability to detect viruses, avian influenza A (H4N6) and A (H9N2) virus, in blood samples with limit of detection of 0.0269 HAU and 0.0331 HAU respectively.

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.

The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not wellcharacterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400-2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9D4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9. INTRODUCTIONFowl adenoviruses (FAdVs) have a worldwide distribution, and some are important poultry pathogens that cause inclusion body hepatitis and hydropericardium syndrome in chickens (Adair & Fitzgerald, 2008). FAdV serotypes are grouped into five species (Fowl adenovirus A-E), all belonging to the genus Aviadenovirus, family Adenoviridae (Benkő et al., 2005). Complete nucleotide sequences are available for the genomes of FAdV-1 (CELO virus) and FAdV-9 (strain A-2A) (Chiocca et al., 1996;Ojkic & Nagy, 2000), and partial nucleotide sequences are also available for the left and right ends of the genomes of FAdV-2, -4, -8 and -10 (strain C-2B) (Corredor et al., 2006(Corredor et al., , 2008. None of the early genes at the left and right ends of the FAdV genomes are homologues of the E1, E3 and E4 genes of mastadenoviruses, which infect mammals Chiocca et al., 1996). In this study, the importance of ORFs at the left end of the FAdV-9 genome in virus replication in vitro and in vivo was investigated by analysing deletions, as a first step towards elucidating the roles of the early genes in this region. RESULTS Generation of deletionsThe ORF arrangement within the left-end genomic region of FAdV-9 and the features of all deletions are depicted in Fig. 1(a, b), respectively. Nine deletions were generated in the region within nt 250-4200 of the FAdV-9 genome as shown: FAdV-9D1 (D1194-2342), FAdV-9D2 (D854-2782), FAdV-9D3 (D854-2817), FAdV-9D4 (D491-2782), FAdV-9D5 (D491-2980), FAdV-9D6 (D491-4200), FAdV-9D7 (D400-2782), FAdV-9D8 (D330-2782) and FAdV-9D9 (D250-2782). Viable viruses were recovered following transfection of CH-SAH (chicken hepatoma) cells without complementation with only the FAdV-9D1, -9D2, -9D4 and -9D7 genomes at 5-7 days post-transfection (p.t.). The first viable deletion mutant, FAdV-9D1, was generated by Supplementary methods and a supplementary figure and table are available with the online version of this paper.