Pyrosequencing analysis for a rapid classification of fowl adenovirus species

Figures & data

Table 1.  FAdV reference strains used in the present study.

Species	Serotype	Strains
A	FAdV-1	CELO
B	FAdV-5	TR22, IBH-2A
C	FAdV-4	KR5, 506, J2-A
	FAdV-10	C-2B
D	FAdV-2	685, P7-A
	FAdV-3	SR49, 75, 75-1A-1
	FAdV-9	A-2A
	FAdV-11	380
E	FAdV-6	CR119
	FAdV-7	YR36, X11-A
	FAdV-8a	TR59, 58, T8-A
	FAdV-8b	764, B-3A
Species and serotypes are indicated according to the International Committee on Taxonomy of Viruses current nomenclature system (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fs_adeno.htm#Genus2).

Figure 1.  Schematic representation of the P1 and L1 regions of the FAdV hexon gene and the primers used in this study for PCR reaction and pyrosequencing analysis.

Table 2.  Pyrosequencing report of 22 reference strains representing the five FAdV species (A to E) and the 12 different serotypes.

				Score (%)
Sample	BLASTn results	BLASTn sequence homology (%)	Pyrosequence results	First extraction	Second extraction
75-1A-1	AF339921	100	D, serotype 3	100	100
J2-A	AF339917	100	C	100	100
T8-A	AF339919	100	E	100	100
P7-A	AF339915	100	D, serotype 2	100	100
B-3A	AF339922	100	E	100	100
A-2A	AF339918	100	D, serotype 9	100	100
C-2B	AF339923	100	C	100	100
X11-A	AF339924	100	E	100	95.3
YR36	AF508955	100	E	95.3	100
58	AF508957	100	E	95.3	100
75	AF508949	100	D, serotype 3	100	100
506	AF508950	100	C	100	100
685	AF508947	100	D, serotype 2	100	100
764	AF508958	100	E	100	100
CELO	AF339914	100	A, serotype 1	100	100
KR5	AF508951	100	C	100	100
TR22	AF508953	100	B, serotype 5	69.1	100
380	AF339925	100	D, serotype 11	100	100
SR49	AF508948	100	D, serotype 3	100	100
TR59	AF508956	100	E	100	100
IBH-2A	AF339916	100	B, serotype 5	100	100
CR119	AF508954	100	E, serotype 6	100	100
Pyrosequencing analysis, performed after DNA extraction on two different days (first and second), identified all the different species and at least seven serotypes among those tested. No pyrosequencing reaction was observed for the non-template PCR controls tested either with or without beads and sequencing primer (data not shown). The results column shows the FAdV species and serotype from the sequence library to which the tested samples shares homology in the 30-bp pyrosequencing region. The degree of homology is reflected by the score, which is given as a percentage.

Table 3.  Comparison between the BLASTn and the pyrosequence results of the 27 field isolates.

						Species/serotype
Isolation year	Sample	GenBank accession number	BLASTn results^a	BLASTn sequence homology (%)	Strain	BLASTn	Pyrosequence
2008	1279/08	HM592267	AF508956	100	TR59	E/8a	E
	147/08	HM592268	EF685489	98	–	E	E
	1474/08	HM592269	AF508956	99	TR59	E/8a	E
	132/08	HM592270	AF508950	98	506	C/4	C
	499/08	HM592271	EF685649	100	–	E	E
	829/08	HM592272	U46933	99	CELO	A/1	A/1
	457/08	HM592273	FJ360746	91	TAdV-A	TAdV-A	TAdV-A
2007	5670	HM592274	AF508950	99	506	C/4	C
	5705	HM592275	AF339924	98	X11-A	E/7	E
	5363	HM592276	AF508950	99	506	C/4	C
	6169	HM592277	AF508951	99	KR5	C/4	C
	5341	HM592278	AF508956	100	TR59	E/8a	E
	5630	HM592279	AF339924	98	X11-A	E/7	E
	5441	HM592280	AF508948	96	SR49	D/3	D/3
	5997	HM592281	EU931693	99	–	C/4	C
	5765	HM592282	AF508947	99	685	D/2	D/2
	4587	HM592283	AF508950	98	506	C/4	C
	4158	HM592284	AF508950	98	506	C/4	C
	4220	HM592285	EU979367	99	CELO	A/1	A/1
	5231	HM592286	U46933	100	CELO	A/1	A/1
	5339	HM592287	AF508956	100	TR59	E/8a	E
	3890	HM592288	EU931693	99	–	C/4	C
	2496	HM592289	EU979374	99	TR59	E/8a	E
	2797	HM592290	AF508956	99	TR59	E/8a	E
	2161	HM592291	EU861037	98	CELO	A/1	A/1
	5083	HM592292	AF508949	99	75	D/3	D/3
	3636	HM592293	AF508956	100	TR59	E/8a	E
^aBlastn results were obtained by alignment of the field sample consensus sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Figure 2.  Agarose gel electrophoresis of the 740 to 765 bp PCR products from single FAdV strains representing FAdV serotypes 1 to 11 and control samples using HEV, EDS'76, ILTV, IBV strain M41, AIV subtype H7 and non-template control (K–). MW, molecular weight.

Figure 3.  Phylogenetic tree established with the nucleotide sequences of the approximately 765 bp fragment of the reference strains hexon L1 region. The FAdVs are clustered into five species (A to E) and then divided into 12 serotypes according to the ICTV current nomenclature (ICTVdB Management, 2006). Serotypes:

FAdV-1;

FAdV-2;

FAdV-3;

FAdV-4;

FAdV-5;

FAdV-6;

FAdV-7;

FAdV-8a;

FAdV-8b;

FAdV-9;

FAdV-10;

FAdV-11.

Figure 4.  Example of pyrogram showing the 30-bp sequence of the hexon L1 of a reference strain (506). The pyrophosphate anion (P₂O₇ ^4-, PPi) is released by the hydrolysis of ATP into AMP when a nucleotide is incorporated into a growing DNA strand by the polymerase. Peak height is proportional to the amount of PPi and thereby to the number of incorporated nucleotides.

Figure 5.  IdentiFire report of 27 field samples collected in the north-east of Italy between 2007 and 2008. This approach also allowed the identification of a Turkey adenovirus A isolate (sample number 457/08) of a distinct genogroup. No pyrosequencing reaction was observed for the non-template PCR controls (K–). The results column shows the FAdV species and serotype from the sequence library to which the tested samples shares homology in the 30-bp pyrosequencing region. The degree of homology is reflected by the score, which is given as a percentage.

ICTVdB Management 2006 . 00.001.0.02.001. Fowl adenovirus A C. Büchen-Osmond ICTVdB—The Universal Virus Database (version 4) . New York : Columbia University .

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