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

Here, we report the complete genome sequence of fowl adenovirus D (FAdV-D) isolated from a preserved 24-year-old pancreas sample of a broiler chicken embryo. The results of the sequence showed that the viral genome is 44,079 bp long.

ABSTRACT

Here, we report the complete genome sequence of fowl aviadenovirus D (FAdV-D) isolated from a preserved 24-year-old pancreas sample of a broiler chicken embryo. The results of the sequence showed that the viral genome is 44,079 bp long.

ANNOUNCEMENT

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).

TABLE 1.

Characteristics of the full-length genome of FAdV-D isolate GA/1358/1995

Gene name	Strand directiona	Start codon position	End codon position	No. of codons	Closest viral homology (GenBank accession no.)	Amino acid identity (%)
ORF0	R	524	808	94	FAdV-2 (ANJ02325.1)	100
ORF1	R	848	1,339	163	FAdV-2 (ANJ02326.1)	100
ORF1B	R	1,501	1,731	76	FAdV-3 (ANJ02402.1)	100
ORF1C	R	1,679	1,879	66	FAdV-3 (ANJ02403.1)	100
ORF2	R	1,953	2,756	267	FAdV-9 (NP597818.1)	100
ORF7	L	2,348	2,668	106	FAdV-11 (QFR45452.1)	100
ORF24	L	2,839	3,519	228	FAdV-11 (QIM09468.1)	99.56
		16,294	16,299
ORF14	L	3,538	4,224	230	FAdV-9 (AP000373.1)	100
		16,294	16,299
ORF13	L	4,263	5,249	330	FAdV-2 (ANJ02370.1)	100
		16,294	16,299
ORF12	L	5,245	6,162	307	FAdV-9 (AP000375.1)	100
		16,294	16,299
IVa2	L	6,131	7,351	406	FAdV-9 (NP050280.1)	99.01
DNA polymerase	L	7,348	11,262	1,304	FAdV-2 (QGQ62975.1)	99.39
pTP	L	11,259	13,220	655	FAdV-11 (ANJ02596.1)	99.23
		16,294	16,299
52k	R	13,259	14,467	402	FAdV-11 (AIS19821.1)	100
pIIIa	R	14,454	16,229	591	FAdV-11 (AKR76192.1)	100
Penton base	R	16,310	17,947	545	FAdV-11 (AIS19822.1)	100
pVII	R	17,987	18,223	78	FAdV-11 (AKR76194.1)	100
pX	R	18,458	19,057	199	FAdV-9 (NP050285.1)	100
pVI	R	19,187	19,873	228	FAdV-9 (NP050286.1)	100
Hexon	R	19,985	22,834	949	FAdV-2 (QGQ62983.1)	100
Protease	R	22,848	23,465	205	FAdV-11 (AIS19826.1)	100
DNA-binding protein	L	23,580	25,009	557	FAdV-11 (AKR76199.1)	98.56
		25,107	25,350
100k	R	25,414	28,398	994	FAdV-11 (AKR76200.1)	93.55
33k	R	28,079	28,409	221	FAdV-11 (AKR76201.1)	99.55
		28,655	28,989
22k	R	28,079	28,624	181	FAdV-11 (ALS87111.1)	99.45
pVIII	R	29,029	29,754	241	FAdV-11 (AIS19829.1)	100
U exon	L	29,638	30,006	122	FAdV-11 (QIM09482.1)	98.36
Fiber	R	30,005	31,717	570	FAdV-11 (ANQ43489.1)	98.60
ORF22	L	31,777	32,349	190	FAdV-11 (AIS19831.1)	100
ORF20A	L	32,353	32,832	166	FAdV-11 (AKR76206.1)	100
		33,846	33,866
ORF20	L	32,833	33,750	312	FAdV-2 (ANJ02355.1)	100
		33,846	33,866
ORF19	L	34,067	36,193	714	FAdV-11 (QGQ63065.1)	99.58
		36,272	36,289
GAM-1	R	37,747	38,580	277	FAdV-2 (QGQ63210.1)	100
ORF17	L	39,674	40,144	156	FAdV-11 (QFR45478.1)	99.36
ORF11	R	40,519	40,879	261	FAdV-2 (QGQ63176.1)	100
		40,957	41,201
		41,261	41,442
ORF23	L	41,675	42,610	311	FAdV-11 (ANJ02619.1)	100
ORF25	R	43,067	43,075	169	FAdV-11 (QIM09485.1)	99.41
		43,154	43,654

^aR, rightward-transcribed strand; L, leftward-transcribed strand.

FIG 1.

Phylogenetic analysis of fowl aviadenovirus isolates based on the complete genome sequences constructed with the maximum likelihood method based on the general time-reversible model in MEGA v.7.0. The tree with the highest log likelihood (−217,618.18) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. A discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories [+G, parameter = 1.4043]). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 17.27% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 31 nucleotide sequences (sequences of goose adenovirus [GoAdV] is included as an outgroup). All positions containing gaps and missing data were eliminated. There were a total of 29,569 positions in the final data set. The isolate used in this study is shown in blue.

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.

Data availability.

The complete genome sequence of isolate GA/1358/1995 of FAdV-D has been deposited in GenBank under the accession number MN711789. Raw data were deposited in the SRA under accession number SRR10500667, BioSample number SAMN13338320, and BioProject number PRJNA590745.