Virology

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

Article Figures & Data

Figures

  • FIG. 1.
    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.

  • FIG. 2.
    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).

  • FIG. 3.
    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

  • TABLE 1.

    Influenza viruses used in this study

    VirusAbbre- viationSegment no.GenBank or Influenza Sequence Database accession no.
    A/Equine/Kentucky/1/98 (H3N8)aKY984AF197241
    A/Equine/Miami/63 (H3N8)aMI634M29257
    5M22575
    6L06580
    7AF001674
    A/Panama/2007/99 (H3N2)bPanama 994ISDNCDA001
    A/Aichi/2/68 (H3N2)cHK681AF348170
    2AF348172
    3AF348170
    4AF348176, J02090
    5X15890
    6U42630
    7AF348198, M63515
    8AF348198
    A/PR/8/34 (H1N1)cPR81ISDN13419
    2ISDN13420
    3ISDN13421
    4ISDN13422
    5ISDN13423
    6ISDN13424
    7ISDN13425
    8ISDN13426
    A/Equine/Kentucky/9/95 (H3N8)aKY954AF197247

    • ↵ a Obtained from T. Chambers.

    • ↵ b Obtained from N. Cox.

    • ↵ c Obtained from E. Kilbourne.

  • TABLE 2.

    Variation in normalized pixel intensities for similar oligonucleotidesa

    SegmentSequenceE-valuePixel intensity
    1 AATCTAATGTCGCAGTCTC −4.00
    1 CTGATGTCGCAGTCTCGCAC −2.77,283
    1 CTGTCAGTAAGTATGCTAGAGTCCC −7.243,161
    1 GGCTGTCAGTAAGTATGCTA −4.57,289
    2 TTGAATGGATGTCAATCCGA −4.50
    2 GAATGGATGTCAATCCGACC −4.50
    4 TTTCTAATATCCACAAAATGAAGGC 0.50
    4 ACCAAAATGAAGGCAAACC −4.07,769
    4 CAGATGCAGACACAATATGT −4.53,314
    4 CAGATGCAGACACAATATGTATAGG −7.21,341
    4 AAACCGGCAATGGCTCCAAA −4.54,842
    4 ACCGGCAATGGCTCCAAA −3.41,581
    4 GATGCAGACACAATATGTATAGG −6.20
    4 CAGATGCAGACACAATATGT −4.53,314
    4 CAGATGCAGACACAATATGTATAGG −7.21,341
    4 AAAGCAGGGGAAAATAAAAACAACC −7.252,256
    4 GCAGGGGAAAATAAAAACAACC −5.552
    4 GCAGGGGAAAATAAAAGCCAC −2.00
    5 TGCGGGGAAGGATCCTAAGAAAAC −4.36,709
    5 GGGAAAGATCCTAAGAAAAC −4.5884
    5 TCCTCTGCATTGTCTCCG −3.458,051
    5 CTCTGCATTGTCTCCGAAG −4.048,211
    7 GTCAGCATCCACAGCACTCTGCTGTTCC −9.010,645
    7 TCGTCAGCATCCACAGCA −3.456,454
    7 GACCAGCACTGGAGCTAGGA −4.536,488
    7 GACCAGCACTGGAGCTAGGG −4.015,976
    7 GGCAAGTGCACCAGCAGAATAAC −6.233,891
    7 GGCAAGTGCACCAGCAGAATAACT −6.748,119
    7 AGCGTAGACGCTTTGTC −3.00
    7 CTGCAGCGTAGACGCTTTGTCCAAAATG −9.07,788
    8 CCCATTCTCATTACTGCTTC −4.554,028
    8 ATAATGTTTTTCTCATTACT 0.91,731
    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)aScoreb
    Type AType BType 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)aScoreb
    HumanSwineAvianEquineOther
    KY98*40.1 (1)13.926.0 (0)8.1 (1)12.0
    MI63*42.0 (1)16.823.3 (0)9.0 (2)8.9
    Panama 99*46.0 (1)15.623.5 (0)5.3 (0)9.6
    PR8*39.7 (0)14.028.7 (0)3.7 (0)13.9
    HK68 (A)*40.5 (1)15.926.1 (0)6.1 (0)11.3
    HK68 (B)*40.7 (0)13.926.7 (0)7.0 (0)11.6
    HK68 (C)*41.1 (0)14.425.8 (0)6.7 (0)12.0
    HK68 (D)46.3 (2)12.023.2 (0)5.8 (0)12.7
    HK68 (E)40.1 (1)14.425.6 (0)6.4 (0)13.5
    HK68 (F)41.8 (1)14.625.9 (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)aScoreb
    Segment 1Segment 2Segment 3Segment 4Segment 5Segment 6Segment 7Segment 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)aScoreb (by HA subtype)
    Subtype 1Subtype 2Subtype 3Subtype 4Subtype 5Subtype 6Subtype 7Subtype 8Subtype 9Subtype 10Subtype 11Subtype 12Subtype 13Subtype 14Subtype 15
    KY98*5.5 (0)0.840.8 (2)5.5 (0)10.7 (0)6.55.6 (0)0.04.4 (0)0.07.40.012.90.00.0
    MI63*8.8 (0)1.136.0 (6)3.3 (0)7.0 (0)3.310.3 (0)5.33.1 (0)3.33.80.08.00.06.6
    Panama 99*5.1 (0)0.948.4 (7)5.6 (0)4.3 (0)4.42.7 (0)0.02.7 (0)0.013.70.012.20.00.0
    PR8*69.6 (2)0.01.2 (0)0.0 (0)8.2 (0)0.00.0 (0)17.80.0 (0)0.03.20.00.00.00.0
    HK68 (A)*4.6 (0)0.844.0 (6)5.7 (0)4.6 (0)4.02.4 (0)0.02.4 (0)7.912.50.011.10.00.0
    HK68 (B)*8.6 (0)1.425.8 (1)7.6 (0)7.6 (0)7.14.3 (0)0.03.9 (0)0.012.20.021.40.00.0
    HK68 (C)*8.0 (0)1.519.2 (1)8.8 (0)6.7 (0)6.84.2 (0)0.04.2 (0)0.021.50.019.20.00.0
    HK68 (D)6.1 (0)1.011.8 (0)7.0 (0)5.3 (0)16.45.5 (0)0.03.2 (0)4.921.10.017.70.00.0
    HK68 (E)10.0 (1)0.841.5 (6)5.3 (0)4.4 (0)3.72.3 (0)0.02.3 (0)7.511.80.010.50.00.0
    HK68 (F)6.6 (0)1.132.2 (1)7.7 (0)3.4 (0)2.25.7 (0)0.02.1 (0)20.213.40.05.40.00.0
    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)aScoreb (by NA subtype)
    Subtype 1Subtype 2Subtype 3Subtype 5Subtype 6Subtype 7
    KY98*0.0 (0)100 (5)0.00.00.00.0
    MI63*2.9 (0)72.5 (6)0.00.012.312.3
    Panama 99*4.3 (0)86.9 (10)0.00.08.80.0
    PR8*97.2 (1)2.8 (0)0.00.00.00.0
    HK68 (A)*0.0 (0)100 (4)0.00.00.00.0
    HK68 (B)*0.0 (0)100 (7)0.00.00.00.0
    HK68 (C)*0.0 (0)100 (7)0.00.00.00.0
    HK68 (D)16.9 (1)83.1 (5)0.00.00.00.0
    HK68 (E)0.0 (0)100 (3)0.00.00.00.0
    HK68 (F)0.0 (0)100 (4)0.00.00.00.0
    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.

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KEYWORDS

Oligonucleotide Array Sequence Analysis
Orthomyxoviridae

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