Phylogenetic analysis and molecular epidemiology
Molecular epidemiology was used to define evidence of virus clustering and thus the occurrence of swine-to-swine transmission. Analysis was conducted on full genomes of all H3N2 swine influenza virus isolates sequenced in our study (Figure 1). The influenza virus sequences were analysed together with other existing Asian H3-subtype swine influenza virus sequences retrieved from the NCBI Influenza Virus Resource (Supplementary table 2) (Bao, 2008) and GISAID (Supplementary table 3) (Shu, 2017). All Asian H3 SIVs with a complete set of segments in full-length were included and identical sequences were collapsed. Each genome segment was aligned separately using MAFFT version 7 (Katoh, 2013) and alignments were then trimmed to coding regions. The resultant data sets contained a total of 353 full genome sequence sets.
The coding regions of gene segments PB2, PB1, PA, HA, NP, NA, MP and NS were concatenated using Geneious 2019.2.1 (http://www.geneious.com). Multiple alignment for the concatenated whole genomes was performed with MAFFT version 7. We inferred the maximum-likelihood phylogenetic trees using IQ-TREE (Nguyen, 2014) and the built-in ModelFinder (Kalyaanamoorthy, 2017) to determine the best-fit substitution model. Branch supports were obtained using approximate likelihood ratio test (aLRT) (Guindon, 2010) to assess the robustness of the phylogenetic tree. Branch support aLRT statistics were shown at major nodes with values larger or equal to 0.8.