Journal of Virological Methods 97 (2001) 13 – 22 www.elsevier.com/locate/jviromet

Identification and subtyping of avian influenza viruses by reverse transcription-PCR Ming-Shiuh Lee a,b, Poa-Chun Chang c, Jui-Hung Shien a, Ming-Chu Cheng b, Happy K. Shieh a,c,* a

Department of Veterinary Medicine, National Chung Hsing Uni6ersity, Taichung, Taiwan b National Institute of Animal Health, Council of Agriculture, Taipei, Taiwan c Institute of Veterinary Microbiology, National Chung Hsing Uni6ersity, Taichung, Taiwan Received 6 November 2000; received in revised form 5 March 2001; accepted 7 March 2001

Abstract Avian influenza viruses have 15 different hemagglutinin (HA) subtypes (H1– H15). We report a procedure for the identification and HA-subtyping of avian influenza virus by reverse transcription-PCR (RT-PCR). The avian influenza virus is identified by RT-PCR using a set of primers specific to the nucleoprotein (NP) gene of avian influenza virus. The HA-subtypes of avian influenza virus were determined by running simultaneously 15 RT-PCR reactions, each using a set of primers specific to one HA-subtype. For a single virus strain or isolate, only one of the 15 RT-PCR reactions will give a product of expected size, and thus the HA-subtype of the virus is determined. The result of HA-subtyping was then confirmed by sequence analysis of the PCR product. A total of 80 strains or isolates of avian influenza viruses were subtyped by this RT-PCR procedure, and the result of RT-PCR gave an excellent (100%) correlation with the result of the conventional serological method. The RT-PCR procedure we developed is rapid and sensitive, and could be used for the identification and HA-subtyping of avian influenza virus in organ homogenates. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Avian influenza virus; Hemagglutinin; Subtype; Reverse transcription-polymerase chain reaction (RT-PCR)

1. Introduction Avian influenza (AI) is a highly contagious disease caused by type A influenza virus, a member of the family Orthomyxo6iridae (Lamb and Krug, 1996). Avian influenza viruses are divided into subtypes on the basis of two surface glyco* Corresponding author. Tel.: + 886-4-22860196; Fax: + 886-4-22872392.

proteins: hemagglutinin (HA) and neuraminidase (NA) (Easterday et al., 1997). Fifteen HA (H1 – H15) and nine NA subtypes (N1 –N9) have been identified (Rohm et al., 1996). All 15 HA subtypes of influenza virus are found in aquatic birds, which serve as the primordial reservoir of all influenza A viruses (Webster et al., 1997). Among 15 HA subtypes, only H5 and H7 are highly virulent in poultry (Alexander, 1995). In humans, though histologically viruses of only H1, H2 and

0166-0934/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 0 1 ) 0 0 3 0 1 - 9

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H3 subtypes have caused pandemics, growing evidence has showed that viruses of other subtypes might infect humans (Webster et al., 1997). For example, an avian H7 virus might be transmitted directly from ducks to humans (Zhou et al., 1996). Most importantly, the H5N1 virus found in Hong Kong in 1997 could transmit from poultry to humans, and cause high mortality in both species (Claas et al., 1998; Suarez et al., 1998; Subbarao et al., 1998; Zhou et al., 1999). The HA and NA subtypes of avian influenza viruses are identified by the hemagglutinin inhibition test and neuraminidase inhibition tests by antisera prepared from 15 HAs and 9 NAs (Beard 1989a,b). Recently, RT-PCR has been used to differentiate H1 from H3 virus (Wright et al., 1995; Stockton et al., 1998), or to differentiate N1 from N2 virus (Stockton et al., 1998). Moreover, RT-PCR followed by sequence analysis of the HA cleavage site was used for rapid determination of the virulence potential of H5 and H7 viruses in birds (Horimoto and Kawaoka, 1995; Senne et al., 1996). It is believed that PCR might serve as a fast and effective alternative to virus isolation for the detection of influenza A virus (Claas et al., 1993; Yuen et al., 1998). However, to date there is no report on differentiating H1– H15 or N1 – N9 of avian influenza viruses by RT-PCR. The molecular basis for different antigenicity of HA and NA subtypes lies in the difference in the amino acid sequences (Wiley et al., 1981). Air (1981) showed that different HA subtypes have amino acid differences between 20% and 74%, whereas the same HA subtype has differences between only 0–9%. Because the amino acid sequences are determined by nucleotide sequences and because wobble is present in the codon usage, the differences in nucleotide sequences between HA subtypes should be more substantial than 20–74%. This serves as the basis for HA-subtyping avian influenza virus by RT-PCR, because the specificity of PCR is determined by differences in nucleotide sequence. We report here a procedure for rapid identification and HA-subtyping of avian influenza virus by RT-PCR. We found RTPCR gave a result that is highly consistent with the serological method; moreover, RT-PCR could be used for the identification and HA-subtyping

of avian influenza viruses directly from organ homogenates.

2. Materials and methods

2.1. Viruses Reference strains of avian influenza virus used, and strains of virus sequenced in this study are listed in Table 1. Reference strains of H1 and H2 subtypes include Taiwanese avian isolates of which the subtypes were confirmed in the AI reference center in the Central Veterinary Laboratory (Weybridge), Addlestone, Surrey, UK. Other strains were obtained from Dr H. Kida at the School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, or from Dr R.G. Webster at St. Jude Children’s Research Hospital, Memphis, TN. Fifty five field isolates of avian influenza virus, isolated from aquatic birds or chickens in Taiwan in 1989–1999, were propagated in embryonated eggs as described (Beard, 1989a). Other avian viruses used in the specificity test of PCR included Newcastle disease virus (strain B1), infectious bronchitis virus (Mass. and Conn. types), infectious bursal disease virus (Bursine-2), infectious laryngotracheitis (Laryngo-Vac), avian reovirus (strain S1133). These viruses were all obtained from SOLVAY Animal Health (Charles City, IA).

2.2. Serological studies Antisera against H1–H15 subtypes were obtained either from Dr R.G. Webster or from Dr H. Kida. Hemagglutination inhibition tests (HI) were performed in microtiter plates as described (Beard, 1989b).

2.3. RNA isolation RNA was extracted using Trizol reagent (Life Technology, Gaithersburg, MD). In brief, 0.1 ml of allantoic fluid or organ homogenate was mixed with 1 ml of Trizol reagent. After mixing completely and being kept at room temperature for 5

M.-S. Lee et al. / Journal of Virological Methods 97 (2001) 13–22 Table 1 Reference strains and strains sequenced in this study Reference strains of avian origins used in this study

Source and accession numbers a

Influenza A/Duck/Yilan/106/86 (H1N1) Influenza A/PR/8/34 (H1N1) Influenza A/Shorebirds/Taiwan/35/98 (H2N3) Influenza A/Singapore/1/57 (H2N2) Influenza A/Duck/Ukrine/1/63 (H3N8) Influenza A/Duck/Czechoslovakia/56 (H4N6) Influenza A/Duck/Hong Kong/820/80 (H5N3) Influenza A/DK/Singapore/3/97 (H5N3) Influenza A/Shearwater/Australia/1/72 (H6N5) Influenza A/Hong Kong/301/78 (H7N1) Influenza A/Turkey/Ontario/6118/68 (H8N4) Influenza A/Turkey/Wisconsin/1/66 (H9N2) Influenza A/Quail/Hong Kong/G1/97 (H9N2) Influenza A/Chick/Germany/N/49 (H10N7) Influenza A/Duck/England/56 (H11N6) Influenza A/Duck/Memphis/546/74 (H11N9) Influenza A/Duck/Alberta/60/76 (H12N5) Influenza A/Gull/Maryland/704/77 (H13N6) Influenza A/Mallard/Gurjev/263/82 (H14N5) Influenza A/Duck/Australia/341/83 (H15N8) Influenza A/Shearwater/West Australia/2576/79 (H15N9)

A B A B B B B C B B B B

Table 1 (Continued) Reference strains of avian origins used in this study

Source and accession numbers a

Influenza A/Chicken/Taiwan/na3/98 A (AF310984) (H6N1) Influenza A/Chicken/Taiwan/ns2/99 A (AF310985) (H6N1) Influenza A/Duck/Taiwan/g9/89 (H11N?) A (AF310986) Influenza A/Pintail Duck/Alberta/114/79 C (AF310987) (H8N4) Influenza A/Mallard Duck/Alberta/357/84 C (AF310988) (H8N4) Influenza A/Red Kont/Delaware/254/94 C (AF310989) (H8N4) Influenza A/Ruddy C (AF310990) Turnstone/Delaware/67/98 (H12N4) Influenza A/Mallard Duck/Alberta/342/83 C (AF310991) (H12N1) Influenza A/Laughing Gull/New C (AF310992) Jersey/171/92 (H12N5) Influenza A/Shorebird/Taiwan/31/99 A(AF311750) (H10N7)

C B B B B B C C C

Reference strains of non-a6ian origins used in this study Influenza A/PR/8/34 (H1N1) B Influenza A/FM1 /47 (H1N1) B Influenza A/Singapore/1/57 (H2N2) B Influenza A/Aichi/2/68 (H3N2) B Influenza A/Swine/Iowa/15/30 (H1N1) B Influenza A/Swine/Shizuoka/1/78 B (H1N1) Influenza A/Sw/IA/8548-1/98 (H3N2) C Influenza A/Equine/Piaque/1/56 (H7N7) B Influenza A/Equine/Miami/1/63 (H3N8) B Strains sequenced in this study Influenza A/Chicken/Taiwan/7-5/99 (H6N1)

15

A (AF310983)

a A, Taiwanese isolates of which the subtypes were confirmed in the AI reference center in Weybridge. B, strains provided by Dr H. Kida. C, strains provided by Dr R.G. Webster.

min, the mixture was extracted with 0.18 ml chloroform/isoamylalcohol (24:1). After centrifugation at 10 000× g for 15 min, the RNA in the aqueous solution was precipitated by adding an equal volume of isopropanol. The RNA precipitate was collected by centrifugation at 10 000× g for 20 min, washed by 75% ethanol and dissolved in 50 ml of RNase-free water.

2.4. Primer design To identify avian influenza virus by RT-PCR, we designed two primers based on conserved sequences of the NP gene of viruses of avian, human, swine and equine origins (Altmuller et al., 1989; Gorman et al., 1990; Shu et al., 1993). The conserved sequences were selected by sequence comparison and sequence alignment of about 100 NP sequences retrieved from the GenBank of the National Center of Biotechnology Information (NCBI), USA. The NP-specific primers we designed by this strategy are

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NP1200 (forward): 5%-CAG(A/G)TACTGGGC (A/T/C)ATAAG(A/G)AC-3%, and NP1529 (reverse): 5%-GCATTGTCTCCGAAGAAATAAG-3% To subtype avian influenza virus by RT-PCR, we designed 15 sets of primers, each based on conserved sequences of a single HA subtype. More than 1000 HA sequences in the GenBank, including all HA subtypes of viruses of avian, human, swine and equine origins, were considered for primer design. The primers designed, and numbers of strains that were considered for primer design, are listed in Table 2.

2.5. Polymerase chain reaction RT-PCR was carried out in a reaction mixture (25 ml) containing 2.5 ml of 10-times reaction buffer (Promega, Madison, WI), 2.5 ml dNTP blend (2.5 mM each of four dNTPs, Promega), 0.2 ml AMV reverse transcriptase (9 units/ml, Promega), 0.3 ml RNase inhibitor (40 units/ml, Promega), 0.5 ml Taq DNA polymerase (9 units/ ml, Promega), 1 ml of each primer (10 pmol each), 1 ml of RNA template (about 1 ng), and 17 ml of water. The PCR condition for the amplification of NP, H2, H3, H4, H11, H14 and H15 was 42°C for 45 min (reverse transcription), 95°C for 3 min, 35 cycles of 95°C for 30 s (denaturation), 55°C for 40 s (annealing) and 72°C for 40 s (extension), followed by 72°C for 10 min (final extension). The PCR condition for the amplification of H1, H5, H6, H7, H8, H9, H10, H12 and H13 was the same as above, except that the annealing temperature was reduced to 50°C.

2.6. DNA sequencing The amplified DNA fragments were purified by using a QIAquick PCR purification kit (QIAGEN Inc., Valencia, CA). Purified DNA fragments were sequenced from both directions (using the same primers that amplify the DNA), by an automatic sequencer (ABI-377, PE Applied Biosystems, Foster City, CA). The nucleotide sequence was compiled using the SEQUMAN program in the LASERGENE package (DNASTAR Inc., Madison, WI). For sequence comparison and identification,

the sequences were searched against the GenBank by the BLAST program (Altschul et al., 1990) provided by NCBI, USA.

3. Results

3.1. Identification of a6ian influenza 6irus by RT-PCR The nucleotide sequences of NP gene are highly conserved in all subtypes of avian influenza viruses. To identify these viruses by RT-PCR, we designed two primers based on consensus sequences of NP genes of avian influenza viruses. The two primers, designated as NP primers, were able to amplify a 330 bp fragment from all reference strains listed in Table 1 (data not shown); these strains include viruses of avian, human, swine and equine origins (Table 1). The identity of the 330 bp fragment was confirmed by sequence analysis and sequence comparison using BLAST search. A total of 91 strains or isolates were tested by this RT-PCR, and all of them gave a 330 bp product (data not shown). We therefore conclude that the consensus regions we chose for primer design are highly conserved in influenza A viruses, and RT-PCR using NP primers could be used to identify these viruses. The specificity of NP primers was examined by RT-PCR using template extracted from other avian viruses, including Newcastle disease virus, infectious bronchitis virus, infectious bursal disease virus, and infectious laryngotracheitis virus. None of the above viruses gave a PCR product after amplification (data not shown), indicating that NP primers are highly specific to avian influenza virus. The sensitivity of RT-PCR using NP primers was also determined by a serial dilution of RNA of avian influenza virus from 1 ng to 0.1 fg, and the sensitivity was found to be between 1 pg and 0.1 pg (data not shown).

3.2. HA-subtyping of a6ian influenza 6irus by RT-PCR To design a set of primers specific to each single HA subtype, we compared nucleotide sequences

M.-S. Lee et al. / Journal of Virological Methods 97 (2001) 13–22

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Table 2 Primers used for HA-subtyping of avian influenza viruses by RT-PCR Primer

Primer sequences a

PCR product (bp)

Total numbers of sequences considered for primer design b

H1-550f H1-1016r H2-422f H2-1083r H3-175f H3-896r H4-8f H4-777r H5-155f H5-699r H6-661f H6-962r H7-12f H7-645r H8-166f H8-597r H9-151f H9-638r H10-521f H10-932r H11-240f H11-689r H12-11f H12-431r H13-203f H13-433r H14-444f H14-986r H15-455f H15-837r

5%-AACAAYAARGRGAAAGAAGT 5%-GGGACDTTYCTTARTCCTGT 5%-GAGAAARTWAAGATTCTGCC 5%-CCAAACAAYCCYCTTGAYTC 5%-CARATTGARGTGACHAATGC 5%-GGTGCATCTGAYCTCATTA 5%-GCAGGGGAAACAATGCTATC 5%-CCWGGYTCTACAATWGTCC 5%-ACACATGCYCARGACATACT 5%-CTYTGRTTYAGTGTTGATGT 5%-AGCATGAATTTTGCCAAGAG 5%-GGRCATTCTCCTATCCACAG 5%-GGGATACAAAATGAAYACTC 5%-CCATABARYYTRGTCTGYTC 5%-GTGGAAACAGAGAAACAT 5%-CCATAAGAARATGATGTCT 5%-CTYCACACAGARCACAATGG 5%-GTCACACTTGTTGTTGTRTC 5%-GGACAAAAYTTCCCTCAGAC 5%-GRAAAGGGAGCTTTGTATTT 5%-TGYTCMTTTGCTGGRTGGAT 5%-CTCTGAACCCACTGCTACAT 5%-AGGGGTCACAATGGAAAAA 5%-GGTGAAATCAAACATCTTCA 5%CCACACAGGAACATAYTGTTC 5%-CTACTGAAWGAYCTGATTCC 5%-TCATCGCCGAACAATTCACC 5%-GCAGTTTCCTATAGCAATCC 5%-GTGCGTGTAAGAGAACAGTG 5%-ATTAGAGCGGAGAAAGGTGG

467

170

662

40

722

585

770

10

545

91

302 634 432 488

5 (3 were sequenced in this work) 62 4 (3 were sequenced in this work) 23

412

3 (1 were sequenced in this work)

450

2 (1 was sequenced in this work)

421

4 (3 were sequenced in this work)

231

4

543

2

383

2

a

Codes for mixed bases position: R = A/G, Y= C/T, M= G/C, D = G/A/T, W= A/T, B= G/C/T, H= A/C/T. Sequences were retrieved from the GenBank provided by NCBI, USA. Accession numbers of representative strains of each serotype are: H1 (AF085413, AF091309, L25071, U46021), H2 (AF116197, J02127, L11125, L11142), H3 (AF079570, D00929, J02109, L31949, M16737), H4 (D90302, M25283, M25291, J02102), H5 (AF084532, M10243, U20460, U79449), H6 (AF100181, D90903), H7 (A24987, AF071775, AF072383, J02111, U20459), H8 (D90304), H9 (AF156373, AF156377, AF156385, D90305), H10 (M21646, M21647), H11 (D90306), H12 (D90307), H13 (D90308, M26089, M26090, K00383), H14 (M35996,M35997), H15 (L43916, L43917). b

of all subtypes of HA genes retrieved from the GenBank. Because subtypes H6, H8, H10, H11 and H12 have only one or two complete nucleotide sequences available in the current GenBank, we sequenced a total of 11 additional strains for the above subtypes for sequence comparison (Table 1). Sequences from regions that are con-

served in only a single subtype were chosen for the primer design. A total of 15 sets of primers were designed by this strategy for RT-PCR amplification (Table 2); all primers are from the sequence of the HA1 portion of HA gene. The specificity of these primers was then tested by RT-PCR using RNA template isolated from all 15

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HA subtypes of avian influenza viruses. As shown in Fig. 1, for a single HA subtype, only one of the 15 RT-PCR reactions gives the product of expected size. The size of PCR product ranges from 231 bp to 770 bp, depending on the HA subtype (Fig. 1). The identities of these PCR products were then confirmed by sequence analysis and sequence comparison using BLAST search. BLAST search gave a maximum sequence homology of

98–100% when the nucleotide sequence of a PCR product was compared to the sequence of HA gene of the same HA subtype. A total of 91 strains or isolates of influenza viruses, including 80 of avian origin and 11 of human, swine or equine origin, were subtyped by RT-PCR. Subtyping results are shown in Table 3. The results of RT-PCR are 100% consistent with those of serological methods (Table 3).

Fig. 1. HA-subtyping of avian influenza viruses by RT-PCR. Each panel is the subtyping result of a single reference strain, of which the subtype is shown on the left top of each panel. The expected sizes of RT-PCR products are shown in parentheses. Lane M, size markers (100 bp ladder, PRO-tech, Taipei, Taiwan); lanes 1 – 15, RT-PCR products after amplification with primers specific to H1–H15 subtypes; lane C, negative control.

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Fig. 1.

3.3. Direct identification and subtyping of a6ian influenza 6irus from organ homogenates To see whether the RT-PCR procedure could be used for the identification and subtyping of avian influenza viruses in organ homogenates, we conducted RT-PCR amplification of 48 organ homogenates collected randomly from 12 different chicken farms in 1999–2000. Among the 48 homogenates, only three (two from respiratory tract, one from spleen), collected independently from three different farms, were found positive

after RT-PCR amplification using NP primers of avian influenza virus (the result of one farm is shown in Fig. 2, lane 16). The HA subtypes of avian influenza viruses in the three homogenates were then determined by RT-PCR, and all were found to be H6 (the result of one farm is shown in Fig. 2, lanes 1–15). The presence of H6 virus in the three homogenates was confirmed by conventional virus isolation and serological subtyping; from the 48 homogenates, exactly the three homogenates that were positive in RT-PCR gave positive results of virus isolation, and the isolates

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Table 3 Comparison of the result of HA-subtyping by RT-PCR and by HI test Numbers of virus tested

Result of subtyping Field isolates

Total

RT-PCR

HI test a

6 3 4 1 2 1 2 4 2 1 2 4 1 1 2

6 1 6 20 0 12 4 1 0 3 1 0 0 1 0

12 4 10 21 2 13 6 5 2 4 3 4 1 2 2

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15

36

55

91

Reference strains

a HI tests of all 36 reference strains, together with 26 out of the 55 field isolates, were conducted in the AI reference center in the Central Veterinary Laboratory (Weybridge), UK. The HI tests of the remaining 29 field isolates were conducted in our laboratory, and the HI titers of viruses against homologous antisera were found to be of 5 or 6 Log2, whereas those against heterologous antisera were B1 or 1 log2.

were all found to be H6. Therefore, in this case, the sensitivity for the detection of avian influenza virus by RT-PCR and by conventional methods is the same. This result indicates that RT-PCR procedure could be used for the identification and subtyping of avian influenza viruses in organ homogenates, and thus circumvents the need for propagation of viruses in tissue cultures or embryonated eggs.

virus (Stockton et al., 1998). However, we are the first to design subtype-specific primers that were able to differentiate H1– H15 viruses. One major advantage of subtyping of avian influenza virus by PCR is the saving of time. Conventional methods for subtyping of the virus require expansion of viruses in tissue cultures or embryonated eggs, followed by subtyping with serological methods

4. Discussion The use of RT-PCR for the detection of influenza viruses is not new; several strategies of RTPCR have been used to detect influenza A viruses (Cherian et al., 1994; Atmar et al., 1996), or to distinguish between influenza A, B or C viruses (Wright et al., 1995; Zou, 1997). Subtype-specific primers have been used to differentiate H1 virus from H3 virus (Wright et al., 1995; Stockton et al., 1998), or to differentiate N1 virus from N2

Fig. 2. Identification and subtyping of avian influenza viruses in organ homogenates. Lane M, size markers (100 bp ladder, PRO-tech, Taipei, Taiwan); lanes 1 – 15, RT-PCR products obtained by using primers specific to H1– H15 subtypes; lane 16, the RT-PCR product obtained by using primers specific to the NP gene of the virus. The sizes of RT-PCR products, 302 bp for H6 and 330 bp for NP gene, are indicated by arrows.

M.-S. Lee et al. / Journal of Virological Methods 97 (2001) 13–22

(HI tests). This procedure might take a week and considerable effort. In contrast, the RT-PCR procedure could identify and subtype the virus directly from organ homogenates without virus expansion, and thus shorten the time to 1 day for the identification and subtyping of avian influenza virus. Another advantage of subtyping of the virus by PCR is that sequence analysis of the PCR product, followed by sequence comparison and phylogenetic analysis, could provide important information on the origin of the avian influenza virus identified. This information cannot be provided by HI tests. A successful PCR amplification relies on a good primer design. We designed subtype-specific primers based on sequences that are conserved in a single HA subtype. Theoretically, the greater the number of sequences considered, the more likely it is that the sequences will be conserved among viruses of the single HA subtype. For examples, for H1 –H5, H7 and H9 subtypes, at least 10 sequences are considered for each subtype; it is therefore more likely that primers we designed will work in RT-PCR amplification of unknown viruses of the above subtypes. In contrast, for each of H6, H8 and H10– H15 subtypes, there are only 1–4 sequences available for each subtype in the current GenBank. Although we have sequenced additional 1 or 3 strains for each of H6, H8, H10, H11 and H12 subtypes, the primers might need to be refined when more sequences are reported. This refinement will further improve the feasibility of HA-subtyping of avian influenza viruses by RT-PCR. Parallel with this study, we have designed nine NA-specific primers for NA-subtyping of avian influenza viruses, but the subtyping result was unsatisfactory; only some strains or isolates could be subtyped by this method whereas others gave no PCR product at all. This might reflect poor primer design due to the limitation of NA sequences available in the GenBank. Although NA has nine subtypes (N1– N9), most sequences in the GenBank are those of N1 and N2 subtypes; only a few sequences of other NA subtypes have been reported. In particular, no complete sequence for N3, N4 and N6 is available in the GenBank. Though we have determined one or

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two complete sequences for each of N3, N4 and N6 subtypes (Lee et al., unpublished data), it is conceivable that primers designed based on such a limited number of NA sequences could not work well in PCR amplification of the N gene of unknown strains. We found by RT-PCR that three out of the 48 organ homogenates contained avian influenza viruses, and all of them were H6 subtype. These organ homogenates were collected from chickens of 12 different farms in Taiwan in 1999–2000. This finding suggests a current endemic of H6 virus in the chicken population of Taiwan. In fact, we and other laboratories have isolated a total of eight avian influenza viruses from chickens in Taiwan in 1997–2000, and seven of them are found to be H6 subtype (H6N1) (Lee et al., unpublished data). Therefore, both RT-PCR and conventional subtyping methods demonstrate a current endemic of H6 viruses in chickens of Taiwan. It was shown recently that a H6N1 virus isolated in Hong Kong in 1997 might be the precursor of the highly virulent H5N1 isolated in Hong Kong (Cauthen et al., 2000; Hoffmann et al., 2000). The presence of H6N1 virus in Taiwan urges further characterization of these viruses to understand the epidemiology of avian influenza viruses in these areas.

Acknowledgements We thank Dr R.G. Webster at St. Jude Children’s Research Hospital, Memphis, TN, and Dr H. Kida at School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, for providing viruses and antisera. This investigation was supported by grants 89-2.2-62 and 89-6.1-56 from the Council of Agriculture, Taiwan, Republic of China.

References Air, G.M., 1981. Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. Proc. Natl. Acad. Sci. USA 78, 7639 – 7643.

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