A 627K variant in the PB2 protein of H9 subtype influenza virus in wild birds

Background Wild birds are gaining increasing attention as gene‐mixing reservoirs for influenza viruses. To investigate the molecular properties of the viruses isolated and epidemiological analysis of H9N2 subtype AIV in wild birds, we studied samples obtained over two years (2014‐2015) from wetlands in Anhui province, China. Methods A total of 4534 samples were collected from migratory waterfowl in Anhui in 2014‐2015, and 8 strains of H9 subtype AIV were isolated. Results Phylogenetic analysis showed different degrees of gene segment reassortment in H9 viruses between the Eurasian lineage and the North American lineage. Most importantly, two viruses harbored the E627K mutation in the polymerase PB2 (PB2) protein. This is the first report of the mutation of this virus from low pathogenicity to high pathogenicity in wild birds. Conclusions The continued surveillance of wild birds, especially migratory birds, is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV along the Eurasian‐Australian migration flyway and the need for more extensive AIV surveillance in eastern China.


| INTRODUC TI ON
Influenza A virus in wild birds includes the 16 HA and 9 NA types, 1 and wild birds are gaining increasing attention as gene-mixing reservoirs for influenza viruses. 2 Wild bird origin avian influenza viruses (AIVs) may gain the ability to infect domestic poultry and humans more effectively after reassortment with one or more AIVs that are already well adapted to domestic birds or have certain amino acid alterations that are adaptive to mammals. 3 Humans and animals are threatened by influenza A virus because of its frequent changes via mutation, recombination, and/or reassortment. H10N8 and H7N9, which may be fatal in human, reassortment among the hemagglutinin (HA), neuraminidase (NA), and internal genes of H9N2 have been observed, along with the E627K mutation in the polymerase PB2 protein. [4][5][6][7] H9N2 is widespread in nature and is sporadically detected in many poultry and even human beings. 8,9 After the isolation of the first strain in 1994, H9N2 AIVs have rapidly differentiated such that more than 102 genotypic variants have now been recognized based on the nomenclature system. 10 In this study, we describe the detection and genetic characteristics of H9N2 viruses in wild birds in Anhui province in 2014-2015. We successfully acquired eight H9N2 viruses, two of which harbored the E627K mutation in their PB2 protein. Our results provide baseline information about the prevalence of H9N2 AIVs in wild birds. This study found 2 | MATERIAL AND ME THODS

| Virus isolation and identification
In 2014-2015, 4534 samples, including feces, oral swabs, and cloacal swabs, were collected from wild birds in Anhui province, China.
The samples were placed in a phosphate-buffered solution (pH 7.0), which included penicillin, streptomycin, and 10% glycerin, and were stored at a low temperature for transport. After the samples were prepared, they were inoculated into 9-day-old specific pathogen-free (SPF) chicken embryos, and allantoic fluid was harvested after 72 hours of culture. The HA activity of the allantoic fluid harvested from each generation was evaluated using an HA test. Hemagglutination inhibition (HI) tests were also carried out with H1-H16 monofactor serum. The HA activity and HI test results were verified by subtypespecific real-time PCR (RT-PCR). NA subtypes were directly analyzed by subtype-specific RT-PCR and sequencing analysis.
Viral RNA was extracted from the allantoic fluid samples that were positive in the HA test using TRIzol (Invitrogen, Carlsbad, CA, USA). RNA was reverse-transcribed into cDNA, which was amplified by PCR with primers complementary to the conserved promoter and noncoding region of each gene segment ( Table 1).
The PCR mix contained 1 μL of cDNA, 1 μL of forward primer and reverse primer, 5 μL of 10× Taq buffer (TaKaRa Bio Group, Dalian, Japan), 4 μL of 2.5 mmol/L dNTPs (TaKaRa Bio Group), 1 μL of Ex Taq (TaKaRa Bio Group), and 37 μL of RNase-free water for a final volume of 50 μL. A single PCR program was used for all primers: initial denaturation at 95°C for 10 minutes; 30 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1.5 minutes; and a final extension at 72°C for 10 minutes. The PCR products were purified using a PCR purification kit (Tian Gen, Beijing, China) and sequenced on an Applied Biosystems DNA analyzer (ABI3500S; Applied Biosystems, Foster City, CA, USA).

| Genetic and phylogenetic analyses
Nucleotide sequences were edited using the SeqMan module of the DNAstar package, and phylogenetic analyses were performed with  Anseriformes; the remaining virus was from a pigeon ( Table 1). Among the 8 strains, one virus which named (AH/L139) was an internal gene supporter for the H10N8 subtypes, as previously reported. 11 In this study, we analyzed the other seven viruses in this study. The complete genome sequences of the seven H9N2 viruses generated in our study were submitted to the GenBank database(MG781063-MG781118).

| Phylogenetic analysis of genes encoding surface proteins
Phylogenetic analysis revealed that the HA genes of the seven H9 isolates could be separated into two geographically distinct lineages, Eurasian (group 1) and North American (group 2), based on a nucleic teal/Finland/10529/10(H9N2)) ( Figure 1A).
The 7 N2 NA genes exhibited greater diversity than the HA genes and clustered into 4 groups based on nucleic identity above

| Phylogenetic analysis of the internal genes
To better understand the evolution of the H9 AIVs isolated from wild birds in Anhui province, all six internal genes of the 7 novel genotypes identifies in this survey were phylogenetically analyzed as whole.
Indications of the reassortment of two geographical lineages were also present in the internal genes. All of the internal genes exhibited a Eurasian lineage, except the NS gene of AH/S93, AH/S102, AH/L258, and AH/L281, which belonged to the North American lineage. The internal genes exhibited diversity. Eight segments of seven viruses comprised six genotypes based on their identity values (>95%) ( Table 2).  (Figure 2A-F).  Figure 3B). Thus, all H9N2 viruses were showed different internal gene segments.

| Molecular characterization of viral genes
Adaptive mutations in viral proteins are important for AIVs to cross species barriers and infect mammalian hosts. 12 To determine whether the H9N2 viruses in this study had acquired genetic markers associated with mammalian pathogenicity, virulence, and adaptation to new hosts, we analyzed the whole-genome sequences The amino acid substitutions Q226L and G228S (H3 numbering, which is used throughout the manuscript) favor the affinity of influenza viruses for human-type receptors. 13,14 Necklace deletion in the NA gene confers enhanced virus lethality in mice. 15 The aforementioned characteristic changes were detected here: two viruses,  16 The seven H9 strains in this study all had no substitutions in PB2-A588V. The amino acid 292V/I is conserved in human and avian isolates 20 ; therefore, 292V in the PB2 protein might be another important residue for the mammalian adaptation of AIVs. 16 Here, two viruses, AH/BLH12 and AH/DGG4, had the 292V variant in the PB2 protein.
None of the seven H9 viruses had a Y436H substitution in the PB1 protein or T515A substitution in the PA protein, which suggested low pathogenicity to mammalian and avian hosts. An N66S substitution was found in the PB1-F2 protein of all the H9 strains in this study, which is associated with the increased virulence of the 1918 pandemic virus and the highly pathogenic AI H5N1 virus in mice and ferrets. 21,22 The mutations N30D and T215A in the M1 protein and some had P42S in the NS1 protein suggest the viruses would exhibit increased virulence in mammals. No amino acid substitutions were found in the M2 transmembrane domain, suggesting that this virus strain is sensitive to M2 ion channel inhibitors. 23 The S31N amino acid substitution in the M2 protein was not present, indicating that these viral strains were sensitive to amantadine inhibitors. 24 The virulence of influenza viruses in humans is related to their resistance to the antiviral effects of cytokines, such as interferon (IFN), and the D92E mutation in the NS1 protein increases resistance to IFN. 25 However, no mutations at residue 92 of NS1 were observed in this study.

| DISCUSS IONS
H9N2 AIV is widely distributed in different regions of China and occasionally jumps hosts and reassorts with other subtypes of influenza virus, posing a severe public health threat. 26 This AIV subtype has an impact that is significantly more severe than that observed in previous years not only because it is widespread but also because it is a donor of internal genes and undergoes extensive reassortment with different subtypes of avian AIVs, including HPAI H5N1 and H7N3. 27 Recently, H9N2 has contributed internal genes to H7N9 and H10N8 viruses that cause severe human respiratory infections. During surveillance, we isolated one strain (AH/L139) whose six internal genes had high identity with H10N8 and H7N9, suggesting that the role of H9N2 AIVs as donors of internal genes cannot be ignored. 11 The PB2-K627 residue is a mammalian marker that is usually present in highly pathogenic AIVs (HPAIVs) and appears in mammalian-adapted viruses. 28   We are also grateful to some other related laboratories for their technical support.