Pathogenic and antigenic properties of phylogenetically distinct reassortant H3N2 swine influenza viruses cocirculating in the United States

Abstract

Swine influenza is an acute respiratory disease caused by type A influenza viruses. Before 1998, swine influenza virus isolates in the United States were mainly of the classical H1N1 lineage. Since then, phylogenetically distinct reassortant H3N2 viruses have been identified as respiratory pathogens in pigs on U.S. farms. The H3N2 viruses presently circulating in the U.S. swine population are triple reassortants containing avian-like (PA and PB2), swine-like (M, NP, and NS), and human-like (HA, NA, and PB1) gene segments. Recent sequence data show that the triple reassortants have acquired at least three distinct H3 molecules from human influenza viruses and thus form three distinct phylogenetic clusters (I to III). In this study we analyzed the antigenic and pathogenic properties of viruses belonging to each of these clusters. Hemagglutination inhibition and neutralization assays that used hyperimmune sera obtained from caesarian-derived, colostrum-deprived pigs revealed that H3N2 cluster I and cluster III viruses share common epitopes, whereas a cluster II virus showed only limited cross-reactivity. H3N2 viruses from each of the three clusters were able to induce clinical signs of disease and associated lesions upon intratracheal inoculation into seronegative pigs. There were, however, differences in the severity of lesions between individual strains even within one antigenic cluster. A correlation between the severity of disease and pig age was observed. These data highlight the increased diversity of swine influenza viruses in the United States and would indicate that surveillance should be intensified to determine the most suitable vaccine components.

FIG. 1.

Infection of 4-week-old pigs with H3N2 cluster I, II, and III swine influenza viruses. (A) Mean percentage of lung surface with macroscopic lesions on day 5 p.i. (B) Microscopic lesions in the medium bronchioles on day 5 p.i. (C) Virus titers in BALF on day 5 p.i. TCID₅₀, 50% tissue culture infective dose. n.d., not done.

FIG. 2.

Medium-sized bronchiole from a 4-week-old pig 5 days p.i. with Sw/TX/98 virus (cluster I). The airway is lined irregularly with immature cuboidal epithelial cells. The lumen contains sloughed necrotic epithelial cells and a few neutrophils. A loose cuff of infiltrating lymphocytes surrounds the bronchiole (magnification, ×149).

FIG. 3.

Lung from a 4-week-old pig 3 days p.i. with Sw/TX/98 virus (cluster I). Immunohistochemical staining techniques identify swine influenza virus antigen in epithelial cells in the bronchioles and in scattered pneumocytes in the alveoli. Light lymphocyte infiltration around infected airways is evident (magnification, ×149).

FIG. 4.

Medium-sized bronchiole from a 4-week-old pig 5 days p.i. with Sw/CO/99 virus (cluster II). The bronchiole is almost completely denuded with the remaining epithelial cells attenuated and irregularly distributed. Low numbers of infiltrating lymphocytes are around the bronchiole and are mixed with neutrophils in adjacent partially collapsed alveoli (magnification, ×149).

FIG. 5.

Medium-sized bronchiole from 4-week-old pigs 5 days p.i. with Sw/OK/99 virus (cluster III). (A) Necrosis and sloughing of epithelial cells have resulted in prominent attenuation of the remaining epithelial cell layer. The lumen contains proteinaceous debris, sloughed necrotic epithelial cells, and a few leukocytes. Lymphocytes surround the airway (magnification, ×149). (B) Higher magnification of a bronchiole. Epithelial layer is severely disrupted by necrosis and sloughing of cells into airway lumen, which contains cellular debris, proteinaceous fluid, and a few leukocytes. At this stage of infection, much of the acute necrosis has already occurred, and attenuation and proliferation of the remaining epithelial cells and underlying connective tissue predominate (magnification, ×204).

FIG. 6.

Infection of 12-week-old pigs with H3N2 cluster I, II, and III swine influenza viruses. (A) Mean percentage of lung surface with macroscopic lesions on day 5 p.i. (B) Microscopic lesions in the medium bronchioles on day 5 p.i. (C) Virus titers in BALF on day 5 p.i. TCID₅₀, 50% tissue culture infective dose.