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Journal of Clinical Microbiology, August 2008, p. 2561-2567, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00466-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Universal Primer Set for Amplification and Sequencing of HA₀ Cleavage Sites of All Influenza A Viruses ^,

Astrid Gall, Bernd Hoffmann, Timm Harder, Christian Grund, and Martin Beer^*

Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald—Insel Riems, Germany

Received 9 March 2008/ Returned for modification 22 April 2008/ Accepted 9 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sequence analysis of the endoproteolytic cleavage site within the hemagglutinin (HA) precursor protein HA₀ is fundamental for studies of the molecular biology of influenza A viruses, in particular, for molecular pathotyping of subtype H5 and H7 isolates. A current problem for routine diagnostics is the emergence of new strains of the H5 or H7 subtype or even other subtypes which escape detection by commonly used reverse transcription-PCR (RT-PCR) protocols. Here, the first pan-HA (PanHA) RT-PCR assay targeting the HA₀ cleavage site of influenza A viruses of all 16 HA subtypes is reported. The assay was assessed in comparison to H5 and H7 subtype-specific RT-PCRs for the HA₀ cleavage site and a real-time RT-PCR detecting the M gene. A panel of 92 influenza A viruses was used for validation. Sequence data for influenza A viruses from 32 allantoic fluid samples and 11 diagnostic swab samples of all 16 HA subtypes were generated by direct sequencing of the PanHA RT-PCR products. The results demonstrate that the new PanHA RT-PCR assay—followed by cycle sequencing—can complement existing methods and strengthen the reliability of influenza A virus diagnostics, allowing both molecular pathotyping (H5 and H7) and subtyping (non-H5 or -H7) within a single approach.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Highly pathogenic avian influenza viruses (HPAIV) cause epidemic disease with high mortality rates in both poultry and wild birds (6) and are biologically characterized by intravenous pathogenicity indices of more than 1.2 (2, 3). Molecular pathotyping by sequence analysis of the cleavage site within the hemagglutinin (HA) precursor protein HA₀ is more rapid and reduces the risk of handling infectious material (2). The presence of multiple basic amino acids (arginine or lysine) at the HA₀ cleavage site, indicating processibility by ubiquitous proprotein convertases, is a molecular indicator for pathogenicity (13, 16). These viruses cause severe systemic infections and have historically belonged to either the H5 or the H7 subtype (2). In contrast, AIV of low pathogenicity (LPAIV) as well as mammalian influenza A viruses show a monobasic composition at this site. Their HA₀ is cleaved extracellularly by tissue-specific, trypsin-like proteases; thus, LPAIV infection remains localized to the intestinal tract of birds or the respiratory tract of mammals. HPAIV consistently have a polybasic cleavage site which is cleaved intracellularly by ubiquitous subtilisin-like proteases (13, 16).

Nevertheless, current strains show high sequence heterogeneity and new strains continue to emerge. At least two isolates of the H10 subtype fulfilled the definitions of the World Organization for Animal Health (Office International des Epizooties) and the European Union (EU) for HPAIV (2, 3, 17) but lacked multiple basic amino acids at the HA₀ cleavage site. In addition, the R-S-S-R motif of H9N2 viruses from birds, swine, and humans (1, 5, 7, 20) was recently proven to have been replaced by an R-S-R-R (20) in an isolate from a quail. Although this virus showed multiple basic amino acids as a molecular determinant for identification as HPAIV, pathogenicity tests of chickens still revealed low pathogenicity. Londt et al. (12) have described a further four H5 and H7 AIV showing incongruency between molecular and biological pathotyping results. Therefore, exact molecular pathotyping, i.e., determination of HA₀ cleavage site sequences, and subtyping of all influenza A viruses are necessary for diagnostics, surveillance, and epidemiological studies as well as for investigations of the significance of the amino acid motifs for pathogenicity.

This report describes a validated one-step pan-HA (PanHA) reverse transcription-PCR (RT-PCR) amplifying a fragment encompassing the HA₀ cleavage site as a new diagnostic tool. Direct sequencing of the RT-PCR products was accomplished for molecular characterization of the HA₀ cleavage site sequences and pathotyping and subtyping of influenza A viruses of all 16 HA subtypes.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Viruses and diagnostic samples. A panel of 92 influenza A virus isolates (see Table S1 in the supplemental material) was obtained in the form of allantoic fluid samples from the Office International des Epizooties and German National Reference Laboratory for Avian Influenza. In addition, 17 field samples, cloacal swabs from wild and domestic birds, were used.

Isolation of RNA. Viral RNA from allantoic fluid samples and cloacal swabs was purified using a QIAamp viral RNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with some modifications, including the addition of 5 µl of in vitro-transcribed internal control (IC) RNA (2 x 10⁵ copies/µl) after lysis of the sample to control the efficiency of RNA isolation and RT-PCR (9).

Real-time RT-PCR. Quantitative RT-PCR detecting the matrix (M) gene of influenza A virus was performed as described previously (15) with some modifications, including integration of an IC system (9). The duplex real-time RT-PCR was performed using a 25-µl reaction volume and a SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). For one reaction, the assay was optimized to 2.5 µl of RNase-free water, 12.5 of µl reaction mix (2x), 2 µl of M-specific primer-probe mix (10 pmol of primer IVA-M_1for/µl, 15 pmol of primer IVA-M1.1rev/µl, 1.25 pmol of probe IVA-M_1-FAM-BHQ/µl) (Table 1), 2 µl of IC-specific primer-probe mix (2.5 pmol/µl each of primers EGFP-12-F and EGFP-10-R and 1.25 pmol of probe EGFP-HEX/µl) (Table 1), 1 µl of SuperScript III RT/Platinum Taq mix, and 5 µl of template RNA. The reaction was carried out using an Mx3000P real-time PCR system (Stratagene, La Jolla, CA) with the following temperature profile: 30 min at 50°C (RT), 2 min at 94°C (inactivation of reverse transcriptase/activation of Taq polymerase), followed by 42 cycles of 30 s at 94°C (denaturation), 30 s at 57°C (annealing), and 30 s at 68°C (elongation). Fluorescence data were collected during the annealing step.

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TABLE 1. Primers and probes used in this study

RT-PCR. (i) The PanHA RT-PCR was performed using a 25-µl reaction volume and a SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase. For one reaction, the assay was optimized to 4.5 µl of RNase-free water, 12.5 µl of reaction mix (2x), 2 µl of PanHA Mix 1.0 [5 pmol/µl each of primers HA-1057.1-F, HA-1057.2-F, HA-1057.3-F, HA-1232.2(555)-R, and HA-1232.1(555)-R] (Table 1), 1 µl of SuperScript III RT/Platinum Taq Mix, and 5 µl of template RNA. The reaction was carried out using an Applied Biosystems 2720 thermal cycler (Applied Biosystems, Foster City, CA) with the following temperature profile: 30 min at 50°C (RT) and 2 min at 94°C (inactivation of reverse transcriptase/activation of Taq polymerase), followed by 45 cycles of 30 s at 94°C (denaturation), 45 s at 50°C (annealing) and 45 s at 68°C (elongation), and 5 min at 68°C (final elongation). Amplicons with a size of 164 to 176 bp were visualized by 3% agarose gel electrophoresis for 1 h at 120 V in Tris-acetate-EDTA buffer. (ii) H5 and H7 subtype-specific RT-PCR assays were performed according to the same protocol. H5-specific primer pair H5-kha-1 and H5-kha-3 and primer pair J3 and B2a as well as H7-specific primer pair GK7.3 and GK7.4 (4) were utilized at a concentration of 20 pmol each/25 µl of reaction volume.

Direct sequencing. PanHA RT-PCR products were purified from agarose gels by use of a QIAquick gel extraction kit (Qiagen) as recommended by the manufacturer and were subjected to automated direct sequencing at Agowa, Berlin, Germany, using a 3730xl DNA analyzer (Applied Biosystems). The products were sequenced utilizing panHA-Mix 1.0-F (5 pmol/µl each of primers HA-1057.1-F, HA-1057.2-F, and HA-1057.3-F) and panHA-Mix 1.0-R [5 pmol/µl each of primers HA-1232.2(555)-R and HA-1232.1(555)-R] as sequencing primers.

Sequence analysis. Sequence data were analyzed and edited with BioEdit version 7.0.9.0 (8). Amino acid sequences were deduced and pathotypes were determined according to properties described previously (16). A BLASTN search was performed to identify the query sequence and to find similar sequences. All sequences generated in this study were submitted to the European Molecular Biology Laboratory (EMBL) database.

Nucleotide sequence accession numbers. The nucleotide sequences obtained in this study are available from EMBL, GenBank, and DDBJ under accession numbers AM922136 to AM922167 and AM930525 to AM930535.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Design of the PanHA RT-PCR. A set of primers (Table 1) was designed and combined in a one-step RT-PCR to amplify a fragment of 164 to 176 bp spanning the HA₀ cleavage site of influenza A viruses of all 16 HA subtypes. A panel of 92 influenza A virus isolates (see Table S1 in the supplemental material) was used for validation. All samples were verified for their content of influenza A virus genome by real-time RT-PCR targeting the M gene (9, 15) and revealed threshold cycle (C_T) values between 15 and 28 (data not shown). With the PanHA RT-PCR protocol described here, 87 influenza A virus isolates showed clearly positive results (see Fig. S1 in the supplemental material). Only A/turkey/Wisconsin/66 (H9N2) and A/pilot whale/Maine/328/84 (H13N2) could not be detected, whereas three recent isolates of the H10 and H13 subtype (A/Mallard/Föhr/Wv1781-82/03 [H10N7], R2609/06 [H13N8], and R2622/06 [H13]) were detected with low sensitivity.

Comparison of the PanHA RT-PCR with H5 and H7 subtype-specific RT-PCR assays. The reliability of the PanHA RT-PCR in detection of influenza A virus isolates of the H5 and H7 subtypes was compared to that of subtype-specific RT-PCRs (H5-specific primer pair H5-kha-1 and H5-kha-3 and primer pair J3 and B2a and H7-specific primer pair GK7.3 and GK7.4) recommended in the EU Diagnostic Manual for Avian Influenza (4). RNA extracted from 12 influenza A virus isolates of the H5 subtype (C_T values between 16 and 22) and 16 influenza A virus isolates of the H7 subtype (C_T values between 17 and 24) was tested using the appropriate primer pairs. It was shown that all samples scored positive when the PanHA primers were used. In contrast, the results obtained with A/Mall/QC/2323-19/2006 (H5N2), A/duck/Alberta/48/76 (H7N3), and A/Mallard/NVP/1776-80/03 (H7N7) remained negative when the EU-recommended subtype-specific primers were used. Furthermore, A/DK/BC/26-6/05 (H5N2) was detected by only one of the two H5-specific primer pairs (data not shown).

Sensitivity of the PanHA RT-PCR for H5 and H7 detection in comparison to the sensitivity of H5 and H7 subtype-specific RT-PCR assays and M gene-specific real-time RT-PCR. Tenfold dilution series of RNA extracted from two European (one HPAIV and one LPAIV) and two North American (LPAIV) influenza A virus isolates of both the H5 and H7 subtypes were prepared in RNA-safe buffer (0.05% Tween 20, 0.05% sodium azide, 50 ng/µl of carrier RNA [poly(A) homopolymer; Amersham Biosciences, Piscataway, NJ]) and amplified using the different protocols. The results are shown in Fig. 1. The sensitivity of the PanHA RT-PCR for the selected European influenza A virus isolates was comparable to or slightly lower than that seen when the EU-recommended PCR methods were used (4). In addition, the PanHA RT-PCR protocol revealed a higher sensitivity than the subtype-specific assays for the selected North American influenza A virus isolates. In contrast to the M gene-specific real-time RT-PCR results, influenza A virus RNA from allantoic fluids with a C_T value of approximately 30 or lower was detected by the PanHA RT-PCR. Therefore, the PanHA RT-PCR protocol was approximately 100- to 1,000-fold less sensitive than the real-time RT-PCR.

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FIG. 1. Assay sensitivity for influenza A virus of subtypes H5 and H7. (A) RNA from four influenza A virus isolates of the H5 subtype was tested in 10-fold dilution series with the PanHA primers as well as H5-specific primer pair H5-kha-1 and H5-kha-3 and primer pair J3 and B2a. (B) RNA from four influenza A virus isolates of the H7 subtype was tested in 10-fold dilution series with the PanHA primers as well as H7-specific primer pair GK7.3 and GK7.4. CT values for the M gene obtained by real-time RT-PCR are shown beneath the agarose gel image. Marker, GeneRuler 100-bp DNA Ladder Plus (Fermentas). ntc, no template control.

Sensitivity of the PanHA RT-PCR using diagnostic samples. RNA from 17 cloacal swabs from wild and domestic birds was proven to contain influenza A virus genome (C_T values between 23.49 and 37.57 in M gene-specific real-time RT-PCR) (Table 2). C_T values for the IC ranged between 28.50 and 31.61, indicating that no inhibitory effects were present in these samples. No C_T values for the IC were observed for the two samples with the highest viral RNA loads, due to competitive inhibition by coamplification of the M product, or for sample R1642/07, due to inefficient RNA isolation or the presence of inhibitors of the RT-PCR. With the PanHA RT-PCR protocol, the influenza A virus genome could be detected in 11 out of 17 samples. All samples with C_T values for the M gene of less than 30 (except for sample R1642/07) and, in addition, sample R2619/07 (C_T value of 33.10) showed positive results. Thus, influenza A virus RNA was detected in diagnostic samples with a C_T value of approximately 30 or lower. Repeated testing of selected samples proved the validity of the observed results (data not shown).

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TABLE 2. Sensitivity of PanHA RT-PCR compared to real-time RT-PCR for influenza A virus in diagnostic samples

Determination of HA₀ cleavage site sequence, pathotype, and HA subtype of influenza A viruses from allantoic fluids. In order to determine HA₀ cleavage site sequences, PCR primer mixes were also used as sequencing primers for subsequent direct sequencing of the purified PanHA RT-PCR products. In a first step, reliability of the novel method was ensured by generation of sequence data for 16 influenza A virus isolates and comparison to sequences deposited in EMBL, GenBank, and DDBJ. All of the sequences obtained (accession no. AM922136 to AM922138, AM922140, AM922145, AM922146, AM922150 to AM922153, AM922156, AM922157, AM922162, and AM922165 to AM922167) exhibited 100% identity with the published sequences. In a second step, HA₀ cleavage site sequence data were also generated for 16 influenza A virus isolates for which no HA sequence data were available from EMBL, GenBank, and DDBJ (Table 3). Pathotypes were determined using the amino acid sequence, and subtypes were determined by a BLASTN search. Fully concordant results were established for 15 out of 16 influenza A virus isolates in comparisons of subtyping results obtained by use of the PanHA approach with the results of molecular analyses of an HA₂ fragment according to the method published by Phipps et al. (14). In summary, sequence data for HA₀ cleavage sites were generated for 32 avian and human influenza A virus isolates of all 16 HA subtypes. Typical motifs for the different subtypes as well as for HPAIV subtypes H5 (A/HongKong/156/97 [H5N1]) and H7 (A/chicken/Brescia/19/02 [H7N1]) were detected. Moreover, some of the investigated LPAIV within the H5, H7, and H9 subtypes were found to have the capacity, following the introduction of single transversional or transitional point mutations, to become highly pathogenic and thus would not depend on insertional mutations (data not shown).

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TABLE 3. Determination of pathotype and HA subtype of influenza A viruses from allantoic fluids

Determination of HA₀ cleavage site sequence, pathotype, and HA subtype of influenza A viruses from the diagnostic samples. HA₀ cleavage site sequence data were generated for 11 influenza A viruses from diagnostic samples which showed positive results by use of PanHA RT-PCR (Table 2 and Table 4). Pathotypes were ascertained by analysis of the amino acid sequence, and subtypes were determined by BLASTN search (Table 4). Subtypes corresponded to results obtained by sequencing of an HA₂ fragment (14) where those results were available.

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TABLE 4. Determination of pathotype and HA subtype of influenza A viruses from diagnostic samples

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subtyping and subsequent pathotyping of influenza A viruses are of utmost significance for initiation of restrictive measures against HPAIV. Molecular methods have the advantage of accelerating diagnostics and reducing the risk of handling infectious material. Here we report a PanHA RT-PCR amplifying a fragment encompassing the HA₀ cleavage site of influenza A viruses of all 16 of the known HA subtypes. Direct sequencing of the PCR products permitted both pathotyping and subtyping within a single approach.

In order to minimize the risk of cross-contamination, a one-step RT-PCR protocol using a commercially available kit (SuperScript III One-Step RT-PCR system with Platinum Taq DNA polymerase) was selected. To ensure proper detection of all 16 HA subtypes, a mix of five degenerated primers was derived and used under cycling conditions that allowed the detection of different influenza A viruses. A panel of 92 influenza A virus isolates—mainly from poultry and wild birds (subtypes H1 to H16)—originating from Eurasia, North America, and Australia was used for validation of the new assay. The panel also contained one isolate from a swine (H1) and six isolates from human hosts (four H1, one H3, and one H5), including H1N1, H3N2, and H5N1 strains. The sensitivity of the new method was sufficient, detecting 87 out of 92 influenza A virus isolates (94.6%) of all HA subtypes; negative results were obtained with only two older isolates (H9N2 and H13N2). Detection results indicating lower sensitivity were evident for three recent isolates (H10N7, H13N8, and H13) for which no HA sequence data were available. However, in contrast to previously published results of studies using "pan" HA primers for full-length amplification of the HA gene (11) or for partial amplification of the HA₂ gene for subtyping (14, 19), the amplification of a product from influenza A viruses of all 16 HA subtypes could be ensured.

All known HPAIV belong to either the H5 or the H7 subtype. Subtype-specific RT-PCR assays allowing the analysis of the HA₀ cleavage site of these subtypes have been described previously (4, 18). When the PanHA RT-PCR assay was used, all influenza A virus isolates tested (12 H5 isolates and 16 H7 isolates) yielded positive results. The results for one recent H5N2 virus from North America remained negative with each of the H5-specific primer pairs, whereas a second was detected by only one of the two primer pairs. For the H7 subtype, an older H7N3 isolate from North America as well as a recent H7N7 virus encountered during wild-bird monitoring in Germany could not be detected using the H7-specific primer pair. Moreover, the sensitivity for the selected North American influenza A virus isolates was higher when employing the PanHA primers than that seen when utilizing the EU-recommended subtype-specific primers. For European influenza A virus isolates, the use of subtype-specific primers resulted in equal or higher sensitivity. Accordingly, the implementation of the PanHA RT-PCR protocol described here is advisable, especially for influenza A virus isolates differing from well-characterized strains chosen for primer design as well as for recent isolates. Nevertheless, since influenza viruses show a high mutation frequency, the primer sequences for the PanHA RT-PCR will also have to be adapted continuously to currently circulating strains.

Compared to diagnostic real-time RT-PCR detecting the influenza A virus M gene (9, 15), which showed an analytical sensitivity of about 10 copies/reaction, the PanHA RT-PCR protocol was approximately 100- to 1,000-fold less sensitive. However, AIV RNA extracted from influenza A viruses from allantoic fluid samples as well as those from cloacal swabs at concentrations yielding C_T values of approximately 30 or lower in M gene real-time RT-PCR was successfully amplified by the PanHA RT-PCR. As the real-time RT-PCR used in this study is very sensitive and targets the conserved M gene, this difference was expected and is tolerable due to the different aims of the two methods. In contrast to real-time RT-PCR, which is used for rapid identification of influenza A virus, the PanHA RT-PCR products are directly sequenced for molecular characterization of the HA₀ cleavage site, allowing pathotyping and subtyping.

Sequence data generated for 16 influenza A virus isolates were compared to the sequences published in EMBL, GenBank, and DDBJ, and all were found to have 100% identity. Thus, the new protocol, utilizing primer mixes to provide sequencing primers which have different melting temperatures and nonviral tails at the reverse primers, is reliable. Subsequently, HA₀ cleavage site sequences and pathotypes were determined for 16 different influenza A virus isolates for which no HA sequence data were available from EMBL, GenBank, and DDBJ. Based on the results reported here, the feasibility of the newly developed PanHA RT-PCR, followed by cycle sequencing, for molecular pathotyping and subtyping of influenza A viruses is evident. In contrast to the method widely used with subtype-specific primers (18) or even real-time RT-PCR specific for the HA₀ cleavage site of HPAIV H5N1 of the Qinghai lineage (10) for pathotyping, the protocol is universal and can be applied to all HA subtypes.

In addition, the reported assay was further validated with 17 influenza A viruses from diagnostic samples and the HA₀ cleavage site of influenza A viruses from 11 cloacal swabs was sequenced. Altogether, HA₀ cleavage site sequence data were generated for 32 avian and human influenza A viruses of all 16 subtypes from allantoic fluid samples and 11 influenza A viruses from diagnostic samples of wild and domestic birds. Nevertheless, results demonstrate that a certain amount of influenza A virus genome material is necessary for successful amplification and sequencing of the HA₀ cleavage site. Therefore, the assay described here may be especially appropriate when utilized for diagnostic samples which yield C_T values in M gene real-time RT-PCRs (15) of approximately 30 or lower.

In conclusion, the assay described here is a useful novel tool that is supportive of rapid diagnostics as well as of investigations of the molecular biology of influenza A viruses.

 
We thank Anne Juengling and the laboratory team of the Office International des Epizooties and German National Reference Laboratory for Avian Influenza for excellent technical assistance. We are grateful to colleagues at the Office International des Epizooties Reference Laboratories for Avian Influenza in Canada, Italy, and the United Kingdom for supplying reference virus isolates.

This work was supported by the Federal Ministry of Food, Agriculture and Consumer Protection, BMELV, Germany (FSI, project no. 1.1.), and the European Union, NoE EPIZONE (Theme 4, WP4.2).

 
* Corresponding author. Mailing address: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald—Insel Riems, Germany. Phone: 49 (0) 38351 7200. Fax: 49 (0) 38351 7151. E-mail: martin.beer{at}fli.bund.de

Published ahead of print on 18 June 2008.

Supplemental material for this article may be found at http://jcm.asm.org/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Clinical Microbiology, August 2008, p. 2561-2567, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00466-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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• Gall, A., Hoffmann, B., Harder, T., Grund, C., Ehricht, R., Beer, M. (2009). Rapid and Highly Sensitive Neuraminidase Subtyping of Avian Influenza Viruses by Use of a Diagnostic DNA Microarray. J. Clin. Microbiol. 47: 2985-2988 [Abstract] [Full Text]  
• Gall, A., Hoffmann, B., Harder, T., Grund, C., Hoper, D., Beer, M. (2009). Design and Validation of a Microarray for Detection, Hemagglutinin Subtyping, and Pathotyping of Avian Influenza Viruses. J. Clin. Microbiol. 47: 327-334 [Abstract] [Full Text]  

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