Comparison of the pathogenic potential of highly pathogenic avian influenza (HPAI) H5N6, and H5N8 viruses isolated in South Korea during the 2016-2017 winter season

Abstract

Highly pathogenic avian influenza (HPAI) A(H5N6) and A(H5N8) virus infections resulted in the culling of more than 37 million poultry in the Republic of Korea during the 2016/17 winter season. Here we characterize two representative viruses, A/Environment/Korea/W541/2016 [Em/W541(H5N6)] and A/Common Teal/Korea/W555/2017 [CT/W555(H5N8)], and evaluate their zoonotic potential in various animal models. Both Em/W541(H5N6) and CT /W555(H5N8) are novel reassortants derived from various gene pools of wild bird viruses present in migratory waterfowl arising from eastern China. Despite strong preferential binding to avian virus-type receptors, the viruses were able to grow in human respiratory tract tissues. Em/W541(H5N6) was found to be highly pathogenic in both chickens and ducks, while CT/W555(H5N8) caused lethal infections in chickens but did not induce remarkable clinical illness in ducks. In mice, both viruses appeared to be moderately pathogenic and displayed limited tissue tropism relative to HPAI H5N1 viruses. Em/W541(H5N6) replicated to moderate levels in the upper respiratory tract of ferrets and was detected in the lungs, brain, spleen, liver, and colon. Unexpectedly, two of three ferrets in direct contact with Em/W541(H5N6)-infected animals shed virus and seroconverted at 14 dpi. CT/W555(H5N8) was less pathogenic than the H5N6 virus in ferrets and no transmission was detected. Given the co-circulation of different, phenotypically distinct, subtypes of HPAI H5Nx viruses for the first time in South Korea, detailed virologic investigations are imperative given the capacity of these viruses to evolve and cause human infections.

Fig. 1. Growth kinetics and attachment of viruses in human lung tissues ex vivo.

Replication of two Em/W541(H5N6) and CT/W555(H5N8) viruses were monitored in Madin-Darby Canine Kidney (MDCK) cells (a), Normal Human Bronchial Epithelial (NHBE) cells (b), Human lung tissue explants and corresponding culture supernatants (c and d, respectively) starting at 12 and 24 hpi intervals thereafter. Growth kinetics were compared to those of the control 2009 pandemic CA/07(H1N1) virus. The titers shown are means ± SD from three independently performed experiments. (*p < 0.05, **p < 0.01, ^†p < 0.001)

Fig. 2. Replication of Em/W541(H5N6) and CT/W555(H5N8) in avian models.

Virus replication was examined in chickens and ducks that had been experimentally inoculated via the intranasal route with 10⁶ EID₅₀/ml of each virus. Oropharyngeal swabs, cloacal swab, and lung titers of chickens are shown for Em/W541(H5N6) (a) and CT/W555(H5N8) (b). Oropharyngeal swabs, cloacal swabs, and lung titers from ducks are shown for Em/W541(H5N6) (c) and CT/W555(H5N8) (d). Mean viral titers (log₁₀ EID₅₀/ml) are shown for each group of birds. The limit of virus detection was 0.7 log₁₀ EID₅₀/ml

Fig. 3. Cytokine and chemokine responses in the lungs of infected mice.

Concentrations of various cytokines/chemokines in BAL fluid from mice at 1, 3, 5, and 7 dpi were measured by protein analysis with the Luminex™ Instrumentation Systems multiplex array reader (Bio-Plex Workstation from Bio-Rad Laboratories). The values shown are means ± SD (error bars) from BAL fluid of three mice per time point tested. (*p < 0.05)

Fig. 4

Histopathology and immunohistochemical staining for influenza virus antigen in mouse (a, b, e, and f) and ferret (c, d, g, and h) lungs at 3 (a, c, e, and g) and 5 dpi (b, d, f, and h) with HPAI Em/W541(H5N6) and CT/W555(H5N8).In the lung, viral antigens are widely presented in the alveolar septum which are mildly thickened with increased cellularity of inflammatory cells and also necrotic cell debris are presented in the lumen (arrows in a, b, e, and f)

Fig. 5. Replication of Em/W541(H5N6) and CT/W555(H5N8) in ferrets experimentally inoculated via the intranasal route with 10⁶ EID₅₀/ml of each virus.

Rectal temperatures and body weights were measured at 1, 3, 5, 7, 9, 11, and 14 dpi (a and b). Individual nasal wash titers are shown for ferrets inoculated with Em/W541(H5N6) and CT/W555(H5N8). To examine transmission, the inoculated animals were individually paired with a direct contact (DC) and a respiratory droplet (RD)-contact animal (1:1:1 setup, triplicate) (c and d). The mean viral titers (log₁₀ EID₅₀/ml) are shown for each group of mammals. The limit of virus detection was 0.7 log₁₀ EID₅₀/ml