Pathogenesis and transmission of swine origin A(H3N2)v influenza viruses in ferrets

Abstract

Recent isolation of a novel swine-origin influenza A H3N2 variant virus [A(H3N2)v] from humans in the United States has raised concern over the pandemic potential of these viruses. Here, we analyzed the virulence, transmissibility, and receptor-binding preference of four A(H3N2)v influenza viruses isolated from humans in 2009, 2010, and 2011. High titers of infectious virus were detected in nasal turbinates and nasal wash samples of A(H3N2)v-inoculated ferrets. All four A(H3N2)v viruses possessed the capacity to spread efficiently between cohoused ferrets, and the 2010 and 2011 A(H3N2)v isolates transmitted efficiently to naïve ferrets by respiratory droplets. A dose-dependent glycan array analysis of A(H3N2)v showed a predominant binding to α2-6-sialylated glycans, similar to human-adapted influenza A viruses. We further tested the viral replication efficiency of A(H3N2)v viruses in a relevant cell line, Calu-3, derived from human bronchial epithelium. The A(H3N2)v viruses replicated in Calu-3 cells to significantly higher titers compared with five common seasonal H3N2 influenza viruses. These findings suggest that A(H3N2)v viruses have the capacity for efficient replication and transmission in mammals and underscore the need for continued public health surveillance.

Fig. 1.

Comparison of A(H3N2)v influenza virus titers recovered from ferret tissues. Three ferrets were inoculated by the i.n. route with 10⁶ pfu of IN/11 virus (A), MN/10 virus (B), PA/10 virus (C), or KS/09 virus (D), and tissues were collected 3 dpi. Titers are expressed as log₁₀ pfu per gram of tissue; the limit of detection was 1.0 log₁₀ pfu/g or mL. Nasal turbinate virus titers are expressed as log₁₀ pfu/mL Each bar represents one ferret.

Fig. 2.

Transmissibility of A(H3N2)v influenza viruses among ferrets in the RD model. Three ferrets each were inoculated i.n. with 10⁶ pfu of IN/11 virus (A), MN/10 virus (B), PA/10 virus (C), or KS/09 virus (D); a naïve ferret was placed in an adjacent cage at 24 h after inoculation to initiate contact. Nasal washes were collected from inoculated (dark bars) and contact (light bars) ferrets, and titers are expressed as log₁₀ pfu/mL. Values for individual ferrets are shown.

Fig. 3.

Binding preference of A(H3N2)v influenza viruses. Dose-dependent direct glycan array binding of MN/10 (A), KS/09 (B), and PA/10 (C) viruses. 3′SLN, 3′SLN-LN, and 3′SLN-LN-LN are representative avian receptors, and 6′SLN and 6′SLN-LN are representative human receptors. The binding signals were determined based on the HRP activity by using the Amplex Red Peroxidase Assay (Invitrogen) according to the manufacturer's instructions. The assays were done in triplicate, and appropriate negative controls were included.

Fig. 4.

Replication kinetics of A(H3N2)v influenza viruses in polarized human airway epithelial cells. The seasonal H3N2 viruses are abbreviated as follows: WI/05, A/Wisconsin/67/2005; PE/10, A/Perth/16/2009; RI/10, A/Rhode Island/01/2010; WI/10, A/Wisconsin/13/2010; and HK/09, A/Hong Kong/34341/2009. Calu-3 cells cultured for 1 wk were inoculated with virus at an MOI of 0.01. Culture supernatants were collected at the times indicated, and virus titers were determined by standard plaque assay. The titer value represents the average for three independent wells. Asterisks indicate statistically significant difference between seasonal H3N2 viruses and four A(H3N2)v viruses at 16, 24, 48, and 72 h, based on Mann–Whitney test (each was P < 0.004).