Efficient isolation of Swine influenza viruses by age-targeted specimen collection

Abstract

The control of swine influenza virus (SIV) infection is paramount for increasing the productivity of pig farming and minimizing the threat of pandemic outbreaks. Thus, SIV surveillance should be conducted by region and on a regular basis. Here, we established a microneutralization assay specific for SIV seroprevalence surveillance by using reporter gene-expressing recombinant influenza viruses. Growth-based SIV seroprevalence revealed that most sows and piglets were positive for neutralizing antibodies against influenza viruses. In contrast, the 90-day-old growing pigs exhibited limited neutralizing activity in their sera, suggesting that this particular age of population is most susceptible to SIV infection and thus is an ideal age group for SIV isolation. From nasal swab specimens of healthy pigs in this age population, we were able to isolate SIVs at a higher incidence (5.3%) than those of previous reports. Nucleotide sequencing and phylogenetic analysis of the hemagglutinin (HA) genes revealed that the isolated SIVs have circulated and evolved in pigs but not have been recently introduced from humans, implying that a large number of SIV lineages may remain "undiscovered" in the global porcine populations. We propose that the 90-day-old growing pig-targeted nasal swab collection presented in this study facilitates global SIV surveillance and contributes to the detection and control of SIV infection.

FIG 1

Neutralizing activities in swine sera against influenza viruses. Sera of pigs from various stages of growth (Table 1) were collected in July (A and B) and November (C and D) 2012 on farm A. The neutralizing activities of the sera against GFP-expressing PB2KO viruses of H1 (A and C) or H3 and H4 (B and D) subtypes were determined by microneutralization assays in AX4/PB2 cells as described previously (13). Yellow and gray regions highlight the data from 90-day-old growing pigs and sows, respectively. H1/ref, A/duck/Alberta/35/76(H1N1); H1/vac, A/swine/Kyoto/3/79(H1N1); H1/iso, A/swine/Mie/R01/2012(H1N2); H1/pdm, A/California/04/09(H1N1); H3/ref, A/duck/Ukraine/1/1963(H3N8); H3/vac, A/swine/Wadayama/3/69(H3N2); H4/ref, A/duck/Czechoslovakia/1956(H4N6).

FIG 2

Nasal swab collection and SIV isolation. Nasal swab specimens were collected from pigs ranging in age from 90 to 120 days on farms B to K (A) in May (top graph) and August to September (bottom graph) 2013 and in farm L (B) in August to November 2013 and subjected to virus isolation in embryonated chicken eggs.

FIG 3

Phylogenetic tree of the HA gene of H3 viruses. The nucleotide sequence of the HA genes from the H3 subtype influenza virus isolated in this study [A/swine/Japan/KU-MD4/2013(H3N2) (MD4 virus), indicated by the black arrow] was phylogenetically analyzed with counterparts from the representative swine (●), avian (△), and human (▩) viruses by using the maximum likelihood method with a bootstrapping set of 100 replicates. The licensed H3 SIV vaccine strain A/swine/Wadayama/3/69(H3N2) is indicated by the white arrow. The scale bar indicates the number of nucleotide substitutions per site.

FIG 4

Phylogenetic tree of the HA gene of H1 viruses. The nucleotide sequences of the HA genes from the H1 subtype influenza viruses isolated in this study [A/swine/Japan/KU-HY5/2013(H3N2) and A/swine/Japan/KU-YG5/2013(H3N2), indicated by the black arrows] were phylogenetically analyzed with counterparts from the representative swine (●), avian (△), and human (▩) viruses by using the maximum likelihood method with a bootstrapping set of 100 replicates. The licensed H1 SIV vaccine strain A/swine/Kyoto/3/79 (H1N1) is indicated by the white arrow. The scale bar indicates the number of nucleotide substitutions per site.