Statistical analysis
We fitted a logistic regression model to predict isolation of influenza A(H3N2) subtype, considering factors such as length of stay at slaughterhouse of the live pigs, transport distance from originating farms to Hong Kong, HAI titer against the reference H3N2 strain (A/swine/Hong Kong/4348/2016) of individual pigs, and H3 seroprevalence of the specific originating farms.
The H3 seroprevalence of each originating farm was calculated as the proportion of seropositive (HAI titer ≥1:40) samples in 2012-2016 and only those samples collected from major source farms (≥20 samples over the study period) were analysed (n = 5,832) (Figure 1). We stratified the individual anti-H3 HAI titer level into sero-negative (<1:40) and sero-positive (≥1:40).
The slaughtered pigs originated from more than 150 farms. While we had the location of each farm to the level of the Province, exact geo-spatial farm locations were not available. We estimated the transport distance based on the originating province and stratified data into 3 groups: a) nearest, from Shenzhen, Zhuhai and other cities of Guangdong Provinces (within 150km from Hong Kong approximately); b) more distant, from Guangxi, Hainan, Hunan and Jiangxi Provinces (approximately 500-700 km), and c) most distant, from Hubei, Hebei, Henan and Zhejiang Provinces (approximately >900 km). All factors were tested in the multivariable analysis. P value <0.05 was considered statistically significant. All statistical analyses were conducted using R version 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria).
We further estimated the force of infection (FOI) in farms and during transportation of swine respectively, based on the analysis of virological and serological results on H3-subtype SIVs in the subset where paired serum and swab samples were available. Prior to importation of live pigs to Hong Kong, pigs from distant provinces were gathered by local traders but without clear records. Therefore, the exact duration of transportation of individual pigs were not available. Hence, we performed the FOI analysis based on pigs from Guangdong only (n = 4,226, 65% of the total samples) (Figure 1) from which pigs were transported directly to Hong Kong from the farms, to minimise discrepancies between actual and assumed exposure duration.
Paired viral isolation and serological data were utilized to deduce the infection history of individual pigs. Previous studies had reported that pigs would begin shedding virus in nasal swabs by 1-3 days post-exposure and virus shedding would last for 4-5 days (Janke, 2013), whereas circulating antibodies become detectable 10-14 days after infection (Detmer, 2013). The time delay between virus shedding period and seroconversion allowed us to deduce where infections had taken place. Specifically, we defined the number of pigs which have experienced 4 possible infection histories: n1, naïve population (negative virological and serological test results); n2, recent primary infection (positive virological and negative serological test results); n3, previous infection (negative virological and positive serological test results); and n4, recent secondary or re-infection (positive virological and serological test results). We estimated the probability of infection during transport (pt) from the virus isolation rate at the abattoir among swine naïve to H3N2 prior to transportation i.e. sero-negative to H3 antigens, so pt= n2 / (n1+n2). The probability of infection in farm (pf) was estimated by the proportion of seropositive samples, i.e., pf = (n3+n4) / (n1+n2+n3+n4), assuming recently infected pigs have not yet seroconverted.
The force of infection (FOI), defined as the instantaneous rate at which a susceptible pig becomes infected, was used to quantify the transmission potential. The relationship between FOI and probability of infection p is given by:
\begin{equation} p=1-exp(-\int_{t_{1}}^{t_{2}}{\lambda\left(t\right)\text{dt}})\nonumber \\ \end{equation}
where t1, t2 are the start and end times of the exposure, λ(t) is the FOI from infectious pigs in farms or transport system. The probability of infection during transport depends on the duration of travelling time for imported pigs. We assumed the exposure duration related to transport (time before slaughtering, including transportation, inspection and holding) is 3 days. The ages of the pigs sampled were 28 to 36 weeks old (no individual data was recorded) and pigs in farms were assumed to have 4 to 8 weeks of protection by maternal antibodies (Markowska-Daniel, 2011). A previous study on maternally derived antibodies suggested that the maternally acquired immunity may only mask clinical illness of the pigs but not protecting them from infection (Loeffen, 2003). Therefore, we considered possible exposure durations of 20, 28 and 36 weeks for the estimation of FOIs in farm.