Serological assays
Serum was extracted from clotted blood samples and stored at -20°C.
Hemagglutination inhibition (HAI) assays were performed on each of the
6,532 serum samples collected, with a representative swine H3 influenza
virus strain: A/Swine/Hong Kong/4348/2016 (BD-like H3N2). HAI tests were
performed following WHO recommendations for animal influenza diagnosis
(World Health Organization, 2011). Serum samples were first treated
overnight at 37°C with receptor destroying enzyme (RDE) (Denka Seiken Co
Ltd., Tokyo, Japan) and were heat-inactivated for 30 min at 56°C next
morning. Serum samples were then serially diluted two-fold (1:10 to
1:1,280) in 96-well plates and 25 µL of virus antigen with four
hemagglutination units was added to an equal volume of each serum
dilution in duplicate. After 1h incubation, 50µL of 0.5% TRBCs was
added to each well and incubated for 30 min. The HAI titer was defined
as the highest serum dilution that inhibited complete hemagglutination.
For calculation of the geometric mean titer (GMT), serum samples with a
titer of <1:10 was assigned a value of 5 and those with
≥1:1,280 were assigned a value of 1:1,280.