New reassortant and enzootic European swine influenza viruses transmit efficiently through direct contact in the ferret model

Abstract

The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2, and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titres in nasal wash and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naïve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titres. Further, the viruses revealed preferential binding to the 2,6-α-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggested that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.

Fig. 1.. Replication kinetics in the upper respiratory tract. Groups of four ferrets were intranasally inoculated with 1 ml 10⁶ TCID₅₀ (0.5 ml per nostril) of the respective viruses. Each virus group contained eight ferrets: four donor ferrets (until 5 days p.i.), two DC ferrets and two AT ferrets. (a) H1avN2hu, (b) H1N2, (c) H3N2 and (d) H1pdmN2sw. Nasal washes were collected on days 1, 3, 5, 7, 9 and 11 p.i. Data are presented as virus titre for individual ferrets (log₁₀TCID₅₀ ml⁻¹) on the indicated day. Limit of detection was 10¹ TCID₅₀ ml⁻¹.

Fig. 2.. Seroconversion of ferrets. Ferrets were tested for seroconversion at 19 and 21 days p.i. using a blocking ELISA detecting antibodies against the NP gene. In the H3N2 AT group, one of the animals died before seroconversion and hence only one animal is above the baseline. Serum samples were tested in duplicate and values are presented as mean±sem per cent seropositive.

Fig. 3.. Comparison of European swine influenza virus titres recovered from ferret tissues. Ferrets were inoculated intranasally with 1 ml 10⁶ TCID₅₀ (0.5 ml per nostril) with H1N2, H1avN2hu, H3N2 or H1pdmN2sw and tissues were collected at day 5 p.i. Titres are expressed as log₁₀TCID₅₀ (g tissue)⁻¹. Each bar represents one ferret. Data are presented as virus titre (log₁₀TCID₅₀ g⁻¹) from the indicated tissue. Limit of detection was 10¹ TCID₅₀ g⁻¹.

Fig. 4.. Lung pathology in ferrets inoculated with one of the four European swine influenza viruses and euthanized 5 days p.i. Lung tissue from ferrets, haematoxylin/eosin; bar, 50 µm. (a) Normal lung tissue from control ferret. (b) Ferret inoculated with H3N2, 5 days p.i. Suppurative bronchiolitis, dysplasia of bronchiolar epithelium and hyperplasia of BALT (asterisk). (c) Ferret inoculated with H1pdmN2sw, 5 days p.i. Suppurative bronchiolitis, dysplasia and desquamation (arrowhead) of bronchiolar epithelium and BALT hyperplasia (asterisk). (d) Ferret inoculated with H1N2, 5 days p.i. Suppurative bronchiolitis and peribronchiolar infiltration of mononuclear cells. Dysplasia, necrosis and desquamation (arrowhead) of bronchiolar epithelium. (e) Ferret inoculated with H1avN2hu, 5 days p.i. Suppurative bronchiolitis, and dysplasia, necrosis and desquamation (arrowhead) of bronchiolar epithelium.

Fig. 5.. Replication kinetics of European swine influenza viruses in different cell lines. Growth curves were obtained by inoculating cells at m.o.i. 0.01 p.f.u. per cell with H1N2, H1avN2hu, H1pdmN2sw or H3N2. Supernatant was harvested and titrated in MDCK cells at 8, 10, 12, 18, 20, 24, 36, 48 and 60 h p.i. Data are expressed as mean±sem log₁₀TCID₅₀ from two independent experiments titrated in quadruplicate.

Fig. 6.. Receptor specificity of four European swine influenza viruses. The receptor-binding specificities of the four European swine influenza viruses H1N2, H1avN2hu, H1pdmN2sw and H3N2 were tested in a dose-dependent glycan array assay against the sialyl glycans 2,6-α-SL, 2,6-α-SLN and 2,3-α-SL.