Genetic characterization of influenza A virus subtypes H11N6, H11N7, and H11N9 isolated from free‐grazing ducks, Thailand

Abstract Influenza A viruses (IAVs) infect avian species and several mammalian species including humans. Anseriformes water birds are an important reservoir of IAVs. In this study, we identified and characterized IAV subtypes H11N6 (n = 5), H11N7 (n = 3), and H11N9 (n = 3) isolated during the influenza surveillance program in free‐grazing ducks from 2012 to 2015 in Thailand. Eleven IAV‐H11 viruses were characterized by either whole genome sequencing (n = 5) or HA and NA gene sequencing (n = 6) for phylogenetic and amino acid analyses. Phylogenetic analysis showed that Thai IAV‐H11 were grouped into Avian Eurasian lineage. Amino acid analysis showed that all Thai IAV‐H11 viruses have low pathogenic avian influenza (LPAI) characteristics and sensitive to Oseltamivir and Amantadine. Novel reassortant viruses (IAV‐H11N7 and IAV‐H11N9) have been observed. The reassortant viruses contained NP, M, and NS gene segments which originate from intercontinental sources which never been reported in Thai IAVs. In summary, this study demonstrated high genetic diversity of IAV‐H11 circulating in free‐grazing ducks. Free‐grazing ducks infected with IAVs generated novel reassortant IAV‐H11. Thus, surveillance of IAVs in free‐grazing ducks should be routinely conducted to monitor novel reassortant viruses and subsequently potential virulence viruses.

IAV-H11 rarely infect humans. For example, IAV-H11 had been reported to infect humans who were exposed to wild birds. 8 The previous study showed that receptor binding site of IAV-H11 has preferential binding to both avian (SA α 2,3-Gal) and mammalian (SA α 2,6-Gal) receptors. 9 In addition, IAV-H11N6 had been reported to infect pigs in South Korea. 10 The IAV-H11 viruses (H11N2, H11N3, and H11N9) have been reported in domestic ducks from live bird markets in China. [11][12][13] In Thailand, IAV-H11 were isolated from free-grazing ducks FGDs are reservoirs of the IAVs, therefore, interspecies transmission of IAVs commonly occurred. [14][15][16][17][18][19] It is also noted that FGDs could be infected with HPAI-H5N1 without clinical signs and can carry virulent viruses to other animals. Thus, novel reassortant viruses with virulence genes can arise. 20,21 In this study, we selected IAV-H11 (H11N6, H11N7, and H11N9) isolated from FGDs in Thailand and performed genetic characterization of IAV-H11 by whole genome sequencing to determine genetic diversity of the viruses and subsequently monitor potential virulence viruses.

| Viruses
The IAVs were isolated from oropharyngeal swabs (OP) and cloacal swabs (CS) collected from FGDs during 2012-2015 influenza surveillance program in Thailand. The FGD flocks were selected based on owner collaboration, and approximately 50 ducks from each flock were randomly selected and sampled. The IAVs were isolated by egg inoculation into 9-to 11-day-old embryonated chicken eggs. 22 The allantoic fluid was tested for influenza virus by hemagglutination (HA) test. The allantoic fluid with the HA titer ≥2 HA unit were interpreted as positive influenza virus. The HA positive samples were confirmed for the presence of IAV by using real-time Reverse Transcriptase -PCR (rRT-PCR) specific to the Matrix (M) gene. 23 In this study, IAVs (n = 11) isolated from FGDs were included for genetic characterization. This study was conducted under the approval of the Chulalongkorn University Animal Care and Use Protocol (IACUC# 2031050 and 2031051).

| IAV detection
RNA extraction from the allantoic fluid was carried out by Nucleospin ® RNA virus (Macherey-Nagel, Germany) according to the manufacturer's instruction. The viral RNA was subjected to IAV detection by rRT-PCR specific to Matrix (M) gene. 23 In brief, the rRT-PCR was performed by using the SuperScript ® III Platinum ® One-Step Quantitative RT-PCR System (Invitrogen ® ). The 30 μl of reagent mixture contained 4 μl of RNA template, 1Â master mix buffer, 0.8 μM of M gene specific primers, 0.2 μM of probe, 0.6 mM MgSO 4 , 1 unit of Superscript III reverse transcriptase, and RNase-free water. Amplification was done by rRT-PCR which contained three steps: (1) reverse transcription at 50 C for 30 min, (2) predenaturation at 95 C for 15 min, and (3) denaturation for 50 cycles of 95 C for 15 s and annealing-extension at 60 C for 30 s. The rRT-PCR results were evaluated by cycle threshold (Ct), and values less than 36 were considered positive, greater than 40 were negative, and between 36 and 40 were interpreted as inconclusive.

| IAV subtype identification
The positive RNA samples were synthesized for cDNA by using Improm-II Reverse Transcription System (Promega, Madison, WI, USA) with universal primer for IAVs. The cDNA samples were subjected to IAV subtype identification. The specific primers of each influenza subtypes, H1-H15 and N1-N9, were used for influenza subtyping by PCR using primers previously described. [24][25][26] In detail, 30 μl of PCR mixture contained 1 μl of cDNA, 1Â master mix buffer (TopTaq ™), 0.8 μM of primers for each subtype, and distilled water.
The PCR conditions were 94 C for 3 min and 35 cycles of 94 C for 30 s, 50 C (for H1-H15) or 45 C (for N1-N9) for 30 s, and 72 C for 30 s. The PCR product was run in 1.2% of agarose gel with Red safe in 0.5Â Tris borate EDTA (TBE).

| IAV characterization
Eight segments of the IAVs were amplified using TopTaq master mix (Qiagen, Hilden, Germany) with specific primer sets and newly designed primers for sequencing. 27 In brief, 30 μl of PCR mixture con- Phylogenetic trees of eight gene segments were generated by using MEGA v10.0 applying neighbor-joining method with 1000 replications for bootstrap analysis. The reference data set for phylogenetic analysis was selected to represent geographic locations (Eurasian and North America lineages) and available sequences of Thai IAV-H11Nx, HxN6, HxN7, and HxN9.

| Amino acid analysis of Thai IAV-H11
In this study, amino acid analysis for the translated genetic sequences of the Thai IAV-H11 was conducted. At the HA cleavage site, the   showed that Thai IAV-H11 contained Q226 and G228 suggesting preferential binding for α 2-3-linked sialic acid receptor or avian receptor. 28,29 Moreover, Thai IAV-H11N7 had single amino acid difference near the receptor binding site (134-138; GVTAS) which is different from that of other IAV-H11 (134-138; GVTAA) ( Table 3). For NA gene analysis, the Thai IAV-H11 did not contain amino acid deletion in the NA stalk region and amino acid substitutions associated with neuraminidase resistance suggesting that the IAV-H11 were sensitive to Oseltamivir. 30,31 For PB2 gene analysis, the PB2-627 of IAV-H11 contained glutamic acid (E), whereas one IAV-H11N9 (CU-12660) contained glycine (G). 32,33 Interestingly, glycine at PB2-627 position is very rare because most amino acid at PB2-627 is 627E in avian viruses and 627K in mammalian viruses ( Table 3).
The analysis of genomic signatures related to host specificity of the internal gene segments of IAV-H11 showed that the IAV-H11N9 (CU14442) contained lysine (K) at position NP109, which is rare in avian viruses (Table 4). For IAV-H11N7 (CU16340), the virus contained four unusual amino acids at NP293 (K), NP305 (H), NP313 (L), and NP455 (E), which NP293 (K) and NP455 (E) were predominantly observed in human viruses. It is noted that NP305 (H) and NP313 (L) were rarely reported in any IAVs in the GenBank (Table 3).

| DISCUSSION
FGD is one of the important reservoirs for influenza virus. FGDs can be infected with IAVs without clinical signs. Thus, FGDs can receive and/or spread IAVs to and/or from wild birds, domestic birds, domestic animals, and humans. 15,17 In Thailand, during HPAI-H5N1 outbreaks, the FGD production system was considered to be an important potential risk pathway for HPAI-H5N1 outbreaks. 14 The HPAI-H5N1 infected ducks showed no clinical signs with low mortality and morbidity. As FGDs can carry IAVs without clinical appearance and their nature of frequent movement among rice fields, these factors contributed to increasing risk of IAVs to widely spread in the country. 16 Since 2008, the HPAI-H5N1 outbreak has not been reported in Thailand; however, surveillance of IAV in FGDs has been routinely conducted to monitor the status of HPAI and LPAI viruses circulating in the country. in Thailand during 2012-2015, we able to identify IAV-H11 (n = 11).
In 2012, the IAV-H11N6 (n = 5) and IAV-H11N9 (n = 2) could be iso- Because most reported IAV-H11 have been isolated from the Anseriformes order and few IAV-H11N9 was detected in Charadriiformes order and rarely in Galliformes order. 41 The FGDs are one of the reservoir species that are frequently infected with IAV-H11. It has been reported that IAV-H11 has zoonotic potential. There is evidence of IAV-H11 exposure in humans, for example, seropositivity of H11 antibody among chicken growers, duck hunters, and wildlife professionals. 8 In this study, Thai IAV-H11 did not contain some virulence determinants, for example, PB2-627 mutation (E627K), which relating to viral replication and more virulence of IAVs in mammals. 42,43 However, the Thai IAV-H11N9 (CU12660) contained glycine (G) at PB2-627, which is rarely reported in avian viruses and never been reported in mammalian viruses and need further investigation. For the analysis of genomic signatures, PA and NP gene segments of Thai IAV-H11 contained identical amino acids in both avian and human viruses.
In summary, this study provided genetic information of Thai IAV-H11 isolated from FGDs. The Thai IAV subtypes H11N6, H11N7, and H11N9 were characterized. Phylogenetic analysis showed that some IAV-H11N9 and IAV-H11N7 are novel reassortant viruses in Thailand. From amino acid analysis, the HA cleavage site and receptor biding sites of IAV-H11 showed low pathogenic characteristics suggesting less potential to be zoonotic or virulence viruses. Novel reassortant IAV-H11N9 and IAV-H11N7 suggested that IAVs originated from several sources are circulating in FGDs in Thailand.

ACKNOWLEDGEMENTS
We would like to thank Chulalongkorn University for its financial support to the Center of Excellence for Emerging and Re-emerging Infectious Diseases in Animals (CUEDIAs) and the One Health Research Cluster. We thank the 90th Anniversary of Chulalongkorn University

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/irv.12960.

DATA AVAILABILITY STATEMENT
The nucleotide sequence data that support the findings of this study are openly available in the GenBank database (https://www.ncbi.nlm. nih.gov/genbank/), under accession numbers MW857483-857534.