A 24-Year-Old Sample Contributes the Complete Genome Sequence of Fowl Aviadenovirus D from the United States

Fowl aviadenovirus (FAdV) is a member of the genus Aviadenovirus within the family Adenoviridae (1). Adenoviruses can infect a wide range of hosts; however, avian adenoviruses are reported to infect only avian species (2). FAdVs have been categorized into 5 species (FAdV-A to FAdV-E) on the basis of their genome structure and are further divided into 12 serotypes, based on a cross-neutralization test (3, 4). FAdVs are widely distributed and cause various degrees of associated clinical disease (5, 6). Some species of FAdVs cause inclusion body hepatitis (1, 7–14), hepatitis-hydropericardium syndrome (15–17), adenoviral gizzard erosions (18, 19), and possibly hypoglycemia and spiking mortality syndrome (H-SMS) (20, 21) in chickens. To date, only a few complete genomes of FAdV-D from the United States are available in public databases (22, 23). In this study, we report the complete genome sequence of an FAdV-D isolate from the United States.

A fecal sample was collected from a broiler chicken with hypoglycemia and spiking mortality syndrome (H-SMS) at a commercial farm in Georgia in 1995 (24). At that time, in order to experimentally reproduce severe H-SMS, a series of embryo-passaged preparations were performed. Briefly, H-SMS was experimentally reproduced by inoculating crude feces to 1-day-old chicks. Virus particles from their intestines, which were collected at 12 to 14 days postinoculation, were banded in a discontinuous Renografin gradient and inoculated into 7-day-old specific-pathogen-free embryonating chicken eggs (SPF ECE). Four days postinoculation, the embryos were harvested, homogenized in sterile phosphate-buffered saline, filtered, and then inoculated into 7-day-old SPF ECE. The embryos from this passage died between 48 and 96 hours postinoculation and then were harvested and processed as described before to create a third passage. The pancreases of the embryos from the third passage were homogenized, filtered, and stored at −70°C for 24 years. In 2019, viral RNA and total nucleic acids were isolated from a preserved pancreas sample using the QIAamp viral RNA minikit and the DNeasy blood and tissue kit (Qiagen, Germany), respectively, after first undergoing DNase treatment with the Turbo DNA-free kit (Ambion, USA) to remove host DNA according to the manufacturer’s recommendations. Sequence-independent single-primer amplification (25–27) was used to produce random amplicons that were processed using the Nextera XT DNA library preparation kit (Illumina, USA). The distribution size and concentration of the prepared library were checked on a 2100 bioanalyzer, using the high-sensitivity (HS) DNA kit (Agilent Technologies, Germany), and on a Qubit fluorometer, using the double-stranded DNA (dsDNA) HS assay kit (Life Technologies, USA), respectively. Two next-generation paired-end sequencing (2 × 150 and 2 ×250 bp) runs were performed on an Illumina MiSeq instrument using the 300- and 500-cycle MiSeq reagent kit v.2 (Illumina), respectively. Sequence data from the two runs were combined, and de novo assembly was performed utilizing MIRA v.3.4.1 (28) within a customized workflow on the Galaxy platform (29); all tools were run with default parameters, as described previously (30, 31). A total of 1,113,717 raw paired-end reads (904,985 and 208,732 reads of 150- and 250-bp reads, respectively) were generated. The de novo-generated contigs of interest were subjected to BLASTn search and aligned with the full-length reference genome MX95-S11 (GenBank accession number KU746335.1) to obtain a draft genome scaffold. The genome consensus was then recalled from 200,733 FAdV reads using BWA-MEM (32) mapping of trimmed but unnormalized reads to the genome scaffold. The median read depth of the assembly was 220, and the maximum depth was 5,226. The final genome consensus of the isolate designated GA/1358/1995 was 44,079 nucleotides long (100% genome coverage) with a GC content of 53.6% and coded 37 putative open reading frames (ORFs) (Table 1). The ORFs were identified using the Geneious 11.1.5 and confirmed by alignment with published FAdV genomes. BLAST comparison to the currently available full-length FAdV genome sequences showed the highest (99.23%) nucleotide identity to the FAdV-D serotype 2 prototype ATCC reference strain P7-A (GenBank accession number MK572866.1) (23, 33) (Fig. 1).

Fowl aviadenoviruses appear to be widely endemic in poultry and have been associated with clinical disease, but full-genome sequences of FAdV-D circulating in the United States are scarce. More complete genome sequence information is necessary to understand how fowl aviadenoviruses contribute to disease in poultry and how to control it.

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This study was supported by USDA CRIS project 6040-32000-072 and USDA APHIS interagency agreement 60-6040-5-009.