Pathobiology of Clade 2.3.4.4 H5Nx High-Pathogenicity Avian Influenza Virus Infections in Minor Gallinaceous Poultry Supports Early Backyard Flock Introductions in the Western United States in 2014-2015

Abstract

In 2014 and 2015, the United States experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus. Initial cases affected mainly wild birds and mixed backyard poultry species, while later outbreaks affected mostly commercial chickens and turkeys. The pathogenesis, transmission, and intrahost evolutionary dynamics of initial Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses in the United States were investigated in minor gallinaceous poultry species (i.e., species for which the U.S. commercial industries are small), namely, Japanese quail, bobwhite quail, pearl guinea fowl, chukar partridges, and ring-necked pheasants. Low mean bird infectious doses (<2 to 3.7 log₁₀) support direct introduction and infection of these species as observed in mixed backyard poultry during the early outbreaks. Pathobiological features and systemic virus replication in all species tested were consistent with HPAI virus infection. Sustained virus shedding with transmission to contact-exposed birds, alongside long incubation periods, may enable unrecognized dissemination and adaptation to other gallinaceous species, such as chickens and turkeys. Genome sequencing of excreted viruses revealed numerous low-frequency polymorphisms and 20 consensus-level substitutions in all genes and species, but especially in Japanese quail and pearl guinea fowl and in internal proteins PB1 and PB2. This genomic flexibility after only one passage indicates that influenza viruses can continue to evolve in galliform species, increasing their opportunity to adapt to other species. Our findings suggest that these gallinaceous poultry are permissive for infection and sustainable transmissibility with the 2014 initial wild bird-adapted clade 2.3.4.4 virus, with potential acquisition of mutations leading to host range adaptation.IMPORTANCE The outbreak of clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus that occurred in the United States in 2014 and 2015 represents the worst livestock disease event in the country, with unprecedented socioeconomic and commercial consequences. Epidemiological and molecular investigations can identify transmission pathways of the HPAI virus. However, understanding the pathogenesis, transmission, and intrahost evolutionary dynamics of new HPAI viruses in different avian species is paramount. The significance of our research is in examining the susceptibility of minor gallinaceous species to HPAI virus, as this poultry sector also suffers from HPAI epizootics, and identifying the biological potential of these species as an epidemiological link between the waterfowl reservoir and the commercial chicken and turkey populations, with the ultimate goal of refining surveillance in these populations to enhance early detection, management, and control in future HPAI virus outbreaks.

Keywords: clade 2.3.4.4; gallinaceous poultry; highly pathogenic avian influenza virus; next-generation sequencing; pathobiology; transmission; virus-host adaptation.

FIG 1

Scatterplots of oral shedding detected by qRRT-PCR from birds inoculated with low (2 log₁₀ EID₅₀/0.1 ml), medium (4 log₁₀ EID₅₀/0.1 ml), and high (6 log₁₀ EID₅₀/0.1 ml) doses of A/Northern pintail/Washington/40964/2014 (H5N2) virus. The limit of detection for H5N2 virus was 1.7 log₁₀ EID₅₀/ml; a value of 1.6 log₁₀ EID₅₀/ml was given to qRRT-PCR-negative samples.

FIG 2

Scatterplots of oral shedding detected by qRRT-PCR from birds inoculated with low (2 log₁₀ EID₅₀/0.1 ml), medium (4 log₁₀ EID₅₀/0.1 ml), and high (6 log₁₀ EID₅₀/0.1 ml) doses of A/Gyrfalcon/Washington/40188-6/2014 (H5N8) virus. The limit of detection for H5N8 virus was 1.9 log₁₀ EID₅₀/ml; a value of 1.8 log₁₀ EID₅₀/ml was given to qRRT-PCR-negative samples.

FIG 3

Virus detection by qRRT-PCR in tissues from birds inoculated with 6 log₁₀ EID₅₀/0.1 ml of A/Northern pintail/Washington/40964/2014 (H5N2) virus or A/Gyrfalcon/Washington/40188-6/2014 (H5N8) virus. The limits of detection were 2.7 and 2.9 log₁₀ EID₅₀/g for the H5N2 and H5N8 viruses, respectively; for statistical purposes, negative samples were given values of 2.6 and 2.8 log₁₀ EID₅₀/g for the H5N2 and H5N8 viruses, respectively. Asterisks indicate statistically different mean viral quantities among tissues at different time points (Kruskal-Wallis test; P < 0.05).

FIG 4

Scatterplot of average numbers of nonsynonymous mutations at a 1% minimum variant frequency in virus subpopulations excreted at 3 dpc from birds inoculated with A/Northern pintail/Washington/40964/2014 (H5N2) virus. Means sharing the same superscript are not significantly different from each other (Tukey's posttest; P < 0.05).