Abstract
Rapid neuraminidase subtyping of avian influenza viruses from diagnostic samples is crucial considering the existence of permanently emerging and evolving strains. Here we report an easy-to-use, low-cost microarray for neuraminidase subtyping following fragment amplification by a generic, neuraminidase-specific reverse transcription-PCR (RT-PCR). This method enables highly specific characterization with a sensitivity equal to that of matrix gene-specific real-time RT-PCR.
Avian influenza viruses (AIV) are classified into 16 hemagglutinin (HA; H1 to H16) and 9 neuraminidase (NA; N1 to N9) subtypes on the basis of antigenic and/or genetic differences of these surface glycoproteins (2, 3, 5). Acknowledging the potential threat of the AIV pool for carrying prepandemic human-pathogenic viruses, exemplified by transspecies transmissions of highly pathogenic AIV (HPAIV) directly from infected poultry to humans, surveillance studies of influenza A virus in wild birds and poultry have been intensified recently (12, 13). Real-time reverse transcription-PCR (rRT-PCR) and other molecular diagnostic techniques are used with preference in such programs because of their superior sensitivity, suitability for rapid and high-throughput analysis, and reduced risk of the operator handling infectious material. However, molecular subtyping of AIV from an rRT-PCR-positive sample, which is required to further understand the epidemiology of these viruses, can be challenging if commonly used conventional RT-PCRs followed by sequencing do not achieve the same sensitivity as the screening method (1, 4, 10, 14).
We recently described a highly sensitive microarray assay for HA sub- and pathotyping (7, 8) which is based on an RT-PCR amplifying an HA gene fragment encoding the HA0 cleavage site of all influenza A viruses (6). The ArrayTube system (Clondiag, Jena, Germany) utilized is a small and inexpensive platform with spot-on low-density microarrays integrated in reaction tubes and with signal amplification by enzyme-catalyzed local TMB precipitation staining. Hybridization and analysis are fast and are easily conducted with standard laboratory equipment complemented by a simple transmission reader. For higher throughput, the system can be operated in a completely automated 96-well microplate format (ArrayStrip and ArrayMate reading device).
Here we present a new, ready-to-use ArrayTube assay for NA subtyping which is based on an RT-PCR amplifying a fragment of the NA genes of all influenza A viruses. In an extensive validation study, the assay was proven to exhibit high specificity and sensitivity.
ArrayTubes with 106 oligonucleotide probes specific for the NA gene were fabricated at Clondiag (Jena, Germany) as described previously (11). Sequence characteristics and print patterns can be found in Table S1 in the supplemental material. NA subtype-specific probes and hybridization controls were spotted in triplicate. The array also contained positive controls for the staining procedure (3′-C-7-amino- and 5′-biotin-modified oligonucleotides) and negative controls (spotting buffer). Influenza A virus isolates (allantoic fluids) as well as field samples (cloacal swabs from wild and domestic birds) were obtained from the repository of the Office International des Epizooties (OIE) and German National Reference Laboratory for Avian Influenza. Viral RNAs were purified with a QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. Specificity tests were conducted with RNAs or DNAs from Newcastle disease virus strain La Sota, reovirus strain 1133 (Intervet), infectious bursitis disease virus D78, infectious laryngotracheitis virus A489 (Intervet), Mycoplasma gallinarum, and Mycoplasma gallinaceum. A universal primer set for amplification of an ∼620-bp fragment of the NA genes of all influenza A viruses was designed. It consisted of 5′-biotinylated (Bio) primers NA886.1Fbio (5′-Bio-ATC GAR GAR TGY TCN TGY TA-3′), NA886.3Fbio (5′-Bio-GTC GAR GAR TGY TCH TGB TA-3′; positions 846 to 865 [GenBank accession no. NC_007361]), and AIV(647)Rbio (5′-Bio-GCA GTA TAT CGC TTG ACA AGT AGA AAC AAG G-3′; positions 1458 to 1446 [GenBank accession no. NC_007361] [nonviral universal tail is shown in italics]). Target RNA was reverse transcribed, amplified, and biotin labeled by a one-step “pan”-NA RT-PCR with the SuperScript III one-step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). The reaction was carried out analogously to a previously published “pan”-HA RT-PCR (6), but using 2 μl NA-specific primer mix panNA-Mix 4.1-bio [10 pmol/μl (each) of primers NA886.1Fbio, NA886.3Fbio, and AIV(647)Rbio]. Amplicons were verified by agarose gel electrophoresis and submitted to microarray analysis without further purification. Microarray hybridization and data analysis were performed as described by Gall et al. (8). An rRT-PCR specific for the matrix (M) gene (15) was performed for comparison purposes. NA subtypes of diagnostic samples were determined by subtype-specific RT-PCRs followed by partial sequencing of the NA gene (4) and/or sequence analysis of the entire NA gene (10).
For validation of the ArrayTube assay, we utilized a panel of 94 influenza A virus reference strains of all NA subtypes (see Table S2 in the supplemental material)—mainly from poultry and wild birds—originating from Eurasia, North America, and Australia. A recent swine-origin influenza virus (H1N1) from a patient in Germany was also investigated as a representative of the ongoing pandemic. NA subtypes were accurately determined by the microarray assay for all strains. In contrast, Newcastle disease virus strain La Sota, reovirus strain 1133, infectious bursitis disease virus D78, infectious laryngotracheitis virus A489, Mycoplasma gallinarum, and Mycoplasma gallinaceum were not detected (not shown).
The sensitivity of the new assay was compared to that of a standard rRT-PCR specific for the M gene (15) by using 10-fold serial dilutions of RNAs from reference strains of the four most relevant NA subtypes in RNA-safe buffer (9) {0.05% Tween 20, 0.05% sodium azide, 50 ng/μl of carrier RNA [poly(A) homopolymer; Amersham Biosciences, Piscataway, NJ]} as templates for both assays. Comparable sensitivities were observed for the selected strains of the N1, N2, and N7 subtypes (Table 1), while for A/Mallard/Germany/Wv677/04 (H2N3) the ArrayTube assay was found to be 10-fold less sensitive than the rRT-PCR.
TABLE 1.
Sensitivity of the new ArrayTube assay for AIV of subtypes N1, N2, N3, and N7
| Virus and dilution | M gene-specific rRT-PCR resulta | RT-PCR resultb | ArrayTube assay resultc |
|---|---|---|---|
| A/whooper swan/Germany/R65/06 (H5N1) HPAIV | |||
| 10−5 | 30.57 | + | ND |
| 10−6 | 33.99 | +/− | + |
| 10−7 | 36.63 | − | + |
| 10−8 | No CT | − | − |
| 10−9 | No CT | − | − |
| A/turkey/Germany/22/96 (H9N2) | |||
| 10−5 | 31.69 | + | ND |
| 10−6 | 35.18 | +/− | + |
| 10−7 | 38.40 | − | + |
| 10−8 | 40.98 | − | + |
| 10−9 | No CT | − | − |
| A/mallard/Germany/Wv677/04 (H2N3) | |||
| 10−5 | 33.21 | + | ND |
| 10−6 | 36.38 | +/− | + |
| 10−7 | 39.20 | − | + |
| 10−8 | 40.73 | − | − |
| 10−9 | No CT | − | − |
| A/chicken/Germany/R28/03 (H7N7) HPAIV | |||
| 10−5 | 31.00 | + + | ND |
| 10−6 | 34.24 | + | + |
| 10−7 | 38.08 | − | + |
| 10−8 | 40.80 | − | + |
| 10−9 | No CT | − | − |
CT (dR) value for M gene-specific rRT-PCR (15).
++ and +, different intensities of the bands by agarose gel electrophoresis; +/−, weak band; −, no band visible.
ND, not determined.
The reported microarray assay was further validated with 76 diagnostic samples, comprising cloacal swabs from poultry and wild birds (threshold cycle [CT] values for the M gene ranged from 14.54 to 38.02 for these samples), including material from an outbreak of HPAIV H5N1 clade 2.2 in Germany in 2008 (Table 2). A total of 74 samples (97.4%) was successfully analyzed, and NA subtypes determined by the ArrayTube assay perfectly corresponded to results obtained by subtype-specific RT-PCRs and/or sequencing (4, 10). Among the nine known NA subtypes, only N4 and N7 were not represented in the panel of samples. In one sample (no. 46), the N8 and N2 subtypes were detected simultaneously, and direct PCR sequencing was unsuccessful due to two superimposed sequences; an H9N2 virus was isolated from this sample. The microarray analysis of the sample with the lowest viral RNA load (no. 58) remained negative.
TABLE 2.
Characterization of influenza A viruses from diagnostic samples
| Sample no. | Virusa | M gene-specific rRT-PCR resultb | RT-PCR resultc | ArrayTube assay result |
|---|---|---|---|---|
| 1 | A/duck/Germany/R4-Lippe/08 (H1N1) | 26.64 | ++ | N1 |
| 2 | A/Anas platyrhynchos/Germany/R91/08 (H3N8) | 31.32 | + | N8 |
| 3 | A/Branta canadanensis/Germany/R107/08 (H11N9) | 34.06 | + | N9 |
| 4 | A/Cygnus olor/Germany/R108/08 (H4Nx) | 36.95 | + | N6 |
| 5 | A/Anser albifrons/Germany/R165/08 | 32.38 | + | N2 |
| 6 | A/Anser albifrons/Germany/R167/08 | 29.08 | ++ | N8 |
| 7 | A/Anser albifrons/Germany/R168/08 | 31.58 | + | N2 |
| 8 | A/Anser albifrons/Germany/R169/08 | 32.74 | + | N2 |
| 9 | A/Anas platyrhynchos/R234/08 (H1N1) | 29.91 | ++ | N1 |
| 10 | A/Anser anser/Germany/R239/08 (H9N2) | 33.96 | + | N2 |
| 11 | A/Anser anser/Germany/R240/08 (H9N2) | 32.34 | + | N2 |
| 12 | A/Anser anser/Germany/R248/08 (H9N2) | 22.33 | ++ | N2 |
| 13 | A/Struthio camelus/South Africa/R296/2/08 (H5N2) HPAIV | 26.35 | +/− | N2 |
| 14 | A/Struthio camelus/South Africa/R296/4/08 (H5N2) HPAIV | 18.98 | ++ | N2 |
| 15 | A/Struthio camelus/South Africa/R296/5/08 (H5N2) HPAIV | 25.86 | ++ | N2 |
| 16 | A/Struthio camelus/South Africa/R296/7/08 (H5N2) HPAIV | 30.50 | − | N2 |
| 17 | A/Callonetta leucophrys/Germany/R641/2/08 (H6N2) | 23.40 | ++ | N2 |
| 18 | A/Anas platyrhynchos/Germany/R665/2/08 (H5Nx) | 36.73 | − | N3 |
| 19 | A/Anas platyrhynchos/Germany/R695/08 (H5Nx) | 28.47 | + | N2 |
| 20 | A/Anas platyrhynchos/Germany/R701/08 (H5Nx) | 34.42 | − | N2 |
| 21 | A/Anas platyrhynchos/Germany/R726/08 (H5Nx) | 29.37 | + | N3 |
| 22 | A/Anas platyrhynchos/Germany/R734/1/08 (H5N3) | 24.23 | ++ | N3 |
| 23 | A/domestic duck/Germany/R786/08 (H5N1) HPAIV | 24.84 | ++ | N1 |
| 24 | A/domestic duck/Germany/R821/08 (H5N1) HPAIV | 36.91 | − | N1 |
| 25 | A/domestic duck/Germany/R836/08 (H5N1) HPAIV | 36.68 | − | N1 |
| 26 | A/domestic goose/Germany/R1110/08 (H6N2) | 30.95 | ++ | N2 |
| 27 | A/domestic goose/Germany/R1125/08 (H6N2) | 30.49 | ++ | N2 |
| 28 | A/mallard/Germany/R1140/2008 (H6N2) | 16.01 | +++ | N2 |
| 29 | A/mallard/Germany/R1142/2008 (H6N2) | 14.54 | +++ | N2 |
| 30 | A/Anser anser/Germany/R1416/08 (H2N9) | 24.31 | +++ | N9 |
| 31 | A/Anas platyrhynchos/Germany/R1419/08 | 30.67 | ++ | N9 |
| 32 | A/Anas platyrhynchos/Germany/R1423/08 (H5Nx) | 33.74 | + | N3 |
| 33 | A/Anas platyrhynchos/Germany/R1430/08 (H10Nx) | 29.02 | +++ | N9 |
| 34 | A/Anas platyrhynchos/Germany/R1433/08 | 33.08 | + | N9 |
| 35 | A/Anser anser/Germany/R1467/08 (H2N9) | 25.24 | +++ | N9 |
| 36 | A/Anas platyrhynchos/Germany/R1516/08 (H9N2) | 36.55 | − | N2 |
| 37 | A/Anas platyrhynchos/Germany/R1521/08 (H9N2) | 33.19 | + | N2 |
| 38 | A/Anas platyrhynchos/Germany/R1522/08 (H9N2) | 30.11 | + | N2 |
| 39 | A/Anas platyrhynchos/Germany/R1523/08 (H11N9) | 28.57 | ++ | N9 |
| 40 | A/Anser aegyptiacus/Germany/R1524/08 | 32.84 | ++ | N9 |
| 41 | A/Anas platyrhynchos/Germany/R1526/08 (H2N3) | 33.97 | + | N3 |
| 42 | A/wild bird/Germany/R1527/08 (H6N2) | 32.47 | + | N2 |
| 43 | A/Cygnus olor/Germany/R1541/08 (H7N3) | 34.55 | +/− | N3 |
| 44 | A/Anas platyrhynchos/Germany/R1594/08 (H11N1) | 28.22 | ++ | N1 |
| 45 | A/Anas platyrhynchos/Germany/R1597/08 (H11N1) | 28.01 | ++ | N1 |
| 46 | A/Anas platyrhynchos/Germany/R1599/08 (H9N2) | 28.65 | +++ | N8, N2 |
| 47 | A/Anas platyrhynchos/Germany/R1601/08 (H5N2) | 32.91 | + | N2 |
| 48 | A/Anas platyrhynchos/Germany/R1603/08 | 30.89 | ++ | N9 |
| 49 | A/Anas platyrhynchos/Germany/R1604/08 | 27.67 | ++ | N9 |
| 50 | A/Anas platyrhynchos/Germany/R1609/08 | 29.11 | ++ | N2 |
| 51 | A/Anser anser/Germany/R1625/08 (H6N2) | 29.73 | ++ | N2 |
| 52 | A/Anser anser/Germany/R1628/08 (H6N2) | 30.35 | ++ | N2 |
| 53 | A/Meleagris gallopavo/Germany/R1649-53/08 (H5N3) | 30.09 | +++ | N3 |
| 54 | A/domestic duck/Germany/R1769/08 (H6N2) | 33.60 | − | N2 |
| 55 | A/domestic duck/Germany/R1774/08 (H6N2) | 29.06 | +/− | N2 |
| 56 | A/domestic duck/Germany/R1779/08 (H6N2) | 32.92 | +/− | N2 |
| 57 | A/Meleagris gallopavo/Germany/R1914-6/08 (H5N3) | 25.84 | +++ | N3 |
| 58 | A/Meleagris gallopavo/Germany/R2040/08 | 38.02 | − | —d |
| 59 | A/Meleagris gallopavo/Germany/R2045/08 (H5N3) | 32.56 | + | N3 |
| 60 | A/Anas platyrhynchos/Germany/R2316/08 | 32.20 | + | N1 |
| 61 | A/Anas platyrhynchos/Germany/R2319/08 | 31.70 | + | N1 |
| 62 | A/Cygnus cygnus/Germany/R2324/08 (H6N5) | 34.22 | + | N5 |
| 63 | A/Cygnus cygnus/Germany/R2325/08 (H6N5) | 33.74 | + | N5 |
| 64 | A/duck/Germany/R2331/0 8 (H5N3) | 27.86 | ++ | N3 |
| 65 | A/duck/Germany/R2342/0 8 (H5N3) | 31.15 | ++ | N3 |
| 66 | A/Meleagris gallopavo/Germany/R2379/08 (H5N3) | 26.88 | ++ | N3 |
| 67 | A/Anser albifrons/R2/09 (H6N8) | 27.10 | ++ | N8 |
| 68 | A/Anser albifrons/R3/09 (H6N8) | 31.99 | + | N8 |
| 69 | A/Meleagris gallopavo/Germany/R23/09 (H3N2) | 34.14 | − | N2 |
| 70 | A/Meleagris gallopavo/Germany/R24/09 (H3N2) | 35.48 | − | N2 |
| 71 | A/Larus canus/Germany/R127/09 | 27.08 | − | N6 |
| 72 | A/Larus argentatus/Germany/R128/09 | 29.24 | − | N6 |
| 73 | A/Larus argentatus/Germany/R129/09 | 29.42 | +/− | N6 |
| 74 | A/Anas platyrhynchos/Germany/R131/09 | 21.21 | ++ | N2 |
| 75 | A/Anas platyrhynchos/Germany/R132/09 | 36.67 | − | N2 |
| 76 | A/Anas platyrhynchos/Germany/R133/09 | 32.28 | +/− | N2 |
Subtyping was not successful for all samples. NA subtypes were determined by subtype-specific RT-PCRs followed by sequencing (4) and/or by sequencing of the entire NA gene (10).
CT (dR) value for M gene-specific rRT-PCR (15).
+++ to +, different intensities of bands by agarose gel electrophoresis; +/−, weak band; −, no band visible.
For sample no. 58, no ArrayTube assay result could be achieved.
The validation data presented here demonstrate the feasibility of a novel ArrayTube assay for highly specific NA subtyping of AIV with a sensitivity comparable to that of rRT-PCR specific for the M gene (15). Within 24 h, the NA subtypes could be ascertained for all virus isolates and the majority of field samples included in the study. The assay clearly surpasses the potential of conventional molecular tools for NA subtyping (1, 4, 10) with regard to sensitivity. Nevertheless, sequencing of NA genes remains an important tool for studies of the phylogeny of AIV.
In conclusion, the established microarray assay addresses the requirements of diagnostic laboratories for a rapid, easy-to-use, and affordable system enabling NA subtyping of influenza A virus-positive samples within surveillance programs of both wild bird and poultry populations. The ArrayTube assay described here is now integrated into the routine diagnostics of the OIE and German National Reference Laboratory for Avian Influenza. It was merged with a previously described assay for HA subtyping and pathotyping (8) to form a single microarray integrated in eight-well strips (ArrayStrip), which is currently under validation and allows processing of large sample numbers for complete HA and NA subtyping.
Supplementary Material
Acknowledgments
We thank Anne Jüngling as well as the laboratory team of the OIE and German National Reference Laboratory for Avian Influenza for excellent technical assistance and Konrad Sachse for the kind introduction to the use of the ArrayTube system. We are grateful to colleagues within the Friedrich-Loeffler-Institut and at the OIE Reference Laboratories for AI in Canada, Italy, and the United Kingdom for supplying reference material. We also thank Stephan Becker (Institute of Virology, Philipps University Marburg) for providing the novel influenza virus strain A/Regensburg/Germany/01/2009 (H1N1).
This work was supported by the Federal Ministry of Food, Agriculture and Consumer Protection, BMELV, Germany (FSI project 1.1), and by the EU Network of Excellence, EPIZONE (contract FOOD-CT-2006-016236).
Footnotes
Published ahead of print on 8 July 2009.
Supplemental material for this article may be found at http://jcm.asm.org/.
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