Avian Influenza H5N6 Viruses Exhibit Differing Pathogenicities and Transmissibilities in Mammals

Abstract

Since 2013, highly pathogenic avian influenza H5N6 viruses have emerged in poultry and caused sporadic infections in humans, increasing global concerns regarding their potential as human pandemic threats. Here, we characterized the receptor-binding specificities, pathogenicities and transmissibilities of three H5N6 viruses isolated from poultry in China. The surface genes hemagglutinin (HA) and neuraminidase (NA) were closely related to the human-originating strain A/Changsha/1/2014 (H5N6). Phylogenetic analyses showed that the HA genes were clustered in the 2.3.4.4 clade, and the NA genes were derived from H6N6 viruses. These H5N6 viruses bound both α-2,3-linked and α-2,6-linked sialic acid receptors, but they exhibited different pathogenicities in mice. In addition, one virus was fully infective and transmissible by direct contact in guinea pigs. These results highlight the importance of monitoring the continual adaptation of H5N6 viruses in poultry due to their potential threat to human health.

Figure 1

Phylogenetic analysis of HA and NA genes. Phylogenetic trees of the HA (A) and NA (B) genes were constructed using the distance-based neighbor joining method in MEGA7.0.21 software. The reliability of the trees was assessed by bootstrap analysis. Horizontal distances are proportional to genetic distances. The H5N6 viruses analyzed in the present study are indicated in blue. Human H5N6 viruses are indicated in red.

Figure 2

Agglutination activities of the H5N6 viruses for various red blood cells. Four types of red blood cells: a Chicken red blood cells (with α-2,3-linked sialic acid receptors and α-2,6-linked sialic acid receptors). b, Chicken red blood cells treated with α-2,3-sialidase (with only α-2,6-linked sialic acid receptors). c, Sheep red blood cells (with only α-2,3-linked sialic acid receptors). d, Chicken red blood cells treated with VCNA (no receptors). Figure 2A,B and C show the agglutination activities of DK01, CK918 and CK165 for the four types of red blood cells, respectively. Figure 2D represents the HA titers of DK01, CK918 and CK165 in the four types of red blood cells.

Figure 3

Pathogenicities of the H5N6 viruses in mice. Five mice per group were intranasally inoculated with the H5N6 viruses at 10⁶ EID₅₀. (A) Their body weights were monitored daily for 14 days. The values represent the average scores of overall body weight loss compared with the initial body weight ± standard deviations (SD). (B) The percentage of survival values were calculated by observing the infected mice. (C) Lungs were collected from the infected mice (n = 3) on the indicated days post-infection (dpi), and viral titers were determined in 9-day-old specific pathogen-free embryonated eggs.

Figure 4

Histopathology analysis. Lungs of the infected mice were fixed with formalin, embedded in paraffin and stained with hematoxylin and eosin. (Arrow a): alveolar wall thickening; (arrow b): epithelial cell shedding; (arrow c) lymphocyte infiltration; (arrow d) alveolar cavity decreasing or even disappearing; (arrow e) epithelial cell swelling and rarefaction of cytoplasmic structures.

Figure 5

Horizontal transmission of the H5N6 viruses between guinea pigs. Groups of three guinea pigs were inoculated with the indicated viruses. The next day, the inoculated animals were paired by co-housing with direct-contact guinea pigs; aerosol-transmission animals were also housed in a wire-frame cage adjacent to the infected guinea pigs. Nasal washes were collected from all the animals for virus shedding detection every other day beginning on day 2 after the initial infection. Each color bar represents the virus titer in an individual animal. Dashed lines indicate the lower limit of virus detection.