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.