Influenza A Virus, H9N2 Subtype

Using standard reverse genetics an H9N2 virus of the G1 lineage, A/chicken/Pakistan/UDL-01/2008 (UDL1/08), was rescued from bidirectional reverse genetics plasmids as previously described47 (link). All other H9N2, H5N1 or H7N1 viruses (including stocks of UDL1/08 and its escape mutants used for downstream antigenic analysis) used were rescued using the HA and NA of the named viral strain with the remaining gene segments from A/Puerto Rico/8/1934 (PR8). Multiple, independently rescued virus stocks of UDL1/08 were propagated in 10 day old specific pathogen free (SPF) embryonated chicken eggs and titrated by plaque assay or TCID₅₀ on MDCKs. To guarantee the genetic purity of all the escape mutants used for downstream antigenic analysis identified amino acid substitution in HA were reintroduced into the reverse genetics plasmids by site directed mutagenesis and mutants were reconstituted through virus rescue as H9N2:PR8 2:6 recombinants. Full length sequencing (see below) of the HA and NA gene segments of all reconstituted escape mutants was performed to verify no additional mutations or reversions appeared after rescue of the reconstituted escape mutants. Viruses were purified by ultracentrifugation through a continuous 30–60% w/v sucrose gradient.
MDCK cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37^oC, 5% CO₂. Human embryonic kidney 293T (293T) cells were maintained in DMEM containing 2% FBS and 1 mM sodium pyruvate at 37^oC, 5% CO₂.

Nucleotide sequences used in our analyses may be obtained from GISAID (www.gisaid.org), Genbank (www.ncbi.nlm.nih.gov/genomes/FLU), and the WHO website (www.who.int/influenza/gisrs_laboratory/h5n1_nomenclature). GISAID acknowledgement tables for laboratory contributions for both H5N1 and H9N2 hemagglutinins can be found in Supplemental Files S1 and S2. The Supplemental Files also contain a listing of virus strain names, accession numbers, data sources, as well as known annotations versus LABEL predicted annotations for all data used in this study. H5N1 clade annotations were obtained courtesy of WHO/OIE/FAO H5N1 Evolution Working Group members [5] . Thanks to advice from working group members and insights gained from our analyses, a few annotations were corrected or updated with respect to the published WHO/OIE/FAO tree datasets, see Supplementary File S1. We excluded laboratory-derived viruses and sequences shorter than 1200 base-pairs. This threshold was more inclusive than the 1,600 nucleotide cutoff used in [5] but still larger than a typical mature HA1 segment (∼960 nts). Collectively, highly pathogenic H5N1 influenza A viruses that share common ancestry with the first isolate (A/goose/Guangdong/1/96) are known as Gs/GD-like while non-Gs/GD viruses include Eurasian and North American low pathogenicity H5 isolates. Multiple sequence entries (i.e., duplicate virus names) and HA sequences with 100% sequence identity were removed using a custom Perl script. For H5 annotation, 2506 sequences were used to create the profile HMMs with 586 of these being further used to train the SVMs. A simplified example of pHMM and SVM training data is shown diagrammatically in Supplemental Fig. S1, steps 2 & 3. SVM training sequences were then removed to test the SVMs. In order to test both the SVMs and pHMMs together, 373 newly submitted GISAID H5 hemagglutinin sequences (1 Feb 2011 to 1 Apr 2012) that satisfied our criteria and were not redundant with any training data were tested. In order to account for H5 lineages outside of the WHO nomenclature studies we used 524 non-Gs/GD lineage H5 HA to create profile HMMs, with 59 being used to train SVMs. We again removed SVM training sequences to test non-Gs/GD H5s, leaving 465 sequences. For H9 annotation, 1592 sequences were used to create HMM profiles, with 342 of them used for SVMs. These were removed to test the SVMs. Most H9 hemagglutinin sequences were from the H9N2 subtype viruses, although H9s paired with other neuraminidase subtypes were also included. For the analysis of partial sequences (fragments) of both H5 and H9 hemagglutinins, shortened sequences were removed if they were more than 5% shorter than the alignment length (ensuring coverage of the region of interest) or if they were redundant with existing sequences in the set.

Mouse myeloma SP2/0 cells and hybridoma cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 15% foetal bovine serum (FBS, Gibco), hypoxanthine and thymidine (HT, Sigma-Aldrich) at 37°C in 5% CO₂. Madin-Darby canine kidney (MDCK) cells and COS-1 cells were maintained in DMEM supplemented with 10% FBS at 37°C in 5% CO₂. All field strains of H9N2 viruses were isolated from poultry in China and grown in 9-day-old embryonated SPF chicken eggs. Allantoic fluid of each virus was harvested at 120 h post-inoculation and stored at −70°C. The pCAGGS plasmid, containing NA gene sequence of a wild-type (WT) A/Chicken/Jiangsu/XXM/1999 (XXM) H9N2 virus, was constructed, as previously reported [23 (link)].

The H9N2 viruses that we isolated in the live-poultry market were distributed in three independent branches; therefore, we performed a spatiotemporal analysis of clades A, B, and C, respectively. In order to reduce the potential sampling biases, we randomly subsampled the database in a stratified manner to create a more equitable spatio-temporal distribution of the HA genome sequences of three branches of viruses. To be precise, sequences in each branch were clustered using the CD-HIT program (Huang et al., 2010 (link)), and identical sequences within the same time and region were removed. The discrete sampling locations of the clade A, B, and C viruses in this study include Guangdong, Yunnan, Jiangxi, Shandong, Shanghai, Fujian, Jiangsu, Hunan, Henan, Hebei, Hubei, Xinjiang, Ningxia, Chongqing, Guizhou, Guangxi, Sichuan, Shanxi, Beijing, Tianjin, Heilongjiang, and Anhui in China, there are also viruses from Vietnam in clade C. Detailed information regarding the subsampled HA gene sequences of the clade A, B, and C H9N2 subtype viruses used in this study is provide in the Supplementary Table 1.
Time-measured phylogenies were inferred using the Bayesian discrete phylogeographic approach implemented in the BEAST package (v1.10.4). We first performed a regression of root-to-tip genetic distances on the ML tree against exact sampling dates using the TempEst v1.5.3 (Rambaut et al., 2016 (link)), which showed a strong temporal signal. Then, we used an uncorrelated lognormal (UCLN) relaxed molecular clock model. In addition, a Bayesian stochastic search variable selection (BSSVS) model with asymmetric substitution was used. For each independent dataset, multiple runs of the MCMC method were combined using LogCombiner (v1.10.4), utilizing 5,000,000,000 total steps for each set, with sampling every 500,000 steps. Subsequently, we used SpreaD3 v0.9.7.1 to develop interactive visualizations of the dispersal process through time and to compute a Bayes factors (BFs) test to assess the support for significant individual transitions between distinct geographic locations (Bielejec et al., 2016 (link)). The BF values >100 indicated robust statistical support, 30 < BF values ≤100 indicated very strong statistical support, 10 < BF values ≤30 indicated strong statistical support, 3 < BF values ≤10 indicated substantial statistical support and BF values <3 indicated poor statistical support (Lemey et al., 2009 (link)). We used QGIS Version 3.28 to create plots showing the results of the BF tests.⁵

Human NP swab samples were transported on wet ice to the Virology Section Lab of NHL, and bird OP, cage, and bioaerosol samples were transported on wet ice to the Yangon Veterinary Diagnostic laboratory at LBVD where they were preserved at −80°C until ready for RNA extraction and molecular study. NHL is one of the WHO‐recognized National Influenza Centers and a member of the Global Influenza Surveillance Network. RNA extraction was performed using the QIAamp Viral RNA Mini Kit (Qiagen) per the manufacturer's instructions. RNA extracts were screened for conserved matrix genes from IAV and IDV using previously published rRT‐PCR assays.⁸, ⁹ Human NP specimens with molecular evidence of IAV were hemagglutinin‐subtyped for H1pdm and H3 using rRT‐PCR (primer and probe sequences available in Table S1). Following this, all IAV‐positive specimens were further studied at St. Jude Children's Research Hospital by culture in embryonated eggs for virus isolation. Extracted RNA from positive samples was subjected to cDNA synthesis using the Superscript IV system (Life Technologies). All eight gene segments were amplified using Phusion high‐fidelity DNA polymerase and universal conserved Uni12/13 primers for IAVs. Amplicons were purified using the GFX PCR DNA and Gel Band Purification Kit (Cytiva, UK). DNA libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina) and subsequently sequenced using an Illumina MiSeq personal genome system. The full genome of A/H9N2 viruses was assembled using CLC Genomics Workbench, version 20 (CLC Bio, Qiagen, Hilden, Germany). A/H9N2 isolates were analyzed by a hemagglutination inhibition (HI) assay against reference antisera raised against A/chicken/Hong Kong/G9/97 (H9N2), A/Hong Kong/308/2014 (H9N2), and A/Anhui‐Lujiang/39/2018 (H9N2) viruses. HI assays were performed per standard protocol with 0.5% turkey red blood cells.¹⁰

Publicly available sequences of the A/H9N2 viruses from Myanmar and reference A/H9N2 strains were downloaded from the Global Initiative on Sharing All Influenza Data (GISAID; accession numbers are available in Table S1).¹¹ BLASTN homology analysis of each segment was performed on the GISAID website, and globally related sequences were downloaded. The evolutionary history of the sequences was inferred by using the Maximum Likelihood method and Kimura 2‐parameter model,¹² which were conducted in MEGA X.¹³ Pairwise sequence analyses were performed with Jalview¹⁴; reference sequences included A/H9N2 sequences collected in Myanmar in 2015, HI anti‐serum strains and other related sequences from the region.