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.

A Turkey-origin H9N2 Avian Influenza Virus Shows Low Pathogenicity but Different Within-host Diversity in Experimentally Infected Turkeys, Quail and Ducks

“…The high complexity of the virus population in oropharyngeal swabs from quail resulted mainly from the preservation and amplification of the original viral diversity, but quail showed also the highest propensity for the generation of novel low-frequency variants. Similar observations were made for Japanese quail infected with clade 2.3.4.4 H5N2 HPAIV as they showed the highest number of polymorphisms in the excreted virus among the tested minor gallinaceous poultry species and chickens [51]. Comparing to chickens and turkeys, the scale of domesticated quail production is small, but it is a common species in mixed backyard holdings, which may support the multipath evolution of the virus once it is introduced into the flock.…”

“…The limited number of studies on the diversity of AIV at the level of individual birds precludes drawing any far-reaching conclusions. Due to the differences in the experimental setup (e.g., infection with HPAI viruses [51,52,54,55]) and the methodology (e.g., data on the virus diversity obtained with deep sequencing [18,51,52], sequencing of amplicons cloned into plasmids [54] or analysis of double peaks in chromatograms [55]), results of these studies cannot be directly compared. However, we show that the within-host diversity of AIV varies between species and noticeable differences may be found even in the same host in various replication sites.…”

Experimental infection of racing pigeons (Columba livia domestica) with highly pathogenic Clade 2.3.4.4 sub-group B H5N8 avian influenza virus

“…The levels of virus shed into the shared environment by the infected pigeons were insufficient to infect contact SPF chickens, indeed the levels of virus shed by the racing pigeons were well below experimentally-determined bird infectious dose 50 (BID 50 ) values for most poultry species, especially for chickens, where 10 3.5 to 10 5.7 EID 50 has been required for infection of depending on the strain of the Clade 2.3.4.4 H5 virus and the chicken breed (Tanikawa et al, 2016;Bertran et al, 2016a;Bertran et al, 2016b;Bertran et al, 2017;Pantin-Jackman et al, 2017;Spackman et al, 2017). Nonetheless, the low viral levels excreted from challenged pigeons appeared sufficient to infect in-contact pigeons.…”

Avian influenza overview September – November 2017

“…Bertran et al (2017) inoculated HPAIV A(H5N8) (A/Gyrfalcon/Washington/40188-6/2014), which they considered representative of the wholly Eurasian A(H5N8) lineage viruses into different species of minor gallinaceous poultry at 4 week of age: Japanese quail (Coturnix coturnix japonica), bobwhite quail (Colinus virginianus), pearl guinea fowl (Numida meleagris), chukar partridge (Alectoris chukar), and ring-necked pheasant (Phasianus colchicus). Bertran et al (2017) inoculated HPAIV A(H5N8) (A/Gyrfalcon/Washington/40188-6/2014), which they considered representative of the wholly Eurasian A(H5N8) lineage viruses into different species of minor gallinaceous poultry at 4 week of age: Japanese quail (Coturnix coturnix japonica), bobwhite quail (Colinus virginianus), pearl guinea fowl (Numida meleagris), chukar partridge (Alectoris chukar), and ring-necked pheasant (Phasianus colchicus).…”

“…Information extracted from the scientific literature Between September and November 2017, there were several articles on experimental inoculation of HPAIV A(H5N8) isolates from the 2014/2015 outbreak into domestic birds. Bertran et al (2017) inoculated HPAIV A(H5N8) (A/Gyrfalcon/Washington/40188-6/2014), which they considered representative of the wholly Eurasian A(H5N8) lineage viruses into different species of minor gallinaceous poultry at 4 week of age: Japanese quail (Coturnix coturnix japonica), bobwhite quail (Colinus virginianus), pearl guinea fowl (Numida meleagris), chukar partridge (Alectoris chukar), and ring-necked pheasant (Phasianus colchicus). Susceptibility varied among species; the least susceptible birds were Japanese quail, which required 3.2 log10 mean bird infectious doses (BID 50 ) for becoming infected.…”