Enhanced virulence of clade 2.3.2.1 highly pathogenic avian influenza A H5N1 viruses in ferrets

Abstract

Sporadic avian to human transmission of highly pathogenic avian influenza (HPAI) A(H5N1) viruses necessitates the analysis of currently circulating and evolving clades to assess their potential risk. Following the spread and sustained circulation of clade 2 viruses across multiple continents, numerous subclades and genotypes have been described. To better understand the pathogenesis associated with the continued diversification of clade 2A(H5N1) influenza viruses, we investigated the relative virulence of eleven human and poultry isolates collected from 2006 to 2013 by determining their ability to cause disease in the ferret model. Numerous clade 2 viruses, including a clade 2.2 avian isolate, a 2.2.2.1 human isolate, and two 2.2.1 human isolates, were found to be of low virulence in the ferret model, though lethality was detected following infection with one 2.2.1 human isolate. In contrast, three of six clade 2.3.2.1 avian isolates tested led to severe disease and death among infected ferrets. Clade 2.3.2.1b and 2.3.2.1c isolates, but not 2.3.2.1a isolates, were associated with ferret lethality. All A(H5N1) viruses replicated efficiently in the respiratory tract of ferrets regardless of their virulence and lethality. However, lethal isolates were characterized by systemic viral dissemination, including detection in the brain and enhanced histopathology in lung tissues. The finding of disparate virulence phenotypes between clade 2A(H5N1) viruses, notably differences between subclades of 2.3.2.1 viruses, suggests there are distinct molecular determinants present within the established subclades, the identification of which will assist in molecular-based surveillance and public health efforts against A(H5N1) viruses.

Keywords: Ferrets; Influenza H5N1; Pathogenesis.

Fig. 1. Mean viral titer in nasal washes of ferrets inoculated with HPAI A(H5N1) influenza viruses

Three ferrets per group were inoculated with 10⁶ or 10⁷ PFU or EID₅₀ of virus. Nasal washes were collected on alternate days p.i. Mean titers for each group are shown as log₁₀ EID₅₀/mL + standard deviation (SD) from three ferrets per group unless otherwise specified. Due to virus lethality, titers day 7 p.i. reflect one (BnSw/HK/10) or two (Dk/VN/672) ferrets only. The limit of detection was 1.5 log₁₀ EID₅₀/mL.

Fig. 2. Viral dissemination of A(H5N1) influenza viruses in ferrets

Three ferrets per group were inoculated with 10⁶ or 10⁷ PFU or EID₅₀ of virus and tissues were collected 3 days p.i. for virus titration. Mean titers for each group are shown as log₁₀ EID₅₀/g or mL + SD. Tissues below our limit of detection (1.5 log₁₀ EID₅₀/g or mL) were assigned a value of 1.5. ND, sample was not collected and not titered.

Fig. 3. Histopathologic evaluation of ferrets infected with A(H5N1) influenza viruses

Representative photomicrographs of hematoxylin and eosin [A, E, F] and immunohistochemically [B-D] stained lung sections from influenza virus-infected ferrets collected day 3 p.i. (A) Moderate bronchointerstitial pneumonia with predominant histiocytic alveolitis and mild bronchiolitis (arrow), Dk/VN/1206, bar =40 µm. (B) Viral antigen in type II pneumocyte, BnSw/HK/10, bar =20 µm. (C) Viral antigen in alveolar macrophages, BnSw/HK/10, bar =20 µm. (D) Viral antigen in bronchiolar epithelium, BD/5487, bar =35 µm. (E) Severe diffuse bronchointerstitial pneumonia with prominent necrotizing alveolitis and severe bronchiolitis, BnSw/HK/10, bar =80 µm. (F) Mild alveolitis with perivascular cuffing with mononuclear inflammatory cells, Dk/VN/1232, bar =40 µm.