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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.

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Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged.

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional proteins, including the 110-kDa protein. The 110-kDa protein was also identified as a glycoprotein by periodic acid-Schiff and silver staining. A single band precipitated against convalescent-phase pig antiserum when the polyethylene glycol precipitate was used in an Ouchterlony plate. Vaccination with CE conferred greater protection against challenge with the homologous serotype than either a commercial bacterin or an outer membrane protein vaccine. Hemolysin-neutralizing titers were higher both pre-and postchallenge in the group vaccinated with the CE compared with in all other groups. We believe that this demonstrates the importance of secreted factors in protection against disease and suggests that the 110-kDa protein is an important immunogen. Actinobacillus (Haemophilus) pleuropneumoniae is a 358