Rde 2

The haemagglutination-inhibition (HAI) assay at Day-1 and 29 (VisMederi srl, Siena, Italy) for all cohorts. Serum was pre-treated with receptor-destroying enzyme (RDE) II (Denka Seiken, Japan) and twofold serially diluted starting from a dilution of 1:10 to 1:2,560 in physiological saline. Virus (A/H1N1/California/04/2009, NIBSC) or (A/Victoria/361/2011) was added to each well at 4 haemagglutination units (HAU)/50 μl for 1 h at room temperature, followed by 0.35% turkey red blood cells for 1 h at room temperature. Reference sheep hyperimmune antisera were provided by NIBSC.
For real-time RT-PCR (rRT-PCR), the WHO protocol “CDC protocol of real-time RT-PCR for swine influenza A (H1N1)” was followed by VisMederi srl using but only the InfA primer designed for the universal detection influenza A viruses. A specimen was considered positive for influenza A if cycle threshold (Ct) values were within Ct values of 40. A virtual quantification tool was applied to the Ct values to allow the conversion of CDC rRT-PCR Ct values to virus RNA copy number, in the absence of a standard curve run in parallel to the samples (Piralla et al., 2013 (link)).

Sera (n = 6) collected from wild boars (Sus scrofa leucomystax) in Japan were used to estimate reaction specificity of HA-ELISA; four of them were antibody positive to H1N1 but negative to H3N2 and H5N1 viruses using our previous VN test, while the others were negative to these three subtypes [13 (link)]. In addition, three sera collected from apparently healthy feral racoons (Procyon lotor) captured in Japan, which were antibody positive to H5N1 (n = 1) [14 (link)] or to H9N2 (n = 2) (our unpublished data), respectively, by VN test, were used for multi-HA-ELISA. Prior to the test, all sera were treated with a receptor destroying enzyme (RDEII; Denka Seiken, Tokyo, Japan) for 16 h at 37 °C followed by inactivation for 30 min at 56 °C. The sera were further treated with kaolin and with MDCK cell pack (1 × 10⁵ cells/μL) for 16 h at 4 °C to decrease non-specific reactions.

HAI antibody titers were measured using 0.5% chicken erythrocytes and 4 HA units of 2 recombinant influenza viruses. Prior to HAI testing, the serum specimens were treated with receptor-destroying enzyme II (RDE II, Denka Seiken Co., Ltd., Tokyo, Japan) at 37 °C overnight followed by 56 °C for 30 min to remove nonspecific hemagglutinin inhibitors and natural serum agglutinins. The HAI assays were finally performed in 96-well microtiter plates according to the manual for GB/T-27535-2011 of the state standard of the People’s Republic of China.
Virus neutralizing activity were determined using a modified neutralization assay from a previously described procedure [37 (link)]. In brief, serum samples were heat-inactivated at 56 °C for 30 min, 2-fold serial diluted and incubated with 100 TCID50/well of swine influenza virus in 96-well microplates. The mixture was incubated at 37 °C for 1 h, the serum-swine influenza virus mixture was then transferred to 96-well microplates containing MDCK cells and incubated for 72 h. Anti-swine influenza VN antibodies were measured using a standard cytopathic (CPE) inhibition, neutralization titers are expressed as the reciprocal value of the highest dilution of the serum which inhibited the production of CPE.

Virus neutralization assays were conducted by using the methodology described in the WHO manual on Animal Influenza Diagnosis and Surveillance (http://www.wpro.who.int/emerging_diseases/documents/docs/manualonanimalaidiagnosisandsurveillance.pdf) with some modifications. Briefly, plasma samples were initially treated with a receptor-destroying enzyme (RDE II, DENKA SEIKEN Co., LTD, Tokyo, Japan) to remove non-specific inhibitors. Two-fold serially diluted plasma samples were mixed with 100 TCID₅₀ of each isolate. The mixtures were incubated for 30 min at 37 °C and were then inoculated into an MDCK monolayer in a 96-well microplate. Cytopathic effect was observed to detect the presence of viral propagation 3 days after inoculation. The reciprocal number of the minimum dilution of plasma needed to suppress the appearance of CPE was used as the neutralization titer.

Hemagglutination (HA)-inhibition (HI) test was performed to detect the anti-influenza D virus antibody in the bovine serum or plasma samples, according to the World Health Organization manual on animal influenza diagnosis and surveillance [13 ]. The samples were treated with receptor-destroying enzyme (RDEII; Denka Seiken, Japan) at 37°C for 16 h, followed by heat-inactivation at 56°C for 30 min. Serially diluted samples were then reacted to the D/OK virus (4 HAU) for 30 min at room temperature, followed by incubation with a 0.5% suspension of turkey red blood cells for 30 min at room temperature before reading the result. The HI titer of each sample was expressed as the reciprocal of the highest sample dilution that completely inhibited HA. The samples showing an HI titer ≥40 were considered as positive. An HI titer of 40 has been commonly used as the threshold for a seropositive result in influenza D virus surveillance [3 (link), 8 (link), 9 (link)]. This threshold authenticates reaction specificity in HI test [14 ].

Ferret sera were treated with receptor‐destroying enzyme (RDE II; Denka Seiken Co., Ltd) at 37°C for 20 hours, followed by RDE inactivation at 56°C for 30‐60 minutes. The treated sera were serially diluted twofold with PBS in 96‐well U‐bottom microtiter plates and mixed with the amount of virus equivalent to four hemagglutination units, followed by incubation at room temperature (25°C) for 60 minutes. After addition of 50 µL of 0.5% chicken red blood cells, the mixtures were gently mixed and incubated at 4°C for a further 45 minutes. HI titers are expressed as the inverse of the highest antibody dilution that inhibited hemagglutination.