Molecular Detection and Identification of Influenza Viruses by Oligonucleotide Microarray Hybridization

Srikumar Sengupta; Kenji Onodera; Alexander Lai; Ulrich Melcher

doi:10.1128/JCM.41.10.4542-4550.2003

DOI: 10.1128/JCM.41.10.4542-4550.2003

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FIG. 1.
Within-slide reproducibility of median pixel intensities. Influenza virus oligonucleotide microarray was hybridized with KY98 cDNA. Minimum (squares) and maximum (triangles) median intensities for quadruplicate spots plotted against the rank order of the average of the median intensities (line) for the four spots.
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FIG. 2.
Experiment-to-experiment reproducibility of pixel intensities. Relative intensities (i.e., normalized median pixel intensity [10⁻³]) from influenza virus oligonucleotide microarray hybridized with HK68 target cDNA in one experiment (experiment A, Table 3) were plotted against the intensities obtained for the same spot with another target cDNA preparation made from the same virus (experiment E, Table 3).
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FIG. 3.
Independence of oligonucleotide normalized median pixel intensities from complementarity to template as measured by BLASTn E-values. Influenza virus oligonucleotide microarray was hybridized with labeled cDNA derived from PR8. Logarithms of E-values were plotted against relative intensity (i.e., normalized median pixel intensity [10⁻³]).

Tables

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TABLE 1.

Influenza viruses used in this study

Virus	Abbre- viation	Segment no.	GenBank or Influenza Sequence Database accession no.
A/Equine/Kentucky/1/98 (H3N8)^a	KY98	4	AF197241
A/Equine/Miami/63 (H3N8)^a	MI63	4	M29257
		5	M22575
		6	L06580
		7	AF001674
A/Panama/2007/99 (H3N2)^b	Panama 99	4	ISDNCDA001
A/Aichi/2/68 (H3N2)^c	HK68	1	AF348170
		2	AF348172
		3	AF348170
		4	AF348176, J02090
		5	X15890
		6	U42630
		7	AF348198, M63515
		8	AF348198
A/PR/8/34 (H1N1)^c	PR8	1	ISDN13419
		2	ISDN13420
		3	ISDN13421
		4	ISDN13422
		5	ISDN13423
		6	ISDN13424
		7	ISDN13425
		8	ISDN13426
A/Equine/Kentucky/9/95 (H3N8)^a	KY95	4	AF197247

↵a Obtained from T. Chambers.
↵b Obtained from N. Cox.
↵c Obtained from E. Kilbourne.

TABLE 2.

Variation in normalized pixel intensities for similar oligonucleotides^a

Segment	Sequence	E-value	Pixel intensity
1	`AATCTAATGTCGCAGTCTC`	−4.0	0
1	`CTGATGTCGCAGTCTCGCAC`	−2.7	7,283
1	`CTGTCAGTAAGTATGCTAGAGTCCC`	−7.2	43,161
1	`GGCTGTCAGTAAGTATGCTA`	−4.5	7,289
2	`TTGAATGGATGTCAATCCGA`	−4.5	0
2	`GAATGGATGTCAATCCGACC`	−4.5	0
4	`TTTCTAATATCCACAAAATGAAGGC`	0.5	0
4	`ACCAAAATGAAGGCAAACC`	−4.0	7,769
4	`CAGATGCAGACACAATATGT`	−4.5	3,314
4	`CAGATGCAGACACAATATGTATAGG`	−7.2	1,341
4	`AAACCGGCAATGGCTCCAAA`	−4.5	4,842
4	`ACCGGCAATGGCTCCAAA`	−3.4	1,581
4	`GATGCAGACACAATATGTATAGG`	−6.2	0
4	`CAGATGCAGACACAATATGT`	−4.5	3,314
4	`CAGATGCAGACACAATATGTATAGG`	−7.2	1,341
4	`AAAGCAGGGGAAAATAAAAACAACC`	−7.2	52,256
4	`GCAGGGGAAAATAAAAACAACC`	−5.5	52
4	`GCAGGGGAAAATAAAAGCCAC`	−2.0	0
5	`TGCGGGGAAGGATCCTAAGAAAAC`	−4.3	6,709
5	`GGGAAAGATCCTAAGAAAAC`	−4.5	884
5	`TCCTCTGCATTGTCTCCG`	−3.4	58,051
5	`CTCTGCATTGTCTCCGAAG`	−4.0	48,211
7	`GTCAGCATCCACAGCACTCTGCTGTTCC`	−9.0	10,645
7	`TCGTCAGCATCCACAGCA`	−3.4	56,454
7	`GACCAGCACTGGAGCTAGGA`	−4.5	36,488
7	`GACCAGCACTGGAGCTAGGG`	−4.0	15,976
7	`GGCAAGTGCACCAGCAGAATAAC`	−6.2	33,891
7	`GGCAAGTGCACCAGCAGAATAACT`	−6.7	48,119
7	`AGCGTAGACGCTTTGTC`	−3.0	0
7	`CTGCAGCGTAGACGCTTTGTCCAAAATG`	−9.0	7,788
8	`CCCATTCTCATTACTGCTTC`	−4.5	54,028
8	`ATAATGTTTTTCTCATTACT`	0.9	1,731
KY	`ATTGACCCCTAACCCACG`		30,727
KY	`CTGCCTGATTGACCCCTAACC`		60,471
KY	`TATCCTGCCTGATTGACC`		18,603
KY	`TTATCCTGCCTGATTGACC`		4,997
KY	`CTTATCCTGCCTGATTGACC`		24,744
KY	`TGCTTATCCTGCCTGATTGACC`		9,453

↵a Values reported with segment numbers are from hybridization of the influenza virus oligonucleotide microarray with PR8 cDNA target, whereas those identified as “KY” are from hybridization of the microarray with KY98 cDNA target. Values are normalized mean net median pixel intensities for quadruplicate spots. Negative values (due to spot intensities being insignificantly less that background intensities) are reported as zeros.

TABLE 3.

Identification of influenza virus type by microarray hybridization

Virus (expt)^a	Score^b
Virus (expt)^a	Type A	Type B	Type C
KY98*	80.3 (55)	14.4 (0)	5.3 (0)
MI63*	83.2 (69)	12.0 (0)	4.7 (0)
Panama 99*	80.8 (88)	13.6 (0)	5.5 (0)
PR8*	86.1 (54)	8.5 (0)	5.3 (0)
HK68 (A)*	89.6 (70)	7.8 (0)	2.6 (0)
HK68 (B)*	82.5 (70)	11.4 (0)	6.1 (0)
HK68 (C)*	80.8 (79)	11.7 (0)	7.5 (0)
HK68 (D)	75.8 (47)	20.8 (1)	3.4 (0)
HK68 (E)	89.4 (63)	7.9 (0)	2.7 (0)
HK68 (F)	91.5 (60)	8.5 (0)	0.0 (0)
Available signature oligonucleotides	(274)	(32)	(1)

↵a Hybridization with HK68 cDNA was performed in six experiments: experiments B and C were done at 42 and 32°C, respectively, while all others were done at 22°C. A high concentration of cDNA was used for experiment D. F was scanned at lower laser (95%) and PMT (90%) settings; all others were at 100 and 95% laser and PMT settings, respectively. *, Experiments included in training set.
↵b The scores are from the weighted oligonucleotide calculation (see Materials and Methods). Values in parentheses are the numbers of signature oligonucleotides that registered positive.

TABLE 4.

Identification of influenza virus source host by microarray hybridization

Virus (expt)^a	Score^b
Virus (expt)^a	Human	Swine	Avian	Equine	Other
KY98*	40.1 (1)	13.9	26.0 (0)	8.1 (1)	12.0
MI63*	42.0 (1)	16.8	23.3 (0)	9.0 (2)	8.9
Panama 99*	46.0 (1)	15.6	23.5 (0)	5.3 (0)	9.6
PR8*	39.7 (0)	14.0	28.7 (0)	3.7 (0)	13.9
HK68 (A)*	40.5 (1)	15.9	26.1 (0)	6.1 (0)	11.3
HK68 (B)*	40.7 (0)	13.9	26.7 (0)	7.0 (0)	11.6
HK68 (C)*	41.1 (0)	14.4	25.8 (0)	6.7 (0)	12.0
HK68 (D)	46.3 (2)	12.0	23.2 (0)	5.8 (0)	12.7
HK68 (E)	40.1 (1)	14.4	25.6 (0)	6.4 (0)	13.5
HK68 (F)	41.8 (1)	14.6	25.9 (0)	5.9 (0)	11.8
Available signature oligonucleotides	(31)	(0)	(2)	(3)	(0)

↵a See Table 3 for the conditions of experiments A to E. *, Experiments included in training set.
↵b The scores are from the weighted oligonucleotide calculation (see Materials and Methods). Values in parentheses are the numbers of signature oligonucleotides that registered positive.

TABLE 5.

Identification of influenza virus segments by microarray hybridization

Virus (expt)^a	Score^b
Virus (expt)^a	Segment 1	Segment 2	Segment 3	Segment 4	Segment 5	Segment 6	Segment 7	Segment 8
KY98*	1.6 (2)	6.9 (1)	3.8 (1)	26.1 (5)	13.4 (12)	17.3 (5)	16.4 (12)	14.5 (9)
MI63*	2.4 (4)	5.4 (3)	6.1 (1)	31.7 (6)	9.7 (9)	12.8 (6)	17.2 (21)	14.8 (9)
Panama 99*	2.1 (5)	2.9 (2)	6.0 (3)	27.0 (7)	9.3 (12)	16.4 (11)	22.9 (29)	13.4 (13)
PR8*	1.6 (2)	6.2 (2)	1.6 (0)	27.0 (4)	13.2 (10)	7.3 (1)	29.1 (22)	13.9 (8)
HK68 (A)*	2.7 (2)	4.2 (2)	4.3 (2)	21.5 (6)	11.6 (13)	14.4 (4)	27.6 (24)	13.6 (11)
HK68 (B)*	2.7 (3)	1.9 (3)	8.0 (2)	25.1 (1)	12.1 (12)	10.0 (7)	26.7 (22)	13.4 (12)
HK68 (C)*	3.2 (3)	1.7 (3)	6.0 (2)	26.7 (1)	10.4 (13)	9.9 (7)	27.2 (29)	15.0 (14)
HK68 (D)	0.8 (1)	1.9 (0)	7.3 (2)	28.2 (2)	11.5 (10)	18.7 (6)	15.5 (8)	16.1 (9)
HK68 (E)	3.8 (2)	4.5 (1)	3.9 (2)	23.5 (7)	10.4 (12)	11.5 (3)	27.4 (20)	15.1 (11)
HK68 (F)	3.2 (3)	3.6 (3)	2.8 (1)	28.4 (3)	10.4 (12)	12.8 (4)	29.2 (19)	9.7 (8)
Available signature oligonucleotides	(7)	(4)	(5)	(122)	(25)	(22)	(41)	(20)

↵a See Table 3 for the conditions of experiments A to E. *, Experiments included in training set.
↵b The scores given are from the weighted oligonucleotide calculation (see Materials and Methods). Values in parentheses are the numbers of signature oligonucleotides that registered positive.

TABLE 6.

Identification of influenza virus HA subtype by microarray hybridization

Virus (expt)^a	Score^b (by HA subtype)
Virus (expt)^a	Subtype 1	Subtype 2	Subtype 3	Subtype 4	Subtype 5	Subtype 6	Subtype 7	Subtype 8	Subtype 9	Subtype 10	Subtype 11	Subtype 12	Subtype 13	Subtype 14	Subtype 15
KY98*	5.5 (0)	0.8	40.8 (2)	5.5 (0)	10.7 (0)	6.5	5.6 (0)	0.0	4.4 (0)	0.0	7.4	0.0	12.9	0.0	0.0
MI63*	8.8 (0)	1.1	36.0 (6)	3.3 (0)	7.0 (0)	3.3	10.3 (0)	5.3	3.1 (0)	3.3	3.8	0.0	8.0	0.0	6.6
Panama 99*	5.1 (0)	0.9	48.4 (7)	5.6 (0)	4.3 (0)	4.4	2.7 (0)	0.0	2.7 (0)	0.0	13.7	0.0	12.2	0.0	0.0
PR8*	69.6 (2)	0.0	1.2 (0)	0.0 (0)	8.2 (0)	0.0	0.0 (0)	17.8	0.0 (0)	0.0	3.2	0.0	0.0	0.0	0.0
HK68 (A)*	4.6 (0)	0.8	44.0 (6)	5.7 (0)	4.6 (0)	4.0	2.4 (0)	0.0	2.4 (0)	7.9	12.5	0.0	11.1	0.0	0.0
HK68 (B)*	8.6 (0)	1.4	25.8 (1)	7.6 (0)	7.6 (0)	7.1	4.3 (0)	0.0	3.9 (0)	0.0	12.2	0.0	21.4	0.0	0.0
HK68 (C)*	8.0 (0)	1.5	19.2 (1)	8.8 (0)	6.7 (0)	6.8	4.2 (0)	0.0	4.2 (0)	0.0	21.5	0.0	19.2	0.0	0.0
HK68 (D)	6.1 (0)	1.0	11.8 (0)	7.0 (0)	5.3 (0)	16.4	5.5 (0)	0.0	3.2 (0)	4.9	21.1	0.0	17.7	0.0	0.0
HK68 (E)	10.0 (1)	0.8	41.5 (6)	5.3 (0)	4.4 (0)	3.7	2.3 (0)	0.0	2.3 (0)	7.5	11.8	0.0	10.5	0.0	0.0
HK68 (F)	6.6 (0)	1.1	32.2 (1)	7.7 (0)	3.4 (0)	2.2	5.7 (0)	0.0	2.1 (0)	20.2	13.4	0.0	5.4	0.0	0.0
Available signature oligonucleotides	(23)		(53)	(1)	(2)		(1)		(1)

↵a See Table 3 for conditions of experiments A to E. *, Experiments included in training set.
↵b Scores given are from the weighted oligonucleotide calculation (see Materials and Methods). Values in parentheses are the numbers of signature oligonucleotides that registered positive.

TABLE 7.

Identification of influenza virus NA subtype by microarray hybridization

Virus (expt)^a	Score^b (by NA subtype)
Virus (expt)^a	Subtype 1	Subtype 2	Subtype 3	Subtype 5	Subtype 6	Subtype 7
KY98*	0.0 (0)	100 (5)	0.0	0.0	0.0	0.0
MI63*	2.9 (0)	72.5 (6)	0.0	0.0	12.3	12.3
Panama 99*	4.3 (0)	86.9 (10)	0.0	0.0	8.8	0.0
PR8*	97.2 (1)	2.8 (0)	0.0	0.0	0.0	0.0
HK68 (A)*	0.0 (0)	100 (4)	0.0	0.0	0.0	0.0
HK68 (B)*	0.0 (0)	100 (7)	0.0	0.0	0.0	0.0
HK68 (C)*	0.0 (0)	100 (7)	0.0	0.0	0.0	0.0
HK68 (D)	16.9 (1)	83.1 (5)	0.0	0.0	0.0	0.0
HK68 (E)	0.0 (0)	100 (3)	0.0	0.0	0.0	0.0
HK68 (F)	0.0 (0)	100 (4)	0.0	0.0	0.0	0.0
Available signature oligonucleotides	(3)	(17)	(0)	(0)	(0)	(0)

↵a See Table 3 for the conditions of experiments A to E. *, Experiments included in training set.
↵b The scores given are from the weighted oligonucleotide calculation (see Materials and Methods). Values in parentheses are the numbers of signature oligonucleotides that registered positive. There were no NA-specific oligonucleotides that reacted with N4, N8, or N9 subtypes in BLASTn searches.