Antigenic Categorization of Contemporary H3N2 Swine Influenza Virus Isolates Using a High-Throughput Serum Neutralization Assay

Abstract

Abstract. In vivo, neutralizing antibodies are critical for viral clearance. A high-throughput serum neutralization (HTSN) assay was developed to antigenically categorize Swine influenza virus (SIV) isolates. Uncategorized viruses were tested using a panel of antisera representing the H3N2 SIV subtypes and the results expressed as a serum neutralization ratio. Antisera were generated against contemporary isolates representing circulating H3N2 SIV subtypes (clusters I, III, IV). Reference viruses and the corresponding antisera were evaluated using traditional hemagglutination inhibition (HI) and the HTSN assays and good correlation (r 5 0.84) was observed between the 2 tests. Categorical clustering of 40 recent (2008-2009) SIV isolates was assessed using the HTSN assay. The H3N2 SIV isolates with amino acid similarity .97% to the commonly used H3N2 cluster IV reference strain A/Swine/Ontario/33853/2005 (ON05) showed strong reactivity with cluster IV antisera. Isolates with ,97% amino acid similarity to ON05 sporadically or completely failed to react with any antiserum. A cluster of 3 isolates with weak reaction with cluster III antiserum may be a potential emerging cluster of H3N2 with moderate genetic similarity to cluster II H3N2 (93% similarity). Potential uses of the HTSN assay include identification of broadly cross-reactive or antigenically distinct SIV isolates for use in vaccine virus selection or as part of surveillance efforts monitoring antigenic drift.

Antigenic Characterization of H3N2 Influenza A Viruses from Ohio Agricultural Fairs

“…One previous study suggested that the protein sequence identity could be used to differentiate antigenic clusters of H3N2 IAVs infecting swine (22). However, our results indicated that the rate of antigenic evolution among H3N2 swine-origin IAVs could be independent of the rate of genetic evolution and that the protein sequence similarity alone does not predict IAV antigenic relationships.…”

“…Phylogenetic analyses of HA genes from H3N2 IAVs isolated from North American pigs after 1998 demonstrate that these trH3N2 IAVs can be classified into at least four genetic groups (clusters I, II, III, and IV) (21) with one lineage (cluster IV) predominating since 2005. Neutralization assays with swine antisera demonstrated that these four genetic clusters are also antigenically distinct (22). Several reassortment events between trH3N2 IAVs, human-origin IAVs, and classical swine influenza virus have occurred since 1998, resulting in multiple gene constellations with various HA and NA genes in combination with the triple reassortant internal gene (TRIG) cassette (which in-cludes PB2, PB1, PA, NP, NS, and M segments) (23,24).…”

Multiple Reassortment between Pandemic (H1N1) 2009 and Endemic Influenza Viruses in Pigs, United States

“…Swab samples were added to MDCK or swine testicle (ST) cells (American Type Culture Collection) as described ( 27 , 28 ). Virus isolates were identified by HA assay, as described in the World Health Organization manual on animal influenza diagnosis and surveillance ( 28 ).…”

Migration of the Swine Influenza Virus δ-Cluster Hemagglutinin N-Linked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

“…The HTSN assay was performed as previously described (11,12). Briefly, antisera were diluted 1:800 in DMEM, combined with 1,000 median TCID 50 of virus, and incubated at 37°C for 1 h. The virus-antiserum mixture was next transferred to a confluent monolayer of ST cells and incubated for 4 days at 37°C.…”