Efficient Isolation of Swine Influenza Viruses by Age-Targeted Specimen Collection

Abstract

hThe 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.

Serological survey of influenza A virus infection in Japanese wild boars (Sus scrofa leucomystax)

“…To detect antibodies against swine IAVs in Japanese wild boars, we examined the subtype‐specific seroprevalence of anti‐influenza antibodies in collected serum samples using a virus‐neutralization test. The following swine IAVs were used for the neutralization test: strain KU‐MI2‐3 possessing hemagglutinin (HA) and neuraminidase (NA) genes originating from the pandemic A (H1N1) 2009 (H1N1pdm09) virus; strain KU‐FK3 possessing HA and NA genes originating from the classical swine H1N2 virus; and strain KU‐MD4 possessing HA and NA genes originating from the human seasonal H3N2 virus . Twofold serum dilutions were tested starting from a dilution of 1:20.…”

“…Virus-neutralization tests were conducted using recently isolated swine influenza viruses of H1 and H3 subtypes and highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype in Japan, specifically A/swine/ Chiba/KU-MI2-3/2016 (H1N1, KU-MI2-3), A/swine/ Chiba/KU-FK3/2016 (H1N2, KU-FK3), A/swine/Japan/ KU-MD4/2013 (H3N2, KU-MD4) and HPAIV strains, A/duck/Kagoshima/KU-116/2015 (H5N8, KU-116), and A/crane/Kagoshima/KU-4/2016 (H5N6, KU-4). [4][5][6][7] In brief, a mixture of twofold dilution of serum and 200 TCID 50 (median tissue culture infectious dose) of the viruses in Dulbecco's Modified Eagle's Medium (DMEM) was allowed to stand for 2 hr. The mixture was then inoculated in MDCK cells seeded into 48 well tissue culture plates.…”

“…The following swine IAVs were used for the neutralization test: strain KU-MI2-3 possessing hemagglutinin (HA) and neuraminidase (NA) genes originating from the pandemic A (H1N1) 2009 (H1N1pdm09) virus; strain KU-FK3 possessing HA and NA genes originating from the classical swine H1N2 virus; and strain KU-MD4 possessing HA and NA genes originating from the human seasonal H3N2 virus. 4,6 Twofold serum dilutions were tested starting from a dilution of 1:20. As presented in Table 1, 15 serum samples (25.4%) were positive for the H1N1 strain KU-MI2-3 with virus-neutralizing antibody titers ranging from 20 to 2560.…”

Host Factors Affecting Generation of Immunity Against Porcine Epidemic Diarrhea Virus in Pregnant and Lactating Swine and Passive Protection of Neonates

“…This corresponded to higher RVC IgG and IgA antibody titers in milk but no differences were observed in serum [159]. In another study, multiparous sows had greater neutralizing activity in serum against swine influenza viruses than primiparous gilts but milk antibodies were not measured [160]. It is likely that for enteric viruses like RV and PEDV, locally produced IgA ASCs and antibodies secreted in the gut traffic to the MG via the gut-MG-sIgA axis and contribute to piglet protection against clinical disease [1][2][3].…”

Distribution of gene segments of the pandemic A(H1N1) 2009 virus lineage in pig populations

“…It is, therefore, necessary to isolate and characterize SIVs circulating in each region in a timely manner to understand the global status of SIVs. In Japan, SIVs belonging to the A(H1N1)pdm09, classical swine H1N2 virus and human seasonal virus‐derived H3N2 virus lineages have been isolated from 2009 to 2013 (Matsuu et al., ; Ozawa et al., ). Here, we genetically characterized 24 SIV strains isolated in Japan together with recent global SIV and human isolates.…”