Phylogenetic analysis and molecular epidemiology
Whole-genome sequencing and phylogenetic analysis were carried out for all 174 H3N2 viruses isolated from both nasal and tracheal swab samples during the study period. Together with 179 reference H3 SIV genome sequences retrieved from the existing databases (GenBank, GISAID), a maximum likelihood phylogenetic tree of 353 concatenated genomes of H3 SIVs was constructed. All tattoo codes indicating farm of origin and sampling dates of the H3 isolates were labelled in the tree (Figure 3). The 174 sequences of the H3N2 virus isolates can be grouped into 9 clusters, numbered 1-9, of closely related virus sublineages within the concatenated phylogenetic tree. Genetically similar viruses were detected in pigs from multiple farms on the same sampling occasion. For example, all H3N2 viruses collected on 3rd July 2014 formed a subcluster of taxa with closely related evolutionary history (subcluster 5). Interestingly, in subcluster 5, 18 nearly identical H3N2 viruses were isolated at the abattoir from pigs originating from 13 different farms in 3 different provinces (Guangdong, Hunan and Jiangxi). Similarly, in subcluster 4, there were 17 highly related H3N2 viruses isolated from pigs originated from 5 different farms in 2 provinces (Jiangxi and Guangdong) on one sampling date 7thJanuary 2016. A similar phenomenon was found in virus subclusters 6, 7 and 8. It was highly improbable that a genetically identical virus was simultaneously circulating in multiple farms and provinces at the same time period. Thus, the phylogeny was strongly suggestive of a virus from a common source which transmitted to swine from other farms during transportation or while being held within the abattoir prior to slaughter.
One of these farms GD101 had similar but not identical viruses isolated on 2nd July 2015 and 7th September 2016, suggesting that this virus sub-lineage was persisting within the farm for over two years.