Molecular Characterization and Pathogenesis of H6N6 Low Pathogenic Avian Influenza Viruses Isolated from Mallard Ducks (Anas platyrhynchos) in South Korea

The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus—KNU2019-48 (A/Mallard (Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))—was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59–69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID 50/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.


Introduction
Avian influenza viruses (AIVs) belong to the Orthomyxoviridae family and are composed of eight single-stranded negative-sense RNA segments. The AIVs have been identified based on the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) glycoproteins. AIVs are categorized into 18 HA (H1-16 in wild birds and H17-18 in bats) and 11 NA (N1-9 in wild birds and N10-11 in bats) subtypes [1,2]. Furthermore, AIVs have

Virus Isolation from Feces
Fecal samples were processed according to our previously described protocol [14]. The collected fecal sample was dissolved in PBS (phosphate buffered saline; pH 7.4) sup-

Virus Isolation from Feces
Fecal samples were processed according to our previously described protocol [16]. The collected fecal sample was dissolved in PBS (phosphate buffered saline; pH 7.4) supplemented with an antibiotic solution (100 U/mL penicillin and 100 mg/µL of streptomycin) (Merck, St. Louis, MO, USA) and centrifuged at 3000 rpm for 10 min at 4 • C. The supernatant was filtered through a hydrophilic polyethersulfone membrane filter having a pore size of 0.45 µm (GVS Syringe, Novatech, Kingwood, TX, USA) to remove bacteria and eukaryotic cells. The filtered supernatant samples were inoculated into the allantoic cavities of 10-day-old SPF (specific pathogen-free) ECEs (embryonated chicken eggs). Next, eggs were incubated at 37 • C for 96 h under humidified conditions, and their status was checked every day. Subsequently, eggs were chilled overnight at 4 • C, and allantoic fluids were harvested and analyzed for the confirmation of viruses based on HA (hemagglutination) activity using chicken erythrocytes.

Bird Species Identification Using the Mitochondrial Gene Cytochrome c Oxidase I (COI) as a DNA Barcode
Host bird species were determined by confirmation of a DNA barcode of a region consisting of 751 base pairs (bp) of the mitochondrial gene cytochrome c oxidase I (COXI) as previously described elsewhere [17]. The identification of the host was discovered using information from Barcode of Life Data system (BOLD; Biodiversity Institute of Ontario, University of Guelph, ON, Canada) in a combination comparison with Basic Local Alignment Search Tool for nucleotides (BLASTn; NCBI, National Institute of Health, Bethesda, MD, USA).

Extraction of Viral RNA for Sequencing
A NucleoSpin RNA kit (MACHEREY-NAGEL, Düren, Germany) was used to extract the viral RNA directly from the allantoic fluid of ECEs according to the manufacturer's instructions. Finally, the extracted RNA was eluted in RNase-free water distributed with 20 units of RNase inhibitor and was stored at −80 • C for further analysis. Conventional real-time RT-PCR was performed to determine the presence of the influenza virus and its subtypes using total RNA following WHO (World Health Organization) guidelines [17].

Next Generation Sequencing (NGS) Analysis
NGS analysis was accompanied by GnCBIO (Dae-Jeon, Korea) on the Illumina Hiseq X platform method as previously reported [16]. Briefly, the extracted viral RNA quality was determined using an Agilent RNA 6000 Pico kit (Agilent, Santa Clara, CA, USA), and total RNA concentration was measured using a nano spectrophotometer. The cDNA library of viral RNA was constructed using a QIAseq FX single-cell RNA library kit (Qiagen, Venlo, Netherlands). Library quality was evaluated via the LightCycler qPCR system (Roche, Penzberg, Upper Bavaria, Germany) and library size was verified using TapeStation HS D5000 ScreenTape system (Agilent, Santa Clara, CA, USA). For cluster generation, the library was loaded into a flow cell where fragments were seized on a lawn of surface-bound oligos complementary to the library adapters. Using bridge amplification, each fragment was amplified into distinct clonal clusters, following which cluster creation templates were ready to be sequenced. For sequencing, data were converted into raw data for analysis. Raw sequence reads were quality-trimmed using "trim galore" (q = 20), and non-influenza virus reads were excluded using Deconseq (iden = 60). The quantity of data was corrected using a Python script up to 600,000 reads. Ephemerally, a database of only segments 4-HA, 6-NA, and 8-NS1 from the influenza virus information from NCBI were generated and aligned to those of the reference using Gsmapper (iden = 70, mL = 40). The ORF (open reading frame) was observed using the obtained consensus and adopted a result with an ORF similar to the reference. As the ORF length varied from that of the reference, sequence error was altered using Proof Read as previously described [18].

Phylogenetic Analysis and Molecular Characterization
Nucleotide blast analysis was used to identify pertinency of the viral genes. Entire reference sequences were downloaded from the NCBI (National Center for Biotechnology Information https://www.ncbi.nlm.nih.gov/ (accessed on 27 March 2022)) and GISAID (Global Initiative on Sharing All Influenza Data https://www.gisaid.org/ (accessed on 27 March 2022)) databases. Data were merged, all duplicated sequences were deleted, and phylogenetic trees were finally constructed using MEGA6.0 software (Molecular Evolutionary Genetics analysis version 6.0, Pennsylvania State University, PA, USA). Phylogenetic trees of all segments (PB2, PB1, PA, HA, NP, NA, M, and NS) of the KNU2019-48 (H6N6) viral isolate were generated by applying the maximum-likelihood with Tamura-Nei model and 1000 bootstrap replicates.
2.7. Determination of 50% Tissue Culture Infectious Dose (TCID 50 ) and 50% Egg Infectious Dose (EID 50 ) ELISA (enzyme-linked immunosorbent assay) was used to measure TCID 50 titers as previously reported [19], MDCK cells (ATCC, Manassas, VA, USA) were grown on 96-well flat-bottom microplates at 37 • C with 5% CO 2 . MDCK cells with 80-90% confluence were washed with 1× concentrated PBS and then inoculated with serial 10-fold dilutions of virus suspensions in media containing 1 µg/mL of TPCK-trypsin. Virus-infected cells were incubated at 37 • C with 5% CO 2 for 72 h. Next, the TCID 50 titers were determined via the Reed and Muench method [20]. To determine EID 50 , the allantoic cavities of 10-day-old SPF ECEs were inoculated with 100 µL serial 10-fold dilutions of the viruses, using 5 eggs for each dilution. The eggs were incubated at 37 • C for 96 h. Allantoic fluid was harvested and tested using HA assays [21], and EID 50 calculation of viruses was completed using the Reed and Muench method [20].

Viral Growth Kinetics in MDCK Cells
The growth kinetics of virus isolates were evaluated in vitro. MDCK cells were infected with the viruses at an MOI (multiplicity of infection) of 0.001 in DMEM medium containing 1% antibiotic and 1 µg/mL TPCK-trypsin. Supernatants were collected every 12 hpi up to 72 hpi. ELISA was performed using anti-influenza nucleoproteins (PAN antibodies) to detect infected cells, and the virus titer (TCID 50 ) of each supernatant was determined [22].

Animal Experiment
The pathogenic potential of the new isolate was determined in mammals using 6week-old female BALB/c mice purchased from Orient (Seongnam, Korea) (n = 11), which were intranasally infected with 10 5 EID 50 /mL of virus. Mice were anesthetized using 1% isoflurane following the manufacturer's instructions (Hana Pharmacy, Hwasung, Korea). The survival rate and bodyweight of mice were observed for 15 days, following which the mice were euthanized, and their lungs (n = 3 mice) were collected at 3, 6, and 15 dpi. The lung tissue was homogenized, and the TCID 50 was determined to analyze the viral titers of homogenate supernatants [23]. For histopathology, the lungs of three mice were collected at 3, 6, and 15 dpi and stored in 4% formaldehyde/saline at 4 • C until further analysis. Hematoxylin and eosin (H&E) were used for histopathological examination of paraffin-embedded lung tissues mounted on glass slides. All sections were observed using a light microscope (magnification ×100). This study was approved by the Animal Ethics Committee of the Wonkwang University (WKU19-64; approved on 25 November 2019), South Korea, and all methods were conducted in accordance with relevant guidelines and regulations.

Statistics
The calculation of mean and SD (standard deviation) and Student's t-test were performed using GraphPad Prism Software Version 5.0 (La Jolla, CA, USA). Results were presented as the mean ± SD. Statistical significance was set at p < 0.05.  Figure 2 shows the phylogenetic analysis of the KNU2019-48 (H6N6) strain for eight gene segments.

Hypothesis for the Reassortment Event of Each Gene Segment
Using evolutionary reassortment tracking analysis of our isolate, HA gene reassortment prevailed from South Korea with links to China A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6) isolates in 2015. This was followed by the transmission of the HA

Hypothesis for the Reassortment Event of Each Gene Segment
Using evolutionary reassortment tracking analysis of our isolate, HA gene re-assortment prevailed from South Korea with links to China A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6) isolates in 2015. This was followed by the transmission of the HA gene from South Korea  Figure 3.
Based on the evolutionary information, whole gene segments of our isolate KNU2019-48 (H6N6) originated from Chinese H6N6 isolates. It is possible that the Asian birds migrated through the East Asia-Australasian Flyway. Detailed information on the evolutionary re-assortment is presented in Figure 4.  Based on the evolutionary information, whole gene segments of our isolate KNU2019-48 (H6N6) originated from Chinese H6N6 isolates. It is possible that the Asian birds migrated through the East Asia-Australasian Flyway. Detailed information on the evolutionary re-assortment is presented in Figure 4.

Molecular Characterization of the KNU2019-48 (H6N6) Isolate
The HA cleavage site of KNU2019-48 (H6N6) contained the PRIETR↓GLF (↓denotes the cleavage site) sequence with a single basic amino acid in the HA cleavage site, which revealed a low pathogenic feature of the isolated virus. This HA receptor-binding site (RBS) suggests that H6 isolates would have a high preference toward α-2,6-linked sialic

Molecular Characterization of the KNU2019-48 (H6N6) Isolate
The HA cleavage site of KNU2019-48 (H6N6) contained the PRIETR↓GLF (↓denotes the cleavage site) sequence with a single basic amino acid in the HA cleavage site, which revealed a low pathogenic feature of the isolated virus. This HA receptor-binding site (RBS) suggests that H6 isolates would have a high preference toward α-2,6-linked sialic acid, which is abundant in the upper respiratory tract of avian species. We compared the HA protein of our isolate with those of the following four other isolated H6N6 strains that originated from birds and swine: (1) a low-pathogenic H6N6 isolate from domestic ducks in central China, labeled as A/duck/Hubei/10/2010 (H6N6); (2) A/swine/Guangdong/K6/2010 (H6N6) isolates, which originated from domestic ducks following whole gene reassortment; (3) isolates from poultry markets in Southern China that are a potential threat to mammals, labeled as A/duck/China/A729-2/2011(H6N6); (4) novel North American-origin avian influenza A (H6N5) virus isolated from bean goose in South Korea in 2018, which was published by our team in 2020, labeled as A/Bean goose/South Korea/KNU18-6/2018(H6N5).
A reasonable analysis of HA RBS at positions 138, 186, 190, 226, and 228 (H3 numbering) shows no mutation in amino acid residues ( Table 2). The single basic residue at the HA cleavage site, with no prominent mutation at the HA RBS, showed that our KNU2019-48 (H6N6) isolates and reference strains, except for Hunan-2011, were low pathogenic H6 viruses. Moreover, the NAs of the H6N6 isolates had no amino acid deletion in the stalk (59-69) regions (Table 2). Mutations in other internal genes, also presented in Table 3, signify an enhanced viral replication efficiency along with virulence in mammals. Particularly, three human host marker mutations (PB1-S375N, PA-A404S, and S409N) were observed in KNU2019-48 (H6N6) strains, along with the mutation S375N in the PB1 gene displayed in our old H6N5 isolates from Korea, which had originated from North America. KNU2019-48 (H6N6) showed variance in certain molecular aspects, such as mutations at Q368R of PB2, K328N of PB1, S37A of PA, T223I of NA, and N30D of M1, each contributing to the potentially increased virulence of KNU2019-48 (H6N6) in mammals.

Growth Kinetics of KNU2019-48 (H6N6) Isolate in Mammalian Cell Culture
Since the molecular characterization shows reassortment via mutation, our viral isolate could increase replication efficiency; therefore, in vitro examination of the viral replication of our isolate along with other isolates was performed. To evaluate the growth kinetics of our isolate, human-origin viruses, A/California/07/2009 (H1N1) and H7N7, which are infectious to mammals, were used as controls. H1N1 replicated more efficiently in MDCK cells as compared to KNU2019-48 (H6N6) and H7N7 ( Figure 5). Raw data of the TCID 50 assay are shown in Figure S1.

Growth Kinetics of KNU2019-48 (H6N6) Isolate in Mammalian Cell Culture
Since the molecular characterization shows reassortment via mutation, our viral isolate could increase replication efficiency; therefore, in vitro examination of the viral replication of our isolate along with other isolates was performed. To evaluate the growth kinetics of our isolate, human-origin viruses, A/California/07/2009 (H1N1) and H7N7, which are infectious to mammals, were used as controls. H1N1 replicated more efficiently in MDCK cells as compared to KNU2019-48 (H6N6) and H7N7 ( Figure 5). Raw data of the TCID50 assay are shown in Figure S1.  (12,24,36,48,60,72, and 84 hpi). The virus titer concentration in cell culture supernatant was determined by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza nucleoprotein (NP) to detect infected cells, and TCID50 was determined in MDCK cells. Data are represented as mean ± SD and calculated from three replicates. **, p < 0.01; ***, p < 0.001.

Pathogenicity in Mice
We further examined the pathogenic potential of mice (6-week-old female BALB/c) that were intranasally infected with 10 5 EID50/50 µL. The H1N1 and H7N7 strains were used as the controls for comparisons. The bodyweight of the mice infected with the positive control H1N1 strain decreased from 3 dpi and was gradually regained after 10 dpi, with the lowest weight (76.15 ± 1.96%) observed at 8 dpi. Similarly, our KNU2019-48 (H6N6) isolates and the H7N7 control strain demonstrated a stable body weight trend for 15 days ( Figure 6A).  (12,24,36,48,60,72, and 84 hpi). The virus titer concentration in cell culture supernatant was determined by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza nucleoprotein (NP) to detect infected cells, and TCID 50 was determined in MDCK cells. Data are represented as mean ± SD and calculated from three replicates. **, p < 0.01; ***, p < 0.001.

Pathogenicity in Mice
We further examined the pathogenic potential of mice (6-week-old female BALB/c) that were intranasally infected with 10 5 EID 50 /50 µL. The H1N1 and H7N7 strains were used as the controls for comparisons. The bodyweight of the mice infected with the positive control H1N1 strain decreased from 3 dpi and was gradually regained after 10 dpi, with the lowest weight (76.15 ± 1.96%) observed at 8 dpi. Similarly, our KNU2019-48 (H6N6) isolates and the H7N7 control strain demonstrated a stable body weight trend for 15 days ( Figure 6A).
All the infected mice survived the entire experimental period and showed no difference in the survival mortality between the three groups ( Figure 6B). Virulence in mice lungs after 3, 6, and 15 dpi were analyzed by TCID 50 assay. Based on the experimental results, control group H1N1 showed maximum virus titer in the lungs in comparison with the KNU2019-48 (H6N6) and H7N7 strains at 3 dpi (4.62 ± 0.17, 3.76 ± 0.38, 2.71 ± 0.08 log 10 TCID 50 /mL, respectively), and these viral loads gradually decreased at 6 dpi (4.26 ± 0.44, 3.80 ± 0.63, 1.80 ± 0.05 log 10 TCID 50 /mL, respectively) ( Figure 6C). In contrast, no virus was detected in all groups at 15 dpi, indicating that these viruses were not yet well-adapted for murine infections. Further, histopathological analyses were conspicuously shown in the lungs of the virus-challenged mice. Infected lungs at 3 and 6 dpi show a condensed penetration of neutrophils into the alveolar air spaces (Figure 7). All the infected mice survived the entire experimental period and showed no difference in the survival mortality between the three groups ( Figure 6B). Virulence in mice lungs after 3, 6, and 15 dpi were analyzed by TCID50 assay. Based on the experimental results, control group H1N1 showed maximum virus titer in the lungs in comparison with the KNU2019-48 (H6N6) and H7N7 strains at 3 dpi (4.62 ± 0.17, 3.76 ± 0.38, 2.71 ± 0.08 log10 TCID50/mL, respectively), and these viral loads gradually decreased at 6 dpi (4.26 ± 0.44, 3.80 ± 0.63, 1.80 ± 0.05 log10 TCID50/mL, respectively) ( Figure 6C). In contrast, no virus was detected in all groups at 15 dpi, indicating that these viruses were not yet well-adapted for murine infections. Further, histopathological analyses were conspicuously shown in the lungs of the virus-challenged mice. Infected lungs at 3 and 6 dpi show a condensed penetration of neutrophils into the alveolar air spaces (Figure 7).

Discussion
The H6N6 subtype AIV is widespread in poultry, and its host range extends to mammals such as pigs. Moreover, it has become an endemic disease in domestic animals [55]. Our isolated KNU2019-48 (H6N6) strain was co-circulated in China, Vietnam, and

Discussion
The H6N6 subtype AIV is widespread in poultry, and its host range extends to mammals such as pigs. Moreover, it has become an endemic disease in domestic animals [61]. Our isolated KNU2019-48 (H6N6) strain was co-circulated in China, Vietnam, and Korea for half a decade. These H6N6 subtype gene segments were derived from the East Asia-Australasian flyway lineages. Poultry birds may play an intermediate host role in the cross-species transmission of the influenza virus from domestic birds to humans [62]. However, this is the first isolation of H6N6 strains during our 2018 surveillance program in South Korea, and there are no detailed evidential molecular and pathological studies on H6N6 isolates from South Korea. In particular, other subtypes such as H7N3 and other H7 subtypes of AIV were pre-vailed/isolated during the surveillance period [16,63].
According to the molecular analysis of the H6N6 strain in our study, the HA cleavage site sequence PRIETR↓GLF was subtyped into a low-pathogenic virus. According to the results of phylogenetic analysis, the HA gene was closely related to A/mallard/Korea/M219/2014 (H6N2), while the genes of PB2, PA, NP, M, and NS were closer to the Chinese and Vietnamese isolates (A/duck/China/330D17/2018 (H6N6) and A/Muscovy duck/ Vietnam/LBM755/2014 (H5N6)). The PB1 gene was similar to that of the Chinese isolate, A/duck/Guangdong/7.20_DGCP015-C/2017 (H6N6), and the NA gene was similar to that of co-circulated Chinese isolates A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6) and A/chicken/Guangdong/8.30_DGCP022-O/2017(H6N6), respectively. These analyses strongly indicate that the H6 subtype virus has been frequently detected in migratory ducks in China [48,64,65].
Furthermore, we re-confirmed the pathogenicity of our KNU2019-48 (H6N6) isolate using in vitro experiments. We monitored the growth kinetics of KNU2019-48 (H6N6) replication in MDCK cells as follows: the virus replicated promptly and exhibited no obvious signs of illness. A maximum virus titer (2.84 log 10 TCID 50 /mL) was achieved at 24 hpi, and there were no changes until 72 h post-infection. Similarly, our isolate strongly exhibited low pathogenicity in the lungs of mice at 3 and 6 dpi compared to the positive control H1N1, which originated from humans. Additionally, we performed body weight and survival percentage analysis. Low pathogenicity was confirmed in intranasally-infected mice; mice lungs weighed the highest at 3 dpi, whereas at 6 and 15 dpi, no variance was observed, and the lung weight was normal ( Figure 6D). Although our H6N6 isolate is a low-pathogenic isolate, its movement should be monitored frequently to detect incidences of infection in humans and a potential genetic reassortment event.
In conclusion, this is the first study that broadens the knowledge of Korean H6N6 isolates from evolution to mammalian cell expressions and in vivo characterization. Continuous monitoring and molecular characterization of the H6N6 virus will be required for a better understanding of the evolutionary dynamics of the virus, which can further assist in improving control measures.

Conflicts of Interest:
The authors declare no conflict of interest.