Virus Genes DOI 10.1007/s11262-014-1065-9
Molecular characterization and phylogenetic analysis of H3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China Haibo Wu • Nanping Wu • Xiaorong Peng • Changzhong Jin • Xiangyun Lu • Linfang Cheng Hangping Yao • Lanjuan Li
•
Received: 17 December 2013 / Accepted: 1 April 2014 Ó Springer Science+Business Media New York 2014
Abstract In 2013, 15 avian influenza viruses (AIVs), H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2), were isolated from domestic ducks in Zhejiang Province in China. These strains were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. Analysis of the neuraminidase (NA) gene indicates that a re-assortment event between H3 and H9N2 AIV occurred in these ducks. The molecular markers analyzed over the genome of all viruses indicated that these strains were low-pathogenic AIVs. Although there was no evidence of re-assortment in subtype H3 AIVs among the avian species’ and mammalian hosts in this study, continued surveillance is needed considering the important role of domestic ducks in AIV re-assortment. Keywords Avian influenza viruses Subtype H3 Domestic ducks Re-assortment
Haibo Wu and Nanping Wu contributed equally to the work.
Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1065-9) contains supplementary material, which is available to authorized users. H. Wu (&) N. Wu X. Peng C. Jin X. Lu L. Cheng H. Yao L. Li (&) State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, Zhejiang, China e-mail: [email protected] L. Li e-mail: [email protected]
Introduction Avian influenza viruses (AIVs) are members of the Orthomyxoviridae family and contain eight segments of singlestranded RNA with negative polarity [1]. Based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, AIVs are classified into 18 HA and 11 NA subtypes [2, 3]. Most subtypes of AIVs have been identified in aquatic birds, including domestic ducks, which are considered a natural reservoir for AIVs [4]. Though domestic ducks do not usually display symptoms when they are infected with AIVs, they provide an environment for the re-assortment of H5 and H7 subtype low pathogenic avian influenza (LPAI) viruses, which can serve as progenitors of highly pathogenic avian influenza (HPAI) viruses [5, 6]. Since 2003, the H5N1, H7N2, H7N3, H7N7, H7N9, and H9N2 AIV subtypes have caused disease outbreaks in poultry world-wide and have infected humans in many countries [7–12], and thus these AIV subtypes have garnered great world-wide attention. However, information regarding the molecular characteristics of the H3 and other AIV subtypes is limited. During the surveillance of poultry for AIVs in Zhejiang Province, Eastern China in 2013, 15 AIVs, H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2), were isolated in embryonated chicken eggs from domestic ducks. To better understand the genetic relationships between these strains from Eastern China and from wild birds in Asia, all gene segments of these strains were sequenced and compared with sequences available in GenBank.
Materials and methods Five-hundred and five cloacal swabs were collected from apparently healthy Shaoxing pockmark ducks (Anas
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Virus Genes
platyrhyncha var. domestica, from commercial farms), in live poultry markets (LPMs; n = 6) in Zhejiang Province, Eastern China, from June 2013 to December 2013. For virus isolation, cloacal swab material from domestic ducks was inoculated into embryonated chicken eggs as previously described [13]. After incubation at 37 °C for 72 h, the allantoic fluid was harvested and tested by HA assay. The subtypes of the virus isolates were determined by conventional HA inhibition and NA inhibition assays, according to the Office International des Epizootics manual [14]. Aliquots of virus allantoic fluid stock were stored at -80 °C before use. RNA extraction was performed using the Viral RNA Mini Kit (Qiagen, Limburg, The Netherlands), according to the manufacturer’s instructions. All segments were amplified with primers as previously described [13]. The PCR products were cloned into pGEM T easy vectors, and three pGEM clones were sequenced per amplicon. Fragment sequencing was carried out using the Big Dye Terminator V.3.0 Cycle Sequencing Ready Reaction Kit (Life Technologies, Carlsbad, CA, USA) and 3730 DNA Analyzer (Life Technologies), according to the manufacturer’s instructions. The sequences were analyzed using BioEdit version 7.0.9.0 DNA analysis software. Molecular markers associated with virulence, host adaptation, and drug resistance were analyzed in HA, NA, M2, and PB2 proteins. Phylogenetic trees were generated using neighbor-joining analysis (bootstrapped with 1,000 replicates) with the Tamura–Nei model in the MEGA program (version 5.05) as described elsewhere [15, 16]. The sequence data obtained in this study were deposited into GenBank; the accession numbers are KF357794–KF357821, KF357854– KF357857, and KJ439814–KJ439901.
Results and discussion We isolated 37 strains of AIVs: subtypes H1 (n = 2), H2 (n = 3), H3 (n = 15), H4 (n = 6), H5 (n = 3), H7 (n = 4), H9 (n = 2), H10 (n = 1), and H11 (n = 1) from domestic ducks in LPMs in Zhejiang Province, China. In this study, all eight gene segments of the subtype H3 strains H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2) were sequenced. Based on the deduced amino acid sequences of the HA gene, the HA cleavage site pattern, PEKQTR/GL, all of the 15 H3 AIVs, displayed features of a monobasic cleavage site and indicated that these strains were LPAI viruses. It is known that the addition of multiple amino acids to the cleavage site, such as arginine (R) and lysine (K), may convert an LPAI into the HPAI subtypes
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H5 and H7 [17]. Amino acids at receptor binding sites (RBSs) were also analyzed, and they were well conserved (Table 1; Fig. S1) [18, 19]. The RBS of these strains, Q226 and G228, was similar to all H3 AIVs in both Eurasian and North American lineages suggesting that this strain would preferentially bind to alpha 2–3 linked sialic acid receptors predominant in avian species [18, 19]. There was no mutation of interest in the RBS, which can increase affinity toward alpha 2–6 linked sialic acid receptors. The results of phylogenetic analysis showed that all genes, HA, NA, PB2, PB1, PA, NP, M, and NS, of H3 AIVs were clustered in the Eurasian lineage (Figs. 1, S2). For HA, six potential glycosylation sites, sites 24, 38, 54, 181, 301, and 499, were detected in these strains identified in this study. The specific polypeptide for N-linked glycosylation is defined as (Asn-X-Ser/Thr), where X can be any amino acid, except proline or aspartic acid [20]. Furthermore, HA glycosylation is associated with virulence and the viral affinity for the influenza virus receptor [21, 22]. The potential glycosylation site is at position 24, and some of the viruses in this study, namely, 4613, 4625, 4637, 6D4, 6D7, and 4812, have lost position 24 glycosylation, and whether this alters viral affinity for the receptor in these strains needs further investigated. In the NA of the D11 and D13 (H3N2) viruses derived from the Y280-like virus, there were deletions of three amino acid ‘‘TEI’’ (sites 63–65) at the NA stalk region. As previously described [23, 24], the deletion of the ‘‘TEI’’ amino acid sequence at the NA stalk region also occurred in the H1N2 virus [13]. Previous studies have shown that the 3-amino acid deletion in the NA stalk region combined with some HA substitutions may significantly increase the virulence of AIVs in multiple hosts and provide a selective advantage [25, 26]. Furthermore, stalk deletion in the H9N2 virus appears to provide an HA–NA compatibility advantage and acts as a significant adaptation in poultry. It was showed that the NA of D11 and D13 (H3N2) viruses shared a higher sequence similarity with H9N2 AIVs than other N2 AIVs. No mutations associated with resistance to NA inhibitors (oseltamivir and zanamivir) or amantadines were observed in the NA and M2 proteins, respectively. Previous reports showed that the PB2 protein mutation Glu627Lys is associated with H5N1 AIVs pathogenicity in mice [27], and Thr271Ala plays a key role in enhanced polymerase activity of influenza viruses in mammalian host cells [28] but neither was observed in the PB2 of H3 AIVs. The HA phylogenetic tree suggests that four different H3 genetic groups are co-circulating in Zhejiang Province.
Virus Genes Table 1 Genetic analyses of amino acids at HA cleavage sites and receptor binding sites (RBSs) of HA genes from 15 H3 AIVs and reference H3 viruses Virus (abbreviation)
HA cleavage
RBS
324–331
98
153
155
183
190
Left edge of RBS
Right edge of RBS
194
224–229
134–138 GGSGA
A/duck/Zhejiang/4613/2013(H3N2) (4613)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/4625/2013(H3N2) (4625)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/4637/2013(H3N2) (4637) A/duck/Zhejiang/D11/2013(H3N2) (D11)
PEKQTRGL PEKQTRGL
Y Y
W W
T T
H H
E E
L L
RGQSGR RGQSGR
GGSGA GGSNA
A/duck/Zhejiang/D13/2013(H3N2) (D13)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA GGSGA
A/duck/Zhejiang/6D4/2013(H3N2) (6D4)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/6D7/2013(H3N2) (6D7)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/D16/2013(H3N3) (D16)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D17/2013(H3N3) (D17)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D18/2013(H3N3) (D18)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D1-1/2013(H3N6) (D1-1)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA GGSGA
A/duck/Zhejiang/D1-2/2013(H3N6) (D1-2)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/D1-3/2013(H3N6) (D1-3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/D1-6/2013(H3N8) (D1-6)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/4812/2013(H3N8) (4812)
PEKQTRGL
Y
W
T
H
E
L
RGQSGS
GGSGA
A/duck/Zhejiang/5/2011(H3N3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/bantam/Nanchang/9-366/2000(H3N3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Nanchang/8-174/2000(H3N6) A/duck/Beijing/33/04(H3N8)
PEKQTRGL PEKQTRGL
Y Y
W W
T T
H H
E E
L L
RGQSGR RGQSGR
GGSNA GGSNA
A/mallard/Maryland/615/2005(H3N2)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Chiba/8/2006(H3N8)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/mallard/Altai/1208/2007(H3N6)
PERQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/Guangdong/322/2010(H3N2)
PEKQTRGI
Y
W
T
H
D
L
RNIPSR
GTSSA
A/Hong Kong/CUHK10297/2005(H3N2)
PEKQTRGI
Y
W
T
H
D
L
RNIPSR
GTSSA
A/swine/Fujian/43/2007(H3N2)
PEKQTRGI
Y
W
H
H
D
V
RNIPSR
GTSSA
Bold residues indicate changes from the consensus alignment
One group is formed by D11, D13, D16–18 strains that come from the same LPM, the second group is formed by the 4812 strain that comes from the second LPM, the third group is formed by 4613, 4625, 4637 (from the third LPM), 6D4, and 6D7 (from the fourth LPM) strains, while D1-1, D1-2, D1-3, and D1-6 strains belong to the fourth group that comes from the fifth LPM. In the NA (N2) phylogenetic tree, the two H3N2 AIVs (D11 and D13) belong to the Y280-like category. The NA (N8) phylogenetic tree suggests that the two H3N8 AIVs (4812 and D1-6) belong to different NA genetic groups. The N3 and N6 of H3 AIVs were clustered in the Eurasian lineage, and the phylogenetic trees suggest that the internal genes, PB2, PB1, PA, NP, M, and NS, also belong to the Eurasian lineage. Furthermore, they had a high nucleotide similarity to different AIV subtypes from aquatic birds and poultry in Eastern
Asia, especially those from domestic ducks in LPMs in Zhejiang Province. We chose the isolate D11, which represents two H3N2 AIVs. The sequence comparisons of D11 (H3N2) with its closest genetic relatives are shown in Table 2. The HA and NA genes of D11 show the highest nucleotide similarity to A/aquatic bird/Hong Kong/399/99 (H3N8) and A/chicken/ Hong Kong/YU148/2012 (H9N2), respectively. According to phylogenetic relationship analysis, the D11 strain was a re-assortment virus and derived its genes from different subtypes of viruses from aquatic birds and poultry in Eastern Asia. This finding indicates that a re-assortment event between H3 and H9N2 occurred in ducks. Since February 2013, a novel H7N9 AIV associated with human death has emerged in Eastern China [11, 29], and some investigations indicate that the virus is a re-assortment
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Virus Genes Fig. 1 Phylogenetic analysis of HA and NA (N2, N3, N6, and N8) genes of H3 and other AIVs. The tree was generated using neighbor-joining analysis (bootstrapped with 1,000 replicates) with the Tamura–Nei model. The H3 AIVs characterized are highlighted by a triangle. The scale bar represents the distance unit between sequence pairs
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Virus Genes Fig. 1 continued
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Virus Genes Fig. 1 continued
of H7, N9, and H9N2 AIVs [11, 29, 30]. The phylogenetic trees based on the internal genes of H3 AIVs (Fig. S2) revealed that the H3 AIVs were not the closest common ancestor of human H7N9 viruses. Aquatic birds represent the major natural LPAI virus reservoir and have a higher prevalence of influenza virus than other species. Domestic ducks have been regarded as birds that can perpetuate most AIVs since they generally do
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not show clear clinical signs when they are infected. Our previous study reported that the re-assortment event between H1 and H9N2 AIVs occurred in domestic ducks in LPMs of Eastern China [13, 31]. Considering that the reassorted H3N2 viruses were created in domestic ducks in this study, it is possible that these ducks play an important role in re-assortments that generate AIVs. LPMs are considered a major source of influenza virus dissemination and
Virus Genes Fig. 1 continued
potentially for influenza virus re-assortment. It is generally accepted that the H3 AIVs is a possible progenitor of the 1968 Hong Kong influenza pandemic virus [19, 32, 33]. Although there was no evidence of re-assortment in subtype H3 AIVs among the avian species and mammalian hosts in the present study, continued surveillance of domestic ducks should be used as an early warning system for avian influenza outbreak in poultry and humans.
In conclusion, phylogenetic analysis of all eight viral genes showed that these strains clustered in the Eurasian lineage of influenza viruses. These results indicate that a re-assortment event between H3 and H9N2 AIVs occurred in ducks. None of the major molecular markers linked to increased virulence to chickens and mammals or adaptation to mammalian host and drug resistance were identified in HA, NA, M, or PB2 genes of the studied viruses.
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Virus Genes Fig. 1 continued
Table 2 Sequence homology of the whole genome of A/duck/Zhejiang/D11/2013 (H3N2) strain compared to nucleotide sequences available in the GenBank database Genes
Positions
Virus with the highest percentage of nucleotide identity
GenBank accession numbers
Homology (%)
PB2
1–2,280
A/green-winged teal/Xianghai/430/2011 (H5N2)
JX570867
99
PB1
1–2,275
A/chicken/Jiangsu/RD5/2013 (H10N9)
KF006412
99
PA HA
1–2,151 1–1,701
A/whooper swan/Mongolia/1-21/2007 (H3N1) A/aquatic bird/Hong Kong/399/99 (H3N8)
JN029564 AJ427297
99 98
NP
1–1,497
A/Duck/Hong Kong/P54/97 (H11N9)
AF250474
99
NA
1–1,410
A/chicken/Hong Kong/YU148/2012 (H9N2)
KF259552
98
M
1–987
A/spot-billed duck/Korea/KNU SYG06/2006 (H5N3)
JF800145
99
NS
1–838
A/tundra swan/Shimane/3211A001/2011 (H5N2)
AB830632
99
Acknowledgments This work was supported by Grants from the National Key Technologies R&D Programme for the 12th Five-Year
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Plan of China (2012ZX1000-004-005) and the State Key Laboratory of Independent Task (2010ZZ04).
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Molecular characterization and phylogenetic analysis of H3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China Haibo Wu • Nanping Wu • Xiaorong Peng • Changzhong Jin • Xiangyun Lu • Linfang Cheng Hangping Yao • Lanjuan Li
•
Received: 17 December 2013 / Accepted: 1 April 2014 Ó Springer Science+Business Media New York 2014
Abstract In 2013, 15 avian influenza viruses (AIVs), H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2), were isolated from domestic ducks in Zhejiang Province in China. These strains were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage. Analysis of the neuraminidase (NA) gene indicates that a re-assortment event between H3 and H9N2 AIV occurred in these ducks. The molecular markers analyzed over the genome of all viruses indicated that these strains were low-pathogenic AIVs. Although there was no evidence of re-assortment in subtype H3 AIVs among the avian species’ and mammalian hosts in this study, continued surveillance is needed considering the important role of domestic ducks in AIV re-assortment. Keywords Avian influenza viruses Subtype H3 Domestic ducks Re-assortment
Haibo Wu and Nanping Wu contributed equally to the work.
Electronic supplementary material The online version of this article (doi:10.1007/s11262-014-1065-9) contains supplementary material, which is available to authorized users. H. Wu (&) N. Wu X. Peng C. Jin X. Lu L. Cheng H. Yao L. Li (&) State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, Zhejiang, China e-mail: [email protected] L. Li e-mail: [email protected]
Introduction Avian influenza viruses (AIVs) are members of the Orthomyxoviridae family and contain eight segments of singlestranded RNA with negative polarity [1]. Based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, AIVs are classified into 18 HA and 11 NA subtypes [2, 3]. Most subtypes of AIVs have been identified in aquatic birds, including domestic ducks, which are considered a natural reservoir for AIVs [4]. Though domestic ducks do not usually display symptoms when they are infected with AIVs, they provide an environment for the re-assortment of H5 and H7 subtype low pathogenic avian influenza (LPAI) viruses, which can serve as progenitors of highly pathogenic avian influenza (HPAI) viruses [5, 6]. Since 2003, the H5N1, H7N2, H7N3, H7N7, H7N9, and H9N2 AIV subtypes have caused disease outbreaks in poultry world-wide and have infected humans in many countries [7–12], and thus these AIV subtypes have garnered great world-wide attention. However, information regarding the molecular characteristics of the H3 and other AIV subtypes is limited. During the surveillance of poultry for AIVs in Zhejiang Province, Eastern China in 2013, 15 AIVs, H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2), were isolated in embryonated chicken eggs from domestic ducks. To better understand the genetic relationships between these strains from Eastern China and from wild birds in Asia, all gene segments of these strains were sequenced and compared with sequences available in GenBank.
Materials and methods Five-hundred and five cloacal swabs were collected from apparently healthy Shaoxing pockmark ducks (Anas
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Virus Genes
platyrhyncha var. domestica, from commercial farms), in live poultry markets (LPMs; n = 6) in Zhejiang Province, Eastern China, from June 2013 to December 2013. For virus isolation, cloacal swab material from domestic ducks was inoculated into embryonated chicken eggs as previously described [13]. After incubation at 37 °C for 72 h, the allantoic fluid was harvested and tested by HA assay. The subtypes of the virus isolates were determined by conventional HA inhibition and NA inhibition assays, according to the Office International des Epizootics manual [14]. Aliquots of virus allantoic fluid stock were stored at -80 °C before use. RNA extraction was performed using the Viral RNA Mini Kit (Qiagen, Limburg, The Netherlands), according to the manufacturer’s instructions. All segments were amplified with primers as previously described [13]. The PCR products were cloned into pGEM T easy vectors, and three pGEM clones were sequenced per amplicon. Fragment sequencing was carried out using the Big Dye Terminator V.3.0 Cycle Sequencing Ready Reaction Kit (Life Technologies, Carlsbad, CA, USA) and 3730 DNA Analyzer (Life Technologies), according to the manufacturer’s instructions. The sequences were analyzed using BioEdit version 7.0.9.0 DNA analysis software. Molecular markers associated with virulence, host adaptation, and drug resistance were analyzed in HA, NA, M2, and PB2 proteins. Phylogenetic trees were generated using neighbor-joining analysis (bootstrapped with 1,000 replicates) with the Tamura–Nei model in the MEGA program (version 5.05) as described elsewhere [15, 16]. The sequence data obtained in this study were deposited into GenBank; the accession numbers are KF357794–KF357821, KF357854– KF357857, and KJ439814–KJ439901.
Results and discussion We isolated 37 strains of AIVs: subtypes H1 (n = 2), H2 (n = 3), H3 (n = 15), H4 (n = 6), H5 (n = 3), H7 (n = 4), H9 (n = 2), H10 (n = 1), and H11 (n = 1) from domestic ducks in LPMs in Zhejiang Province, China. In this study, all eight gene segments of the subtype H3 strains H3N2 (n = 7), H3N3 (n = 3), H3N6 (n = 3), and H3N8 (n = 2) were sequenced. Based on the deduced amino acid sequences of the HA gene, the HA cleavage site pattern, PEKQTR/GL, all of the 15 H3 AIVs, displayed features of a monobasic cleavage site and indicated that these strains were LPAI viruses. It is known that the addition of multiple amino acids to the cleavage site, such as arginine (R) and lysine (K), may convert an LPAI into the HPAI subtypes
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H5 and H7 [17]. Amino acids at receptor binding sites (RBSs) were also analyzed, and they were well conserved (Table 1; Fig. S1) [18, 19]. The RBS of these strains, Q226 and G228, was similar to all H3 AIVs in both Eurasian and North American lineages suggesting that this strain would preferentially bind to alpha 2–3 linked sialic acid receptors predominant in avian species [18, 19]. There was no mutation of interest in the RBS, which can increase affinity toward alpha 2–6 linked sialic acid receptors. The results of phylogenetic analysis showed that all genes, HA, NA, PB2, PB1, PA, NP, M, and NS, of H3 AIVs were clustered in the Eurasian lineage (Figs. 1, S2). For HA, six potential glycosylation sites, sites 24, 38, 54, 181, 301, and 499, were detected in these strains identified in this study. The specific polypeptide for N-linked glycosylation is defined as (Asn-X-Ser/Thr), where X can be any amino acid, except proline or aspartic acid [20]. Furthermore, HA glycosylation is associated with virulence and the viral affinity for the influenza virus receptor [21, 22]. The potential glycosylation site is at position 24, and some of the viruses in this study, namely, 4613, 4625, 4637, 6D4, 6D7, and 4812, have lost position 24 glycosylation, and whether this alters viral affinity for the receptor in these strains needs further investigated. In the NA of the D11 and D13 (H3N2) viruses derived from the Y280-like virus, there were deletions of three amino acid ‘‘TEI’’ (sites 63–65) at the NA stalk region. As previously described [23, 24], the deletion of the ‘‘TEI’’ amino acid sequence at the NA stalk region also occurred in the H1N2 virus [13]. Previous studies have shown that the 3-amino acid deletion in the NA stalk region combined with some HA substitutions may significantly increase the virulence of AIVs in multiple hosts and provide a selective advantage [25, 26]. Furthermore, stalk deletion in the H9N2 virus appears to provide an HA–NA compatibility advantage and acts as a significant adaptation in poultry. It was showed that the NA of D11 and D13 (H3N2) viruses shared a higher sequence similarity with H9N2 AIVs than other N2 AIVs. No mutations associated with resistance to NA inhibitors (oseltamivir and zanamivir) or amantadines were observed in the NA and M2 proteins, respectively. Previous reports showed that the PB2 protein mutation Glu627Lys is associated with H5N1 AIVs pathogenicity in mice [27], and Thr271Ala plays a key role in enhanced polymerase activity of influenza viruses in mammalian host cells [28] but neither was observed in the PB2 of H3 AIVs. The HA phylogenetic tree suggests that four different H3 genetic groups are co-circulating in Zhejiang Province.
Virus Genes Table 1 Genetic analyses of amino acids at HA cleavage sites and receptor binding sites (RBSs) of HA genes from 15 H3 AIVs and reference H3 viruses Virus (abbreviation)
HA cleavage
RBS
324–331
98
153
155
183
190
Left edge of RBS
Right edge of RBS
194
224–229
134–138 GGSGA
A/duck/Zhejiang/4613/2013(H3N2) (4613)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/4625/2013(H3N2) (4625)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/4637/2013(H3N2) (4637) A/duck/Zhejiang/D11/2013(H3N2) (D11)
PEKQTRGL PEKQTRGL
Y Y
W W
T T
H H
E E
L L
RGQSGR RGQSGR
GGSGA GGSNA
A/duck/Zhejiang/D13/2013(H3N2) (D13)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA GGSGA
A/duck/Zhejiang/6D4/2013(H3N2) (6D4)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/6D7/2013(H3N2) (6D7)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/D16/2013(H3N3) (D16)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D17/2013(H3N3) (D17)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D18/2013(H3N3) (D18)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/duck/Zhejiang/D1-1/2013(H3N6) (D1-1)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA GGSGA
A/duck/Zhejiang/D1-2/2013(H3N6) (D1-2)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
A/duck/Zhejiang/D1-3/2013(H3N6) (D1-3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/D1-6/2013(H3N8) (D1-6)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Zhejiang/4812/2013(H3N8) (4812)
PEKQTRGL
Y
W
T
H
E
L
RGQSGS
GGSGA
A/duck/Zhejiang/5/2011(H3N3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/bantam/Nanchang/9-366/2000(H3N3)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Nanchang/8-174/2000(H3N6) A/duck/Beijing/33/04(H3N8)
PEKQTRGL PEKQTRGL
Y Y
W W
T T
H H
E E
L L
RGQSGR RGQSGR
GGSNA GGSNA
A/mallard/Maryland/615/2005(H3N2)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSGA
A/duck/Chiba/8/2006(H3N8)
PEKQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/mallard/Altai/1208/2007(H3N6)
PERQTRGL
Y
W
T
H
E
L
RGQSGR
GGSNA
A/Guangdong/322/2010(H3N2)
PEKQTRGI
Y
W
T
H
D
L
RNIPSR
GTSSA
A/Hong Kong/CUHK10297/2005(H3N2)
PEKQTRGI
Y
W
T
H
D
L
RNIPSR
GTSSA
A/swine/Fujian/43/2007(H3N2)
PEKQTRGI
Y
W
H
H
D
V
RNIPSR
GTSSA
Bold residues indicate changes from the consensus alignment
One group is formed by D11, D13, D16–18 strains that come from the same LPM, the second group is formed by the 4812 strain that comes from the second LPM, the third group is formed by 4613, 4625, 4637 (from the third LPM), 6D4, and 6D7 (from the fourth LPM) strains, while D1-1, D1-2, D1-3, and D1-6 strains belong to the fourth group that comes from the fifth LPM. In the NA (N2) phylogenetic tree, the two H3N2 AIVs (D11 and D13) belong to the Y280-like category. The NA (N8) phylogenetic tree suggests that the two H3N8 AIVs (4812 and D1-6) belong to different NA genetic groups. The N3 and N6 of H3 AIVs were clustered in the Eurasian lineage, and the phylogenetic trees suggest that the internal genes, PB2, PB1, PA, NP, M, and NS, also belong to the Eurasian lineage. Furthermore, they had a high nucleotide similarity to different AIV subtypes from aquatic birds and poultry in Eastern
Asia, especially those from domestic ducks in LPMs in Zhejiang Province. We chose the isolate D11, which represents two H3N2 AIVs. The sequence comparisons of D11 (H3N2) with its closest genetic relatives are shown in Table 2. The HA and NA genes of D11 show the highest nucleotide similarity to A/aquatic bird/Hong Kong/399/99 (H3N8) and A/chicken/ Hong Kong/YU148/2012 (H9N2), respectively. According to phylogenetic relationship analysis, the D11 strain was a re-assortment virus and derived its genes from different subtypes of viruses from aquatic birds and poultry in Eastern Asia. This finding indicates that a re-assortment event between H3 and H9N2 occurred in ducks. Since February 2013, a novel H7N9 AIV associated with human death has emerged in Eastern China [11, 29], and some investigations indicate that the virus is a re-assortment
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Virus Genes Fig. 1 Phylogenetic analysis of HA and NA (N2, N3, N6, and N8) genes of H3 and other AIVs. The tree was generated using neighbor-joining analysis (bootstrapped with 1,000 replicates) with the Tamura–Nei model. The H3 AIVs characterized are highlighted by a triangle. The scale bar represents the distance unit between sequence pairs
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Virus Genes Fig. 1 continued
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Virus Genes Fig. 1 continued
of H7, N9, and H9N2 AIVs [11, 29, 30]. The phylogenetic trees based on the internal genes of H3 AIVs (Fig. S2) revealed that the H3 AIVs were not the closest common ancestor of human H7N9 viruses. Aquatic birds represent the major natural LPAI virus reservoir and have a higher prevalence of influenza virus than other species. Domestic ducks have been regarded as birds that can perpetuate most AIVs since they generally do
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not show clear clinical signs when they are infected. Our previous study reported that the re-assortment event between H1 and H9N2 AIVs occurred in domestic ducks in LPMs of Eastern China [13, 31]. Considering that the reassorted H3N2 viruses were created in domestic ducks in this study, it is possible that these ducks play an important role in re-assortments that generate AIVs. LPMs are considered a major source of influenza virus dissemination and
Virus Genes Fig. 1 continued
potentially for influenza virus re-assortment. It is generally accepted that the H3 AIVs is a possible progenitor of the 1968 Hong Kong influenza pandemic virus [19, 32, 33]. Although there was no evidence of re-assortment in subtype H3 AIVs among the avian species and mammalian hosts in the present study, continued surveillance of domestic ducks should be used as an early warning system for avian influenza outbreak in poultry and humans.
In conclusion, phylogenetic analysis of all eight viral genes showed that these strains clustered in the Eurasian lineage of influenza viruses. These results indicate that a re-assortment event between H3 and H9N2 AIVs occurred in ducks. None of the major molecular markers linked to increased virulence to chickens and mammals or adaptation to mammalian host and drug resistance were identified in HA, NA, M, or PB2 genes of the studied viruses.
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Virus Genes Fig. 1 continued
Table 2 Sequence homology of the whole genome of A/duck/Zhejiang/D11/2013 (H3N2) strain compared to nucleotide sequences available in the GenBank database Genes
Positions
Virus with the highest percentage of nucleotide identity
GenBank accession numbers
Homology (%)
PB2
1–2,280
A/green-winged teal/Xianghai/430/2011 (H5N2)
JX570867
99
PB1
1–2,275
A/chicken/Jiangsu/RD5/2013 (H10N9)
KF006412
99
PA HA
1–2,151 1–1,701
A/whooper swan/Mongolia/1-21/2007 (H3N1) A/aquatic bird/Hong Kong/399/99 (H3N8)
JN029564 AJ427297
99 98
NP
1–1,497
A/Duck/Hong Kong/P54/97 (H11N9)
AF250474
99
NA
1–1,410
A/chicken/Hong Kong/YU148/2012 (H9N2)
KF259552
98
M
1–987
A/spot-billed duck/Korea/KNU SYG06/2006 (H5N3)
JF800145
99
NS
1–838
A/tundra swan/Shimane/3211A001/2011 (H5N2)
AB830632
99
Acknowledgments This work was supported by Grants from the National Key Technologies R&D Programme for the 12th Five-Year
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Plan of China (2012ZX1000-004-005) and the State Key Laboratory of Independent Task (2010ZZ04).
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