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J Clin Microbiol. Sep 2008; 46(9): 3048–3055.
Published online Jul 2, 2008. doi:  10.1128/JCM.02386-07
PMCID: PMC2546769

Subtyping of Avian Influenza Viruses H1 to H15 on the Basis of Hemagglutinin Genes by PCR Assay and Molecular Determination of Pathogenic Potential[down-pointing small open triangle]


Serious concern about the worldwide transmission of the Asian H5N1 highly pathogenic (HP) avian influenza (AI) virus by migratory birds surrounds the importance of the AI global surveillance in wild aquatic birds and underscores the requirement for a reliable subtyping method of AI viruses. PCR is advantageous due to its simplicity, lower cross-reactivity, and unlimited reagent supply. Currently, the only available hemagglutinin (HA) subtyping primer set that can subtype H1 through H15 is not fully evaluated and, since it only targets HA1, is unavailable for molecular pathotyping of AI viruses. Our preliminary experiments found that these primer sets were cross-reactive and missed some recent AI viruses. In this study, we developed new primer sets against HA cleavage sites for subtyping H1 to H15 genes and for molecular pathotyping. Our primer sets were subtype specific and detected 99% of previously identified HA genes (115/116, 1949 to March 2006), and the correct amplifications of HA genes were confirmed by sequence analyses of all 115 PCR products. The primer sets successfully subtyped most of the recent AI viruses isolated in Japan (96% [101/105], October 2006 to March 2007). Taken together, our primer sets could efficiently detect HA genes (98% [216/221]) of both previously and recently identified HA genes or of both American (29/29) and Eurasian (187/192) lineages. All 38 H5 and 13 H7 viruses were molecularly pathotyped by sequencing analyses of the HA cleavage site. In contrast, despite efficient detection of previously identified strains (98% [114/116]), the published primer sets exhibited lower specificity and lower detection efficiency against recent AI viruses (80% [84 of 105]). These results indicate that our primers are useful not only for HA subtyping but also for molecular pathotyping of both previous and recent AI viruses. These advancements will enable general diagnostic laboratories to subtype AI viruses for the surveillance in wild aquatic birds.

The H5N1 highly pathogenic (HP) avian influenza (AI) viruses have been enzootic in Asian countries since 2004 (26), raising serious worldwide concern about their pandemic potential. Although wild aquatic birds usually harbor only low pathogenic (LP) AI viruses, fatal infections of aquatic birds by the H5N1 HPAI virus first occurred in Hong Kong parks in late 2002 (3, 21, 22) and then at Qinghai Lake in China in 2005 (1, 13), followed by reports of infections in European and African countries in 2005 (27, 28). These events imply that wild aquatic birds may play a critical role in carrying the H5N1 virus long distances through migration (7). Global surveillance is required to understand the present prevalence of H5N1 viruses in wild aquatic birds.

Since the virus isolation test is the most reliable and sensitive method, use of the test is essential for accurate surveillance for H5N1 virus infection in wild aquatic birds. However, when it is used, many LPAI viruses are isolated from these natural reservoirs. Lack of a subtyping method broadly useful of many AI viruses may hinder the surveillance. Serological subtyping of AI viruses by the hemagglutination inhibition (HI) test is the most often performed at only reference laboratories due to limited supply of antisera. In addition, the HI test exhibits weak reactivity against antigenically different viruses within the subtype and occasionally exhibits cross-reactivity against different subtypes. Thus, a reliable hemagglutinin (HA) subtyping method easily applicable for general diagnostic laboratories is needed.

One possible method is the subtyping of HA genes by reverse transcription PCR (16, 20) . Real-time PCR is highly specific, and H5 and H7 detection methods have been previously developed (2, 6, 19). Loop-mediated isothermal amplification is rapid and sensitive, and a system for H5 gene detection has been reported (8). A DNA microarray technology has also been applied for analysis of human H1N1, H3N2, and H5N1 viruses (12, 25). Although these genetic subtyping methods remain to be evaluated for AI viruses, a subtyping system applicable for all HA subtypes and easily available in general diagnostic laboratories is desirable. PCR is useful due to its easy applicability, unlimited reagent supply, and use of standard equipment. At present, only one previously published series of primer sets is used to subtype H1 to H15 genes (11); however, we found that several recently identified duck viruses could not be subtyped by the primer sets. Thus, more reliable HA subtyping primer sets are needed.

The AI virus is classified into two pathotypes, the HP and LP viruses (18, 24), and quick determination of these pathotypes is also an important factor in surveillance. All HPAI viruses identified to date belong to the H5 and H7 subtypes, and these HA proteins have multiple basic amino acids or insertion of amino acids at the HA cleavage site. Thus, the HA cleavage site, which is not detected by the previously developed subtyping primer sets (Fig. (Fig.1),1), is useful as a target region for HA subtyping by PCR for simultaneous pathotyping of AI viruses.

FIG. 1.
HA subtyping primer sets and corresponding HA gene regions.

In the present study, we developed PCR primer sets to detect the HA cleavage site for each of the H1 to H15 subtypes, and these primer sets were evaluated with more than 200 AI viruses. The usefulness of the primer sets for HA subtyping and molecular pathotyping of Eurasian and American AI viruses in the AI global surveillance are discussed.



A total of 221 AI viruses were used in the present study, including 116 previously (1949 to March 2006) and 105 recently (October 2006 to March 2007) identified AI viruses. The 116, which included 27 H5 (12 HP and 15 LP) and 11 H7 (5 HP and 6 LP) viruses, were composed of 74 Japanese and 42 foreign strains or 90 Eurasian and 26 American lineage strains. These viruses were derived from ducks (79 strains), chickens (18 strains), turkeys (7 strains), swans (4 strains), budgerigar (2 strains), mynah (2 strains), gull (1 strain), quail (1 strain), shearwaters (1 strain), and tern (1 strain). HA subtypes of these AI viruses were previously determined by the HI test. These were used to determine the sensitivity and cross-reactivity of HA subtyping primer sets. The 105 recently identified AI viruses were composed of 101 migratory duck strains isolated from October 2006 to March 2007 in Japan and 4 H5N1 HPAI Qinghai Lake lineage strains isolated from chickens in Japan in 2007 (M. Mase et al., unpublished data). These were used to determine the applicability of primer sets for subtyping of HA genes.

The AI viruses were propagated in chicken embryonated eggs for 1 to 4 days, and the allantoic fluids were frozen at −80°C until use.

The following viruses were kindly provided as indicated: 4 HPAI viruses (A/chicken/Puebla/8623-607/94 [H5N2], A/chicken/Queretaro/14588-19/95 [H5N2], A/chicken/Texas/298313/04 [H5N2], and A/chicken/Chile/184240-2/02 [H7N3]) and 2 LPAI viruses (A/chicken/NJ/15086-3/1994 [H7N3] and A/chicken/NY/119055-7/01 [H7N2]) were from D. Suarez and D. Swayne, 14 LPAI wild duck strains (1 H4N5, 2 H8N4, 3 H10N4, 2 H10N5, 4 H11N9, and 2 H12N5) were from Y. Sakoda and H. Kida (Hokkaido University), 3 Thailand H5N1 HPAI viruses (A/chicken/Suphanburi/1/04, A/duck/Angthong/72/04, and A/quail/Angthong/71/04) were from A. Chaisingh (National Institute of Animal Health, Thailand), A/chicken/Italy/330/97 (H5N2) and A/turkey/Italy/4580/99 (H7N1) were from I. Capua (Istituto Zooprofilattico Sperimentale delle Venezie), A/chicken/Korea/ES/05 (H5N1) (10) was from the National Veterinary Research and Quarantine Service (Korea), and A/duck/Akita/714/06 (H5N2), and A/duck/Akita/212/05 (H7N1) (9) was from K. Takehara (Kitasato University). Two HPAI viruses (A/chicken/Netherland/2586/2003 [H7N7] and A/chicken/Pakistan/447/95 [H7N3]) were transferred from I. Capua after obtaining the original holder's permission.

cDNA synthesis.

Viral RNA was extracted from 250 μl of allantoic fluid by using 750 μl of TRIzol solution (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The viral RNA was resuspended in 20 μl of water and mixed with 4 μl of random primers (6-mer), followed by heating at 95°C for 2 min and immediate chilling on ice. The RNA-primer mixture was used to synthesize cDNA in a 50-μl reaction volume with PrimeScript reverse transcriptase of murine leukemia virus (Takara, Kyoto, Japan). The mixture was incubated at 30°C for 10 min, followed by 42°C for 1 h and 70°C for 15 min. Successful cDNA synthesis was confirmed by PCR amplification of the nucleoprotein (NP) gene with a previously described primer pair (11): NP1200, 5′-CAGRTACTGGGCHATAAGRAC-3′, and NP1529, 5′-GCATTGTCTCCGAAGAAATAAG-3′.

Primer design.

A total of 515 complete nucleotide sequences of HA genes were downloaded from the Influenza Sequence Database (http://www.flu.lanl.gov) (14). Seventy-five HA genes were sequenced in the present study, and these were used to design new primers. These HA nucleotide sequences were aligned for each HA subtype or for all of H1 to H15 subtypes. Forward and reverse primers were designed on the subtype-specific, conserved regions in HA1 and HA2, respectively, to amplify the HA cleavage sites of the H1 to H15 genes (Fig. (Fig.1).1). Each primer shared more than 95% identity for each nucleotide among sequences.


A MicroAmp optical 96-well plate (ABI) and the GeneAmp PCR System 9700 (ABI) were used. The PCR mixture (10 μl per reaction) contained 1 μl of cDNA, 0.5 μl of each primer (20 pmol each), and 5 μl of premixture ExTaq (Takara) in 20 mM Tris-HCl (pH 8.0) containing 50 mM KCl, 2 mM MgCl2, and 0.2 mM concentrations of each deoxynucleoside triphosphate. PCR amplification was performed as follows: denaturation for 1 min at 94°C; followed by 35 cycles of PCR amplification, with each cycle consisting of 30 s of denaturation at 94°C, 30 s of annealing at 50°C, and 30 s of elongation at 72°C; and with one final cycle of elongation at 72°C for 7 min. Then, 2-μl portions of the products were separated by electrophoresis on a 2.0% agarose gel.

Nucleotide sequence analysis.

Portions (20 μl) of the PCR products were purified by using a QIAquick PCR purification kit (Qiagen, Inc., Valencia, CA), and the purified DNA fragments (elusion volume, 30 μl) were used for sequencing using the forward and reverse primers. The nucleotide sequencing reaction mixture (20 μl per reaction) contained 13 μl of purified product, 1 μl of primer (20 pmol), 4 μl of BigDye Terminator v3.1 (Cycle Sequencing Kit; ABI, Foster City, CA), and 2 μl of 5× buffer, according to the manufacturer's instructions. The sequence reaction was performed as follows: denaturation for 1 min at 94°C, followed by 25 cycles of PCR amplification, with each cycle consisting of 10 s of denaturation at 96°C, 5 s of annealing at 50°C, and 4 min of elongation at 60°C. The PCR products were purified with a Sephadex G-50 column and sequenced (ABI-3100; PE ABI, Foster City, CA). The sequence data were subjected to BLAST search analyses using the NCBI database.

Phylogenic analyses.

To characterize the amplified HA gene fragments, the nucleotide sequences were analyzed with the GENETYX-WIN sequence analysis software version 8 (Software Development, Tokyo, Japan). The tree was displayed by using the unweighted pair-group method with arithmetic averages method.



Several primers were designed and empirically tested for sensitivity for detection of HA genes of homologous HA subtype by PCR. The 116 HA genes from previously identified AI viruses (1949 to March 2006) were as follows: 1 H1, 3 H2, 5 H3, 14 H4, 27 H5 (12 HP and 15 LP viruses), 12 H6, 11 H7 (5 HP and 6 LP viruses), 4 H8, 7 H9, 9 H10, 16 H11, 4 H12, 1 H13, 1 H14, and 1 H15 HA genes (see Table Table3).3). First, several primer sets with comparatively high sensitivity for the detection HA genes of each homologous subtype were selected. HA genes not detected by the primer sets were sequenced, and the primers were modified. This category of genes included 75 HA genes (Table (Table1).1). The primer modification process was repeated to identify primer sets with high sensitivity and high specificity for each HA subtype. Consequently, we successfully amplified the predicted target regions from 115 HA genes except for the H8 gene (Table (Table3),3), and the primer sequences are given in Table Table22.

Number of HA gene sequences downloaded from the Influenza Sequence Database and those sequenced in this study for primer design
Primers used for subtyping of HA genes of AI viruses
Detection of homologous subtypes HA genes of previously identified (1949 to 2006.3) and recently identified (2006.10 to 2007.3) AI viruses by PCR with our or Lee's HA subtyping primer sets


To assess the cross-reactivity of these HA subtyping primer sets, each primer set was tested for the cross-reactivity using a set of heterogeneous HA genes, including 1 H1, 2 H2, 3 H3, 3 H4, 5 H5, 3 H6, 5 H7, approximately 3 H8-H12, and approximately 1 H13 to H15 HA genes (41 HA genes per primer set). The primer sets did not amplify HA genes of heterogeneous subtypes; the H9 primer set did weakly amplify one H8 gene (1/3), and faint, incorrectly sized, or smeared PCR products were weakly detected at a low frequency. The nonspecific or cross-reactive amplified products were clearly distinguishable from homologous products by agarose-gel electrophoresis. Figure Figure22 demonstrates an example of the specificity of the H5, H7, and H9 subtyping primer sets, and successful subtyping of four HPAI viruses is presented in Fig. Fig.33 (15).

FIG. 2.
Subtype-specific amplification of HA cleavage sites by PCR from H1 to H15 genes with subtyping primer sets for H5, H7, and H9 genes.
FIG. 3.
HA subtyping of H5 and H7 HPAI viruses by PCR.

These results indicate that the HA subtyping primer sets were highly specific for each HA subtype gene.

Molecular analysis of PCR products.

To confirm the specific amplification of HA genes with the primer sets, all 115 PCR products were directly sequenced. Nucleotide sequence data were successfully obtained from all 104 PCR products using the forward and/or reverse primers, and the BLAST search analyses confirmed the HA subtype-specific amplification of HA genes. Also, the nucleotide sequence data of each HA subtype were useful for quick molecular epidemiological analyses of HA genes.

The HA cleavage site has a molecular motif of HPAI viruses. To determine the ability of the primer sets for molecular pathotyping, the amino acid sequences of the HA cleavage sites were analyzed from 27 H5 and 11 H7 AI viruses. As expected, 13 of the 27 H5 and 5 of the 11 H7 gene products had multiple basic amino acids, whereas the remaining 14 H5 and 6 H7 genes had amino acid sequences of the LP type (see Table Table4).4). The LP A/chicken/Pennsylvania/21525/83 (H5N2), which had the HPAI molecular motif (PQKKKR/GLF), and LP A/chicken/Chile/184240-2/02 (H7N3) with nucleoprotein gene sequence insertions at the HA cleavage site (PEKPKTCSPLSRCRETR/GLF; inserted amino acids are indicated in italics) (23), were molecularly pathotyped as an HPAI virus. Other HA subtype primers were also useful for determining the presence of the multiple basic amino acids at the cleavage sites.

Molecular pathotyping of H5 and H7 viruses by sequencing of HA cleavage sites

Applicability for HA subtyping.

To further evaluate the applicability of our HA subtyping primer sets, a total of 105 AI viruses newly isolated from October 2006 and March 2007 in Japan were used for subtyping of HA genes with H1 to H15 primer sets. These viruses included 101 wild duck strains and 4 Qinghai Lake lineage isolates of H5N1 HPAI viruses. Of the 105 AI viruses, 101, including 4 H5N1 HPAI viruses, were successfully subtyped using the primer sets, although 2 H1 and 2 H2 genes were not detected (Table (Table3).3). As mentioned above, most of the AI viruses generated a single, clear DNA band, whereas faint, smeared, or nonspecific extra DNA bands were observed at a low frequency. Also, 3 of 11 H12 genes were cross-amplified with the H7 primer set, but these products were distinguishable from specifically amplified H12 products by agarose gel electrophoresis.

Nucleotide sequence analyses of the PCR products using the forward and/or reverse primers showed that these 101 amplified HA gene products were subtype-specific. A total of 11 H5 viruses, including four Qinghai Lake lineage virus isolates from chickens in Japan in 2007 (A/chicken/Miyazaki/K11/07), and 2 H7 viruses were molecularly pathotyped (Table (Table4).4). The Qinghai Lake lineage viruses had multiple basic amino acids at the HA cleavage site (PQGERRRKKR/GLF).

These results indicated that the HA subtyping primer sets developed in the present study were useful for HA subtyping, as well as molecular determination of the pathogenic potential of AI viruses recently isolated from wintering wild ducks in Japan.

Evaluation of Lee's HA subtyping primer sets.

Since our previous examinations showed that Lee's HA subtyping primer sets were not useful for analyzation of some of the recent AI viruses, the sensitivity of the primer sets was intensively evaluated with 221 AI viruses used above: 116 previously (1949 to March 2006) and 105 recently identified strains (October 2006 to March 2007). Although 197 of 221 HA genes (89%) were detected with Lee's primer sets these primers exhibited differences in sensitivity between previous (114/116 [98%]) and recent (84/105 [80%]) strains (Table (Table3),3), and the sensitivity also varied among HA subtypes. HA genes undetected with Lee's primer sets were the H1, H6, H10, and H12 genes of recent wild duck strains and were detected at rates of 0, 69, 70, and 0%, respectively.


Wild migratory aquatic birds may play an important role in H5N1 virus transmission, and these viruses have been repeatedly isolated from wild aquatic birds in Asia, Europe, and Africa since the first isolation in Chinese Qinghai Lake in 2005 (1, 13). The risk of exposure of chickens or humans to H5N1 viruses from wild aquatic birds is evident, underlying the importance in evaluating the prevalence of H5N1 HPAI viruses in wild aquatic birds. For this surveillance purpose, a reliable and simple HA subtyping method is required for quick assessment of many AI viruses (4, 17). The HI test remains a valuable tool for HA subtyping of avian influenza viruses; however, the supply of the necessary antiserum is limited. Furthermore, the antisera are occasionally weakly or unreactive against antigenically different AI viruses within a subtype. The antiserum against virus particles is cross-reactive against AI viruses of relevant NA subtypes. Therefore, subtyping of HA genes by PCR is predicted to be an easily applicable method for use in general diagnostic laboratories. The primer sets designed by Lee's group, which were only available for the subtyping of H1 to H15 genes (11), were not evaluated against recent strains. Preliminary experiments found that some recent viruses were not detected. In the present study, we comprehensively evaluated the usefulness of Lee's primer sets with 116 previously identified and 105 recently identified AI viruses and found that Lee's primer sets could only detect 80% of recent AI viruses (84/105); the H1, H6, H10, and H12 genes were poorly detected (0, 69, 70, and 0%, respectively) (Table (Table3).3). In contrast, our primer sets are HA subtype specific and highly sensitive against H1 to H15 genes of both previous (99% [115/116]) and recent (96% [101/105]) viruses (Table (Table3)3) or of both Eurasian (187/192) and American (29/29) HA genes. Furthermore, the generated PCR products were shown to be useful for molecular epidemiological analyses of the H1 to H15 genes. These results indicate that our HA subtyping primer sets are useful for global surveillance of the AI viruses in wild aquatic birds.

Quick determination of the pathogenic potential is an important factor for AI surveillance.

The HA cleavage site contains the pathogenicity motif of AI viruses: all HPAI viruses detected thus far have multiple basic amino acids [R/K-(-)/R/K-R/K-R/K/GLF] or an insertion of viral sequences at the HA cleavage site. Notably, our primer sets target the HA cleavage site of the H1 to H15 HA subtypes, whereas Lee's primers target only the HA1 region (Fig. (Fig.1).1). In the present study, all 38 H5 viruses (16 HP and 22 LP) were molecularly pathotyped (Table (Table4),4), including A/chicken/Pennsylvania/21525/83 (H5N2), which was molecularly HP but biologically LP due to the presence of a glycosylation site beneath the cleavage site. Also, all 13 H7 viruses (5 HP and 8 LP) were also molecularly pathotyped (Table (Table4).4). The A/chicken/Chile/184240-2/02 (H7N3) HPAI virus had an insertion of NP gene sequences at the HA cleavage site (PEKPKTCSPLSRCRETR/GLF; inserted amino acids are indicated in italics) (23). The British Columbia H7N3 HPAI strain (not used in the present study) has a similar insertion at the HA cleavage site (PENPKQAYRKRMTR/GLF), which likely occurred by RNA recombination between the HA and M genes (5). These results indicate that our primers are useful for the molecular pathotyping of H5 and H7 viruses and for the determination of amino acid sequences of HA cleavage sites of other HA subtypes.

The HA gene pool perpetuated in wild aquatic birds is divided into Eurasian and American lineages and exhibits wide diversity in each lineage. Mixed nucleotide number in each primer is a key factor for the efficient detection of highly diverse HA genes. Theoretically, increased numbers of primer nucleotides may expand the detection spectrum but may also decrease the sensitivity and specificity of the PCR. Optimal numbers of mixed nucleotides in primers have yet to be determined for HA genes. In the present study, we designed primers with 95% identity in each nucleotide and then empirically selected primers with high sensitivity and high specificity using 221 HA genes from 179 Japanese viruses and 42 foreign viruses. The selected primers were used for nucleotide sequencing. The actual mixed nucleotide numbers in each primer varied from 0 to 9 (Table (Table2),2), which were higher than expected. This empirical approach may be useful for the development of NA subtyping primer sets. Our study also indicates that our primer sets are useful not only for Eurasian HA genes (187/192) but also for American HA genes (29/29), implying the applicability of these primer sets against diverse HA genes. Nevertheless, further improvement may be required for detection of new HA genes, especially those of American lineages. Preparation of second primer sets for each HA subtype may be a reasonable approach for improved coverage of the widely diverse HA genes perpetuated in wild aquatic birds. The H1, H2, and H8 primer sets, which missed several H1 (2/6), H2 (2/11), and H8 (1/4) genes (Table (Table3),3), should be the first targets for the second primer sets. The H9 and H7 primer sets were weakly cross-reactive to some H8 (1/4) and H12 (3/11) genes, respectively, and also warrant further improvement. Nevertheless, these findings demonstrate that the HA gene subtyping system will be useful for AI global surveillance, as well as quick molecular analysis of AI viruses in wild aquatic birds.


We thank D. Swayne and D. Suarez (Southeast Poultry Research Laboratory, U.S. Department of Agriculture), H. Kida and Y. Sakoda (Hokkaido University), K. Takehara (Kitasato University), I. Capua (Istituto Zooprofilattico Sperimentale delle Venezie), Y.-J. Lee (National Veterinary Research and Quarantine Service, Korea), and A. Chaisingh (National Institute of Animal Health, Thailand) for kindly providing AI viruses. We also thank H. Ikeda at our institute for critical discussion and Chizuko Fujisawa and Makiko Shishido for excellent technical assistance.

This study was supported by a grant-in-aid from the Zoonoses Control Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan.


[down-pointing small open triangle]Published ahead of print on 2 July 2008.


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